Molecular cytogenetics in prenatal diagnosis

Transcription

1 Molecular cytogenetics in prenatal diagnosis Adriana Rosalia Maria Van Opstal

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3 Molecular cytogenetics in prenatal diagnosis Moleculaire cytogenetica in de prenatale diagnostiek Proefschrift Ter verkrijging van de graad van doctor aan de Erasmus Universiteit van Rotterdam op gezag van de rector magnificus Prof. dr P.W.C. Akkermans M.A. en volgens het besluit van het college voor promoties De openbare verdediging zal plaatsvinden op woensdag 4 maart 1998 om uur Door Adriana Rosalia Maria Van Opstal geboren te Hoogstratcn

4 Promotie -COllullissie Promotor: Overige leden: Prof. dr I-I. Galjaard Prof. dr J.\V. \Vladimiroff Prof. dr N.l Lcschot Prof. dr P.J. Willems Co-promotor: Dr F.J. Los Omslag: Anllancio P.G. Braat The studies described in this thesis were perfonned at thc Department of Clinical Gelletics of the University I-Iospital Dijkzigt and the Erasmus Ulliversity Rotterdam. This project was financially supported by the Rotterdam Foundation of Clinical Genetics. Prillted by Copynomie, Rotterdam

5 Contents Chaptel' I Intl'oduction I. Traditional prenatal cytogenetic diagnosis Amniocentesis Chorionic villus sampling Number of amniotic fluid (AF) and chorionic villus (CV) samples rcccivcd 14 between 1970 and 1996 IA Limitations of traditional c)1ogenetic studies in (AF) and (CV) celis Chromosomal mosaicism in AF cell culturcs Confined placentallllosaicislll ~vfolecular cytogenetics 22 Chaptcl' II Application of fluorcsccnt in situ hybridization (FISH) in pl'enatal diagnosis I. Aim of the experimental work rvlaterials and lllcthods ofthe FISH technique Slide preparation and prctreatmcnt DNA probes and labelling Probe hybridization and detection Evaluation oftlle slides Results and discussion FISI-I tor idcntification of structural chromosome aberrations Intcrphase FISH [or the rapid detection ofnulllcrical Crn0I110S01l1C 46 aberrations in uncultured alllniotic tluid cells Ultrasound abnormalities Cytogenetic abnonnality in previous chorionic villus sample Otller indications Interphase FISH for studying chromosomai mosaicislll Mosaicism in amniotic fluid ceii cultures Mosaicisl11 in semi~direct chorionic villus preparations FISH for detection ofmicrodeletions 63 Chapter III Fetal uniparental disomy with and 'without confineu placenta I Jtlosaicism 1. Aim ofthe experimental work 2. Results and discllssion 2.1 lncidence ofuniparental disomy associatcd with confined placental mosaicism 2.2 Uniparental disomy without confined placental mosaicism: a model for trisolllic zygote rescue Concluding rcmal'lis :lilu futul'e pl'ospccts Rcfcl'cllcCS Summary Samenvatting Abbl'eviations

6 CUl'l'iculuJU yitac List of publications Dankwoord Appendix publications I-VIII Publication I D. Van Opstal, H.l. Eussen, J.O. Van Hemel, E.S. Sachs (1993). Application of fluorescent in situ hybl'idization for 'de 1l0YO' anomalies in prenatal diagllosis, Prenat. Diagn. 13, Publication 11 F.J. Los, C. van den Berg, AP.G. Braat, F.K. Cha'ban, lm. Kros, D. Van Opstal (1996). Ring chl'omosome 18 in amniotic fluid ce lis of a fetus witit only facial dyslllol'phisms, Am. 1. Med. Genet., 66, Publicationlll D. Van Opstal, 10. Van Hemel, E.S. Sachs (1993). Fetal aneuploidy diagnosed by fluorescence in situ hybridization within 24 houl's aftel' amniocentesis, Lancet, 342, 802. Publication IV D. Van Opstal, J.O. Van Hemel, B.H.l. Eussen, A. van der Heide, C. van den Berg, P.A In 't Veld, F.l. Los (1995). A chl'omosollle 21 specific coslllid cocktail fol' the detection of clll'omosoillc 21 aberrations in intcl'phase nuclei, Prenat. Diagn., 15, Publieatioll V D. Van Opstal, C. van den Berg, M.GJ. Jahoda, H. Brandenburg, FJ. Los, P.A. In 't Veld (1995). Retl'ospective study of tl'îsomy 18 in chol'ionic Yilli witit fluorescent in situ hybridization on archival dil'cct pl'cparatiol1s, Prenat. Diagn., 15, Publication VI J.O. Van Hemel, C. Schaap, D. Van Opstal, M.P. Mulder, M.F. Niermeijer, lh.c. Meijers (1995). Recul'rellce of DiGeo!'ge s)'ndl'ome: antenatal detection h)' FISH of a molecula!' 22q11 deletion, l Med. Genet., 32, Publication VII D. Van Opstal, C. van den Berg, \V.H. Deelen, H. Branclenburg, T.E. Cohen-Overbeek, D.J.J. Halle)', AM.W. van den Ouweland, P.A. In 't Veld, F.J. Los. Prospective prenatal invcstigations on potcntial uniparental disollly in cases of confined placental trisomy, Prenat. Diagn., in press. Puhlication VIn F.J. Los, D. Van Opstal, C. van den Berg, AP.G. Braat, S. Verhoef, E. \Vesbcy-van Swaay, A.M.\V. van den Ouweland, D.J.J. Halley. Uniparental disomy with and without confined placental Illosaicism: a model fol' tl'isomic zygote l'escue, Prenat. Diagn., in press

7 Chapter I Introduction

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9 1. Traditional prenatal cytogenetic diagnosis Prenatal diagnosis (PD) startcd in the fifties when variotls groups indicated the possibility of prenatal sex determination in amniotic fluid (AF) cells (SeIT et al., 1955; Fuchs and Riis, 1956; Dewhurst, 1956). After the first succesful attempts at AF cell cultivation and karyotyping by SteeIe and Breg (1966) and T1tiede et al. (1966), the first small series of prcnatal chromosomc analyses were presented (Jacobsoll aud Barter, 1967), aud the ficst chromosome aberrations in cultured AF cells were detected (Valenti et al., 1969). AF cells could also be used for prenatal detcction of inham errors of metabolism (NadIer, 1968). Our eentre made an important international contribution towards experienee with a large number of biochemical assays, and the devclopment of ultramicrochemical techlliques penllitting a rapid PD (Galjaard, 1972; Niermeijer, 1975; Galjaard, 1976a, 1979, 1980; Galjaard et al., 1977; K1eijer, 1990). AtlOther important contribution to PD was the finding by Broek and Sutcliffe (1972), that the alpha-fetoprotein level in AF is increascd when the fetus has an open neural tube defect. Amniocentesis is generally perfonlled in the second trimester of pregnancy. The major disadvantage is a midtrimester tcrmination of the prcgnancy in case of a fetal chromosome abnonnality. This may cause serious psychological stress and burden for the parents (ThomassenMBrepols, 1985). Advances in the techllology associated with amniocentesis using high resolution real time ultrasound and improved laboratory methods have made it technically possible to perfofm amlliocentesis prior to 15 weeks of gestatioll, but the safety and accuracy of this "early amniocentesis l1 teclmique has yet to be establishcd (reviewed by Wilson, 1995). The first attempts at first trimester PO were made by Kullander and Sandah1 (1973) and Hahneman (1974) who used long-term cultures of chorionic villi (CV) from fust trimester abortiolls for fetal karyotyping. In 1975, Yamamoto et al. described the use of spontaneous mitoscs in CV for chromosome analysis after induced abortions. That same year, a Chinese group (Atlshan Iron and Steel Company, 1975) reported fetal sex prediction by sex chromatin analysis in direct preparations of CV during early prcgnancy. The origin of spontaneous mîtoses in CV was investigated by \Vatanabe et al. (1978) who found them to be derivcd from the Langhans cells of the cytotrophoblast and not to be present in syncytiotrophoblast and mesodermal core of CV. In 1983, Simoni et al. described all efficient method to obtain metaphases from chorionic villi cytotrophoblast tissue within a few hours after sampling, making a rapid prenatal cytogenetic diagnosis possible in the first trimester ofprcgnancy. This led to the first PD of trisomy 21 at 11 weeks of gestation within five hours aftel' sampling (Brambati alld Simoni, 1983). In the Netherlands, CVS was first employed in Rotterdam in 1983 (Jahoda et al., 1984; Galjaard, 1985; Sachs et al., 1985). M Nowadays, second-trimester amuiocentesis and firsl ll'imester chorionic villus sampling are two widely used invasive tcchniques for PD of chromosome abcrrations. Invasive implies that specimens arc obtained from the fetus or from associated fetal structures Of products by needie pullcture or biopsy technique, in contrast with non-invasive techniques, such as ultrasound investigation. 9

10 1.1 Amnioccntesis Seeond-trimester amnioeentesis is defined as an amniocentesis peformed at 15-HJ weeks of gestation with a cytogcnetic result available after days (Wilson, 1995). This gestational age for the procedure was established in the seventies, when various reports noted that AF could be obtained at this time of pregnancy with an aceeptable degree of safety and aecuracy (Nationallnstitutc of Child Health and Human Developmcnt, 1976). AF is remm'ed from the intrauterine gestational sac by needie aspiration t1l1der continuous ultrasound guidanec. About 20 mi of AF is aspirated, which rcpresents 12,5 % of thc total volmne at 16 weeks (Finegan, 1984). During pregnaney the total number of AF eells per mi increases, although the percentage ofviablc cells declines, being the highest at weeks of gestation (24 %) and it decreases to 9-10 % at weeks of gestation (Galjaard, 1976b). A number of studies have been perfoflllcd on the different ecu types present in uncultured (Huisjes, 1978; Papp and Bell, 1979; T)'dén et al., 1981) as well as cultured AF (reviewed by Gosden, 1983; Chang and Jones, 1988). Tydén et al. (1981) studied the origin of cells in midgestational AF hl' using scatuling electron microscopl', both of the surface ultrastmctural morpholog)' of the AF cells and the fetal surfaces exposed to the AF. The)' idenlified four cell populations derived from periderm, umbilical eord, orai and nasal lllucosa, and from the vagina, respcctivell'. Non-shedding epithelia, whieh do not eontributc a significant number of cells to the AF, are observed in the respiratof)' tract, the urinary bladder, aud the amniotic memhrane. The cells prcsent in the AF have to be cultured, bcfore chromosome stides can be made. The preparation of these eclls for karyotyping eau be carried out aecording to two main priuciples: the n in situ" preparation techniquc, allowing karyotyping of individual cell colonies, and the "flask tuethod l1 requiring trypsinizatiol1 of the eells before harvesting, wwch obviously disrupts colon)' integrity (Roone)' and Czelpulkowski, 1992). According to our protocol, the ceu suspcnsion of 20 mi of AF is seeded over five culture dishes, and the cultures are harvested usîng the in situ method. Trypsill-Giemsa staining is routinell' used for banding of the chromosomes (Seabright, 1971), 8nd we investigate 16 cell colonies per patient. 1.2 Chol'ionic villus sampling (CVS) In our PD centre, CV are mainly s8mpled transabdominally at wceks of gestation. There are two reasons for discarding sampling at an earlier gestational age. Firstll', older women (;;>:36 years), who represent the major indication group for PO, have a higher spontaneous abortion rate in early pregnancl' before 12 wecks of gestation which affected the post-procedure fetal loss rate (Jahoda et al., 1987, 1990; Cohen-Overbeek et al., 1990 ). Seeondly, there might be an association bet ween early CVS during {he critical stage of development of various organ systems and some vascular disruptive syndromes (Firth et al., 1991, 1994; Jahodaetal., 1993; Los et al., 1996). CV for prenatal investigatiolls are biopsied from thc chorion frondosum. Thel' consist of tluee 10

11 major components: (1) an outer Jayer of honnonally actî\'e syllcytiotrophoblast, (2) a middle Jayer of cytotrophoblast from which the syllcytiotrophoblast is derived, and (3) an inner mesodermal core containillg blood capillaries (Figure l.i). A mean of mg vhli is usually obtained alld is llecessary for a succesful chromosome preparatioll. Additiollal biochemical or DNA-illvestigations require all extra mg of CV. Figure 1.1 Cross section through a chorionic viiilis, showing Us major compollellts., s)'llc)'liolrophobbs\ -- + CyloIRll'hob!a,t There are two ways of processing CV for cytogelletic studies: the direct and long-term preparatioll teclmique: -the direct preparatioll tecjmique, first described by Simoni et al. (1983), utilizes the spontaneously dividillg Lallghans ceils in the cytotrophoblast of CV. Figure 1.2 shows a cross section through a CV sho\villg the presence of spontancotls mitoses in the cytotrophoblast. Since the introduction of dus technique, some modifications were introduced by several investigators (Gibas et al., 1987; Terzoli et al., 1987; Simoni et al., 1990). We use the semidirect preparation techniquc described by Gibas et al. (1987). 11

12 Figurc 1.2 Cross section through a chorion Ic "mus, showing a spontaneolls mitosis in the cytotrojlhoblast. A) magni(ication of250 x, and B) of750 x. B -the IOllg-term preparatioll l1iethod consists of a trypsin (0,05%)-EDTA (0,02%) and a eollagenase treatment of the villi before setting up cultures, as described by Smidt-Jensen et al. (1989). \Vhereas the metaphases in the direct chromosome preparations are derived from the cytotrophoblast, those in long-term cultures are assumed to be predominantly of mesenchymal origin. Since cytotrophoblast and mesenchymal core of CV have a different embryogenic origin (Crane and Chellng, 1988; Bianchi et al., 1993), karyotyping bath cell types represents the investigation of two different compartments.îherefore, most centres for PD pref er to use the direct and long-term preparation method simuitaneously to improve the accuracy ofpd on CV, which wiii be discussed extensively in section This approach, however, requires all amollnt of CV of at least 20 mg. As the weight of thc CV samples in our centre did generally not exeeed mg during the study period, we could only perform one of both methods. As culturing of CV is very laboureotls and time-consuming, delaying the reporting time of a cytogenetic result to the parents, and therefore obviate the main advantage of CVS for prenatal diagnosis, and because of the risk of maternal eeu contamination, we prefered the use of the semi-direct method only, eventually followed by amniocentesis for n.111her investigation of uncertaill cytogenetic results. Since January 1997 we use bath preparation methods for samples of at least 20 mg. Chromosome analysis is routinely perfornled using trypsin-giemsa staining (Scabright, 1971). For lllost indieations, we analyse cight eells and eount another eight. A metaphase of average quality and its karyotype are ShOWll in figure

13 Figure 1.3 Mctaphase spread (A) and karyotype (B) of aycrage qualhy in chorion ie "Hli semi-direct preparation. A " '\f I (,V 1~,>ry "J.04. h(i ~~\ B l5 'ij ',' i i IJ rl ~~ :~J "!i \t~ 3, 5 4) t~ ;';.:jj ~I:l 'IJ t~ ~;a!~ 6, 9 JO i?~ h ~rt!{,~ ~1 ~~ t) c\ 13 " is "i,-. 't t?'} " - ",.. ~,. (;{ X Y 13

14 1.3 Numbcr of AF and CV samples recei"ed in thc pcriod Between 1970 and the end of 1996, 25,073 AF and 1l.l40 CV samples wcrc recieved in our laboratory [or prcnatal chromosome analysis (Niermeijer ct al., 1976; Galjaard et al., 1982; Sachs ct al, 1982; lahoda et al, 1984, 1985; Galjaard 1985; Sachs et al, 1985, 1990), The evolution of the numbers of AF and CV samples during that time period is shown in t1gure 1.4. After thc introduction of CVS in 1983, the number of AF samples decreased with a concomitant increase in the numher ofcvs. Howevcr, sincc 1992, a decrease in the llull1bcr of CV samples occured, together with an increase of AF samples, due to the yet unsolvcd problem of inducing fetai vascular disruptive syndromes by CVS, as mentiolled abovc, and because of the limited representativity of un abnormal karyotype in CV for the actual fetal karyotype, which \ViII be discussed in section Figure 1.4 Number or allllliotic nuid ajul ehorionie vim samples reeci\'cd during the time period , N 2000,---- _ Chorionic vuli 1500 '- c=j Amniotic fluid 1000 '- 500 '- '70 '75 '80 '85 '90 '95 y", 1.4 Limitations of traditional eytogenetie nnalysis of AF nud CV eells Traditional cytogenetic analysis of AF and CV eells has a few limitatiol1s: 1) chromosome abnonnalities with indis/ijlel bandillg palferl1s, such as marker ehromosomes or de-nova stmehlral rearrangements, are aften difficult to interpret. The additional use of different staining techniques is important for their identit1cation (Saehs et al., 1987), but can 14

15 not always give a definite diagnosis. 2) thc main limitation of alllniocentesis is the time-consumblg cell cultlll'ing which is required far generating sufficient high-quality chromosame spreads, so that a result is anly achieved twa ta tluee weeks after sampling. This long waiting-time [or aresuit may be a burden an the [uture parents, especially if the pregnancy is at high genetic risk. In the past, several attempts have been made ta use unculturcd AF eells for fetal sex determinatian in pregnancies at risk far X-linked diseases, by demonstratian of X and Y ehromatin in interphase nuclei (Pearson et al., 1970). However, this method turned out to he unreliahle (Gosden, 1983). 3) chromosomal ljlosaicism, defllled as the presence of at least two karyotypically different celllines within an individual (Gosden et al, 1995), may cause interpretation problems, as it may represent true fetal mosaicism ar pseudomosaicisl11 (culture artefacts withaut clinical significanee). Discriminatian between bath phenomena is therefore critical and requircs the analysis of a large number af cells, whieh is often difficult due ta limited time and sample size. 4) the detection of a (non)-masaic chromosome aberration in CV sometimes poses an interpretation dilemma, as the chromosome aberratian may be eonfined to placental tissue (cojiflned placental IJ/osaicism (CP.J\l) and nat be present in the fetus itseif. If CPM is suspeeted, follow-up investigatians are nccessary, including ultrasound examinatian af the fetus as weil as karyatyping AF cells or fetallymphocytes, which severcly deiay the reporting time. 5) although most ehroillosome aberrations that are detected prenatally are numerical abnormalities (trisamy 13, 18, 21, triploïdy, sex-chromosomal aneuploidies), structural cluamasame rearrangements account for a smal! but significant proportion of the abnormal karyatypes, and their detectian is limitcd by the resolutioil of fhe light micl'oscope. Today, cluomosome methodalogy in classical cytogcnetics has reached a stage of resolution that allows the detection of chromosome rearrangements invalving about 6x I 0 6 base pairs (Ferguson-Smith, 1988). Smaller changes, such as microdeletians, are difficult or impossible to identify with classical cytogenetic techniques. 6) maternal cell col1tamil1atiol1 (MCC) of the sample may lead to incorrect sex prediction alld potentially to a false negativc diagnosis. It is regularly found in CV long-term cultures (frequency of 1 % 10 3 %) (Ledbetter et al., 1992a; Smidt-Jensen et al., 1993; ACC working Party on Chorianic Villi in Prenatal Diagnosis, 1994), although it is extremely rare in AF ccll cultures (frequency of 0 % to 0,3 %) (Benn et al., 1983; Hsu aud Perlis, 1984; Bui et al., 1984; \Vorton and Stern, 1984) and CV semi-direct preparations (frcquency of 0 % to 0,4 %) (Simoui et al., 1986; Ledhetter et al., 1992a; ACC Working Party on Chorionic Villi in Prenatal Diagnosis, 1994). Limitatians 3 and 4 will be further discussed in detail in sections and 1.4.2, respectively. 15

16 1.4.1 Chromosomal mosaicism in AF eell cultures The accurate diagnosis of chromosomal mosaicisl11 in AF cell cultures represents a problem in PD. True fetal mosaicism may go undetected if an insufficient number of AF cells is analysed. A false positive diagnosis is also possible, since a chromosome abnormality ean arise in~vitro and may not reflect the actual fetal chromosome constitution; this situation is designated pseudomosaicislll. True chromosomal mosaicisll1 is generally defined as the presence of an identieal chromosome abnormality in at least lwo independently cultured dishes, whereas pseudomosaicism involves multiple eell colonies with the same chroll1osoll1e aberration restricted to one culture dish (pseudomosaicism type C), one colony with a chromosome abnormality with the other colonies of the sample being normal (pseudomosaicism type B), or a partial abnonnal colony (pseudomosaicism type A)(Boué ct al., 1979). Four large collaborative studies (Hsu aud Peri is, 1984; \Vorton and Stern, 1984; Bui et al., 1984; Hsu et al., 1996) provide probably the best data available on mosaieislll and pseudomosaicisll1 in AF cell cultures. The combined frequencies of chromosome mosaicislll and pseudol11osaicislll ranged from 3,4 % to 8,5 % in these four large surveys. Due to the relatively high incidence ofpseudomosaicislll and the fear ofll1issing a case oftme chrom.osoll1e mosaicism, most laboratories use a two~stage approach in the work~up of AF eeu culturing and karyotyping for the diftèrentiation of tme mosaicislll from pseudomosaicism (Hsu and Perlis, 1984; Cheung et al., 1990). In most eytogenetic laboratories the routine karyotyping involves thc examination of I O~ 16 ceu colonles from multiple in situ culture dishes. If all cells show a normal karyotype, a normal result is reported. If one or more ceil colonies in one culture dish show an abnormality, additional colonies from otller dishes are studied. This two~stage approach has been considered more efficient and more cost~effective in work-up for chromosome mosaicism than routinely analysing a large number of ceus in every AF sample (Cheung et al., 1990). The number of additional ccu eolonles that need to be evaluated to exclude tme chromosome mosaicism at a given confidence level can be derived from tables proposed bl' Hook (1977), or those developped by Cheung ct al. (1990), Featherstone et al. (1994), and Sikkcma-Raddatz et al. (1997). Hsu et al. (1992) proposed time different levels of \Vork-up for the exc1usion of potential mosaicislll, depending on the chromosomc abnonnality involved, based on available karyotype/phenotype coitelation data: extensivc, moderate, and no additional work-up (tabie 1.1). Extensive aud moderate additional work~lip mean the analysis of24 and 12 eell eolonies, respectively, from multiple in situ culture dishes, not including the colonies from the culture dish in whieh the abnormal colony/colonies were found. According to the tables of Hook (1977), this auows the detectioll of 12 % and 24 % mosaicism at a 95% confidencc level, respectively (Hsu et al., 1992). Thc t\vo~stage approach offers a high degree of confidenee in excluding mosaicisll1. Benn et al. (1984) made a cmde estimate ofthe extent to which true mosaicisl11 might be interpreted as pseudol11osaicism, or cntirely missed, based on data from the US survey (Hsu and Pedis, 1984). It was concludcd that at the most 4,5 % of cases of truc mosaicism may be completely missed and up to 7 % could be misdiagnosed as pseudomosaicisl11. Examples of both 16

17 situations are described in the literature (\Vo1stenholme et al., 1988; Cheung et al., 1988; Terzoli et al., 1990; Voeldey et al., 1991; Sehneider et al., 1994; Hanna et al., 1995; Wolstenholme, 1996). Table 1.1. Propose<l gui<lclincs for work~ujl of possible pseudomosaicism/mosaicism, using fhe in situ method (Hsu ct al., 1992) A. Indications fol' extensi\'e work~up I. Autosomal trisomy involving ehromosomes 8, 9, 12, 13, 14, 15, 18,20,21, or 21. (Seo, Meo) 2. Unbalanced structural rearrangement (MCo) 3. n. larker chromosomc (1""fCo) B. In<lications for moderate work~up 4. Aulosomal trisomy involving chromosomes 1,2,3,4,5,6,7,10,11,16, [7, or 19 (Seo, Mco) 5. Uilbalanced structural rearrangement (SCo) 6. Marker chromosome (SCo) 7. Extra sex ehromosome (SCo, MCo) 8. 45, X (SCo, MCo) 9. Balaneed struetural rearrangement OVICo) 10. r-,'ionosomy (other than 45,X) (SCo, t\'fco) C No additional work~up 11. Balanced struetural rearrangement (SCo) 12. Break at eentromere with loss ofone arm (SCo) 13. All single eell abnormalities Note.- Seo: single eolony/single dish; MCo: multiple co[ollics/sillgle dish 1.4,2, COllfincd placcntal mosaicislll Since fetus and placenta originate from thc same zygote, their chromosomal complement is expected to be the same. I-Iowever, in 1-2 % of via bic pregnancies studied by CVS, the cytogenetic constitution of fetus and placenta is diftèrent. Firstly, a (non-) nlosaic chromosome abnonnality may be confined to the placenta and be not present in the fcttls. This phenomcnon is called confined placcntal lllosaicism (CPM). It was first described by Kalousek and Dill (1983) in term placentas of infants bom with unexplained intrauterine growth rctardation (IUGR) and it was soon recognised in first trimester CV aftel' the introduction of CVS tbr prenatal cytogenetic studies (Simoni et al., 1985; Mikkelsen et al., 1985; Vejersle\' anct Mikkelsen, 1989; Lesehot et al., 1989; Saehs et al., 1990; MRC Working Party on the Evaluation ofchorionic Villus Sampling, 1991; Lcdbetter et al., 1992a; Teshima et al., 1992; Breed, 1992; Association of Clinical Cytogeneticists Working Party on Chorionic Villi in Prenatal Diagnosis, 1994; Wang et al., 1994; \Voistenholme et al., 1994; Pittalis et al., 1994; Leschot ct al., 1996; Hahnemann and Vejersle\', 1997). Three different types of CPM, according to the compartmcnts of the cv involved, ean occur: type I (confinemcnt of chromosome abnonnality to cytotrophoblast «semi)-direct preparations) of CV), type 11 (confinement of abnormality to mesenchymal core (long-term cultures) of CV), alld type III 17

18 (both cytotrophoblast and mesenchymal core abnonnal) (Kalousek, 1990; Kalousek et al., 1992). Secondly, the converse pattern, normal CV results and a (l1on-) mosaic abnonnal karyotype in thc fettls, has also been observed, although it is extremely rare and it seems ta be l11uinly restricted to the (semi-) direct preparation method (Martin ct al., 1986; Simoni et al., 1987; Leschot ct al., 1988; lvliny et al., 1988; Ledbetter et al., 1992; Piualis et al., 1994; Hahnemann and Vejerslev, 1997). To the best of our knowjedgc, only two cases of tàlse negatî\lc results of both methods «semi-) direct and long-term preparation method) were dcscribed by Pindar ct al. (1992) and Pittalis et al. (1994). Earl}! embrvonic development DUrÎllg early embryogenesis. a complex sequence of events will lead to the formation of distinct embryonic and extra-embryonic components (figure 1.5). Thc inner cellmuss (lcm) and cytotrophoblast ean first be distinguished in the late momla (32 eells). Blastocyst formation is apparent at the 64-cell stage. The blastocyst consists of an outer layer of trophoblast cejls, whieh wiil give rise to the cytotrophoblast of the placenta (studied in semidirect CV preparations), and an lcm, now rcpresented by approximately 16 eells, of which three to four cells wiil develop into the embryo, whereas the remaining cells will form the extraembryonic mesoderm (studied in long-term CV cultures) (Markert and Petters, 1978; Crane and Cheung, 1988; Bianchi et al., 1993). Chromosomal mosaieism is eauscd by abnormal eell divisian arising in early embryanic development. \Vhile early divisian errors may eause generalized l11asaicism, later errors, affeeting speeific eell lineages, will lead ta a masaic cancephls with masaicisl11 confined to either the placenta or the embryo/fetus. Sa time and plaee of a postzygotic ll1itotic eltor in a chromosomal normal or ahnormal situation will determine the pattern of mosaicism. Pittalis et al. (1994) proposed a detailed classificatioll of all theoretical combinations of karyotypes in the various placenta! and fetal compartments (eytotrophoblast, mesenchymal core, and fetus/embrya), and they werc grouped in 11 categories. All but one were found in a consecutive series of 4860 CVS diagnoses, demonstrating a considerabie cytogenetic variability alang the trophoblast-embryo axis. Origin o[trisomv CP.1\! CPM involvillg a trisomy, representing about 50 % of all CVr"I eases, may result from a soma tic duplication of a whole chromosome in placental progenitor cells originatil1g from a diploid zygote (mitotic CPivD, or from a trisoluic concephls with loss of the extra chromosome in embryonic, but not in placelltal progenitor eells (meiotic CPM); this phenomenon is cal1ed trisomic zygote rescue (Kalousek and Vekemans, 1996). A mitotic origin of cpr"i likely results in low levels of mosaicism (\Volstenholme, 1996; Robinson et al., 1997), whereas meiatic Crrvf was shown to be significantly correlated with high levels of abnormal cells in both placelltal lineages (Robinson et al., 1997). t\/leiotic CPÎvl ean theoretically be associated with the phenomenon of fetal uniparental disomy (UPD), i.c., both chromosomes of a chromosome pair derived from one parent oni)' (Engel, 1980). The chance is 66,7 % that one of the chromosomes from thc parent eontributing two copics wiii disappear, and 33,3 % that the one from the parent contributing one copy wiii be lost; in this last situation both cluomosomes left are derived from one parent oniy (Engel and Dclozier B1anchet, 1991) (figure 1.6). 18

19 Figurc 1.5 Early cmbryonic developmcnt, according to Crane and Chcung (1988), and Bianchi et al. (1993). Moruln (32 eell st.:tge) Blaslocysl (64 cell sutge) EPib~< ~ ~------> mb'yook ''''''=}--- Embryonic meso<lcnn Embryonic endo<lenn Amniotic eclodenn Embryo Amnion '" Morula Inner ecu ll'la.."5 (lcm) Hypoblast --c Amniotic mesodenn Embryonic blood cells? extra-cmbryonic meso<lenn (EEM) Mcsenchymal core (CYS cultures) Trophoblast Cytotrophoblast (Semi direct CYS preparations) ~ Syncytiotrophoblas! Chorion Villi / Plncent.:t

20 Depending on the mciotic division in which the non~disjunctional error occurred, and on the extent of crossing~over between thc homologues of the ciuomosome pair, prior to that meiosis, UPD can be heterodisomic, isodisomic, or combined hetero/isodisomic (Engel, 1980; Engel and Delozier-Blanchet, 1991). Fetal col1tirmatiol1l'ale ofcvs Illosaicism If chromosomal mosaicism is detected in first trimester CV, the overall fetal confirmation rate is about 10 % (PiUalis et al., 1994; Phillips et al., 1996) [compared with % when fol!nd in AF cell cultures (Hsl! et al., 1992)]. It has been demonstrated that the risk of fetal mosaicism is related to two factors: the cell type in whieh the abnonnality is seen and the type of cluol11osome abnormality. Firslly, it has repeatedly been shown that cultures of CV mescnehymal core are more likely to reflect the fetal chromosomal constitution than the direct cytotrophoblast preparatians, which is explained by the cmbryonic madels proposed by Crane and Cheung (1988) and Bianchi et al. (1993). PiUalis et al. (1994) found that the predictive value for all abnormal fetal karyotype rised from 12,1 % for mosaic anomalies observed in direct preparations, to 27,3 % for those observed in thc culture method, to 66,7 % for mosaic karyotypcs detected in bath lllcthods. Therefore, many investigators advocate the use of both direct cytotrophoblast preparations and mesenehymal core cultures to improvc accuracy of fctal chromosome studics in CV preparations (Saehs et al., 1990; Ledbctter et al., 1992a; Teshima et al., 1992; Pittalis et al., 1994). Secondly, the illcidenee of generalized mosaicism also varicd according to the type of chromosome abnormality. A mosaic polyploidy on CVS was confirmcd in about 4 % of cases. \Vhen a marker chromosome was involved, it was confirllled in the fetus in 27 % of cases. Mosaicism involving thc common trisomies (13, 18, 21) were foulld in fetal tissues in 19 % of cases in contrast to uneommoll trisomies (3, 5, 7, 8, 9, 16, 20, 22), which account for about 40 % of placental mosaicislll and which were confirmed in the fetus in onl)' 3 % ofcases (Phillips et al., 1996). Pregl1ancv ou/come ijl cases OfCP1H I f chromosomal mosaicislll is detected in CV, follow-up studies arc necessary to verify the fetal karyotype. However, even if the chromosome abnonnality is found ta be absent in AF celis, a normal pregnancy outcomc can not be ensured. Although thc majority of pregnancies with CPM proeeed uneventfully (Leschot et al, 1996), a numbcr of cases were faund to be associated \\1th lugr (Kalousek et al., 1991; Kalousek, 1993; Wolstenholme et al., 1994), fetalloss (Goldberg et al., 1990; Breed et al., 1991; Wapner et al., 1992), or poor perinatal Ol!tcome (Johnson et al., 1990; Brandenburg et al., 1996). This ma)' be explailled b)' three mechanisms: Cl) placental function may be disturbed by the presence of cytagenetically abnonnal cells (Kalousek, 1993; \Voistenholme, 1994), as also indicated by thc rcported associatioll between all abnonnal profile ofmaternal serum markers and CPM, especially CPM 16 (Vaughan et al., 1994; Zillllllerman et al., 1995; Tantravahi et al., 1996) (2) UPD ma)' be present in the disomic cell line in case of trisomy CPM, and depending on the chromosame involved, it may be harmfu! ta tbe tètus. UPD may affect human 20

21 Figure 1.6 $chematic presentation oftrisomic zygote rcscue, resulting in confined placental trisomy and fetal uniparental disomy in ODe third ofthe cases D==v= Fenilization Trisomic zygote N Trisomie zygote rescue l-..-.._.._.. ~ l Inner eell mass CID-CO"CID I l I I l I Fet::tl biparcntal disomy Feta! unip.ll'cntal disomy Trophoblast (lid I I l Contincd placent.il trisomy

22 developl11ent if imprinted regions, known to exist on some chromosomes, are involved (Ledbetter and Engel, 1995), It mayalso lead to recessive disorders in case of (partial) isodisomy, or autosomal dominant diseases in case ofheterodisomy, ifthe parent contributing both holllologues of a chromosome pair is affected by such a disorder. (3) the abnol'll1al cellline is in fact not confined to the placenta, but also present in the fetus. Several cases of placentalmosaicism with normal results in AF cells, but with confirmation of the chromosome abnormality in fetal tissues, have been described (Miny et al., 1991; Ledbelter et al., I 992a; Phillips et al., 1996), Some factors are likely to be ofsignificance in terins ofpredicting the effects ofcpm: I) Robinson et al. (1997) found a significant correlation behveen a meiotic origin ofplacental trisomy and adverse pregnallcy outcome. Because a mciotic origin was found to correlate with high levels oftrisomy in both placentallineages and UPD, it is difficult to distinguish whether an abnoflllal outcome is due to the UPD in fetal and/or placental tissues itself or to the presence of excessive trisomic cells in the placenta. 2) the involvement of some specific chromosomes mayalso be of significance in predicting fetal outcome, Leschot et al. (1996) advocated a careful clinical follow-up in cases of CPM involving the cluol11osomcs 13, 16, and 22. Trisomy 16 is one ofthe most prevalent chromosome aberration involved in CPM, and is associated with maternal UPD 16 in one third of such cases (Kalousek et al., 1993). CPM 16, with or without fetal UPD, is associated with fetal loss later in pregnancy or IUGR, but can be compatible with a viabie pregnancy (\VoIstenholme, 1995). Trisomy 7 occurs at similar frequencies as trisomy 16 at CVS. However, Kalousek et al. (1996) found that most cases of CPM 7 seem to be the re sult of somatic duplication within the placentallineage, and only to be associated with IUGR in case of meiotic CPM 7 with UPD 7 in the fetus. Recently, Shaffer et al. (1996) reported on nine cases of CPM for trisomy 2, two of which were associated with severe IUGR. A biparental origin of (he fetai chromosomes 2 was established in all cases. CPM 2, like CPM 16, seems to be associated with IUGR oniy if high levels of trisolnic cells are present in the placenta, regardless of the paren tal origin of the remaining chromosome pair in the fetus. Further cytogenetic and molecular evaluation of cases with CPM for each chromosome wilt finally result in an improved prediction of pregnancy outcome in cases of prenatally diagnosed CPM. 2. Molecular cytogenetics Most ofthe limitations of traditional prenatal cytogenetic analysis, as described in section 1.4, tau be overcome with a molecular cytogenetic technique known as in situ hybridiz.::'1tion (lsh). It involves the detectioll of aspecific nucleic acid sequence, either RNA or DNA, in a chromosome preparation. Early work with this technique, which was developed by Gall & Parduc (1969) and John ct al. (1969), made use of DNA probes labelled with a radioactive isotope (radiolabelled in situ hybridization, RlSH). In the 1980s, the ISH techllique became accessible to routine cytogenetic laboratories by replacement of the radioative labels by a 22

23 fluorescent label (fluorescent in situ hybridization, FISH) (Cremer et al., 1986; Hopman et al., 1986). The principle of FISH is depicted in figure 1.7. Briefly, target-dna (in nuclei aud/or chromosomes) and labelled cbromosome specific probe-dna are denahired (the DNA becomes siugle-stranded). Probe DNA is applied to the chromosome slide, to allow hybridization ofprobe-dna to target-dna. After removing any remuants ofprobe-dna that is unbound or bound with poar holuology, the targetwdna-probe-dna-complex is subsequent1y detected. FISH allows the detection of chromosomal rearrangements at the submicroscopicallevel, such as microdeletions (Desmaze et al., 1993; Lowery et al, 1995), as weil as identification of chromosomes or chromosomal segments Ofullkllown origill (Saehs et al., 1992; Van Hemel et al., 1992; Callen et al., 1992). However, the major advantage of this technique is its application ta interphase nuclei, whieh is called interphase cytogenetics (Hopman et al., 1988; Cremer et al., 1988), and it was first introduced in the field of cancer research, since most tumors have low mitotic indexes. Fluorescent detection of chromosome specific probes enabled the recognition of the centromeres on the chromosames as clearly localized and brightly fluorescent spots in metaphase spreads and in nuclei. This provided the means of rapidly enumeratîng the copy number of chromosomes in interphase nuclei. Since interphase FISH can be applied to nuclei of cultured as weil as uneultured eells, it soon became clear that this technique had the potentialof improving speed of prenatal diagnosis in AF cells, allowing a rapid cytogelletic evaluation of the fetus (Klinger et al., 1992; Zhellg et al., 1992; Ried et al., 1992; Ward et al., 1993; Divane et al., 1994; Philip et al., 1994). As pointed out in previous seetions (1.4.1 and 1.4.2), the deteetion of ehromosomal mosaicism in AF cel! cultures as weil as in CV may pose an interpretation dilemma. Differentiation between pseudomosaicism in AF cultures or local mitotic division errors in CV, and mosaicism requires the evaluation of a large 11l1mber of cells in the sample, which is often difficult or impossible due to time constraints and a limited amount of metaphases that is suitable for analysis. An elegant and effieient way for differentiation between both situations is the use of FISH (Schwartz et al., 1993; van den Berg et al., 1997). It can rapidly provide infonnation on the presenee of a specific cllfomosome aherration in a large number of eells, sinee it allows the analysis of metaphases of lesser quality, not suitable for chromosome analysis, as weil as interphase eells, nonmaceessible for karyotyping. Discrimination between CPM and generalized mosaicism may require follow-up investigations, suelt as cytogenetie analysis of AF and lor fctal blood cells. These foliowmup studies prolonge severely the definite reporting time. Sincc FISH allows analysis of uneultured eells, it makes a rapid differentiation belween eonfined and generalized mosaieism possible. If the chromosome aberration is found to be confined to the placenta, and if it involves a trisomy, study of the parental origin of bath homologues of the involved ehromosome pair in AF cells is essential, since CPM may be associated with fetai UPD (Kalousek and Barrelt, 1994). Sinee FlSH does not permit differentiation between the paternal and maternal inherited chromosome, other moleeular techniques have to be used. Polymerase chain reaction (PCR) amplification of polymorphic microsatellite repeat markers permits the investigation on the parental origin of both homologues of a ehromosome. Moreover, it may also allow the identification of the parental origin of an unbalaneed ehromosome aberration as weil as of the extent of a deleted chromosome segment, if relevant FISH probes are not available. 23

24 Figure 1. 7 Principa 0!:~~~~~~lf~nll~o~re~sc~ell~'"~I=SI==~ /~. 'tu hybndlza.. tion( ~ <rf~is~h2:.)' I. Iknaturat!On t IUIIII,nlh <:> XXXX o XXX IJ / )Jllnl'UI"1 I. Hybridizauon W!I I t,m, F,oore'''r d,"'u," k... h, 24

25 Chapter II Application of FISH for prenatal cytogenetic diagnosis

26

27 1. Aim of the experimelltal work Traditional cytogenetic analysis of chorionic villi (CV) and amniotic f1uid (AF) samples has a few limitations which \Vere described in sectiol1 1.4 of chapter 1. The experimental work, presented in this chapter, was aimed at thc solution of some of these limitations, and represented the introduction of the tluorescent in situ hybridization (FISH) technique in our laboratory as an adjunctive prenatal diagnostic tooi. Fitst!y, we introduced FISH for the idenfijicafiol1 of structural chromosome aberrations, such as marker chromosomes and dc~llovo unbalanced chromosome abnormalities, which could not be adequately charactcrized with classica! banding techniques (publications land 11), as weil as for fmther characterization of stmctural chromosome rearrangements, aud for detection of subtie familial chromosome aberrations involving small chromosomal segments or chromosomal parts with similar banding patterns (section 3.1). Secondly, we introduced FISH for the rapid de/ecfioll of chromosome aberrafiol1s in uneul/ured AF eells ifa quick result is necessary (section 3.2). This is the case when (I) feta I allomalies arc detected by ultrasound (US) which are suspicious of chromosomal aneuploidy, and gestational age comes close to 24 weeks (tennination of pregnancy legally not allowed after 24 weeks) or in case of impending birth (publieations Hl, and IV). (2) a chromosome aberration is detected in a CV sample which is suspected to be confined to the placenta, but lllight be present in the fetus as weil (discrimination confined placental l110saicism (CPM)/gcncralized l11osaicism)(publication VII). (3) low-ievel mosaicism is deteeted in all initial AF sample, but the number of cell colollies is too small to confirm true mosaicism. In these cases, FISH is applied to uncultured AF eells of a follow-up AF sample for rapidly verifying the presence of mosaicism. (4) maternal age is L 44 years, iftriple test restdts indicate a risk of Dawn syndrome of L 5 %, or in case of twin pregnancies of advanced gestational age (± 16 weeks) Thirdly, interphase FISH was introduced for studying chromosomal mosaicism in culturcd AF eells, and semi-direct CV slides, espeeially for differentiation bet ween mosaicism and pseudomosaicism or loeal mitotie division errors, respectively (publicatian V; section 3.3); Fout/hly, we introduced FISH far the detection of submicroscopical dele/ions, sueh as at 17p13.3 (type I lissencephaly, Miller-Dieker syndrol11e) (Lcdbetter et al., 1989), 7ql1.23 (Williams sylldrome) (Lowely et al., 1995), and 22ql1 (CATCH22) (Desmaze et al., 1993). In these cases the couple already has a ehild with sllch a çleletion or with the associated syndrome, or one of the parents is a carrier, or US illvcstigations are suspicious for sueh a dcletion (publicatioll VI; section 3.4). After a short description of the FISH procedure, as used in Dur laboratory (seetion 2), the possibilities and limitations of FISH as a diagnostic tooi will be cvaluated for eaeh of the four indications separately, based on our experiencc during four ycars ( ) (seetion3). 27

28 2. Matel'ials and methorls of the FISH technique 2.1 Slidc preparation alld pretreatment Thc stides that we use for FISH ean be the same slides that are used for chromosomc analysis. Thc preparation of these chromosomc slides has been previously described (sections 1.1 and 1.2). Slides of uncultured amniotic fluid cells (AF cells) are specifically prepared for FISH analysis, as described in publication lil, with a small, but essential modification since 1995: after preparation of the slides, the cells arc swollen by a short immersion in 70 % formamide in 2x standard saline citrate (SSe), followed by a 1 min wash in phosphate hutlèred saline (PBS), whieh improved hybridization eftlciency and signal intensity ( J.G. \Vauters, University of Antwerp-U.I.A., Antwerpen, personal col11munication; Fidlerová et al, 1994). Before hybridization, the metaphase and interphase ceus in linstained prepal'atiol1s need pretreatment with RNase and a protease (pepsin) to improve accessibility ofthe target DNA in chromosomes and nuclei for the probe DNA (pubications I, and IV). Tlypsin-Giemsa slaîned slides are destained prior to hybridization, according to Klever et al. (1991), and do not need this pretreatment. 2.2 DNA probes ond labclling Thc probes used in the experimental work are of three general categories: repeat-sequel1ce probes, whole clzroljlosome pl'obes, and IOclis-specific probes. -The repeat-sequellce pl'obes wc use are ahnost all chromosome specific centromere probes, detecting DNA sequences that are tandemly repeated several hundred to several thousand times in the (peri-)centromeric regions of the chromosomes. Most of these sequences are in the alpha-satellite. or satellite lil families. -The whole clzroljlosome probes are chromosomc specific col11posite probes whose individual elements have sequellce homology at mally sites along one specific chromosome, enabling the fluorescent stailling of an entire chromosome. These probes are referred to as tlpaintingl! probes or \VCPs (whole chromosome paints), since they are used to paint whole chromosomes. These pro bes contain families of repeat sequences (Alu and Kpn) that are shared by other chromosomes, r~sultillg in aspecific hybridization signais. To achieve specific staining these sequences are prevented from hyhridizing by a preannealing step with unlabelled blocking DNA [total human genomic DNA or Cot-I DNA (Boerhinger Mannheim GmbH. Mannheim, Gennany)] before adding the probe-dna mixture to the target-dna. This method is referred to as chromosome in situ supprcssion (CISS) hybridization (Lichter et al., 1988; Pinkel et al ). -The loclis-specific pj'obes are typically single copy probes homologeous to specific targets 28

29 ranging in size from 15 Kb to > 500 Kb. According to the insert length, different vectors can be used, with eosmids, able to harbour 35 to 45 Kb of foreign DNA, being the most popttlar for FlSH. Preannealing with blocking DNA is necessary to prevent hybridization of repeat sequences in these probes. They produce small signals which are sometimes difficult to detect in uncultured cejls. By using cosmid contigs (overlapping cosmids) or cosmid cocktails (nonoverlapping cosmids) signal intensity may be improved. Most ofthe repeat-sequence and cosmid probes that we USC, are kind gifts from their creators. The painting probes that we used until 1994, were the Bluescribe plasmid libraries from Dl'. J. W. Gray (Laurenee Livermore National Laboratory, Livennore, CA, USA)(Collins et al., 1991). Nowadays we use commerciahy available painting probes (Cambio Ltd., Cambridge, UK). The probes with their chromosomal localization, and their reference or source are listed in table 2.1. Tlibie 2.1 Probes uscd for FISH, their chrolltosomallocalization, :md reference or source Probe A. Reneaf-Se((lIelieg pmher puci.77 p«3.5 pg-a 16 pct?! I «p& phur98 ph8 plciia p«12h8 DI2Z3 LI.26 c23? ptra-20 ptra-25 DI5Z1 phuri95 pl7h8 Chromosoma[ localizatioll lqhet 3 een!, 5,19 een 7 een 8 een 9 qhet 10 een I! een 12 een 12 een 13 and21 een 14 and 22 een 15 een 15 een 15psatIW 16qhet 17een Referenee or souree Cooke and Hindley (1979) Waye and Willard (1989) I-fulsebos et al. (1988) Waye et al. (l987a) DonJon et al. (1986) Moyzis et al. (1987) Devilee et al, (1988) Wa)'c ct al. (1987b) Looijenga et al. (1990) Oncor, Gaithersburg, MD, USA Devilee ct al. (1986) C. Meijers, Institule of Paedia!ric Surgery, Emsmus University, Rotterdam Choo et al. (1990) Choo et al. (1990) Higgins et al. (1985) Moyzis et al. (1987) Waye and Wi[lard (1986) 29

30 Table 2.1 continucd Probe LI.84 p3,4 D20Z1 p pbamx5 pdp97 RPNI305X CEPXlCEPY(satlll) r521 B. Puilltillg fjtohes WCpJ 1 22, X, Y M28 C. Loclts-specific nrobes WSCR (Elaslin Wi11iams Syndrome chromosomal region) lei7q cfk2,6 cw23, and cw9d te120p CB2IcI ccmp21.a M51 cos39 scl L1 sc M69 Chromosomal loealization 18 een 20een 20 een 22 een X een Yeen Yql2 XcenIYq12 NORs , X, Y 12p 7q q36-qter 13q14-q21 16p p pl3-pter 21qll 2Iq22.2-q qll 22qll 22qll 22qll 22ql1.2 22q13 Rcfcrencc or source Devilee et al. (1986) Wayc and Willard, (1989) Oncor, Gaithersburg, MD, USA Rocchi ct al. (1994) Willard et al. (1983) Woltè et al. (1985) Lau (1985) Vysis, Downers Grove, IL, USA B. Bakker, Dpt. of Clinical Geneties, University llospital Leiden Cambio Ltd., Cambridge, UK Zhang et al. (1989) Oneor, Gaithersburg, MD, USA Oneor, Gaithcrsburg, MD, USA F. Kooy, Medische Genetica Groningen European Chromosome 16 Tuberous Sclerosis Consortium (1993) Ledbctter et al. (I 992b) Oncor, Gaithersburg, MD, USA Van Opstal et al. (1995) Zheng et al. (1992) Mulder et al. (1995) Aubryetal.(1993) Halford et al. (I993) Carey et al. (1992) LekalHle Deprez et al. (1995 Mulder ct al. (1995) 30

31 Table 2.1 continucd Probe Chromosomal Reference or sourcc localizatioll Arcos2,4 Xqll ql2 J. Trapman, Dept. Of Pathology, Erasmus University, Rotterdam cal24 Xp21.2 Blonden et al. (1989) cpq23.1 Xpter and Ypter L. Blonden, Dpt. Human Genetics c7b2 Xq28 M. D'Urso, Internationaiinstitute of Genetics and Biophysics, Naples, Haly pdpi05 Yp/(Yq)' Disteche et al. (1986) LOR2.6.4 Yqll Ma et al. (1992) p49r Yqll.2 Ngo et al. (1986) Notc. 1 satlll satellite 111 DNA in short arnl of chromosome 15; 2 NOR nucleolus organizer region in p ann of all acrocentric chromosomes; JWCP= whole cluomosomc paint;4signals on Yp (interval 3) and Yq (interval 6), with the latter mostly not observed with FISH rite ultrasound ljrobe set \Ve used a specific set of probes for dctection of the most conullon aneuploidics (trisomy 13, 18, 21, triploidy, sex~chromosomal aneuploidies) in uilcultured AF cclls of pregnancies complicated by fetal anomalies (the so called US (liltrasolilld) probe se/)(iable 2.2) (see section 3.2.1). Table 2.2 Ultrasound (US) probc set used for FISH on uncllltllred amniocytcs in cases of ultrasound abnormalities dllrillg thc ycars Prohe pbarnx5 RPNI305X CEPXI Ll.26 cfk2,6 Ll.84 CB21cl ccmp21.a (chromosome) (X) (Y) CEPY (13/21) (13) (18) (21) (21) Near (X/Y), 1993,,, 1994 end of 1994, 1995 end of 1995,,,, 1996,,, jul)' 1996, Note.- see table 2.1 ror localization and refercnce ofthe probes 31

32 The compositioll of this set ehanged durillg the years, as some probes, after a while, turned out to be unreliable for interphase eytogeneties. Two probes were used during the whole 4-year period: CB21cl and LI.84, for ehromosome 21 and chromosome 18 deteetion, respectively. \Ve investigated extensively the utility of CB21cl on a series of uncultured AF and blood samples, since a chromosome 21 spccific probe was not yet available at the time we started interphase cytogenetics (see appendix publication IV). L1.84 hybridizes to an alpha satellite repeat sequence in the centromeric region of chromosome 18 (Devilee et al, 1986). Centromere probes are often applied far illterphase cytogenetics as they produce bright signais. However, the copy 11lllnber of the repeat sequellce that they detect is a highly polymorphic trait and sometimes appcars to be tod small ta produce any signal, which may potentially lead to a false negative result. However, we oniy had some minor problems with L1.84 (see section 3.2.1). One of the changes involved suhstitutian of LI.26 (13/21 centromere probe), whieh we initially used far chromosome 13 detcctian, by a chromosome 13 specific probe (cfk2,6) (a kind gift of Dr. Frank Kooy, Medische Genetica Groningen). The reason for this substitution was the recurrent finding of false positive and false negative FISH results, due ta polymarphisms in the centromeric regions ofthe acrocentric chromosomes (sec section 3.2.1; Verma and Luke, 1992; Mizunoe and Young, 1992; Cacheux et al., 1994; Verlinsky et al., 1995). A separate hybridization of X centromere and Y heterochromatine probe was replaced by a simultaneous dual-calor hybridization of X and Y probe reep (chromosome enumeration probe) XlCEPY (satiii) probe mixture] (VYSIS, Downers Grove, IL, USA). This system allows an accurate identification of maternal cehs in thc AF sample, as weil as of all exceptional45)xl46,xy mosaic (sec section 3.2.1, and 3.2.2). During one alld a halfyear, we used two pro bes for chromosome 21 detection [CB2Icl, hl'bridizing to 21ql J (see appendix publication IV), and ccmp21.a, mapping to the Down specific region (Zheng et al., 1992)], in order to minitnize the risk of a misdiagnosis in case of all exceptional unbalanccd reciprocal translocation of chromosome 21 with breakpoints distal to 21qll. However, as we experienced progressive technical problems with ccmp21.a, we prec1l1ded this probe from further FISH illvestigations. Probes are labelled bl' nick translation using either biotin-ll-dutp (Gibco BRL, Gaithersburg, MD, USA), biotin-16-dutp or digoxigenin (DIG)-II-dUTP (Boerhinger MaJlIlheim GmbH, Mannheim, Germany). The commercial probes are purchased already labelled with either biotin or DIG, for indirect detection with tluorescently labelled antibadies, or with a fluorochrome for direct detection. 2.3 Pl'obe hybl'idization and detection The commercial probes are au processed according to the manufacturer's instmctions. For the non-commercial probes we use the hybridization protocol described in publication I (whole chromosome plasmid libraries, centromere probes)) and publication IV (cosmid probes). Fluorescent detection of the probes depends an probe labeling: detection of biotin labelled probes is described in Pllblications I and IV. DlG labelled probes are detected with one layer of anti-dig-fitc (I :40 to I: I OO)(Boehringer Mal1l1heim GmbH, Mannheim, Gennany) in 32

33 0,5 % blocking milk in 0, I M Tris/O, 15 M NaCI/0,05 % Twecn-20. Mounting of the slides is described in publicatioll I. The concentration of thc fluorescent counterstains propidiul11 iodide (PI) and 4',6' Diamidino-2-Phenylindole (DAPI) varies according to cell type and probe (0,05 ~g-0,5 flg/ml PI and 0,0 15 ~g-o, 15 ~g/ml DAPI) 2.4 Evaluatioll of the slides Slides are exumined under a LeÎca Aristoplan epifluorescencc equipped microscope and, images are captured with the Genetiscan ProbeMaster system (Perceptive Scientific International Ltd. (PSI), Chester, UK) incillding a Xybion cooled CCD 24-bit colour camera. According ta our own standards, metaphase FISH ullalysis requires the examination of 10 ceiis. Interphase FISH analysis involves the counting of at least 50 nuclei of uncultured eells, and 200 nuclei of cultured ceus (including CV eells in semi-direct preparations). \\fe use the Înterphase scoring criteria as proposed by Hopman et al. (1988): (I) nuclei should not overlap (2) cells should not be asymmetrically covered by cytoplasm (3) minor hybridization spots should not be countcd (4) fluorescent spots or patclles of fluorescence may only be included when the signa Is are completely separated from each other (5) spots in a paired arrangement (split spots), close to each other, are couilted as one For interpretation of interphase FISH results, a statistical approach is necessary. Therefore, signal distribution profiles of the most commonly used probes in our laboratory were generated on a series of CV alld uncultured AF samples with normal karyotypes. Statistical analysis of these data was used to determine the 95 % confidellce interval of the olle-sidcd upper reference limit (97,5 %) for the proportion of cells with an abnormal number of signals for each ofthe probes and for each tissue, according to Lomax et al. (1994). This cut-off level is used to discriminate the nonnal state from the lowest detectable level of monosomy and trisomy mosaicislll (tabie 2.3). For probes that are onl)' occasionally used, a normal control sample, simujtalleously processed with the test sample, is used for intcrprctation of the resuits. 33

34 Table 2.3 Statistical analysis of normal diploid controls: one-sided upper reference limit (97,5 %) and corresponding 95 %. confidence interval (Cl) for tbe proportion of uneultured AF and CV eells witb an abnormal signal number Probe Neontrols N signals One-sided upper reference (97.5 %) limit (95 % Cl) AF CV AF CV pa: ,6 (2,2-5,0) 4,9 (2,7-7,0) pa:?tl ,6 (3,2-5,9) 4,0 (2,5-5,6) phur ,9 (3,5-6,3) 2,2 (12-3,3) plciia (0-0) 1,8 (0,9-2,7) pa:12h (l, 1-4,0) cfk ,0 (4,3-5,8) 6,0 (3,6-8,4) Ll ,4 (153-21,6) phur ,9 (3,2-6,5) M (4,6-8,5) L ,6 (3,0-4,0) 2,7 (2,0-3,5) CB21c ,7 (75-10) 3.3 (1,1-5,4) M ,9 (9,5-26,3) 10,6 (75-13,7) 3 11,7 (4,8-18,6) 5,7 (3,7-7,8) pbarnx5 ~: ,6 (10,8-16,4) 93 (6,5-12,1) 3 4,0 (2,8-5,1) 2,9 (l,8-4,1) 0": ,1 (3,0-5,2) 7,6 (3,6-11,6) CEPXICEPY(satlll) ~: (3,9-8,6) 17,4 (9,7-25,1) 3 0(0-0) 2.6 (1.3-3,9) cf: ,6 (3,0-6,1) 3,3 (0,8-5,8) 3 0(0-0) 0(0-0)

35 3. ResllIts and discussion 3.1 FISH fol' identification of stl"uctural Chl'OIllOSOIllC abcrratiolls Appendix publieation 1 Cytogenetic problems in PD arise when de navo unhalanced structural chromosome aberrations or marker chromosomes, i.e., extra chromosomes of unknown origin, are present. COllvcntional banding techniques, sneh as Ag-NOR (nucleolus organizer region) banding, C (centromere) banding, and DA-DAPT (Dystamycinc- 4',6'Diamidino-2-Phcnylilldolc) stai-ning are important (Saehs et al., 1987), but eun not always givc a dcfinitc diagllosis. FISI-I has made further identit1cation of these chromosome abnormalities possible, as showll [or a rew cases in appelldix publicatioll I. These identification studies allowed a more detailed counsejling ofthe future parents, and a better understanding ofthe fetal pathology. Behveen 1993 and the end of 1996, we used the FISH technique for identificatiol1 of extra chroll10s0ll1al material in the karyotype (3.1.1). Additionally, FISH silowed to be a useful tooi for characterization of the breakpoints of a structural chromosome rearrangel11ent, and for confirmation of an uncertail1 cytogenetic result (because of pool' chromosome quality) (3.1.2), and for the dctcction of subtie familial chromosome aberrations in semi-direct CV preparatiolls (3.1.3) Identificatioll of extra chromosomul material of unlmown origin (tables 2.4 and 2.5) Extra chromosomalmaterial of unknown origin may be the result of an unbalanced structural chromosome aberration (trallslocation, duplication, il1sertion) or a marker chromosome. The latter pose a problcm in prenatal counselling procedures, as they present a helerogeneous group of chromosomes with varying phenotypic consequences, depending on their chroillosomal odgin: they may cause severe anomalies, but also may have no phe~lotypic effect on the carrier. Before the introduction of FISH, the chroll10somal origin of most markers remained obscure and thc risk of phcnotypic abnonllality has been estimated in relatian to size, staillîllg propertjes, level of mosaicislll, and familial occurence (Buckton et al., 1985; Saehs et al., 1987; Warburtoll, 1991). The risk ofabllonnal de\'elopment in familial cases was predieted ta be very sm all (Gardner and Sutherland, 1989; Sachs et al., 1987), whcrcas the risk in de nova cases has been estimated to be 13 % (\Varburton, 1991). The introduction of FISH has providcd the passibility of rcsolving the heterogeneity of this group of chromosomes (Callen et al., 1990a and b, 1991, 1992; Crolla et al., 1992; Blennowet al., 1993, 1994; Plaltner et al., 1993; I.eana-Cox et al., 1994; Brondum-Nielsen and I'vlikkclsCll, 1995). These studies led ta the identification of the most collullonly obscn'cd marker chromosomes in PD [in\' dup (15), i(l8p), i(l2p), and del(22q)] and their associated phenotypes (Robinsou et al., 1993; Callen et al., 1990a; SehinzeI, 1991; McDennid et al., 1986), facilitating gcnctic counselling of the prospective parents. 35

36 Table 2.4 FISH for identification of marker chromosomes Case Probes' r521, ap8, phur98, ph8. plc 11 A. pa 12H8, phur195. p17h8. LJ.84. pg-ai6. p3.4 FISH result ISH der(16)(phuri95+) 2 3 US probe sef (pbamx5, RPN1305X. Ll.26. Ll.84. CB2Icl). WCPI2, M28, D1223. pa12h8 US probe set (pbarnx5, RPNI305X, Ll.84. CB2lcI), M5J, sc4.1, Ll.26 ISH i(12)(qio)(wcpi2+.m28+.di2z3-.pai2h8-)[17]1 del( 12)( q 1 O)(WCPI2+.M28+.D 12Z3-.pa 12H8-)[ 6], ISH der( 13 or 21 )(L 1.26+) 4 r521. L1.26. c237. ptra-20, P ISH mar(r521+) Note.- jsee table 2.1 törchromosomaliocajization äiia references ofthe pröbes~ ~ abnormalities (see table 2.2): 3 sec Los et al. (1995) USprobe set=: set ofprobes used to screen uncultured AF ceiis in cases ofljitrasound(us)

37 Table 2.5 FISH for identification of ad extra chromosomal segment Case Cytogenetic result Probest FISH result 1,2 21ps+ r52! rsh 21ps+ (r521++) 3,4,5,6 22ps+ r521 ISH 22ps+ (r521 ++) 7 13ps+ r521 ISH 13ps+ (r521++) 8 15ps+ r521 ISH 15ps+ (r52 1++) 9 add (7q) WCP7,7qter ISH dup(7)(wcp7+.7qterx I) 10 add (Iq) WCPI ISH dup(i)(wcpi+) I1 add (Sp) WCP7. WCPS. WCPIS ISH der(s) t(7;s)(pi3;p23)(wcps+,wcp7+) 12 add (16q) WCPI6. WCPII. WCPI2 ISH der(i6) t(11 ori2;16)(wcpi6+. mix (WCPII.WCPI2)+) 13 add (Xp) WCPX. WCpy, cpq23.1. cal24, ISH inv(x)(p22.3q26)(wcpx +,cpq23.1 stcal24mv,pbamx5mv,c7b2st) pbamx5. c7b2 Note.~ I see table 2.1 for chromosomallocalization and references ofthe probes

38 Fïgure ps+. A) partiaj karyotype of cullurcd AF cel Is. IJ) FISH signals 011 :111 acrocentr;c chromosomes, inc1udillg 21 ph (arrow), aftcr hybridiza lion with r521, dcfccting NORs in th e p-arm of the acroecntril' ehromosolllcs. " Figure 2.2 in\'(x)(ll22.3q26), A) parlial karyotyll e of CV eclls, and C) of AF eells, showing in,'(x) on thc righl. 0) mctapha se spread of CV with FISH signals on normal ehromosoilic X (arrowhead) and in\'(x) (arrow) afler hybridizalion whh WCP\': Iwo :lspecific sigmt ls are seen on dim~rcnt chromosome anus (Xpier and XqI J) of thc normaj, and on the same ehrolllosome arm of th e abnorlllal X chromosome, indicati"e for an irn'crsion. 0) melaphasc spread of AF eells: FISH signa Is on normal (arrowhead) alld abuormal (a rrow) X chrolllosome affer simullaneotjs hybridizalion wilh poamxs (X centomere), e702 (Xq28), and ('AL24 (XIl21,2), sho\\'ing Ihal ('702 and ('AL24 hybridize 10 Ihe samc ('hromosome arm of Ih e :l IJllormal X chromosome, confirming :In in\'crsion iullle X ehromosome. A,- h c r 38

39 However, identification of marker chromosomes llsing FISH with clrromosome specific probes, as we did, has limitations. Of the ten cases of marker chromasomes that were encountered during four years, FISH could oniy elucidatc the chromosamal origin in four cases (tabie 2.4). In dayly practice, we were confronted with twa problems. Firstly, the number of available cluomasome slides and the time necessary ta perform FISH studies showed to be the main limitating factors. In most of the larger prenatal studies, marker cllfomosomes were identified retrospectively, without time constraints and without a limitation of the number of available chromosome preparations (Blennowet al., 1994; Brondum-Nielsen and Mikkelsen, 1995). Secondly, whenever the marker silowed not to be one ofthe four mast common markers, the counselling ofthe parents still remained uncertain, and based on the classical characteristics such as size, staining properties, mosaicism, alld familial occurence, than on the FISH resllit. For instanee, the marker in case one (tabie 2.4) was found to be positive with the 16 heterochromatin probe, but the presence of auy euchromatine could not be excluded. Therefore, the parents decided to terminate the pregnancy on the basis of this uncertainty. The probe that silowed to be very useful for counselling procedures was r521, detecting nucleolus organizer regions (NORs) in the short arm of all acrocentric chromosomes. It demonstrated whether the marker was satellited or not, irrespective of the active or inactive state of the satellites in contrast with the Ag-NOR banding technique, with satellited markers having a better prognasis than non-sateliited markers (Warburton, 1991). Similarly, FISH with r521 showed the presence of a normal polymorphism in eight cases showing extra clrromosomal material in the short arm of one of the acrocentric chromosomes (tabie 2.5) (ligure 2.1). On the basis of these results, the prospective parents could be reassured. The remaining five cases of table 2.5 all displayed an unbalanced struetural chromosome aberration, indicative for a duplication or a translocation. Our identification strategy involved FISH with the relevant whole chromosome paint (\VCP), and if a duplication was excluded, \VCPs of other chromosomes were applied, depending on the banding pattern ofthe extra chromasomal part. The advantages offish are clearly illustratcd in case 15. Chromosolllc analysis of CV semi-direct preparations revealed an add(xp) eluomosome (figure 2.2A). FISH with \VCPX resulted in a fluorescent staining of the whole abnormal X chromosome, initially indicative for a duplication. Further hybridization with \VCPY indicated tlie presence of, an inversion in cluomosome X; two aspecific \VCPY hybridization signals were seen on the normal X chromosome at Xpter and Xq13, as described by the probe supplier (Cambio Ltd., Cambridge, UK), but the abnormal X chromosome showed both aspecific signals on the same duomosome arm (figure 2.2B). FISH with locus specilic X probes (cpq23.1, cal24, pbamx5, and c7b2), applied to cultures ofa subsequent AF sample, conlirmed an inversion (X)(p22.3q26)) (ligure 2.2C and D). Recently, ncw FISH techniques have been developed that allow a more efficient and accurate idcntitication of auy extra chrornosomal material in the karyotype. Firstly, the mîcro-fish technique, whieh involves the microdissection of one or a few markers, followed by an amplification of the dissected fragments by a degeneratc oligonucleotide-prirned polymerase chain reaction (DOP-PCR), and hybridization of the PCR product to normal metaphases (Viersbach et al., 1994; Müller-Navia et al., 1995; Engelen et al., 1996). Another approach combines chromosome isolation by fluorescence activated ceh soliing (FACS) with the DOP PCR teehnique (Blennowet al., 1992; Carter et al., 1992). Secondly, a ncw technique, 39

40 Table 2.6 FISH (or confirmation and furthcr charactcrization of a cytogenctically detcctcd structural chromosome rearrangcmcnt Case Cytogenetic result Probes' FISH result i(xq) pbamx5 ISH idic(x)(p 10)(pBarnX5'2) 2 i(x) WCPX. pbamx5. cal24, Arcos2A ISH idicx(p lla)(wcpx+,cal24-.pbamx5x2,arcos2ax2) 3 i(yp) and del(y) pdpi05, pdp97, LOR2.6.4, p49f, RPN1305X ISH idic(y)(qll)(pdpi05'2,pdp97x2,lor2.6.4-, p49f-,rpn 1305X -)[ 14]ldel(Y)(pDPI 05x 1,pDP97' I,LOR2,6,4-, p49f-,rpni305x-)[161' 4 i(i)(qio) WCPI ISH i(i)(qio)(wcpi+) 5 idic(9) WCP9, phur98 ISH idic(9)(qi2)(wcp9+, phur98+) 6 inv(9) WCP 9, phur98 ISH inv(9)(p24q 12)(WCP9+,pHuR9S'2) 7 inv(y) pdp105, L0R2.6.4, RPNI305X ISH inv(y)(pll.2qll.2)(pdp105st.lor2.6amv.rpni305xst) S t(l:2) wcn WCn, puc!.77 ISH t(i;2)(pl!.2;qll.2)(wcp2+,pucl.77+;puc!.77 -,WCPI +) 9 t(i;i3) WCPl, r521 ISH t(l:13)(q31 :pi2)(wcpi+.r521 +;WCPI +.r521 +) 10 t(5;13) WCP5. WCP13 ISH t(5; 13)(qI3;qI2)(WCPI3+;WCP5+) 11 inv(9)(pl!.2qi3)t(9;10) WCP9, WCPIO, phur98, phs ISH inv(9)(pl1.2q13)t(9;10)(q21.32:pl1.23) (pio;qio) (phur9s+, WCPI 0+; WCP9+,pH8+) 12 t(ii;22)(q25;qi3.1) WCPI L WCP22. P190.22, M51. 58B12. M69 ISH t(ll :22)(q23.3:ql1.2)(WCP22+.M69+,58B 12+; WCP11+,PI90.22+M51+) 13 der(21;21 )(q I O;qIO) WCP2l. CB21cl. ccmp21.a ISH der(21 :21)(ql O:qlO)(WCP21 +,CB21cl x2, ccmp21.ax2) 14 der(16p) WCPI6, WCP9 ISH t(9; 16)( q32;p 13.1 )(WCPI6+; WCP9+) 15 del(7) WCP7 ISH del(7)(wcp7+) 16 del(y) pdp105, pdp97, L0R2.6.4, p49f, RPNI305X ISH del(y)(ql1.2)(pdpi05+, pdp97+, LOR2.6.4+, p49f-, RPNI305X-) 17 r(18) WCPIS, L!.84 ISH r(ls)(wcpis+, L!.S4+)' IS monosomy21 WCP21 ISH 21(WCP21'1)' Note.- I see table 2.1 for chromosomallocalization and references ofthe probes: 2see in 'tveld et al. (1995): J see appendix publication Ir: 4 see Joosten et al. (1997).

41 developed in the field of cancer cytogenetics, alld called jpeclral kwyotyping Ol' SKY, involves the hybridization of 24 tluorescently labelled clliomosome painting probes allowing the simultaneous and differential colour display of all human chromosomes (Schröck et al., 1996; Veldman et al., 1997). Thirdly, another revolutionary technique is comparalive gellomic hybridizatiojl (CGH) (Kallioniemi et al, 1992), which allows to screen the entirc genome for genetic Josses and gains (in contrast to conventional lllolecular techniques). It has already been succesfully appl1cd for cytogenctic analysis of tumors (Steenman et al., 1997). CGH involves the isolation of DNA from a test sample and ft normal contral sample, followed by a simultaneous hybridization of bath differentially labelled genomic complements with norl1lal metaphases. The analysis of fluorescence intensity ratios along the target chrol1losomes by a digital image analysis system reflects the ratio between tested and reference genomes, and can detect gains and losses of clliolllosome material in the test sample. This technology is especially useful if l1letaphases arc not available or are difticult to obtain Detcrmination of thc breakpoints of a rearrangcd chromosomc alld rnpid confirmatioll of an unccrtaill cytogenetic l'csult (tabie 2.6) During tour years, we uscd FISH for identification of the breakpoints of an isochromosome (cases 1-3,5), an inversion (cases 6, and 7), a translocation (cases 8-12), or a deletion (case 16). In cases 11 and 12, the translocation breakpoints SilO wed to be diftèrent from what was suspected on the basis of cytogenetic analysis. FISH results in case 9, invojving a t(i;i3)(q3i;pi2), are shown in figure 2.3. In cases 4, 13, and 15, FISH was used for verification of the cytogenetic result, which was uncertain due to the poor quality of the metaphases. The benefits of FISH are clearly iilustrated by case 14, in whieb chromosome analysis was only suggestive of an abnonnal chromosome 16, with all other clliomosomes in the karyotype showing a normal banding pattern. FISH rcvcaled the presence of a balanccd translocation of chromosomes 9 and 16, with breakpoints in 9q32, alld 16p13.1. The derivativc chromosome 9 was not distinguishable from the nonnal chromosome 9 on the basis ofbanding pattern alone (figure 2.4). In case 17, FISH confinned the chromosome 18 origin of a ring chromosome, which is described and cxtensively discussed in appendix pubiication ti. FISH studies perfoflllcd in the rare case of Illonosomy 21 (case 18) showed that pure monosomy 21 does exist indeed (Joosten et al., 1997). In most of these cases, FISH provided lis with a better undcrstanding of the banding pattern of the abnormal chromosome(s), and, thcrefore, it improvcd genetic counsclling. In cases of pooi' chromosome quality, FISH sllowcd to be a powerflll tooi for rapid vcrification ofwhat was suspected on the basis of traditional chromosome analysis Detcction of suotle familial chromosome rearrangcments (tabie 2.7) The aimlysis of subtlc familial clliomosome rearrangements poses a problem in PD. Carriers of such a chromosome aberration are at high risk for unbalanccd offspring, making an early PD in CVS important. However, these clliomosome abnonnalities are diftlcult to detect with c1lfomosome banding techniques, due to thc 5111all size or si mil ar ballding pattern of the exchanged cllfomosome fragments, espccially in metaphasc5 of lesser quality as thase directly prepared trom CVS. During the timc pcriod , we sucessfully investigated scven cases of subtie familial chromosome rcarrangments in semi-direct CV preparations using 41

42 Figure 2.3 t(l; 13)(q31 ;1'12). A) partial karyotype of AF ce lis. 0) FJSH signals 011 der(l) (arrowhead) and dcr(l3) (arrow) aftcr hybridization wilh r521, detecling NORs in p-arm of the ncrocentric chromosomcs, re\'ealing thc breakpoint in 13pl2. A Figurc 2,4 t(9; 16)(q32;p13.1). A) partial karyotype of A FeelIs. 0) FISH signals on 1I0rmai chromosome 16, del'(16) (arrowhead), and der(9) (arrow) aftel' hybridizalion wilh WCPI6. showing a balanced 1(9;16). 42

43 FISH (tab Ie 2.7). In tlu-ee cases (cases 3, 5, and 7), FISH showed the presence of an unbalanced karyotype, resultillg in a partial trisomy 12, 11, and 16, respectively. FISH results in case 3 are shown in figure 2.5. In the remaining four cases, FISH results were indicativc for a normal karyotype (cases 1 and 4), alld for a balanced translocation (cases 2 and 6). Tablc 2.7 FISH foi" detectioll ofsubtlc ramilial chromosome rearrallgements Case Fmnilial chromosome Probcs l FISH result aberration inv ins(18;5) \Veps, ISH 5(WCP5+)18(WCPI8+) (q21.3;pi4pt 3.I)p"' WCPI8 2,(1 ;22)(q24.I;q 13.1)01"' WCPI, ISH.(1 ;22)( q24.i;q 13.1 )m,'(wcp22+; WCP 1+) WCP22 3 ins(18; 12)(pl I.3;q 13q 15)mat WCPI2, ISH der(l8)ills( 18; 12)(1' 11.3;q 13qI5)m"'(WCPI2+) WCPIS 4 '(8; 11)( q 121'23 ;q21 )01" \Veps, ISH 8(WCP8+) I I(WCPI 1+) WCPII 5 in\' ins(5;11)(pi4;q24q 14)mat \Veps, rsh der(5)inv ins(5;11)(pi4;q24qi4)mat(wcpi 1+) WCPll 6 '( 12;18)(pI2.3;q21.2)p"' WCPI2, ISII '(12; 18)(1' 12.3;q21.2)p,'(WCPI8+;WCPI2+) WCPI8 7.(l6;18)(p 13.3;1'11.23)01" WCPI6, ISII der(l8)'(l6; 18)(pI3.3;pI1.23)1l1l1'(WCpI6+) WePI8 Notc.- 1 Sec table 2.1 for chromosomai localization and references ofthe probes In all these cases, FISH cnabled a rapid PD in first trimester CV semi-direct slides, dcspite the poor quality of these lllctaphases, aljowitlg a prcgnancy termination in the first trimester of pregnancy in casc of an unbalanced karyotype (Speleman et al., 1992; Mangelschots et al., 1992; Fuster et al., 1997). Hybridization ofthe relevant \vcps ta melaphases of the carrier of the structural,rearrangement bcfore PD, especiajly when slllau chrolllosomal fragments arc involvcd" showed to be essential in terms of testing whether FISH is able to vistmlize thc involvcd chromosome abermtion. PISH may soll.1etimes not be possible if relevant probes are not available. In sueh cases, polymcrase chail1 reaction (PCR) amplification of polymorphic microsatellite repeats (\Veher and ivlay" 1989) may be a powerful tooi tbr further characterization of an unbalanced structural chromosome aberration, as shown in appendix publication 11. A de novo ring chromosolllc 18 [r(18)] was dctccted in AF cells of a 39-ycar-old pregnant woman. Ultra sound (US) investigations shawed a slightly abnormal facial profile. FlSH with \VCP 18 and 18 ccntromcre probe contlnllcd the chromosome 18 origin of the ring chromosame, but could not reveal any deletions of 18p or 18q material in r( 18). 43

44 Figure 2.5 ins (18;12)(pI1.3;CJI3qI5). A) partial karyotype orcv. 11) FISH signals on Ilormul chrolllosomes 12 and der(18) (arrow) arter hybridization whh WCPI2, and C) FISH signals on normal chromosome 18 and der(18) (arrow) aftcr hybridizatioll w ilh WCPIS, bolh showing all unbalanced ins(18;12), rcsulting in a parliallrisollly 12. Subtelomeric probes were not yet available, and, therefore, we pcrf0n11cd per ajlalysis of variolis mierosatellite markers on chromo same IS to establish potent ia I deletions and determine the parent of origin.thcsc DNA investigations indicated that r(is) was of paternal origin and displayec.l a small ISp c.lelction of undclcnnined size, together with a large 18q delelion, al least del(1 S)(q2 1.33). Thesc DNA data were important for genelic counselling, as weil as for getting more insight into the relationship bctwecn genotype and phenotype. Another example is shown in figure 2.6. Amniocentcsis was pcrfornled at 20 weeks of geslat ion because of abnormal maternal serum serum markers. Chromosomc analysis was suspicious tor a lerminal deletion of ISq. DNA studies were perlormed in order la unambiguously confirm the subtie cytogenetic resull. PCR produels of same microsalellite repeats on chromosome 18 (D 18S59, D18S42, MIlP, and D 18S70) all showed an informative pattern, and revcalcd a patcrnal deletion of at least lsq23, on Ihe basis of which the parcnts decided la terminate the pregnancy. In conclusion, PCR amplification of DNA polymorphisms may sometimes be a useful adjullctivc 1001 for further characterization of de nova subtie siructural chromosome rearrangemenls, if cytogcnetic results rcnlain undcfined and relevant FlSH probes are not available. Today, we would prefer to usc FlSH with clrromosome specific subtelomeric probes, although this would nol elucidale the parent of origin. 44

45 Figure 2.6 Cytogenetic and DNA analysis of del(18)(q23). A) partial karyotype of AF eells. B) ideogram of chromosome 18 with localization ofthe tested microsatellite repeats. PCR analysis of D18S59, D18S42, MSP, and D18S70 shows absence of a paternal alleje at loci MSP, and D18S70 in AF eells, indieating a deletion of at least 18q22.3 A B I, ' :',,:,,\,~:, 1~\0 ~ ',,! " "& p 11,23 -:-:-:::-e::::: ~D18S59 ~ =. - AI t-'!if =,i,~ A' Al ~,1$' A' AC> AJ... v. II 2U2 q -',0' _ D18S MEP -D18S70 Dl8S59, ~._ - AI - AC> - AJ... - MBP M Dl8S42 ~ 7.t--=~ - AJ ~ DI8S70

46 3.2 Intcrphase FISH for thc rapid dctcctioll of chromosome abcnations in uilcultured AF cells Ultl'asound abnol'malitics In appendix publication lil, we describe our first succesfui investigations of uncultured AF cells from 20 high-risk pregnancies by using FISH whh a chromosome X, Y, 18, and 21 specif1c probe. \Vithin 24 hours after sampling, a triploïdy was detected in one sample, showing that FISH is a valuable additional tooi for PD, specifieally for pregnajlcies at high genetic risk, in which a quick result is important. Between 1993 and the end of 1996 we reeeived a total of 622 AF samples for karyotyping because of ultrasound (US) abnormalities, and FISH was applied in 196 cases, whieh were selected on the basis of fetal anomalies, which had to fit one of the most common chromosome aberrations (trisomy 13, 18,21, triploidy, sex chromosomal aneuploidy), and on the basis of gestational age. \Ve screened the cells with a chromosomc X, Y, 13, 18, and 21 speeific probe (the US probe set, see table 2.2). This focus is based on the observation that aneuploidies involving these chromosomes are reported to account for the great majority of all clrromosome aberrations found in chromosomally abnormal fetuses showing sonographically deterl11ined anomalies (\Vladimiroff et al., 1995). For interpretation of the FfSH resltits we used the statistical approach as suggested by Lomax et al. (1994), since it provides a lllethod for establishing the level of tissue lllosaicism that can be detectcd using FISH, whereas othel' analytical approaches are only suitable for detcction of non-mosaic aneuploidies (Klingel' et al., 1992; Ward et al., 1993). FISH results in 196 cases that we investigated are shown in table 2.8. Alllong 196 AF samples that we selected, 56 (29 %) showed a chromosome abnormality of whieh 46 (82 %) were theoretically detectable whh FISH, since they involved chromosomal parts identified by the probes. In 43 of these 46 cases, abnonnal signal distributions. consistent with aneuploidy of one or more ehromosomes, were indeed found (tabie 2.9). In addition to thc common aneuploidies (trisomy 13, 18, 21, 45,X, triploidy, and sex-ehromosomal aneuploidies) (figure 2.7), we were able to detect two unbalanced Robertsonian translocations [der(l3;14)(qlo;qio), and der(l4;21)(qlo;q10)], and a rare mosaic case involving a eellline with an extra dicentric clrromosome 21 and a ceu line with all extra dicentrie and deleted clrromosome 21. The latter case as weil as three cases oftrisomy 21 are included in appendix publication IV. One ofthe two triploidy cases is described in publication lil Signal distributions in abnormal cases were clearly distinct from those of diploid controls (tabie 2.9). However, in two cases of ruil trisomy 18, hybridization patterns were suggestive of a mosaic trisomy 18 as only 55 % and 29 % ofthe analysed cells showed tlrree signals (95 % eonfidenee interval (Cl) of the one-sided upper referenee limit (I): 3%-4%). Maternal eell contamination (MCC) of the AF sample lllay be all explanatiol1. However, hybridization of L1.84 to metaphases in both cases revcalcd the presence ofa very small signa! on one ofthe three chrolllosomes 18. probably due to polylllorphism. This small signalmight have been 46

47 Table 2.8. FISH resuus in cases with ultrasound abnol'lnalilies that were in\'estigated during four years ( ) Year N FISH results Nonnal Abnonnal Non-infommlive (false negati\'e) (false positive) (0) 9 (4) (I ') 9 (2) 2' I (0) 20 (I) 2' (0) 12 (0) 2' Total (I) 50 (7) 6 Nole.-! trisomy 13, not detected wilh L1.26; 2 45,X, nnd lrisomy 21 ; 3 both nomtal karyotypes; 4 low mosaic -13/r(13), and one nomlal karyotype overlooked in the unculhued AF cells. explaining the relatively small proportion of 3-signal containing nuclei in both cases. In thc nine non-mosaic 45,X cases thc percentage of nuclei with two sigllals varied hetween 0-10 %. These nuclei may represent maternal cells. which leak into the AF at the time ofthe atlliliocentesis procedure, as was suggested by \Vinsor et al. (1996). They found astrong correlation hetween visible detection of blood contamination and the presence of fenmie nuclei in the karyotypically male samples. The samples we receive in Dur laboratdry are only rarely contaminated with blood, and an exceptional blood stailled sample is excluded from FISH analysis. In this way we minimize the risk of a false negative result due to MCC. Three detectable chro1l1osome aberratiolls were missed. \Ve had OIlefa/se negative resull with L1.26, whieh failed to detect a trisomy 13. In two other cases (a case of 45,X, and one of trisomy 21). resllits \Vere uninformative due to technical failures (hybridization failure of Y probe in controi46,xysample, and weak hybridization signals of2l-probe, respeetively). In seven cases we had a false posilive I'esult (tabie 2.10). Two cases involved the 13/21 centromere probe, L1.26 (see Materials and methods, section 2.2). In case 6, a false positive result was achieved with the X centromere probe, indicating the presence of 45,X. FISH was also applied to culhlred AF cells, revealing the presence of a very weak signaion one of the two X chromosomes. which was not visible in uncultured cells (figure 2.8). Of the seven false positive results, six were encountered during the first two ycars of our investigations, and one during the last lwo years. ContimlOlIs adjustment of the us probe set, with sllbstitlltion of llnsuitable probes (see Materials and methods, section 2.2), led to a decrease in false positive results. Non-infonnative I'esltlls in six cases. including two detectab!e chromosome aberrations (45,X.nd trisomy 21), oue uou-detect.ble abnorm.lity (45,XY,-13/46,XY,r(l3)),.nd three normal karyotypes, originated from technical problems, such as weak or absent hybridization signais, increased background fluorescence, and hybridization with less than 50 scorable nuclei. 47

48 Table 2.9 FISH signal distributions in Ullcultured alltniocytes in cases with uhrasound abllormalities showlng a chromosome aberration Chromosome abnormality N Probe Mean % (range) ofnuc1ei with 1->3 signals 2 3 >3 Trisomy LI I (0-10) (7-58) (29-93)' (0-5) Trisomy CB21cl I (0-6) (7-30) (64-93) (0 9) 45,X 9 pbamx (90-100)' (0-10) 69,XXX 2 pbamx (0-12) (88-100) LI (2-30) (70-98) CB21c (15-33) (67-85) 68,XX pbamx LI CB21cl ccmp21.a ,XXY pbamx RPNI305X 100 LI CB21cl ccmp21.a 2 98 Trisomy 13 cfk2, ,XXY CEPXfCEPY 99' + der(13;14)(qi0;qlo) cfk2, der(l4;21)(qlo;qi0) ccmp21.a ,XY,+dic(21)(q I 1),+dcl(21)(ql1)[54])! CB21cl ' 47,XY,+dic(21)(q 11)[21]!46,XY[25] Note.- I In two cases, the % of nuclei with 3 signals was 29% and 55%, indicalive for a mosaic Irisomy 18. Karyotyping revealed a full trisomy Screening wîth the Y-specific probe was negative in all cases. J Nuclei with 3 signals silowed two red (X) and one green (Y) signal. The one nucleus with two signals silowed one green (Y) and one red (X) signa!. 419, 11, and 13 % of nuclei silowed 4, 5, nnd 6 signais, respectively. 48

49 Figllre 2.7 Trisolll)' 18. A) Illetaphase spread anc..l D) partinl karyotype of cultured AF ce lis, alld C) interphase nuclei of IIIlCllltllrcd AF ce lis, showing Ihree chrolllosome 18 signals aftel' hybridization with Ll.84 (18 cenlomere probe). A B Figllre 2.8 FISH results in casc 6 aftl.'r hybridizalion of pdalllx5 (X cenlroml.'re probe) 10 1I0rmai 46,XX AF ce lis (1Illcultured and cultured), showing thai Ihe repcall.'d scquence dell.'c!ed b)' pdamx5 is a polymorpllic Irail. A) ulleultured AF eells showing OlJl.' chrolllosome X signal. D) interphase nuclei of cullured AF cells, showing olie brighl aml olle small chrolllosomc X signa!. C) IIll.'laphasc spread wilh ol1e X chrqmqsolllc showing a brighl signal, anc..l one wilh a slllall, weak signal (arrow). 49

50 Table 2.10 False Ilositi\'c FISH results Case Probe % of nuclei with Karyotype 1-6 signals (chromosome ) L1.26 (13/21) ,XX 2 L1.26 (13121) ,XX,+i( 12)(q 10)[17JI 4 7,XX, +del( 12)( q 1 0)[6]146,XX[ 1 J 3 RPNIl05X (Y) ,XY 4 RPN 1l05X (Y) ,XX 5 CB21cl (21) ,XY 6 pbamx5 (X) ,XX 7 pbamx5 (X) ,XX The first major prospective clinical study colnparing FISH analysis of uncultured AF cells with classical cytogenetics of the celi cultures was published in 1993 (Ward et al., (993), They investigatcd a total of 4500 samples, ll1ainly of pregnatlcies of advanced gestational age, and the)' found 146 aneuploidies of which 107 were identified with nsb. The)' had seven faise negative results (5 %, coll1pared to 2 % in Dur series) and in the rell1aining 32 abnormal cases results were non-infonnative (22 %, compared to 4 % in Dur series). This relatively large I1llluber of non-informative abnormal specimens is the consequence of stringent rcporting criteria they dcsigned, in order to minilnize the risk of false positive alld false negative rcsults. In eight of the 43 abnormal cases we found (three cases of trisomy 21, one triploidy, and fom cases of trisomy 18) hybridization pattcrns did not meet these repoltil1g criteria. However, we believe that the combination of abnormal ultrasound findings and abnormal FISH results, whieh titted thc fetal anomalics, justified the report of an abnormal result in these eight cases, Moreover, their stringent criteria do not allow a diagnosis of mosaieism. The rare mosaic case with the dicentric and dcleted chromosome 21 of our series would, therefore, have gone undetected. In earlier years, cordocentesis was the preferred method for PD in cases of ultrasoul1d abnonnalities in our laboratory, as chromosolllc analysis of fetal Iymphocytcs can be complcted within one week. I-Iowever, fetal blood can only be used to study the karyotype of the fetus, but is unsuitable for metabolic studies which sometimes need to be performed. In additiol1 to same obstetrie contra-indications, such as small-for-gestational-age fetuscs alld umbilical cord obstmctions, cordocentesis may fail in case of oligohydramnios or polyhydramnios. Moreover, fetal blood eau not be stored for potential future studies, and it does not always accurately rcfleet the fetal karyotype in cases of fetal mosaicism. Thcrefore, FISH on uncultured AF cells became thc prefered method for rapid fetal chromosomc analysis during the last years. The Ilumber of cordocenteses in cases of ultrasound abnormalities decreased from 53 in 1993 to onl)' 18 in 1996, \Ve collclude that FISH on uncultured AF cells providcs a rapid and accurate method for 50

51 prenatal identification of chromosomal aneuploidies in pregnancies eomplicated by fetal anomalies. However, the reliabiiity, and therefore, the clinical utility, will stand or fall with specificity and hybridization efficiency of the probes. A tèw probe changes were necessary to reduce the number of false positive and false negative resuits th at were cncountered during the first two years of our investigations. The US probe set that we use today has proven to be highly reliable. Therefore, we believe that abnollnal prsh findings justify the report of an abnonnal result to the parents in pregnancies with US abnormalities. I-Iowever, normal FISI-I results should always be eomplemented by a fidl cytogenetic analysis of the cell cultures, since 18 % of chromosome aberrations in thc group of US abnormalities ean not be detected with probes tor chromosomes X, Y, 13, 18, and 21, as they involve struetural chromosome aberrations, such as marker ciuomosomes and translocations, as weil as aneuploidies of other ChrolllOSOlllCS than those detected by the US probe set. Thercfore, FISH ean not be eonsidered to be a stand-alane diagnostic test Cytogenetic abnol"luality in previous chol"ionic villus sample (CVS) Ir a chromosollle abnonnality, other than non-mosaic trisomy 21, triploidy, 47,XXY, 47,XYY or 47,XXX, is detectcd in CV semi-direct preparations, in thc absence of earl)' US abnormalities, it may represent confined placental mosaieism (CPi"!). Therefore, follow-up investigations in AF eells for verification of the fetal karyotype ánd of potential uniparental disomy (UPD) in case of eonfined placental trisomy, may be nccessary (see section of chapter I). Tablc 2.11 Reasons for not pcrfonning mnniocentesis after linccrtain abnormul H'sulls in CVS Rcason US abnomtalities + TOP FISH on semi-direct CV preparations nonnal TOP on parents request, without fmther investigations Continuation ofprcgnanc)', without further investigations Structural chromosome aberration offnmîiial origin l IUD before amniocentesis N II Total Notc.- US: ultrasound; TOP: tennination of pregnanc)'; IUD: intrauterine death;l two balanced trans[ocations, two inversions, nnd one marker chromosome 37 In appendix publication VII we describe UPD studies in AF ceils in cases of confined placental trisol11)' fotlnd during a four-year period ( ) (see chapter IlI). We tlsed FISH on uncultured AF eeus for diseriminatiol1 between erf"i and gencralized mosaicislll in order I) to get a quick result, and 2) ta preclude generalized mosaicism collcealed in eell 51

52 Table 2.12 Cases of (mosaic) alleuploidy, deteded in CYS, in whieh confirlllatory FISH studies,,,ere perforfllcd on subsequellt ullcultured AF cclls Abnormal cellline Cases of11on Cases of high level Cases of low level Total mosaicism mosaicism (> 33.3 %) mosaicîsm (d3.3 %) J.Common aneup/aidies trisomy trisomy trisomy ,X ,XXY UII/{s/{al trisamies trisomy trisomy trisomy trisomy trisomy trisomy trisomy trisomy X 0 0 trisomy 7,13,20, trisomy 13, J.Strl/etl/raJ aberratiojls + der(21 ;21}( q lo;q IO} der( 13; 13}(q IO;qIO} Te/rap/aid)' Totol cultures. FISH results were in agreement with cytogcllctic results in 13 out of 14 cases that were invcstigated. In one case of full trisomy 16 in CVS, a discrepancy was found with 26 % of uncultured AF cells showing three clrromosome 16 signais, whereas the ecu cultures showed a normal karyotype. Postmortem examinatioll of the fetus after intrauterine death at 33 weeks ofgestation, revealing several eongenital malformations fitting a mosaie trisomy 16 phenotype, and postnatal FISI-I studies, support the presence of generalizcd mosaicism. On the basis of these results we believe that FISH is areliabie mcthod for rapid differentiation bet ween CPM alld generalized l11osaicism, and that it mal' even better reflect the actual fetal 52

53 karyotype than cluomosome analysis of the cultured cel1s, since uncultured cells are not affected by culture induced selection mechanisms. Bctween 1993 and the end of 1996 a total of 110 cases ofpatential CPM were detected amang 3499 CV samples. In 37 cases a follow-up amniocentesis was not performed for various reasons (tabie 2.11); in IQ out of these 37 cases [nlne cases oflow-mosaic 45,X (two out of30 metaphases), and one case of Iow mosaic tetraploidy], FISH on CV semi-direct preparatiolls did not confirm the presence of mosaicism, and normal results were reported 10 the parents without follow-up investigations (see section 3.3.2). In the remaining 73 cases a follow-up amlliocentesis was perfonncd: in 21 cases, AF cells were only studied c)10genetically, because of the involvement of a structural chromosome aberration which was not detectable with FISH, and in 52 cases, FISH was applied 10 uncultured AF cells for rapid verification of the fetal karyotype. The chromosome aberrations initially found in CVS and the number of cases are shown in tablc FISH results in 52 cases are summarized in table In 48 out of 52 cases, FISH resuits were in agreement with cytogenetic results, including 12 abnormal (generalized mosaicism) and 36 normal (confined piacetltal masaicism) cases. Inconsistent FISH and cytogenetic resuits were encauntered in tluee cases, and in ane case, showing a normal karyotype, FISH results were not infarmative. Tablc FISH on unculturcd AF eells for \'erifiealion of a chromosome abllormallty dcteeled in a pre\'ious CVS: number of cases in\'csligatcd from 1993 until1996, and FISH resulls Year N FISH results Nomlal AbnoffiHlI Non informativc (false negative) (false positive) (0) 3 (I) (0) 5 (1) (1) 4 (0) (0) 2 (0) 0 Tota! (1) 14 (2) The twelve cases of generalized mosaicism, encountered by bath methods, are shown in table In II of these 12 cases, FISH correctly identiticd the presence of a non-mosaîc ór mosaic chromosome aberration; only in case 10, FISH results were suggestive of a mosaic trisomy 21, whereas the cell cultures revealcd a fuil trisomy 21. lvlaternal cell contamination of the AF sample may be an explanation for the relatively high proportion of disomic uncultured AF cells. The 111'0 fa/se posifive FISH l'esults, and the fa/se l1egatil'e FISH jinding are shawn in table In cases 1 and 2, FISH on uncultured AF eells confirmed the presence of an abnonnal ceillinc, whereas the cell cultures SIlO wed a normal karyotype in seven and 15 cell colonies, 53

54 Table 2.14 Cases of generalized mosaieism: karyotypes in CVS and eultured AF eells, and FISH s~nal distributions in uneultured AF eells Case Karyotype in CVS Probe % of nuclei with 1-4 signals Karyotype in cultured AF eells [number of eells] [number of eeu eolonies] N ,XX,+18[18] LI ,XX,+18[1] 2 47,XY,+18[30] LI ,XY,+18[4] 3 45,X[31] X+yl ,X[3]/46,XY[20] 4 45,X[30] X+Y ,X[13] 5 47,XX,+9[30] phur ,XX,+9[7]/46,XX[30] 6 47,XY,+22[17] 58B ,XY,+22[5] 7 46,XX,der(21 ;21)( q I O;q I 0)[21] CB21cl ,XX,der(21 ;21)(qI0;ql 0)[2] 8 47XY,+18[18]/48XY, LI ,XY,+18[5] + 18,+20[13]!46,XY[3] Te! 20p ,XY,+ 18[27]/46,XY[3] LI ,XY,+18[21] 10 47,XX, + 21 [24]/46,XX[ 6] CB21el ,XX,+21[19] II 47,XXY[6]!46XY[2] pbamx ,XXY[3]!46XY[6] 12 45,X[6]!46,XX[24] pbamx ,X[I]/46,XX[15] Note.- j X+Y: mixture ofpbamx5 and RPN1305X

55 respectivcly, excluding a mosaic of 35% and 19% with 95% confidcncc, respectively (Hook, 1977). It is passible that the abnormal ccils in bath cases had a proliferative disadvantage as compared to normal cells, which may cxplain their absence in the cell cultures, as was shown for trisomy 9 mosaicism (van den Berg et al, 1997). On the other hand, the FISH results may represent true false positive results due to aspecific hybridization of the 16-prabe to another chromosome, and hybridization failure of the X centromere prabe in the other case. However, whether the FrSH results in the trisomy 16 case were false positive indeed is questionable, considering the pregnancy outcome, postmortem examination ofthe fetus, and postnatal FISH studies in this case (sec appendix publication VII). Tahlc 2.15 Discrepant FISH nnd cytogelletic results: karyotypes in CVS and cultured AF rells, FISH signa! distributions in uncultured AF ceus, and pregnancy outcome Case Karyotype in CVS Probe % ofnuelei with Karyotype in Pregnaney [numberofcells] 1 4 signals cultured AF eells outeome [number of eell eolonies] 2 J 4 47,XX,+16 [301 phur J 46,XX[7] IUD 33 wks., 845g, MCA' 2 45,X[15]146,XX[7] pbamx ,XX[15] healthy!fl: J 45,X[JO] pbamx5 9 9t ,XI5]146,XX[34] healthy ~2 Note.- IUD: intra-uterine death; r.. ICA: multiple eongenital abnomlalities;l see appendix publication VII~ mosaicism neonatally confimled in lymphocytes: 45,X[4Jf46,XX[46J In addition to two false positive results, we had alle fa/se llegative FJSHjillding.ln case tluee of table 2.15 a non-mosaic 45,X was tàund in CVS. FISH analysis in llncultured AF cells showed normal signal distributions with the X centromere probe indicative [or a normal female karyotype. CllItured cells showed a low mosaie 45,X[5]146,XX[34], whieh was postnatally eonfirmed in l)'mphoc)'tes. In general, it will be diffielilt to deteet low level mosaicism due to the braad ranges of nuclei exhibiting one, two, and three signa Is in normal samples. Therefore, the sensitivity of FISH [or detection of small subpopulations of monosomic or trisomic cells is limited by the frequenc)' at which they oecm in normal contral samples (Eastmond and Pinkel, 1989). On the other hand, we were able to detect two other cases oflow levelmosaicism (13 % and 7 %) involving a 45,X cellline, with 16 % and 24 % ofthe interphase nuclei showillg one X signa!, respectively (cases 3 and 12 in table 2.14). On the basis of these results we believe that the distribution of normal and abnormal cells in AF cell cultures does not necessarily reflect the pattern found in uncultured AF cells (Kromer et al, 1996; Bryndorf et al, 1997), which may be explained by select ion in favour of normal or abnonnal cells during cell cuituring. In conclusion, FISH on uncultured AF cells allows a rapid differentiation between CPNI and generalized mosaicislll, shortening the reporting time of the fetal karyotype to the parents. However, discrepancies between FISH (uncultured cells) and cytogenetic (cell cultures) results may occur, and may callse au interpretation dilemma. Firstly, false positive FISH filldillgs may sometimes be the result of technica I failure of the probe. However, selection 55

56 against abnormal cells in the cell cultures may be another explanation, with cytogenetic results, in f..1ct, representing false ncgative results. Generally, it is believed that in mosaic cases Ul1cultured cells better retlect the fetal chromosome constitution than cultured cells, due to selecth'e in vitro gro\\1h (Lomax et al., 1994; Bryndorf et al., 1997), Secondly, false negative FISH resltlts ma)' occur if the level of 1110saicism is below the detection level of interphase FISI Dfher indications Other indications for performing FISH on uncultured AF cells are: I) previous AF sample abnormal 2) abnormal triple test ClT) resldts (risk of Down syndrome " 1120) 3) advanccd matel11al age (:?: 44 years) 4) twin pregnancy ofadvanced gestational age (± 16 weeks) 5) X-linked l11ental retardation or biochel11ical disease In all these cases a quick result is important because of an increased risk for a chrol11osome aberration (indications 1-3), or for pregnancy complications in case of selective termination aner 17 weeks of gestat ion (indication 4). For indication 5, a rapid verification of fetai sex is Ilecessary, as filliher prenatal biochemical or DNA investigations are only illitiated when the fetus is male. Indication 1. Between 1993 and the end of 1996, we applied FISH to ul1eultured AF celis of a second AF sample in seven cases, because onc or a iew eell colonies were found to show a chromosol11e abnormality in the tlrst sample, and Ihe tolal number of eell colonies was too sillall to exclude true mosaicism, according to Hsu et al. (1992). Cytogenetic and FISH results are shown in table FISI-I results were concordant with cytogenctic results in all seven cases. FISH confirmcd the presence of a mosaic cl1romosome aberratiol1 in two cases (cases 3 and 4). Subsequent chromosome analysis confinned the FISH result in case 4, although it revealed a higher mosaie trisomy 18 (66 %) than was found with FISH (I8 %). This is in contrast with the more comlllon observation of a larger proportion of abnormal eells in an uncultured specimen as compared to the cultured tissue, most probably due to a proliferative advantage of normal cclls over abnormal cells (Lomax et al., 1994). In case 3, chromosome analysis was not performed, but FISH was simultancously applied to celis of different compartments (AF cells, umbilieal cord Iymphocytcs, anel CV), all revealing a collsiderable number of trisolllic cells (lymphoc)1es and CV results not shown), which, together with slightly abnormal US findings, made a diagosis of generalized true 1l10saicism tor trisomy 9 ccrtain (van den Berg et al., 1997). Aner termination ofthe pregnancy, thc prenutal findings were confinned in different fetal and placental tissues in bath cases. In the other five cases, FISH on the sccoild sample did not show the prcscnce of a significant proportion of abnonnal eells, which was confirmed by cluomosomc analysis of the eeil cultures. 56

57 Table 2.16 FISH on uneultured eells of a seeond amniotie fluid sample (AF2) for verification ofa chromosome aherration in a previous AF sample (AFI) Case Karyotype AF I Probe % of nuclei with 1-3 signals in AF2 Karyotype AF2 N ,XX,+8[1]/46,XX[10] "p ,XX[2S] 2 47,XY,+9[1]/46,XY[S]' phur II 8S 4 46,XY[17] 3 47,XY,+9[4]146,XY[26]' phur , 4 47,XX, + 18[3]/4 7,XX,+ 16[1]/46,XX[ 14] L ,XX,+ 18[19]/46,XX[8] phur S,X[I]/46,XX[7] pbamx ,XX[24] 6 46,XX[ 13]l46,XY[2] CEPx/CEPY ' 0 46,XX[IS] 7 92,XXYY[ I ]/46,XY[9] L ,XY[16] phur Note.- 1 and ~: cases 5 and 3, respectively, in van den Berg et al. (1997): ) karyotyping was not performed; 4 99 % of AF cells showed two red (X) signals

58 lndications 2-5. Behveen 1993 and the end of 1996, 25 cases were investigated within indication groups 2-4. Cclls were screened with the US probe sct (see materials and l11ethods section 2.2, and table 2.2). 'fhis revealcd two chromosol11c aberrations (trisomy 21 and 47, XXY), and 23 normal results by both methods. Additionally, 14 cases were investigated with a chrol110some X and Y specific probe for determination of fetal gender (indication 5). No discrepancies were found behveen FISH and chromosome analysis. 3.3 Intcrphase FISH fol' studying chroillosomal mosaicisill Mosaicism in UIlllliotic fluid (AF) ceu cultures Several prenatal cases of presumed pseudomosaicism in AF cell cultures have resulted in the birth of children affected with true mosaicism (Camurri et al., 1988; Cheung et al, 1988; Vockley et al., 1991). Differentiation behveen both phenomena is therefore essential, and requires the analysis of a large number of cells (Hsu et al., 1992). Between 1993 and the end of 1996, we uscd FISH in nine cases of pseudomosaicism in AF cell cultures, all involving one abnormal colony, in order to test the reliability of interphase FISH fol' discrimination between pscudomosaicislll and true mosaicisl11 (tabie 2.17). FISH was applied to cclls that were left on the bottom of the l1affected" culture dish (dish with abnormal colony), so excluding the abnormal colony from analysis, andjor to remaining cells of other culture dishes of the same sample. These cehs \Vere trypsinized and seeded on glass coverslips before FISH analysis. In all cases, except for case 4, FISH did not reveal the presence of a trisoillic cel! line, confirming the cytogenetic diagnosis of pseudomosaicism. A diagnosis of pscudomosaicism type A could be made in case 3 (partial abnonnal single colony), whereas the others showed a pseudomosaicism Iype B (abnormal single colony) (Boué el al., 1979). In case 4, involving one trisomy 9 ceu colony and five colonies with a normal karyotype, FISH applied to cells of the "affected l1 dish revealed a significant proportion of trisomic cells. This implicates that at least one additional colony in the affected dish showed a trisomy 9 karyotype. FISH signal distributions in the two other dishes were nonnal, cstablishing a diagnosis of pseudomosaicism type C (more than one abnormal colony rcstricted to one culture dish). In all nine cases FISH results were in agreement with the cytogenetic diagnosis of pseudomosaicism. Pseudomosaicism was confirmed by follow-up investigations in a repeat AF sample in two cases (cases 2 and 4. See tablc 2.16, cases 1 alld 2, respectively), and by the birth ofhealthy children in the olhers. In conclusion, these results suggest that whenever the number of analysable celt colallies is too smal! to exclude truc mosaicislll, iuterphase FISH cau be used to determiue whether ally additional eeu colonies have an abnormal karyotype, so that a secolld amlliocentesis for confirmatory studies ean be obviated. In all cases, FISH was applied to trypsinized ceu colonies left on the bottom of the culture dishcs after removal of the coverslip canying the 58

59 Table 2.17 Differentiation between pseudomosaicism and true mosaicism in AF cell cultures usin2 interphase FISH analysis Case Karyotype Probe Dish' % of nuclei with 1-4 signals Prenatal diagnosis N XY.+8[1]/46.XY[33J "p8 A pseudomosaicism type B 2 47.XX +8[ 1 JI46.xX[ 1 OJ "p8 N pseudomosaicism type B 3 47.XX.+9[IJ'146.XX[18J' phur98 N pseudomosaicism type A 4 47.XY.+9[IJI46.XY[5J' phur98 A pseudomosaicism type C N N XY.+ 16[IJI46.XY[17J phur195 A pseudomosaicism type B N XX.+ 17[IJI46.XX[16J pl7h8 A pseudomosaicism type B N N XY.+ 18[1 ]/46.XY[28J LI.84 A pseudomosaicism type B N XY.+21 [1]/46.XY[23] ccmp21.a A pseudomosaicism type B N N XX.+21 [IJI46.XX[23J CB2lci N pscudomosaicism type B Note.- I A- dish with abnonnal eeu eolony (the abnonnal eolony was NOT inc\uded in the FISH slide, and hence in tbe scoring), and N- dish with nonnal cell colonies: 2 colony includes nonnal and abnonnal cells: J, 4: cases 4 and 5, respectively, reported by van den Berg et al. (1997)

60 eell eolonies that are used for cytogenetic analysis. Nowadays, we pref er to destain previously trypsin-giemsa stained slides prepared for chromosome analysis (one normal slide of each AF container, and the liaffected slide ll ), and use them for FISH analysis as wcll, in order to augment the number of actually investigated cell colonies with those not suitabie for chromosome analysis l\'Iosaicism in semi-direct choriollic "illus prepuratiol1s Interphase FISH on semi-direct CV preparations may add to the cytogenetic data in two instances: I) if non-mosaic or 1110saic tris0111y 18 is eneountered 2) iflow level 111osaicis111 ( :<>:33.3 % abnormal eelis) is detected 1) In appendix publication V we silowed that iftrisomy 18 is found in CV semi-direct slides, interphase FISH mal' contribute to the results of classical chromosame anall'sis in terms of predicting the fetal chromos0111e constitution. Mosaic and non-1110saic tris0111l' 18 mal' be confined to the placenta and mal' not represent the fetal karyotype (see section 1.4.2). Therefore, confirmatory studies in CV long term cultures and AF ëells' mal' be necessarl'. Sinee CV cultures were not established during the study period ( ), we investigated the use of interphase FISH as a potential tooi of rapid verification. Thirtl' cases of trisomy 18 (22 non-mosaic and 8 mosaic cases), encouutered during au 8-year period ( ) were, retrospectively, iuvestigated. The onl)' non-confirmed case of full trisoml' 18 had a significantll' smaller nllmber of interphase nuclei displaying three signals in semi-direct preparatiolls than the real, confirmed cases of trisomy 18. In cases of mosaic trisoml' 18, the application of FISH also contributed to the results of traditional Illetaphase analysis: higher levels of tllfee signal containing nuclei were found in the three confirmed l110saic cases as compared with the four non-confirmed cases. If the percentage of nuclei with three signals was smaller than 66 %, trisomy 18 was not eonfirmed in fetal eells. In general, the FISH data were better able to predict the fetal chromosome constitution than cytogenetic analysis of CV semi-direct slides, although FISH yielded ambivalent results in some cases. Between 1993 and the end of 1996, another eight cases of trisomy 18 (fom non-mosaic and four mosaie cases) werc studied with FISH (tabie 2.18). In seven out of eight cases, FISH results were suggcstive for trisoml' 18 in the fetus in tivc cases (cases 1,2, 3, 5 and 6) and CPM in two cases (cases 7 and 8), which was confirmed b)' follow-up investigations. In one case ofnon-mosaic trisomy 18 (case 4), ambivalent FISH resldts were achieved, which could correspond to eïtlwf situation of non-mosaic trisomy 18 in the fetus, generalized mosaicism, or CPM. Fetal karyotyping after termination of the pregnancy, on the basis of US abnonnalities and cytogenetic results, confirmed a non-mosaic trisomy 18 in the fetus. In conclusioll, interphase FISH results on CV semi-direct slides of (noll)-mosaic trisomy 18 cases can aid in the collnselling procedures, although a final result can onll' be achieved bl' (FISH or eytogenetic) analysis ofa subsequent AF sample. 60

61 Table 2.18 FISH on CV semi-direct prcparations in cases of (noll)-mosaic trisomy 18, and pregnancy outcomc Case Karyotype in CVS % ofnucjei Follow-up and prcgnancy [number of cellsj with 1-3 signals outcome ,XX,+18[18] TOP after amnioc. (case I in table 2.14) 2 47,X-,+ 18[18] TOP (US abnomlalities); tris. 18 confirmcd in fetal skin 3 47,XY,+18[30] TOP after amnioc. (case 2 in tablc 2.14) 4 47,X-,+ 18[18] TOP (US abnormalities); tris. 18 confimled in fctal skin 5 47,XY,+ 18[27]/46,XY[3] TOP after amllioe. (case 9 in table 2.14) 6 47,X Y, + 18[ 18]/48,XY,+ 18,+ 20[ 13]146,XY[3] TOP after anmioe. (case 8 in table 2.14) 7 47,XX,+ 18[2]146,XX[28] Continuatioll after amnioe.; hcalthy 'i!, SGA 8 47,XX,+ 18[2]/46,XX[28] Continuatioll after amnioc.; healthy'i! Note.- TOP- tennillation ofpregllancy; US- ultrasound; SGA smal! for gestatiollal age 2) If a chromosome abnormality is encountered in CV semi-direct preparations, in the absence of early US abnormalities, it may represent CPM and follow-up investigations, such as amlliocentesis, may be necessary for verification of the fetal karyotype. Between 1993 and the end of 1996 we encountered IlO cases ofpotential CPM (3,1 %) among 3499 CV samples, aud amuiocentesis was perfonned in 73 cases, representillg a second invasivc procedure in 2,1 % of women undergoing CVS. Low level mosaicism (.:<>: 33.3 % abnormal cells) was encountered in 37 (34 %) cases, with :0;;: 10 % abnonnal cells in 25 of these cases. The question is whether these cases represent mosaicism, justifying follow-up studies in a secolld sample, or wether the abnormal cells are the consequence of alocal mitotic division error. In order to discriminate between both phenomella. we performed fish in these 37 cases; FISH confinned the presence of mosaicism in 13 cases (two out of 25 cases with.:<>: 10 % abnonnal cells, and 11 out of 12 cases with > 10 % abnormal cells) (tahle 2.19), and normal results were found in the remaining 24 cases. Since FISH analysis involves {he scoring of a few hundreds of cells, we believe that it hetter reflects the rcal level of mosaicislll than traditional clll'omosome analysis. Therefore, it is likely that the few abnormal metaphases in the 24 cases with normal FISI-I results represented local mitotic errors instead of mosaicism. 61

62 Table 2.19 Abnormal FISH distributions in CV semi-direct slides in cases of low level mosaicism (:::: 33.3 % abnormal cells) Case No. Abnormal cell line %abnormal Probe % of nuclei with 1-4 signals cc11s J.Common aneuploidies S,X( 45,x/46,XX) 20 pbarnx , 0' ,X(45,X/46,XY) 13 CEPX/CEPY 20' ,XXX 30 pbarnx Unusual trisomies 7 trisomy 3 17 po: trisomy 7 16 po:7ti trisomy plcll.a Note"~ I 20 % ofthe nuclei showing only one red (X) signa!. and 80 % showing one red (X) and one green (Y) signa!.

63 In 12 of these cases, a mosaic 45,X/46,XX or 45,X146,XY was involved. The two 45,X ceus (on a totalof 30 metaphases) in all these cases mayalso have been incomplete normal cclls, rather than monosomy X celis, since incomplete metaphases are often observed in CV semidirect preparatiolls. However, low level masaicism, belaw the detection level of interphase FISH, can not defillitely be excludcd in these cases. \Vhen FISI-I is applied ta normal diploid control samples, a small pcrcentage of ceus wil! exhibit only one signal, due to technical factors such as inef±1cicnt probe hybridization, an inefficient detection of hybridized probe, or overlapping signais. Another small proportion will show three signais, due to aspecitlc probe hybridization or the so-called "split spots" caused by DNA replication in G2 cclls (Eastmond et al., 1995). Therefore, the sensitivity of interphase FISH is limited by the frequency of oneand tluce signal containing cells in norl11al samples (Eastmond and Pinkel, 1989) (see table 2.2). During the first years of our investigations, follow-up studies in AF cells were perf0fj11ed in masaic cases with normal FISH results. However, after recurrent normal findings in AF cells and the birth of healthy children, an amniocentesis was no longer perfornled in 12 out of the 24 cases with normal FISH findings, all ofwhich resulted in the birth ofhealthy children. In conclusion, FISH adds ta the c)1agenetie result in CV semi-direct slidcs in cases of lo\\' level mosaicislll, since it enables a rapid differentiation between real mosaicism and local mitotic errors, apparently without clinical significance, for which follow-up investigations, such as amnioccntesis can be omitted. 3.4 FISH for dctcctioll of microdclctions In appendix publication VI, we report on the first PD by FISH of a familial 22q 11 microdclction, associated with a spectmm of malformations (Cardiac defects, Abnormul facies, Thymic hypoplasia, Cleft palate, Hypocalcaemia) covered by the acronym CATCH 22 (Wilson et al., 1993). The deletion was known in a previous child with symptoms of the classical DiGeorgc syndrollle, who died two weeks after birth, and turned out to be present in the physically norlllal tàther, who s)lowed same psychiatrie problems. The deletions were not visible C)1agenetically. FISH with a probe from the DiGeorge syndrome critical region (Mulder et al., 1995) silowed to be areliabie and rapid method for their detection. Betwecn 1993 and the end of 1996, a total of 48 prenatal cases at risk for a lllicrodcletion syndrome were studied with FISH. The syndromcs involvcd wcre CATCH22 (chromosome 22q 11), tviiller-dieker syndrome (chromosome 17p 13.3), Williams syndrome (chromosome 7q11.23), and Tuberous Sclerosis Complex 2 (TSC2) (chromosome 161'13.3). Such lllicrodclctions are difficult to detect or cannat be detcctcd at au with classical banding techniques, even with high resolution banding. The isolation of FISH probes from the critical rcgion of these microdelctioll syndromcs enabled their rapid and reliable detection (Van Tuinen et al., 1988; KlIwano ct al., 1991; Desmaze et al., 1993; Ewurt et al., 1993; European Chromosalllc 16 TlIberous Sclerosis Consortium, 1993). In most cases the indication for PD was a previous child with the dcletion alld/or the 63

64 associated syndrome. FISH was perfonned irrespective of the PISH results on the parents' Iymphocytes, taking into account the small possibility of gonadal mosaicism. In all but one case (described in publication VI), normal FISH results were achieved, as could be expected. 64

65 Chapter 111 Fetal uniparental disomy (UPD) with and without confined placental mosaicism (CPM)

66

67 1. Aim of thc experimental work Confil1cd placental 1l10SaIClSI11 (CPN!) involving a trisom)' ma)' be associatcd with fetal uniparental disomy (UPD), i.e. both chromosomes of a cluomosome pair dcrived from one parent ani)', as was described in section of chapter 1. Briefty, in such sîtuation, the tri som)' in the placenta has a meiotic origin, and fetal UPD m"îses through loss of Qnc partictilar copy of the set of three chromosomes (namel)' thc chromosome from the parent contributing ouly anc copy) in embryonic progenitor celis, so that the two chromosomes left are from ene parent only. The process of removai of one chromosome from the trisoll1ic conception is called "trisomic zygote rcscuc". The aim of the cxpcrimentai \Vork described in this chapter was ta investigate the incidence of fetal UPD in a collsecutive series of chorionic villi (CV) samples collected during four years ( ), in order to get more insight into the origin of placenta confined trisomy, and in the mechanism oftrisomic zygote reseue. 2. Results and discussioll 2.1 Incidcllcc of unipal'ental disoll1y associatcd with confined placclltalmosaicism (appendix publication VII) Among 3958 CV samples that we investigatcd cytogenetically during four ycars using the semi-direct preparation technigue, 69 cases (1,7 %) ofcpm (type [or type 111) were lound. Of these 69 cases, 29 cases (42 %) involvcd a trisomy. Parental origin studies could be perfornled in 23 of these cases, l'evealing UPD (maternal heterodisomy 16) in only one case. There arc tllfee factors that seem to be important in predicting the disomic state of the fehls: (I) the ehromosome involvcd (2) the level of 1110saicism (3) the type ofcpm The incidence of UPD associated with CPi,,! is expected to be one in three, if CP?..,! is the result of trisolnic zygote rcscuc, and 10ss or removal of one of the three ehromosomcs is a random proeess (see figure 1.6). This theoretical figure has been established for ehromosomcs 16 and 22 (Kalousek et al., 1993; Wolstenholme, 1995, 1996), but not for some other chromosomes, sueh as ehromosomes 2, 3, 7, and 8 (Robinson et al., 1995; Kalousek et al., 1996; Shaffer ct al., [996; Wolstenholme, 1996; Robinson et al., 1997). A different origin of the placenta confined trisomies involving these chromosomcs, bcing predominanti)' meiotic for ehromosomes 16 and 22, but mitotic for the othel' chromosomcs, explaills the difference in frequency of UPD depending on the cluomosome involved (Robinson ct al., 1997). Likewise, a high level of mosaicislll in both placental cell lineages (cytotrophoblast and mcsenchymal core) was expcctcd and shown to be significantly correlated with a meiotic origin of thc trisomy, and therefore with feta I UPD (\Voistenholme, 1996; Robinson et al., 1997). The low 67

68 incidence (one in 23) that we found, could theretàre be expected as iillllost cases Iow level mosaicislll was found in semi-direct CV prcparations, most probably having a mitotic origin (Cranc anel Cheung, 1988; Wolstenholme, 1996; Robinson et al., 1997). One other group investigated the incidence of UPO in a series of 94 cases of CPiv[ and found UrD in 17 cases, illcjuding 13 cases ofupd 16 (RobillSOll et al., 1997). Howe"er, their study poptilation might not be considered a random sample of CP~\'1 cases cllcountered during prenatal diagllosis (PO), because of inclusion of postnalal cases ascertained through illtrauterine growth retardatiol1 l10ted at birth, alld because of a large lllllllber of trisomy 16 cases, since they were the initial focus of their research. Both f:1ctors may contribute to an overestimation ofthe incidence ofupd. In conclusion, the incidence ofupd associated with CPM (type I or type In), in a consceutivc series of CV samples collcctcd during four years, is very Iow, indicating that illlllost cases the trisolllîc cell line most probably originates from somatic duplication, whieh is supported by the low level of mosaicislll illlllost of these cases. The norlllal pregnancy outcollle of the UPD 16 case we found, fmther supports the hypothesis that the impaired feta I gro\\1h, encountered in most cases ofupd 16, may not be the result of the UPD itseif, but is miher due to a malfunctioning placenta, caused by high levels of trisolllîc cells in the placenta (Kalousek et al., 1993; Kalollsck and Barrett, 1994; \Voistenholme, 1995; Brandenburg et al., 1996; Robinsoll et al, 1997). However, a dysfunctional placenta can not explain thc tètal congenital malformations observed in same cases of UPD 16. We hypothesize that in symptomatic cases, trisomy 16 cells are in fact not confined ta the placenta, but that a masaic trîsamy 16 is also present in the fehls, despite a normal karyotype in aml1iotic fluid (AF) cell cultures. This is illustrated by ultrasound and pathalagical observatiolls of the fehls, and FISl I studies, indicating a mosaic trisomy 16 in unculhlred AF cells, perfonned in a CPtvI 16 case without UPD of our series. \Ve advise the analysis of uncultured AF ce Us for verit1catian of (mosaic) trisomy 16 in fetal ce lis, since the trisomic cell line may be complctcly lost in AF cell cultures due ta selection. Finally, we showed th at the obstetrical eomplications, found in seven out of 23 Cp:rv[ cases of the present series, are not thc consequence of fetal UPD. 2.2 Ullipurental disomy,,,ithout collfincd placenta I Illosaicism: a moûel fol' tl'isomic zygote rescuc (appendix publication VIII) Although fètal UPD is prenatally lllainly suspcctcd and observed in cases of confined placenta I trisoj11y, the incidence ofupd associated with CPiv[, investigated during a four-ycar period ( ), showed to be low (one in 23 cases of confined placental trisomy). Surprisingly, during that same time period, we encountered two cases of UPO which were both associatcd with a normal karyotype in semi-direct CV preparations: one (UPD 15) was only discovcred postnatally after birth of a child with Pradcr-\Villi syndrome, alld the ot her (UPD 16) was prenatally encountcred in thc course ofprenatal DNA analysis ofthe hlberous sclerosis complex 2 regioll at 16p cse findings suggest that a normal karyotype in CV 68

69 Illight not be an exception in cases ofupd. Of all mechanisms potentially leading to UPD, that have been proposed bl' Engel (1993), gamete complementation and trisoinic zygote rescue seem to be thc most likely causes of UPD in these two cases. Engel (1980) calculated the potential incidcnce of UPD, originating from gamete complementatiol1, i.e., union of a disomic gamcte with a gamete nullisomic for the homologue, based on data from cytogenetic studies of spontancolls aboliions. The expected incidence was calculated as 2,8 in conceptions. However, so far, no convincing proof of UPD by gamete complemcntation has been reported. Therefore, trisomic zygote rescue seems to be the most Iikely cause. The precise mechanism of trisomic rescue is not known. Anaphase lagging (AL) and noi1- disjunction (ND) in an earll' postzl'gotic cell division have been proposed (figure 3.1) (Kalousek, 1994). However, both seem not to be perfect: anaphase lagging will give rise to one disomic and one trisomic daughtcr ecu, and l1011-disjunetion will produce one disomic and alethal quadrisomie eeli, reducing the number of blastolllcrcs signifieantly, especially when trisolnic zygote rescue takes place in the flrst two ecu divisiolls, which may hamper further normal developmcnt (Tarin et al., 1992 ). Therefore, wc propose an alternative correction mode, whieh we call ehromosome demolition (CD), and which ii1volves the deshuetion and removal of one of the set of three ehromosomes during ecu divisioll resulting in two disomie daughter cells (figure 3.1). Figurc 3.1 Mcchanisms of trisonlic zygote rcscue: A) chromosome delllolition, B) noil-disjunctioll, flnd C) flllaphase lagging \Ve would likc to present a model for the arising of the various combinations of karyotypes in semi-direct CV preparations (cl'totrophoblast of CV), long-term cultured CV (LTC CV) (mesenchymal core of CV), and the fctus frolll trisomic zygote reseue by these three different correction modes (figures 3.2). Takillg into account the embryogcllic modcls proposcd by Crane and Cheung (1988) and Bianchi et al. (1993) (sec figure 1.5), we further assume the eorrection to take place in the fint four ecu divisions, with a subsequently unkllown 69

70 distribution ofnormal and abnormal eells among the progenitor eells oftrophoblast and inner cell mass (lcm) unti! the 16-cell stage. In case of a reduced numher of blastomeres by ND eorreetioll, we assume that colupellsatory reallocatioll may oeem between trophoblast and rcm. From the 16~cell stage ollwards, at which the separation between trophoblast and lcm occurs, we assullle that celis within the rcm can still allocate their daughter cells freely to the colnpartlllent of the fehls, to that of the extraembryonic mesoderm (EEM), or one daughter cell to each colllpartment. As shown in figures 3.2 aud 3.3, trisolnic zygote reseue by CD and ND can explain all combinations of karyotypes in trophoblast, EEM, and fetus that have been described in cases of fetal UPD associated with CPM or normal karyotypes in both placental cell lineages, as weil as in cases of generalized mosaieislll with UPD in the disomic cell line. During the first two eell divisions we believe that CD is the preferred method for correction, since ND wil1 critically reduce the number of blastomeres, and AL can not produce fetai UPD in combination with normal karyotypes in all compartments. From the third cell division onwards, their is not much difference bctweell the tlrree types of correction. The frequently observed eombination of a full trisomy in both placental celllineages and a normal karyotype but UPD in the fetus can only be produced by ND or AL in the fourth cell division. Of all theoretical combinations of karyotypes in the various compm1ments as deseribed by Piltalis et al. (1994), CPM 11 (abnormal cells confined to LTC CV) does not occur in this hypothetical model. This is in agreement with the observation that, up till IlOW, fetal UPD has never been described in association with this type of CPM. Therefore, CPM type II most probably has a mitotic origin, as suggested by Wolstenholme (1996). Most Jlublished cases ofupd showed to be associated with CPM type III (abnormal cells in both semi-direct CV preparations and LTC CV). However, theoretically, it may be found in cases of CPM type I (abnormal cells confined to semi~direct CV preparations), involving either a 100 % trisomy, or high level mosaicism (> 67 %). This was actually show11 by two cases (one ofupd 10, and one ofupd 9), described by Jones et al. (1995) and Wilkillson et al. (1996), respectively. Robinson et al. (1997) found a significant correlation between a meiotic origin of the trisomy and high levels of trisomic cells in both placelltal eell lineages, especially in the trophobiast. Our model shows that, at least theoretically, trisomic zygote rescue in the first two cell divisions through CD may result in Iow level Jl10saicism (about 33 %) in the cytotrophoblast. 70

71 Figure 3.2 Theoretical distributions of trisomie and disomie eells at the 2-, 4-, 8-, and 16-eell stage after trisomic zygote reseue through chromosome demolition (CD) and anaphase lagging (AL). The resulting karyotypes in long-term cultured CV (LTC-CV) and fetus, originating from the lcm, and in semîdirect CV preparations (short-term cultured CV=STC-CV), originating from trophoblast, are shown. A= abnormal, N= normal, M= mosaie...., COIT"'hOn mode 2-<:etl <k,"... "!G-<:cU Rtsulting I:ar)totyp,,, ""d ecu d!vision '''''' co, ~ ~,., CO" AC" com -- AC" - co >V AUV -- I odisomic.trî$omic I 't:o~e sta!:",t:i~c , - 'CM Tropbobh,-" Fcru, LTCvUli STC"WJ,,, -re -@ -, M :<I~\1 M M """" A,..A!I! 0 - lii8s8 N.M.A M M 11!!888 M.A M M, A liiis88 A A M -,, A M M """" "" A, ",," M M..A M.A M M, A - A A M N.MA M A M.A M M, A..A A A M - MA M A -e, A - - A A M -., - e - - e - -

72 Figure 3.3. Theoretical distributions of trisomic and disomic ceus at the 2-, 4-, 8-, and 16-cell stage after trisolllïc zygote rescue through non-disjunction (NO), with alld without compensatory realloeation ofthe cells between inner cellmass (lcm) nnd trophoblast. The rcsulting karyotypes in long-term cuuured CV (LTC-CV) and fetus, originating from the lcm, and in semi-direct CV preparations (short-term cultured CV=STC-CV), originating from trophoblast, are shown. A=abnormal, N= normal, 1\1= mosaic. Celldi,l,l~" "ral!",.~ "4-çellmt~ &"ç.ll'ijt~ 16" «:1,IJg' R<>u1tîngW)Njr<' T',,>,',,,'-'h,! Fe!", LTC,ilIi STC\illi -SJ N N N N N N JIIO 00 N N N S88 " '* - -ij' N N A OO~ N.\\ A N 00" liiil KMA.. " ~ ma MA M N A... " li8il A A " 000 N N " ~ N.\I M A N " M M '''' N A - A A M 00 l1li8 N N M ~ N A M M A " N.. - A A " A '".. - -* >V.. I odisomic.1risomic " - ~ N,MA.. ~ MA - - ~ N.\I MA A l1li8 A A O N N A - M,A A MA A - " M N A " N " A A M M N A A " N A A M M A N A " A N A " A N A " " A " A " 72

73

74

75 COllcludillg l'emal'l<s alld futul'c pl'ospects In Dur efforts te overcomc limitations of traditional c)1ogelletic studies in umuiotic Duid and chorionic villi cells, the FISH technique provcd to he all Îndispcnsable tooi for prcnatal diagnosis. We use FISH as all adjunctive tooi in about 8 % (about 200 samples per year) of all prcnatal samples that we rcccive in Dur laboratory. In addition to the tour indications discussed in this thesis, wc naw also llse FISH [or cytogenetic confirmation of all prenatally dctcctcd (non)-mosaic aneuploidies in uncultured fetal ajld/or placental tissues after tcnnination ofpregnancy. Continuous development ofnew FISH prohes, such as, recenti)', that ofchromosome-specific subtclomcric probes (National Institutes of Health and Institute of Molecular lvledicine Collabomtion, 1996), wil! further improve the detection mtc of chromosame aberrations with FISH at metaphase as weil as intcrphase level. Comparativc genol11ic hybridization (CGH) (KaIIioniemi ct al., 1992) may significantly add to interphase cytogcnetics, as a technique for screening the entire genome for losses and gains of DNA. ft ma)' theoretically improve the detection rate of ehromosome aben'ations in unculhlred amniotic fluid cells of pregnancies at high genetic risk (Bryndorf et al., 1995), and may allow thc cytogenetic investigation of early cleavage embryos, which will be important for understanding the arising of chromosolllal mosaicism during early embryogencsis (Delhanty et al., 1997). The development of ncw techniques, sueh as SKY (Schröck et al., 1996; Veldman et al., 1997) and micro-fish (Viersbach ct al., 1994; Engelen et al., 1996) will flirther improve the efficiency and accuracy of chromos01l1c idcntificatian. The introduction of FISI-I also added to preimplantation diagnosis (PID) of genetic discases. The first applications ofpid werc ta avoid X-linked discases by selection offemale cmbryos, and cmbryo gender was determined by using PCR amplification of X and Y chromosolllc specijlc sequences (Handyside et al., 1990), However, at the bcginning of the 1990s, FISH with chromosome X alld Y specific probes became the methad of choice for embryo sexing (Griffin ct al., 1992), since FISH is not affected by sample contamination and it allows to detect the chrolllosome X copy Ilumber as weli, avoiding the transfer of embryos with sex chromosomal aneuploidy. In addition to further improvelllent of the diagnosis of single gene defects and extension of the range of diseases amenablc to specific diagnosis, PID research is currently focllsing on the diagnosis of chromosomal aneuploidy in preimplantation embryos of infertiie women of advanced maternal age undergoing in vitro feliilization (IVF) in order to improve delivery rates. ivlulticolar FIS I-I has already been used tor screening of IVF embryos (fvlunné et al., 1993; 1995a), as web as of first and second polar bodies, as an alternative approach to PID (Verlinsky et al., 1996). However, scveral difficulties must still be addressed before PID of chrolllosome aberrations can routincly be applied in women of advanced reproductive age undergoing IVF (Reubinoff and Shushan, 1996). FISI-I also opened the possibility to non-invasivc prenatal diagnosis. Efforts have been made to develop such technalogy, since amniocentesis and chorianoic villus sampling are both associated with a low risk of feta I mortality and morbidity. A potential future non-invasive technique may be the analysis of fetal cells recovered from the maternai circulatioll. Several 75

76 groups were alrcady successful in detecting tètal aneuploidy in preparations ofmaternal blood bl' using FISH with ehromosome specific DNA probes (Priee et al., 1992; Bianehi et al., 1992; Gänshirt-Ahlert ct al., 1993;.lansen et al., 1997). Since fetal cells are extremely rare in maternat blood, the devclopment ofthis technology has mainly been hampered by problems in isolation and enriclullcnt of these cells. Therefore, introduction of this technique into clinical practice is still some way off. 76

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89 Simani, G., Fraccaro, M., GimeIli, G., Maggi, F., and Dagna Bricarelli, F. (1987). False-positive and falsenegative findings on chorionic villus sampling, Prenal Diagll, 7, Simoni, G., TerzoIi, G., and Rossclla, F. (1990). Direct chromosome preparation and culture using chorionic villi: an evaluation ofthe two techniques, Am J Med Genel, 35, Smidt-Jensen, S., Christen sen, 8., and Lind, A. M. (1989). Chorion ic villus culture for prenatal diagnosis of chromosome defccts: reduction ofthe long-term cultivation time, Prenal Diagn, 9, Smidt-Jcnsen, S., Lind, A. M., Permin, M., Zachary, J, M., Lundsteen, C., and Philip, J. (1993). Cytogenetic analysis of2928 CVS samples and 1075 amnioccntcses from randomized studies, PreI/at Diagll, 13, , Speleman, F., Vim Roy, N., Wiegant, J., Verschraegen-Spne, M. R., Bcnoit, Y., Govaert, P., Goossens, L., aod Leroy, J. G. (1992). Detectioo of subtie reciprocal translocations by fluorescence in situ hybridization, Clin Genel, 41, , Stee1e, M. W., Breg, W,R. (1966). Chromosome atlalysis ofhuman amniotic fluid cells, Lancet, i, Steenman, M" Redeker, B. de Meulemeester, M., Wiesmeijer, K., VoGte, P.A., Westerveld, A., Slater, R., mannens, M. (1997), Comparativc genomic hybridizatioo aoalysis of Wilms tumors, Cy/ogene/ Cell Genet, 77, Tantravahi, U., Matsumoto, c., Delach, J., Craffey, A., Smeltzer, J., Benn, p, 91996). Trisomy 16 mosaicism in amniotic fluid culturcs, Prena/ Diagn, 16, TaTIn, J. J., Conaghan, J., Winston, R.M.L., Handyside, A.H. (1992). Human cmbryo biops)' on the 2nd day after insemination for preimplantation diagnosis: removal of a quarter of embryo retards cleavage, Ferti/ Steril, 5, Terzoli, G, L., Lalatta, F., and Gilbert, F. (1987). Chorionic viilus sampling: improved method for preparatioo of karyotypes after short-term incubation, PreI/at Diagn, 7, 389~94. Terzoli, G" Rossclla, F., Biscaglia, M., and Sillloni, G. (1990). True fetal mosaicism revealed by a single abnormal colon)' in amniocyte culture, Prenal DiagIl, 10,273-4, Teshima, I. E., Kalousek, D, K., Vekemans, M. J., Markovic, V" Cox, D. M., Dallaire, L., Gagne, R" Lin, J. c., Ra)', M., Sergovîch, F, K, ct al. (1992), Canadian multicenter randolllized clinical trial of chorion villus sampling and amniocentesis, chromosollle mosaicism in CVS and amniocentesis samples, PreI/ai Diagn, 12, Thicdc, H.A., Creasman, \V.T., Metcalfe, S. (1966). Antenatal analysis of the hu man chromosomes, Am J Os(et G)'ltecol, 94, Thomassen-Brepols, L.J. Psychosocial aspects of prenatal diagnosis, thesis, Erasmus University, Rotterdam, Tjio J.H., and Levan A. (1956). Thc chromosome number of man, Heredilas, 42, 1-6, Tydén, 0., Bergstrom, S" and Nilsson, 8. A. (I 981). Origio of amniotic fluid ce lis in mid-trimester pregnancies, BI' J abs/el G)'l1aecol, 88,278-86, Valenti, C., Schutta. E. J., aod Kehaty, T. (1969). C)10genetic diagnosis of Down's syndrome in utero, JAMA, 207, Van den Berg, c., Ramlakhan, S.K., Van Opstal, D., Brandenburg, H., Halley, DJ.J., Los, F.J. (1997). Prcnatal diagnosis oftrisomy 9: cylogenetic, FISH, and DNA studies, Prenat. DiagIl., 17,

90 Van Hemel, J. 0., Eussen, B., Wesby-van Swaay, E., and Oostm, B. A. (1992). Molecular detection of a translocatîon (V; 11) (q 11.2;q24) in a 45,X male with signs of Jacobsen syndrome, Hum Genel, 88, Van Tuinen, P., Dobyns, W.B., Rich, D.C., Summers, K.M., Robinson, 1'.1., Nakamura, V., Ledbetter, D.H. (1988). Molecular detection of microscopie and submicroscopic deletions associated with Milier-Diekcr syndrome, Am. J. Hum. Genet., 43, Vaughan, J., Ali, Z., Bower, S., Bennen, P., Chard, T., Moorc, G. (1994). Human matemal uniparental disomy for chromosome 16 and fetal development, Prenal Diagn, 14, Vejcrslev, L. 0., and fvlikkelsen, M. (1989). Tbe European collaborative study on Illosaicism in chorionic villus sampling: data from , Prel1al Diagn, 9, Veldman, T., Vignon, c., Schrock, E., Rowley, J. D., and Ried, T. (1997). Hidden chromosome abnom1alities in haematologicalmalignaneies detected by multieolour spectral karyotyping, Nal Genel, 15, Vcrlinsky, V., Ginsberg, N., Chmura, M., Freidine, t't'1., White, M., Strom, C., and Kuliev, A. (1995). Crosshybridization of the ehromosome 13/21 alpha satellite DNA probe to chromosome 22 in the prenatal screening of common chromosomal aneuploidies by FISlf, PreI/ai Diagl/, 15, 83 I -4. Verlinsky, V., Cieslak, J., Freidine, M., Ivakhnenko, V., Wolf, G., Kovalinskaya, L., White, M., Lifchez, A., Kaplan, Moise, J., ValIe, J., Ginsberg, N" Strom, c., and Kuliev, A. (1996). Polar body diagnosis of common aneuploidies by FISH, JAssist Reprod Genel, 13, Verllla, R. S., and Luke, S. (1992). Variations in alphoid DNA sequences escape detection of aneuploidy at interphase by FISII technique, Gellomics, 14, Viersbach, R., Schwanilz, G., and Nothen, M. M. (1994). Delineation of marker chromosomes by reverse chromosome painting using only a small number of DOP-PCR amplified microdissccted ehromosomes, Hum Gel/el, 93, Vockley, J., Inserra, J. A., Breg, W. R., and Vang-Feng, T. L. (1991). "Pselldomosaicism" for 4p- in amniotic fluid cell culture proven 10 be truc mosaicism after birth, Am J Met! Genel, 39, Wang, B.T., Peng, W., Cheng, K.T., Chiu, S.f., Ho, W. Khan, V., Wiuman, M., Williams, J. (1994). Chorionic villi sampling: laboratory experienee with 4000 consecutive cases, AII/ J j\led Gel/el. 53, Wapner, R. J., Simpson, J. L., Golbus, M. S., Zachary, J. M., Ledbetter, D. H., Desnick, R. J., Fowler, S. E., Jackson, L. G., Lubs, H., l\ lahony, R. J., cl al. (1992). Chorion ic mosaicism: associalion wilh fetalloss but not with adverse perinatal outcome, PreI/aI Diagn, 12, Warburton, D. (1991). De nova balanced chromosome rearrangements and extra marker chromosomes identilied at prenatal diagnosis: clinical signilicance and distribution of breakpoints, Am J Hum Gellet, 49, Ward, B. E., Gersen, S. L., Carelli, M. P., t... 1cGuire, N. M., Dackowski, W. R., Weinstein, r't'i., Sandlin, c., Warren, R., and Klinger, K. W. (1993). Rapid prcnatal diagnosis of chromosomal anellploidies by fluorescence in situ hybridization: clinical experience with 4,500 specimens, AIII J HUIII Genel. 52, Watanabe, ~!I.,lto. 1'., Vamamoto, ~L Watanabe. G. (1978). Origin oflhe mitotic cells ofthe chorion ic villi in direct chromosome analysis, lil/lil. GeneI., Waye, J. S., and Willard. H. F. (1986). f\!o!ecular analysis of a deletion polyl1lorphisl1l in alpha satellile of human chromosome 17: evidence for holllologous unequal crossing-over and subscquent IÏxation. N"c1 Acid.~ Res,

91 Waye, J. S., England, S. 8., and Willard, H. F. (1987a). Genomic organization ofalpha satellile DNA on hu man chramosome 7: evidence for two distinct alphoid domains on a single chromosome, Mol Cell Biol, 7, Waye, J. S., Creeper, L. A., and Willard, H. F. (I987b). Organization and evolution ofalpha satellite DNA from human chromosome 11, Chromosoma, 95, Waye, J. S., and Willard, H. F. (1989). Chromosame specificity of satellite DNAs: short- and long-range organization of a diverged dim eric subset of human alpha satellite from chromosome 3, Chromosoma, 97, Weber, J. L., and May, P. E. (1989), Abundant class ofhuman DNA polymorphisms which can be typed using Ihe polymerase chain reaction, Am J Genet, 44, Wilkinson, T. A., James, R. S., Crolla, J. A., CockwelI, A. E., Campbell, P. L., and TempIe, I. K. (1996). A case of maternal uniparental disomy of chromosome 9 in association wilh confined placenta I mosaicism for trisomy 9, Prenat DiagIl, 16,371-4, Willard, H. F., Smith, K, 0., and Sutherland, J, (1983).lsolation and characterization ofa major tandem repeal family from the hu man X chromosome, Nucl Acid~ Res, 11, WHson, D. 1., Bum, J., Scambler, P., nnd Goodship, J. (1993). DiGeorge syndrome: part of CATCH 22, J Med Genet, 30, WilsOIl, R. D. (1995). Early nmnîocentesis: a clinical review, Prenat Diagn, 15, Winsor, E. J., Silver, M, P., Theve, R" Wright, rv!., and Ward, B. E. (1996), Matemal cell contamination in uncultured amniotic fluid, Prellat Diagn, 16, \Vladimiroft~ J. W., Bhaggoe, W, R., Kristelijn, M" Cohen-Overbeek, T. E., Den Hollander, N. S., Brandenburg, 11" and Los, F. J. (1995), Sonographically detemlined anomalies and outcome in 170 chromosomally abnonnal feluses, PreI/aI Diagn, 15, Wolfe, J., Darling, S. M., Erickson, R. P., Craig, I. W., Buckle, V, J., Rigby, P. W., Willard, H. F., and GoodfelIow, P. N. (1985), lsolation and characterization of an alphoid centromeric repeat fnmily from the human Y chromosome, J Mol Biol, 182, Wolstenholme, J" Cracker, M., and Jonasson, J. (1988). A study ofchromosomal aberrations in amniotic fluid cell cultures, PreI/at Diagn, 8, Wolstenholme, J., Rooney, D. E" and Davison, E. V. (1994), Conflned placenta I mosaicism, IVGR, and adverse pregnancy outcome: acontrolled retrospective V.K, coilaboratî\'e survey, PreI/at DiagIl, 14, \Voistenholme, J. (1995), Au nudît oftrisomy 16 in man, PreI/af Diago, 15, Wolstenholme, J. (1996). Confined placentnl mosaicism for trisomies 2, 3, 7, 8, 9, 16, and 22: theîr incidence, likely origins, and mechanisms tor ceillineage compartmcntalization, Prenat Diagn. 16, Worton, R. G., alld Stern, R. (1984), A Cnnadi~n cultures, Prenat DiagIl, J collaborntive stud)' of mosaicism in amniotic fluid cel! Yamamoto, M., Fujimori, R" Ito, T., Kamimura, K., Watnnabe, G. (1975). Chromosome studies in 500 induced nbortions. Humangenetik. 29,9-14. Zhang, J., Ivtnrynen, p" De\'riendt, K., Fryns, J. P., Van den Berghe, H., and Cassiman, J. J. (1989). Molecular analysis of the isochromosome 12P in the Pallister-Killian syndrome. COllstruction of a mousc-human hybrid celiline containing an i( 12p) as the sole human chromosame, Hil/Jl Genet, 83,

92 ZhclIg, Y. L., Ferguson-Smith, M. A., Wamcr, J. P., Ferguson-Smith, M. E., Sargent, C. A., and Carter, N. P. (1992). Analysis of chromosome 21 eopy l1umber in uneultured ajnnioc)1es by fluorcseenee in situ hybridization using a eosmid contig, Prellat DiagIl, 12, Zheng, Y. L., Carter, N. P., Priee, C. M., Colman, S. M., Milton, P. J., Hackett, G. A., Greaves, M. F., and Ferguson-Smith, M. A. (1993). Prcnatal diagnosis from matemal blood: simultaneous Îmmunophcnotyping and FISH of fctalnucleated el)1hroc)1es isolated by ncgalive magnetie eeli sorting, J Med Genet, 30, Zinunemtanll, R., Lauper, V., SIreicher, A., Hueh, R., Hueh, A. (1995). Elevuted alphu-fetoprotein and human ehorionie gonadolropin as a marker for plaecntaltrisomy 16 in the seeolld trimester, PreI/af DiagIl, 15,

93 M Summal'y Amniocentesis and chorion ic villus (CV) sampling are two widely used invasive techniques tor prenatal diagnosis (PD). Their possibilities al1d!imitations fol' cytogenetic studies arc discussed in chapter I. The main Iimitations of classica! cytogcnetie analysis in amniotic fluid (AF) and CV cells are: ~ the correct interpretation of chromosome aberrations with indistinet banding patterns (marker ehrolllosomes, de novo structural chromosame aberrations) ~ the til11e~col1suming AF ecu eulturing, with aresuit about the fetal karyotype only available two to three weeks aller sampling the deteetion of a (non)-mosaic ehromosome aberration in CV, which is potentially eonfined to the placenta [confined placenta! mosaicism (CP~'J)] and not present in the fetus, and which may requirc follow-up investigations in AF eeils, delaying the reporting time - the interpretation of low level mosaicism in AF and CV cells, which may represent true mosaicism, but also pseudomosaicism (culture artetàcts without clinical significance) or localmitotic division errors, respectively - the limited resolution ofthe light microscope, with some ehromosome rearrangelllents, slleh as microdeletions, going undeteeted - the possibility of maternal cell contamination of the sample In 1993 we introduced the fluorescent in situ hybridizatioll (FIS I-I) tcchnique in our laboratory as all adjunctive tooi 10 classica I chrolllosome analysis in order to overcome same of these limitations. The main scope of this thesis is lo evaluate the merits and demerits of this techniquc after implementation for di agnost ie purposes during four years in our laboratory. \Ve have tour main indications tor FISH, which arc discussed separately in chapter Il. Indicatiol1 I (section 3.1) involvcs the applicatioll of FISI-I tor identification of structural cluomosome aberrations. We experienccd that FISH may be a uscful taal for characterization of the breakpoints of cytogenetically detectcd structural chromosome rcarrangements. This may be important tor lmderstanding the ballding pattern of the abnormal chromosome. Ivloreover, wc liscd FISH tor rapid verification of an unccrtain c)1ogenetic result (because of pool' quality of the chromosomes), and fol' Jllst dctcction of subtie familial cluomosolllc aberrations in thc first trimester of pregnancy. Howevcr, FISI-I with chrol11osome~specil1c probes may be a time-consuming technique for identification of extra chromosoma! matcrial, such as marker chromosomes and de novo unbalanced ehromosomc rearrangements. Recenti)', new FISH tcchniqucs (micro-f1sh, SKY, CGH) have been devclopped, which will enable a much more efl1eicnt and accurate idcntitication than traditional FISI-I in the near future. PCR analysis of polymorphic microsatellite rcpeats showed to be a powerful tooi lor deterlllination ofthc extent ofa deleted chromosomal segment, irrelevant FISH probes are not available. 93

94 Indication 2 (section 3.2) involvcs thc rapid detection ofnumerical chromosome aberratiol1s in uneultured AF cells in pregnancics at high genetic risk in which a fast result is important. If tètal anomaiies, suspicialls t'or one ofthe most common chromosome aberrations (trisomy 13, 18, 21, triploidy, 45,X) are detcctcd by 1Iltrasolind (US), a rapid result is necessary when gcstational age comes close to 24 wccks, ancr which termination of pregnancy is prohibited by law, or when impending birth dcmands decisiolls concerning obstetric and perinatal poliey (scctiol ). \Ve scrccned ulleuitured AF cells with a set of chroll1osome X, Y, 13, 18, and 21 specific probes (the so-callcd US probe set) in 196 cases and we were able to detect 43 (77 %) of 56 chro1l10s0111c aberrations l'ound among these samples. Thirteen ciuomosomc aberrations went lindcteeted, because other chromosomes than X, Y, 13, 18, or 21 were involved (10 cases), or becallsc of technica I problems (3 cases). \Ve conciudc that FISI-I on uncultured AF cc Us is a fàst (within two days aftel' sampling) alld highly rciiablc method 1'01' rapid detection of chromosome aberrations if appropriate probes arc tlsed. False positive (7 cases) and fàlse negative results (one case) were mainly eneollntered during the first two years of our investigations, alld could filfther be prevented by improving the technique. Since in 18 % of the ehromosome aberratians in this study at her ehromosomes than X, Y, 13, 18, alld 21 werc in"olved, a normal FISH result should always be complemented by cytogenetic analysis afthe eell cultures. 11' a chromosoll1c aberration other than full trisomy 21, triploïdy, 47,XXX, 47,XXY, or 47,XYY, is detected in semi-direct CV preparations, in the absence of US abnonnalities, it might bc coni1ned to the placenta and a follow-up alllllioeentesis 1'01' verification of the fetal karyotype may be necessary, especially if cultureel CV are not available. This severely prolongs the reporting time. FISH on uneultureel AF cells for rapid differentiation bctwecl1 CPrd anel generalized Illosaicism (section 3.2.2), was found to be an accurate lllcthod, since in 48 out of 51 cases FISI-I correcti)' identified the feta I karyotype. Howe"er, tàlse positive (two cases) and false negativc (one case) results may occur. They Illay oecasionally be the result of ineffïcient or ul1specifïc probe hybridization. although we found evidenee that [alse positive flsh findings may arise from select ion against abnonnal eells in the cel! cultures, with cytogenetic resuits in fact representing false llegative results. Application of FISH on uncultured AF cells in same ot her situations rcquiring a rapid result (low level mosaicislll deteetcd in a previous AF sample, matcrnal agc L 44 years, triple test reslilts indicativc f'or a high risk (;?: 5 %) of Downs s)'ndromc, twin pregnancies of advanced gestational agc, sex determination in pregnaneïcs at risk for X linked diseases) (section 3.2.3) silowed ta be highly reliabie, since na discrepancies were fotll1d between FISI-I and cytogcnctie results in all 46 cases that we investigated. In section 3.3 we deseribe Dur experience with FISI-I tor studying low level mo~aicisl11 in AF cell cultures and CV semi direct preparatiolls in order to discriminate betweell pseudolllosaicism (in AF cultures) or local mitotic division ettors (in CV) and ll10saicism (indication 3). Differentiation between both phenomena requires thc analysis of a large number of cells which lllay be difticult due to a limited nulllbcr of metaphases. Interphase FlSH on cultured AF cells of samples showing one abnormal colony was found to be an efficient and accurate tooi for determination whether any additional cells in the different culture vessels of one sample showed the chromosome abnormality. When pselldomosaicism 94

95 is established, a second amniocentesis lar col1lirmatory studies ean be obviated. If chromosomal Illosaicislll is encountered in semi-direct CV preparations, a t'ollow-up alllniocentesis may be necessary lor verilication or the fetal karyotype. This meant a second invasive procedure tor 2,1 % of women undergoing CVS dming the time period However, the question is whethcr a follow-up amniocentesis is really always necessary, since in many cases low level 1110saicisl11 (:0;; 33.3 % abnol"lnal ceils) is encountered which may represent low level Jllosaicism. although the rew abnormal cells mal' also result from Iocal errors during mitosis. \Ve used interphase FISH in 37 cases of low levelmosaicism: 13 cases silowed abnormal and 24 cases silowed norm al FISH resuits, indicating local mitotic division errors. In 12 of these 24 cases, a follow-up amniocentesis was performed, all with normal results. Healthy children were born in all 24 cases. We believe that FISH results better reflect the real level of Illosaicism than traditional cytogenetic analysis, since FISH analysis involves the scoring of a few hundreds of ceils. This means that a norillal karyotype can be reported to the parents when FISI-I results arc normal and follow-up investigations can be omitted in these cases. I I' non-mosaic or mosaic trisomy 18 is ellcotilltered in scmi-direct CV slidcs, follow-up investigations in cultured CV alld/or AF cells are neeessarl', sinee the trisomy mal' bc contined to the placenta. Since CV cultures were not perfonned in our laboratory during lllany years, wc investigated the use of FISH on semi direct CV prcparations as a potcntial taal of rapid veritlcatioll. \Ve fotll1d that interphase FISH mal' acid to the cytogenctic data, in terms of predicting the fetal karyotype, since a higher percentage of thrce signa I containing nuclei were found in the contirmed cases as compared to the non-confirmed cases. However, since FISH yielded ambivalent results in some cases, we conclude that a final result can only be achieved by analysis of 11 subsequent AF sample. In section 3.4, we describe our experience with FISH for the detection of microdeletions associated with specific syndromes (indication 4). These microdeletions can not be identitied with conventional bnnding tcchniques, and FISI-I showed to be an accurate met had tàr their detection. irrespcctîve of thc quality of the chromosomcs. Among 48 cases Ihat wc invcstigatcd, one 22q 11 deletion was encountered, which was known to be present in a previous child with the classical Di George syndrome. Aner the prenatal I1ndings, the deletion was also fotmd in the j~lther, confirming that this syndrome mal' be sporadically transmitted as an autosomal dominant trait. FISH resuits in all other cases were 110rmnl. Charter 111 deals with the phenomenon ofcp~vi involving a trisomy which nmy potentially be associated with fetal uniparental disomy (UPD) (both chromosomes of a chromosame pair derived from one parent oniy), ir thc trisomy has a l11eiotic origin and one particular copy of the set of three chromosomes is lost (trisomic zygote rescue). Fetal UPD may explain the perinatal complications occasionally fotll1d in cases oeepl'vi. \Ve investigated the incidence of UPD associatcd with CPivf in a consecutive series of CV samples collected dming lour years in order to get more insight into the OI"igill of CPi\,f. aud into the mechanisl11 of trisolllîc zygote rescue. Twenty-nine cases of CPM involving a trisoill)' were làulld between 1992 and the end of 1995, alld DN/\ studies were perlànned in 23 cases. Only anc case of tètal UPD (l1pd 16) was lound. indicating that in most cases the trisomic cell line most probably 95

96 originates from somatic duplication. This is supported by the low level of mosaicisll1 in most of these cases. Furthennore, the normal prcgnancy outcome in this case furthcr supports the hypothesis that the impaired feta I growth encauntcrcd in most cases of UPD 16, and the congenitallllatànnations found in same ofthem, are probably not the result ofthe UPD itsclf. ~vforeover, the obstetrical complications encountcred in scvcn out of 23 CPivl cases showcd not to be the consequence of fetal UPD. During the same study period, another two cases of UPD (one case of UPD 15 and one of upn 16), both associated with a normal karyotype in CV, were accidently found. Despite the absence of tri som ic cells in CV, trisomic zygote rcsclle lllight be thc cause of UPD in these cases. The mechanisl1l of tri som ic zygote rescue is not known, but anaphase lagging (AL) as weil as non-disjullction (l\td) in an early postzygotic cell division have been proposed. I-Iowever, AL CUIl not explain the combinatioll of feta I urd alld a norm al CV karyotype, and ND will significantly reduce the number of blastomcres of the early embryo, which may ham per its Ilormal development. Therefore, wc propase all alternative correction mode, chromosome demolition (CD), which involves the destructioll anel removal of anc of the set of three chromosomes, resulting in two disomic daughter celis. We present a model for the arising ofthe various combinations of karyotypes in semi-direct CV slidcs, cultured CV, and fetus from trisomic zygote rescue with each of the different correct ion modes. All cases of UPD associated with CP~vl or with a normal karyotype in CV semi-direct preparations tit this model. It further shows that trisolnic zygote rescue does not necessarily result in a 100 % trisomy or high level mosaicism in semi-direct CV preparations, as was suggesteel by some investigators, but it may theorctically rcsult in a low level of Illosaicism. 96

97 Samenva tting Vruchtwaterpunctie (amniocentese) en vlokkentest (chorion villus biopsie) zijn twee wijdverspreide invasieve technieken voor prenatale diagnostiek. De mogelijkheden en beperkingen van cytogenetisch onderzoek in vl1lchtwater (VW) en chorion villi (CV) cellen worden besproken in hoofdstuk 1. De belangrijkste beperkingen van het klassieke chromosoomonderzoek in V\V eil CV cellen zijn: - de moeilijke interpretatie van chromosoomafwijkingen in geval van een onduidelijk bandenpatroon (marker chromosomen, de navo structurele chromosoomafwijkingen) - de tijdrovende V\V celkweek, waardoor het foetale karyotype pas twee à drie weken na de punctie bekend is - de detectie van een chromosoomafwijking in CV die beperkt kan zijn tot de placenta [confmed placental ll10saicism (CPM)] en dus niet aanwezig is in de foetus; dit vereist eventueel een vervolgonderzoek in V\V cellen, wat de wachttijd voor een definitieve uitslag aanzienlijk verlengt - de juiste interpretatie van een laag mozaïek chromosoomafwijking, als een mozaïek of een pseudomozaïek (kweekartefact zonder klinische betekenis) in V\V cellen, dan wel een l11ozal'ek of een locale mitotische delingsfout in CV cellen - de beperkte resolutie van de lichtmicroscoop, waardoor sommige chromosoomveranderingen, zoals microdcleties, kunnen gemist worden - de mogelijkheid val1l11aternale cel contaminatie van V\V en CV specimen In 1993 hebben we de 'fluorescentie in situ hybridisatie' (FISI-I) techniek gcintroduceerd in ons prenataal cytogenetisch laboratorium als een aanvullende test op het klassieke chromosomen onderzoek, met het doel een aantal van de bovenvermelde beperkingen te overbruggen. De belangrijkste doelstelling van dit proefschrift is de evaluatie van de voor- en nadelen van FISH, nadat we deze techniek gedurende vier jaren hebben gebruikt voor diagnostische doeleinden. \Ve onderscheiden vier indicaties voor fish die afzonderlijk worden besproken in hoofdstuk 11. Indicatie I (paragraaf 3. I) betreft de toepassing van FISH voor identificatie van stmcturele chromosoomafwijkingen. Onze ervaring heeft geleerd dat FISI-I een nuttige techniek kan zijn voor het vaststellen van breukpunten van deze chromosoomafwijkingen, wat van belang kan zijn om het banderingspatroon van het afwijkende chromosoom beter te interpreteren. Tevens gebruiken we FISH voor de snelle bevestiging van cen onzeker cytogcnctisch resultaat (onzeker vanwege een onvoldoende chromosoomkwaliteit), en voor snelle detectie van subtiele f..1miliaire chromosoomafwijkingen in het eerste trimester van de zwangerschap. Echter, FISH met chromosoom-specifieke probes is potentieel een zeer tijdrovende techniek voor de identificatie van extra chromosomaal materiaal, zoals marker chromosomen en de llovo Ollgebalanceerde chromosoomafwijkingen. Recent zijn nieuwe FISH technieken (micro- 97

98 flsh, spectral karyotyping, comparative genomic hybridization) ontwikkeld, die in de nabije toekomst een meer efficiente en accurate identificatie mogelijk zullen maken. Indien relevante FISH probes niet beschikbaar zijn, bleek PCR analyse van polymorfe microsatelliet repeats een nuttige aanvullende techniek te zijn voor het bepalen van de grootte van een gedeleteerd chromosomaal segment. Indicatie 2 (paragraaf3.2) betreft de snelle detectie van numerieke chromosoomafwijkingen in ongekweekte V\V cellen van zwangerschappen met een groot risico op een chromosoolllafwijking indien een snel resultaat belangrijk is. Dit is het geval wanneer echografisch vastgestelde foetale afwijkingen verdacht zijn voor een van de meest voorkomende chromosoom-afwijkingen (trisomie 13, 18, 21, triploidie, 45,X) en de zwangerschapsduur 24 weken nadert of een spoedige geboorte wordt verwacht. Zwangerschapsterminering na 24 weken is wettelijk verboden. Indien een geboorte dreigt zijn snelle beslissingen omtrent obstetrisch en perinataal beleid nodig (paragraaf 3.2.1). We onderzochten 196 ongekweekte VV\' monsters met een set van chromosoom X, Y, 13, 18, en 21 specifieke probes (de zogenoemde US (ultrasound) probe set) en we waren in staat om 43 (77 %) van de 56 chromosoomafwijkingen op te sporen. Dertien afwijkingen werden niet gedetecteerd, omdat er andere chromosomen dan X, Y, 13, 18, of21 bij waren betrokken (10 casus) of omwille van technische problemen (3 casus). We concluderen dat FISH op ongekweekte V\V cellen een snelle (binnen twee dagen na V\V punctie) en zeer betrouwbare methode is voor snelle detectie van chromosoomafwijkingen indien geschikte probes worden gebruikt. Fout positieve (7 casus) en fout negatieve (I casus) resultaten werden voornamelijk gevonden tijdens de eerste twee jaren van het onderzoek en konden verder worden voorkomen door voortdurende verbetering van de techniek. Aangezien in 18 % van de chromosoomafwijkingen in deze studie andere chromosomen waren betrokken dan X, Y, 13, 18, en 21, zal een normaal rrsi-i resultaat steeds moet worden aangevuld met karyotypering van gekweekte cellen. Indien een chromosoomafwijking anders dan trisomîe 21, triploidie, 47,XXX, 47,XXY, of 47,XYY wordt gevonden in semi-directe CV preparaten, kan velvolgonderzoek in V\V cellen noodzakelijk zijn voor verificatie van het foetale karyotype, vooral als gekweekte CV niet beschikbaar zijn. Uiteraard verlengt dit de wachttijd voor een definitief resultaat aanzienlijk. Het gebruik van HSH op ongekweekte VW cellen na eerdere afwijkende bevindingen in vlokken bleek een accurate methode te zijn voor een snelle differentiatie tussen CPlvl en gegeneraliseerd mozaïek (paragraaf 3.2.2). In 48 van de SI onderzochte casus werd het foetale karyotype correct geidentificeerd middels FISH. Echter, fout positieve (2 casus) en fout negatieve (l casus) resultaten kwamen voor. Zij kunnen af en toe het resultaat zijn van inefficiente of niet-specil1eke probe hybridisatie, alhoewel wij aanwijzingen vonden dat fout positieve FISH bevindingen ook het gevolg kunnen zijn van selectie ten voordele van normale cellen in de celkweken, waardoor het cytogenetische resultaat in gekweekte cellen in feite een fout negatieve bevinding is. Het gebruik van FISH op ongekweekte V\V cellen in nog enkele andere situaties die een snelle uitslag vereisen (laag mozaïek in een voorgaand V\V sample, maternale leeftijd :;:..44 jaar, afwijkende triple test met een L 5 % risico op Down syndroom, tweeling zwangerw schappen indien de zwangerschapsduur ongeveer 16 weken bedraagt, geslachtsbepaling in 98

99 zwangerschappen met een verhoogd risico op een geslachtsgebonden ziekte) (paragraaf 3.2.3) bleek zeer betrouwbaar te zijn, aangezien er geen discrepanties werden gevonden tussen FISI-I resultaten en karyotype in alle 46 casus die we onderzochten. In paragraaf 3.3 beschrijven we onze ervaring met de toepassing van FrSH voor het bestuderen van laag mozaïeken in V\V cclkweken en CV semi-directe preparaten, teneinde een onderscheid te maken tussen pseudomozaïeken of locale delingsfouten en mozaïeken (indicatie 3). Deze differentiatie vereist analyse van een groot aantal cellen/clonen, wat soms bemoeilijkt wordt door de aanwezigheid van slechts een beperkt aantal analyseerbare metafasen. Interfase FISH op gek '""weekte V\V cellen in geval van één chromosomaal afwijkende clone, bleek een zeer efficient en accuraat middel te zijn om na te gaan of in de verschillende kweekbakjes nog meer cellen met dezelfde chromosoomafwijking aanwezig waren. Op deze manier kan, bij bevestiging van de aanwezigheid van een pseudomozaïek, een eventueel vervolgonderzoek in een tweede vruchtwatermonster worden voorkomen. Indien een mozaïek chromosoompatroon wordt gevonden in semi-directe CV preparaten, is vervolgonderzoek in vruchtwater soms nodig voor verificatie van het foetale karyotype. In de periode betekende dit een tweede invasieve ingreep voor 2,1 % van alle zwangeren die een vlokkentest ondergingen. De vraag is of dit vervolgonderzoek ook echt altijd nodig is, aangezien er vaak een laag mozaïek (:0;;; 33 % abnormale cellen) aanwezig is, wat echt een CP!v! kan betekenen, doch mogelijk ook het gevolg kan zijn vall een locale mitotische delingsfout in de CV. \Ve gebruikten inter.t:1se FISH in 37 casus met een laag mozaïek, en vonden in 13 casus een afwijkend FISI-I resultaat, maar in de overige 24 casus bleken de FISH resultaten normaal te zijn. In 12 van deze 24 casus werd nog een vervolgonderzoek gedaan in vruchtwater, en in alle gevallen werd een normaal karyotype gevonden. Alle 24 zwangerschappen met een nonnaai FISH resultaat eindigden met de geboorte van een gezond kind. Omdat interfàse FISH het onderzoek betreft van enkele honderden cellen, geven de FISI-I resultaten een beter beeld van de werkelijke hoogte van het mozaïek dan traditioneel cytogenetische resultaten. Dit betekent dat als FISH resultaten nonnaai zijn, een vervolgonderzoek in V\V eellen achterwege kan blijven. Als (mozaïek) trisomie 18 wordt gevonden in semi-directe CV preparaten, is vervolgonderzoek in gekweekte CV, en eventueel in V\V cellen, noodzakelijk, omdat de afwijking beperkt kan zijn tot de placenta. \Ve onderzochten de bruikbaarheid van interfase FISH op semi-directe CV preparaten als potentieel middel voor snelle verificatie van het foetale karyotype, vooral omdat CV kweken niet voorhanden waren gedurende de studieperiode. \Ve vonden dat FISH resultaten een predictieve waarde hadden voor het foetale karyotype, in die zin dat een hoger percentage cellen met drie signalen werd gevonden in die casus waarbij de trisomie 18 werd bevestigd in foetale cellen in vergelijking met de niet-bevestigde casus. Echter, wegens het af en toe voorkomen van ambivalente FISH resultaten, moetell we concluderen dat een detlnitieve uitslag slechts kan worden verkregen door een vervolgvruchtwateronderzoek. In paragraaf 3.4 beschrijven we onze ervaring met FISH voor de detectie van microdeleties die geassocieerd zijn met speeifieke syndromen (indicatie 4). Deze microdeleties kunnen niet worden geidentit1ceerd met behulp van conventionele banderingstechnieken. \Ve 99

100 onderzochten 48 easus met behulp van FIS I-I cn vonden één 22q 11 deletie, waarbij recds cerder eenzelfde deletie was aangetoond in een voorgaand kind met het klassieke Di George fcnotype. De deletie werd na de prenatale bevindingen ook gevonden bij de vader, wat bevestigt dat dit syndroom soms familiair voorkomt. Hoofdstuk III behandelt het fenomeen van trisomie CP~II, wat geassocieerd kan zijn met foetale uniparentele disomie (UFD). UPD betekent dat beide chromosomcn van cen chromosoompaar afkomstig zijn van één en dezelfde ouder. Dit verschijnsel kan zich voordoen als de tri som ie een mciotische oorsprong heeft, en het chromosoom afkomstig van de ouder die er één bijdraagt, verloren gaat. Deze reductie van trisomie naar disomie noemt men 'trisomic zygote rescuc'. Foetale UPD is één van de mogelijke verklaringen voor de perinatale complicaties die af en toe optreden in zwangerschappen met CPwL,\fe onderzochten de tiequentie van foetale UPD geassocîcerd met trisomie crfvl gedurende vier jaren, met de bedoeling meer inzicht te krijgen in het ontstaan van CP~II, en in het mechanisme van trisomic zygote rescuc. In totaal werden 29 casus mct trisomie CPM gevonden, waarvan in 23 DNA studies konden worden verricht. Slechts één casus met UPO 16 werd gevonden, wat aangeeft dat in de meeste gevallcn de tri som ie cellijn hoogstwaarschijnlijk ontstaat als gevolg van somatische duplicatie. Dit wordt ondersteund door de aanwezigheid van cen laag mozaïek in de cytotrophoblast in de meeste van deze gevallen. De normale zwangersehapsuitk0111st in de UPD 16 casus ondersteunt de hypothese dat de foetale groeiachterstand dic optreedt in de meeste, en de congenitale afwijkingen die worden gezien in sommige casus met UPD 16, waarschijnlijk niet worden veroorzaaj...'i door de UPD 16 zelf. Bovendien kunnen we concluderen dat de obstetrische complicaties die zich voordeden in zeven van de 23 onderzochte zwangerschappen met CP!vl niet het gevolg waren van UPD. Gedurende dezelfde studieperiode werden nog twee andere casus mct UPD (één met UPD 16 en één met UPD 15) gevondcn. Beiden waren geassocieerd met een normaal karyotype in CV. Niettegenstaande de afwezigheid van trisome cellen in CV lijkt trisomic zygote resclic toch de meest waarschijnlijke oorzaak van UPD in deze casus. De manier waarop het extra chromosoom verloren gaat, is niet bekend, maar 'anaphase lagging' (AL) en 'non-disjunctie' (ND) tijdens één van de eerste postzygotische celdelingen werden reeds als mogelijke mechanismen voorgesteld. Echter, AL kan nict dc combinatie foetale UPD en nonnaai CV karyotype verklaren, en ND leidt tot een significante reductie van het aantal blastomeren in het vroege embryo, wat een verdere normale ontwikkeling kan bemoeilijken. Daarom stellen wij een alternatieve correcticmcthode voor, 'chromosome demolition' (CD), wat inhoudt dat één van de drie chromosomen wordt verwijderd, resulterend in twee disome dochtercellen. \Ve presenteren cen model voor het ontstaan van alle mogelijke combinaties van karyotypes in cytotrophoblast cellen, gekweekte CV cellen, en foetus als gevolg van trlsomic zygote rescue middels de drie bovengenoemde correctiemcthoden. Alle UPD casus geassocieerd met CPI"I of met een normaal CV karyotype passen in dit model. Het toont verder aan dat trisomic zygote rescue niet noodzakelijk rcsulteert in een 100 % of hoog mozaïek tri som ie in semidirecte CV preparaten, zoals eerder werd gesuggereerd door sommige onderzoekers, lllam dat het theoretisch ook kan leiden tot veel lagere mozaïcken in de cytotrophoblast. 100

101

102

103 Abbl'eviatiolls AF AL CCD CD CGH CPM CV CVS DNA EEM FISH lcm ISH IUGR IVF MCC ND PCR PD SKY UPD US V\V \VCP amniotic fluid anaphasc lagging charge-coupled device chromosome demolitioll comparative genonuc hybridizatioll confined placental mosaicism chorionic "illi chorionic villi sampling deoxyribonucleic acid cxtraembryollic mcsodel111 fluorescent in situ hybridization inner eeil mass in situ hybridization intrauteril1c growth retardation in vitro fertilization maternal ecu contamination l1on-disjunction polymerase chain reactioll prenatal diagnosis spectra! karyotyping uniparental disomy ultrasound vruchtwater whole chromosome paint 103

104

105 Curriculum vitae -16 januari juni oktober september oktober juli juli 1987 geboren te Hoogstraten (België) eindexamen Algemeen Secundair Onderwijs (moderne humaniora, wetenschappelijke B), instituut Spijker te Hoogstraten, België aanvang studie Landbouwwetenschappen, Katholieke Universiteit Leuven (KUL), België Kandidaat Lanbouwkundig Ingenieur, KUL aanvang studie Biologie, KUL Kandidaat in de Wetenschappen, groep Biologie, KUL Licentiaat in de \Vetenschappen, groep Dierkunde, optie Fysiologie en Moleculaire Biologie, KUL -Hoofdvakken: bijzondere morfologie. embryologie. vergelijkende fysiologie, en moleculaire biologie -Keuzevakken: cellulaire immunologie, tulllorîlnmuniteit -Verhandeling: "Onderzoek naar de NPYinnenrutie van de visuele cortex bij Felis catus: retrograde tracertechnieken gecombineerd met immunocytochemische localisatie" bij Prof. Dr. Vandesande -sinds I oktober 1987 okt sept sept dec sinds januari 1998 werkzaam bij de Stichting Klinische Genetica Regio Rotterdam t.b.v. prenatale cytogenetica aanstelling als analist aanstelling als wetenschappelijk onderzoeker aanstelling als cytogelleticus op het prenataal c}1ogenetisch laboratorium los

106

107 List of (lllblicatiolls Van Opstal, D., Eussen, H.J., Van Hemel, J.O., Sachs, E.S. (1993). Application of fluorescent M in situ hybridizatioll for "dc I10VO" anomalies in prenatal diagnosis. PreJ1at Diagn, 13, Van Opstal, D., Van Hemel, J.O., Sachs E.S. (1993). Fetal aneuploidy diagnosed by fluorescence in situ hybridization within 24 haurs atler amniocentesis, Lancet, 342, 802. Van Opstal, D., van den Berg, C., Jahoda, ~vlg.j., Brandenburg, H., Los, F.J., In 't Veld, P.A. (1995). Rctrospective study of trisom)' IS in chorionic vuli with fluorescent in situ hybridization on archival direct preparations, Prenat DiagIl, 15, 51 M55. Van Hemel, J.O., Schaap, C., Van Opstal, D., Mulder, M.P., Niermeijer, M.F., Meijers, lh.c. (1995). ReculTence of DiGeorge syndrome: antenatal detection by FISH of a molecular 22qll deletion, J Med Gel/el, 32, Van Opstal, D., Van Hemel, J.O., Eussen, B.HJ., van der Heide, A., van den Berg, C.,In 't Veld, P.A., Los, F.J. (1995). Achromosome 21-specific cosmid cocktail for the dctcctioll of chromosome 21 aberrations in Înterphase nuclei, Prenat DiagIl, 15, Los, F.J., Van Opstal, D., Schol, M.P., GaiIIard, llj., Brandenburg, H., In 't Veld, P.A.(l995). Prenatal diagnosis of ll10saic tetrasol11y l2p/trisomy l2p by fluorescent in situ hybridization in amuiotic fluid cells; a case report ofpallister-killian syndromc, Prel1al DiagIl, In 't Veld, P.A., Van Opstal, D., van den Berg, C., van Ooijen, r\'i., Brandenburg, H., Pijpers, L., Jahoda, M.GJ., Stijnen, T.H., Los, F.J. (1995). Increased incidence of c)'togenetic abnormalities in chorionic viili samples from pregnancies cstablished by in vitro fertilization and embryo transfer (IVF-ET), Prellal DiagIl, 15, Los, PJ., van den Berg, C., Braat, A.P.G., Cha'ban, F.K., Kras, J.1'v1., Van Opstal, D. (1996). Ring chromosome 18 in amuiotic fluid cells of a fetus with only tàcial dysmorphisl11s, Am J Med Gel/el, 66, Van Opstal, D., Los, P.J., Ramlakhan, S., Van Hemel,J.o., van den Ouweland, A.M.\V., Brandenburg, H., Pieters, M.H.E.C., Verhoen~ A.,Venneer, M.C.S., Dhont, M., In 't Veld, P.A. (1997). Detenllination of the parent of origin in ninc cases of prcnatally detected chromosome aberrations found aftel' intracytoplasl11ic spenn injcction, Hum Repl'od, 12, Joosten, A.l"1.S., De Vos, D., Van Opstal, D., Brandenburg, H., Gaillard, J.L.L.,Vefllley-Kccrs, C. (1997). Pull monosom)' 21, prcnatally diagnosed by fluorescent in situ hybridization, PreI/ai DiagIl, 17, Van Opstal, D., van den Berg, C., Deelen, \V.H., Brandenburg, Il., Cohen-Overbeek, T.E., Halle)', D.JJ., van den Ouweland, AJvL\V., In tt Veld, P.A., Los, F..I. Prospective prenatal investigations on potential uniparental disom)' in cases of confined placental tri som)', Prenat DiagIl (in press). Van den Berg, c., Ral11lakhan, S. K., Van Opstal, D., Brandenburg, H., Halle)', DJJ., Los FJ. (1997). Prenatal diagnosis of trisomy 9: c)'togenetic, FISH, and DNA studies, PreI/at DiagIl, 17,

108 Los, F.J., Van Opstal, D., van den Berg, c., Braat, A.P.G., Verhoer, S., \Vesbey-van Swaay, E., van den Ouweland, A.M.\V., HaIley, DJJ. Uniparental disomy with and without confined placental mosaicism: a model for trisomic zygote rescue, Prenat Diagn (in press). Los, F.l, van den Berg, C., Van Opstal, D., Noomen, P., Braat, A.P.G., Galjaard, R.J.H., Pijpers, L., Cohen-Overbeek, T.E., Wildschut, H.U., Brandenburg, H. Non-normal cytogenetic results in semi-direct chorionic viiii: certainty-rates, prenatal follow-up investigations, and predictive values, submitted. 108

109 Dankwoord Eindelijk is het dan zover! Het boekje is klaar. Graag maak ik van de gelegenheid gebruik om een aantal mensen te bedanken zonder wiens hulp en steun dit proefschrift er misschien nooit zou zijn geweest. Op de eerste plaats Prof. Dr. E.S. Sachs. U ben ik zeer erkentelijk voor de kans die U mij destijds heeft geboden om naast het dagelijkse lahwerk ook wat onderzoek te doen, waarvan de resultaten leidden tot mijn eerste publicaties. Frans, zonder jouw enthousiaste begeleiding, jouw vertrouwen in mijn praktische en theoretische kennis, je bewonderenswaardige belezenheid, en heldere inzicht in bepaalde prenatale vraagstukken, zou dit boekje er op zijn minst minder fraai hebben uitgezien. Bcdank'i voor je onvoorwaardelijke steun in de afgelopen maanden. Ik ben ontzettend blij onze samenwerking te kunnen continueren. 1vlijn promotor Prof. Dr. H. Galjaard ben ik zeer dankbaar voor het vertrouwen dat hij steeds in mij heeft gesteld en voor de kansen die hij mij heeft geboden om mij als analist verder te ontplooien. Evenals de overige leden van de promotie-commissie, Prof. Dr. J. \V. \Vladimiroff, Prof. DI'. NJ. Leschot en Prof. Dr. P.J. \Villems, ben ik hem zeer erkentelijk voor het snelle beoordelen van het manuscript en voor de nuttige adviezen t.a.v. inhoud en stijl waardoor het uiteindelijk beter werd. Alle stafleden en hun vele medewerkers van de 24': verdieping wil ik bedanken voor de plezierige sfeer waarin ik de atf~elopen jaren heb mogen werken. Jan, jou wil ik in het bijzonder bedanken voor je morele steun, die ik, vooral in de beginperiode, af en toe nodig had, en uiteraard ook voor al je wetenschappelijke hulp bij o.a. het schrijven. Peter (in 't Veld), dankje wel voor jou bijdrage aan mijn onderzoek en voor je hulp bij mijn publicaties. Ik wens je vcel succes in het diabetes onderzoek! Dicky, Ans en Ben, bedankt voor jullie gastvrijheid in de tijd dat we nog geen FISHlab hadden en we dankbaar gebruik konden maken vanjullie ruimte, apparatuur en andere spullen. Dicky en Ans, bedankt voor jullie bereidheid om steeds hulp en advies te bieden als DNA technieken nodig zijn yoor het oplossen yan een c)1ogenetisch probleem. Dit heeft reeds geleid tot een aantal publicaties waarvan drie in dit proefschrift.,vim, bedankt voor het ter beschikking stellen van de mimte die we nodig hadden voor het inrichten van een FISH lab. Dit proefschrift kon alleen maar tot stand komen dank zij de tomeloze inzet en de stcun van mijn collega's van het prenataal cytogenetisch laboratorium. Dit is ook jullie boekje! Cardi,joll ben ik veel dank verschuldigd voor het vertrouwen datje de afgelopen jaren steeds in mij hebt getoond, en voor je enorme steun als ik het even niet meer 7--,,1g zitten. Jij hebt mij destijds de chromosomen leren kennen en de liefde voor het vak bijgebracht. Ik heb grote bewondering voor de manier waarop jij je als hoofäanalist inzet voor het lab (zowel de mensen als de werkzaamheden), en ben er trots op datje mijn paranimf wilde zijn. Armulldo (ABart), bedankt voor alle prachtige dia's, foto's, posters die je doorheen de jaren voor mij hebt gemaakt en voor alle adviezen t.a. v. lay-out e.d. (volgens mij heb je je roeping echt gemist). Ook al was ik niet altijd je gemakkelijkste klant ("veel te veel voor één dia", "veel te weinig tijd"), toch maakte je er steeds weer iets moois van. In het bijzonder bedankt voor alle prachtige illustraties in dit boekje! Petra ("Peppi"), wij zijn tienjaar geleden ongeveer gelijktijdig begonnen bij de vlokken. Verschîllende collega's die kort na ons kwamen, zagen we snel weer vertrekken. Ik ben blij dat jij biectl Annemarie (A1vIS) 109

110 bedankt voor de vrolijke noot waannee je ons dagelijks verblijdt (al zou ik af en toe die knop wel eens willen omdraaien!). Joke,jij bent denk ik de meest veelzijdige werkkracht bij prenataal. Of het nu vlokken zijn, vruchtwaters, AFP's offish, in alles benje zeer bedreven en toegewijd. Dank zij jouw flexibele houding kunnen we daar ook dankbaar gebruik Vallll1aken. Jacqueline, jij hebt zo je eigen stijl. Heel erg bedankt voor al je inzet. Els en Nieole, jullie zijn al lang meer dan collega's. Ik hoop dat onze periodieke etentjes (samen met Locs) nog lang blijven voortduren, zodat we weer eens lekker kunnen bij kletsen over "van alles en nog wat ll Gerda, samen met Joke een oude rot in het vak. Mensen met jouw ervaring zijn onmisbaar voor het goed draaiende houden van een lab. Enje bent echt niet de enige zonder vlokkenervaring, hoor. Simone,je inzet voor een gezonde werkomgeving en goede voorlichting van patient en arts vind ik zeer te bewonderen. Hopelijk komt die brochure er nu eindelijk eens, en zijn je inspanningen niet tevergeefs geweest. Ellen, behalve naar chromosomen gaat jouw hart ook uit naar dieren. Bedankt nog voor de goede zorgen die je Cl\ÎO gaf. Jammer dat hij zo snel al het loodje legde. Japie (de Mosselman), ik ken niemand die zo lekker mosselen kan koken als jij! \Vanneer doen we dat nog een keer over? Bedanld voor je goede humeur. :~vrartin (Flatfish), bedanld voor alle bestellingen die de afgelopen jaren via jou liepen. Ongeloofelijk dat die ronde glaasjes toch steeds weer aanbvamen, ook al leek het er even op dat de firma niet bestond. Constant, Monique en Ragonda, ik heb nog weinig gelegenheid gehad om jullie echt goed te Ieren kemlen. Dat jullie alle drie een aanwinst zijn voor het lab staat buiten kijf. Robe11~Jan, ik weet dat de combinatie research en prenatale diagnostiek je SOI11S zwaar valt, en datje af en toe wat meer tijd zou willen hebben voor experimenten. Daarom waardeer ik je inzet en interesse voor de labactiviteiten en kijk ik ernaar uit onze samenwerking voort te zetten. lvlieke Gij hoort uiteindelijk ook bij prenataal), bedankt voor de plezierige en nuttige werk/privé discussies die we de afgelopen jaren hebben gevoerd, en voorje hulp en vooral geduld bij het efficient in orde brengen van mijn referentielijst (ik besef dat ik een slechte leerling was). r..'lijn collega!s van postnatale cytogenetica o.l.v. Jan en Cokkie ben ik zeer dankbaar voor de prettige samenwerking. Alhoewel we de afgelopen tien jaren fysiek wel uit elkaar zijn gegroeid, vereisen onze werkzaamheden gelukkig een nauw contact, waarin ik mijzelf steeds heel prettig heb gevoeld. Bert, jij bracht mij de grondbeginselen bij van de FISH techniek, maar je bent doorheen de jaren vooral een zeer stimulerende en inspirerende bron geweest. Ik bewonder je tomeloze energie, je aanstekelijke enthousiasme,je betrokkenheid, brede interesse en grote kennis van zaken, en ben dan ook heel blij dat je mijn paranimf wilde zijn. Annet, jou wil ik in het bijzonder bedanken voor je collegialiteit en je vriendschap. SOI11S verlang ik nog wel eens terug naar de tijd dat we slechts met drieën "FISI-Iten", en communicatiestoornissen nog een zeldzaamheid waren. De collega's van DNA diagnostiek o.l.v. Dicky en Ans wil ik bedanken voor de gastvrije en gezellige werksfeer en voor de hulp die ik mocht ontvangen bij het PCR' en. Carola, bedankt voor de precieze manier waarop je mij de techniek hebt geleerd. \Vout, dank je wel voor alle wijze raad, en Sarvan, bedank-t voor al het werk dat je van mij overnam toen ik tijdelijk het RA~lab niet in kon. Alle medewerkers van de sector prenatale diagnostiek en verloskunde, afdeling gynecologie/verlosl'1ll1de van het Academisch Ziekenhuis Rotterdam, m.b. Prof. Dr. J.\V. \Vladimiroff, Dr. M.GJ. Jahoda, Helen, Hajo, Titia, Nicolette, Ernst, en l'vlarja en onze Dordse connectie Dr. Leen Pijpers van de afdeling gynecologie en verloskunde van het ~Aerwede 110

111 Ziekenhuis in Dordrecht wil ik bedanken voor al hun bijdragen aan het onderzoek. Ook alle mensen van wie we in de loop der jaren FISH-probes en -adviezen ontvingen (teveel om op te noemen) ben ik zeer erkentelijk. Zonder anderen te kolt te willen doen, wil ik een speciaal woord van dank richten aan Dr. 1. G. \Vauters (U.LA, Antwerpen). Jij leverde ons de zo fel begeerde 13-probe (naast anderen), waarvan we nog steeds wekelijks dankbaar gebruik maken. En dank zij jouw nuttige tip voor het zwellen van celkernen konden we onze resultaten verder verbeteren. Heel erg bedankt voor je vriendelijkheid! Dan zijn er nog een aantal mensen die indirect hun bijdrage leverden en die ik hier wil bedanken. Eerst en vooral de secretaressen Jaqueline, Jolanda, I-Iennine, Cilesta en Jeannette, voor al jullie hulp bij het f.1xen, post versturen, tekstverwerking, en oplossen van kopiccrapparaat- en printerproblemen, en voor de gezellige babbel(s). Peter van Vuuren voor je hulp als de PC weer eens kuren vertoonde (of ligt het aan de gebruiker!?). Ruud, Tom en 1'Iirko voor het fotografische werk dat jullie gedurende al die jaren voor mij hebben verzet. ~vlelle, Rein en Mieke voor alle bestellingen, en Jopie, Joke, ElI)', Rob, \Vilmu en Louise voor atle keukenactiviteiten. Naast mijn werkkring wil ik ook vrienden en familie bedanken voor hun niet aflatende interesse voor mijn werkzaamheden (al blijft het toch moeilijk voor te stellen wat ik nu feitelijk doe). Pa en ma, bedankt voor de kans die jullie me gaven om te studeren, voor het tijdig bijsturen, en vooral voor jullie vertrouwen en steun bij alle keuzes die ik moest maken. Ik weet dat jullie ontzettend trots op mij zijn. Besef dat dit wederzijds is. John, zonder jouw flexibiliteit en grote loyaliteit was dit boekje cr echt nooit geweest. Lieve Jimi, ik ben blij om samen met jou af en toe weer eens kind te kunnen zijn, om zo het "serieuze" werk even te vergeten. 111

112

113 Appendix publications I-VIII

114

115 Appendix publication I Prena!. Diagn., 13, (1993) Copyright John Wiley & Sons Limited. Reproduced with permission

116

117 Application of Fluorescent In Situ Hybridization for "de-novo" anomalies in Prenatal Diagnosis D. Van Opstal, H.J. Eussen, J.O. Van Hemel, E.S. Sachs SUllllllary Fluorescent In Situ Hybridization (FISH) was ealtied out for 3 cases of.bnormal karyotypes in prcnatal studies. Two COllccl'llcd de-iloyo structul'al anomaiics and the 3 rd a marker chromosome. Thc ol'îgin of the extra mutcl'ial could be defined in all 3 cases ",hieh gives befter insight info the l'clationship betwecn genotype and phcnotype 31ld makes more adequate gcilctic counseling possible. Introdllction Prenatal diagnosis has its limitations. The fetus ean anl)' be seen on ultrasoulld and physical examination is not possible. Cytogenetic problems are caused by de 110VO stmctural anomalics such as deletions, inversions and partial trisomies. The possibility of Fluorescent In Situ Hybridization (FISH) studies have made a more accurate diagnosis of these stmctural anomalies possibie which results in better counseling of the prospeetive parents (Callen et al., 1992, Jauch et al., 1990, Stetten et al., 1992). We report on the identification of three structural cllromosome anomalies in prenatal diagnosis. FISH silowed the origin of a de-nova partial trisomy of cluomosome 11 alld 18, and a partial trisomy of chromosome 15 respeetively. The data provided us a better understanding ofthe fetal anomalies. eelt preparatiol1s Material and lllethods Cultivation and chromosome preparations of amniotie fluid eells, skin fibroblasts and lymphocytes were perf0n11ed aeeording to standard techniques. Amniatic fluid eehs were cultured with the in-situ method on glass-eoverslips. Trypsin-Giemsa staining was used routinely. Additional staining techniques such as DA-DAPI-, NOR- and C-banding were applied in ane case. Unstained preparations used the same day for FISH were put on a hot plate (65 C) for 2 hours. The slides were incubated in 70% aeetie acid for 1 min. After washing in PBS, the eells were treated with RNase (Pharmacia) (100 lig/mi in 2 x SSC) at 3rC for 1 hr, followed b)' a pepsin (Serva) (100 lig/mi in O,OIN HCI) t!'eatment at 3rC for 10 min and finali)' fixed in 3.7% formaldehyde (Merek) in PBS/50 mm MgCI, for 10 min. Dehydration in three ethanol solutions (70%, 90% and 100%) followed before the hybridisation procedure. 117

118 DNA-probes and labelling Whole chromosome libraries pbs-4, pbs-ii, pbs-15 and pbs-18 were a gift from Dr. J.W. Gray (Lawrence Livermore National Laboratory, California), (Collins et al., 1991). LI.26 (Devilee et al., 1986) and p22/1:2.1 (Me Dermid et al., 1986) are alphoid DNA probes localized in the pericentromeric region of chromosomes 13 and 21, and chromosome 22 respectively. CRN189-1 is a eosmid DNA-probe whieh maps to 15qI1.2-qI2. (DonIon et al., 1986; Tantravahi et al., 1989). Fluorescent In Sim Hybridizalion (FISH) and probe deleclion Chromosomal in situ suppression (CISS) hybridization for cluomosome specific libraties was based on the methods described by Pinkel et al., (1988). The hybridization mixture, JO fll total volume eonsisting of 50% formamide (Merek)/2 x SSC, 10% dextran slllphate (Pharmaeia), 5 ~g salmon sperm DNA, 5 flg Cot-I DNA (BRL) and 100 ng biotinylated probe DNA, was denatured at 90 0 e for 5 min and immediately put on iee, followed by 1 to 3 lus preannealing at 37 C. Target DNA was denatured by inullcrsion in 70% formamide 12 x SSC for 3 min at 80 oe and dehydrated in all ethanol series. The hybridization reaction was perfonned at 37 C for 40 hrs. In case ofcrni89-1, 40 lig ofbiotinylated probe was precipitated with 5 flg salmon sperm DNA and 2 flg ofcot-1 DNA and dissolved in JO ~I 50% formamidel2 x SSC/JO% dextran sulphate. Denaturation of probe and target DNA was perfornlcd as described above. The hybridization reaction took place overnight at 37 C. The centromere probes LI.26 and p22/i :2.1 (40 ng in JO fll 60% formamidel2 x SSC) and target DNA were denatured simultaneously for 3 min at 80 C. Hybridization was allowed to proceed ovenlîght at 37 C. After hybridization the slides were washed 3 times in 50% formamidel2 x SSC at 42'C for 5 min, followed by 3 changes of2 x SSC, twiee at 42'C and once at 65'C respectively. For the centromere probes, the last washing step (2 x SSC at 65'C) was replaced by 0, I x SSC at 65 'Co The probe was detected by.itemating layers of fluoresceinated avidin and biotinylated goat anti-avidin (Vector Lab, Burlingamc, USA). The slides were mounted in anti-fade medium with propidium iodide (Sigma) for counter-staining of the cllfomosomcs and examined under a Leitz Afistoplan fluorescencc microscope. Case 1. Resuits Amniotic fluid was sampled in the 16th week of the flest prcgnancy of a 31-year old womrul because of a recurrence risk for neural tube defects. The u-fetoprotein level was within normal range. Cytogenetic alld ma/ecu/al' studies The amlliotic fluid cultures showed a chromosome 18q+ in a femalc fetus in 8 clones. 118

119 Figure 1. FISH signals on normal chromosomes (a rrowhcad) and abnormal chromosomes (arrow). (A) Case I with dup (18)(qJ2.3 - q21.3) hybridizcd with PBS- 18. (B) Case 3 with dclcted chromosomc 15 (PBS.15). (C) Case 2 hybridizcd with PBS-4, and(d) with PBS-Il, both showing th at 4p+ matcrial is dcrivcd from chromosomc 11.

120 Subsequent karyotyping of bath parellts gave normal karyotypes, 46,XX and 46,XY, respeetively. Chromosomc painting was applied to identify thc 18q+ chromosome. The 18 library showed that the extra matcrial in 18q+ was derived from chromosome 18 (fig. IA). On the basis of the G-banding pattern and chromosomc painting, we described the karyotype ofthe fetus as 46,XX, inv dup(l8) (pter ~ q22 ::q21.3 ~ qi2.3::q22 ~ qter) (fig. 2A). Ultrasound screening showed a positional abnormality of one hand. The parents were counseled about the expected anomalies corresponding to trisomy 18q (de Grouchy, 1984) and elected to terminate the pregnancy. A fcu1ale fetus was born with external dysmorphic signs. Thc open eyes showed strabisl11us, the base of the nose was broad, thc Hose flat. :Micrognathia, low-set cars alld a large tongue were present. Thc 2nd fingers were crossed over the 3rd and there were clubfeet. There were no internal anomalies present. This phenotype silowed most signs of trisomy l8q. There was na eeli gro\\1h of fetal skin. C.se 2. Fetal blood was obtained by cordocentesis from a 28-year oid womall in her fust pregnancy in the 28th week. Ultrasound examillation had shown the absence of amniotic fluid caused by bilateral renal agenesis. Cylogenetic Gnd ma/ecu/ar studies. Karyotyping of fetal blood silowed extra material on the short arm of cilfomosome 4. Subseqnent karyotyping of bath parents gave normal karyotypes (46,XX and 46,XY respectively). Chromosome painting with the 4 library left a small terminal segment unstailled on 4 p (fig. 1 C). The origin of this extra 4p material was determined by painting with the 11 library (fig.!d). Same otller probes eould he excluded: pbs-6, 7, 8, 9, 12 and 17 respeetively. On the basis of the G-pattern and chromosome painting the fetal karyotype was estimated to be 46,XY,-4,+ der (4), t(4;ii)(pi6;q22.2) (fig. 2B). Because of the ahnonnalities diagnosed by ultrasound and the de-novo unbalanced fetal karyotype, the parents decided to terminate the pregnancy at 34 weeks. Autopsy was perfonned on the stillborn neonatus. The stilibom had a birthweight of 1232 g (helow IOth percentiie) and a height of34 cm. The diagnosis of renal agenesia with luug hypoplasia was confirmed while external anomalies of the head aud extremities were caused by the oligohydramnion. Death had occurred beeause of respiratory insufficiency. Skin cell cultures eonfirmed the fetal karyotype. Case3 Amniotic t1uid was sampled in the 35th week of the pregnancy of a 25-year old woman because the Dandy Walker syndrome had been diagnosed by ultrasound. Cytagel1elic and ma/ecu/ar studies Eight amniotic fluid eell clones were analysed and showed in all metaphase spreads a feulale 120

121 Figul'e 2. Schematic preselltution of the mutated chrol11osol11cs of case 1 (a) alld case 2 (b). p q q,l.,,,~, '" '",D,,, -;n",,, '" " q P q ~ '" "," " '" lh l.\, 4 11 " im' dup(18) (qi2.3.> q2j.3) A t(4:11) (pi6:q22.2) B Figure 3. Amlliotic mctaphase of casc 3 showing FISH signals with probe LI.26 on the centl'omeric rcgions of chl'ojllosome 13 (large arrowhcad) und of chrol11osol11c 21 (smal! arrowhcad). Thc marker chromosome (arrow) is without hybridizatioll slgnal. 121

122 karyotype with an extra chromosome. The size ofthis marker chromosome was comparable to the length of a no. 20. Additional staining methods, consisting of C-,DA-DAPI- and NORbanding, were negative. The karyotype of the mother was 46,XX. Blood of the biological father from a prcvious relationship was not available. After FISH with the 15 library, whieh painted the normal chromosomes 15, but also showed eross-hybridization to the centromeric regions of the other aerocentric chromosomes (Collins et al., 1991), we found the marker to be totally fluorescent (fig. IB). We excluded the origin of a centromere of chromosomes 13, 14,21 and 22 on the extra cluomosome by FISH with LI.26 and p22/1:2.1, because L1.26 showed signals on the centromeres of chromosomes 13 and 21, but not on the marker chromosome (fig. 3), and aner hybridization with p22/1 :2.1, we found strong signals on the ccntromeres of both clrromosomes 22 and a weaker signaion a number of other chromosomes, such as 14, but no signal was seen on the marker. On the basis of these results we expected the marker to be a deleted clrromosome 15, with the deletion most probably covering 15q , because of the absence of CRN189-1 on the marker in about 15 metaphases in which the normal cluomosome 15 showed 100% hybridization. The karyotype of the fetus therefore results in a partial trisomy 15. A female child was bom spontaneously within a week after aml1ioeelltesis and died the same dal'. Birthweight was 2460 g, height 44 cm. The Dandy Walker syndrome was confinl1ed by the autopsy results. The head had greatly incrcased (39 cm) and the vermix cerebelli was replaced by a cyst of 4 cm. There were 110 other anomalies. Discussion Cytogenetic problcms in prenatal diagnosis arise when de-novo stmctural chromosome anomalies or a marker chromosome are present. Conventional cytogenctic methods, like NOR-bauding, centromere and DA-DAPI staining are important in cases of marker clrromosomes (Sachs et al, 1987), but cannot always give a definite diagnosis. FISH (Fluorescent In Situ Hybridization) has made further identification of small de-novo stmctural anomalies and marker cluomosomes possible. The use of FISH in prenatal diagnosis for the identification of de-novo structural anomalies as in our cases one and two, has to our knowledge not been described before. The prenatal cases in the literature concerned known familial translocations ( Jauch et al., 1990; Klever et al., 1992; Rosenberg et al., 1992; Speleman et al., 1992) whilst the de-novo cases were of liveborns (Sachs et al., 1992; Van Hemel et al., 1992; Hulten at al., 1991; Jauch et al., 1990; Speleman et al., 1991). FISH studies for the identification of marker clrromosomes have been described for pre- and postnatal cases by Callen et al., (1992) and Stetten et al., (1992). For the finiher identification of the marker der (15) in case 3 it would be necessary to use FISH with single copy probes mapping to clrromosomal subregions. However, the occurrence of Dandy Walker syndrome makes a breakpoint at 15q22.3 likely since leshima et al., (1985) described a patient with Dandy Walker malformations caused by partial trisomy of 15q22.3 ~ qter. The autopsy results of case one were in aecordance with partial trisomy 18q. Two cases ofpartial trisomy 11 with renal agenesia as in our case 2 have been described by Francke et al, (1972) and Los et al, (1992). Cytogenetic studies followed by FISH studies can therefore give better insight into the 122

123 relationship between genotype and phenotype which may improve genetic counseling and decision making for prenatal diagnosis. Acknowledgements We acknowledge Dr. L. Pijpers, Merwede Hospital Dordrecht, for referral of the patient in case I. We wish to thank Ruud Koppenol.nd Mirco Kuit for fotographic work.nd Jacqueline du Parant for typing the manuscript. References Callen, D.F., Eyre, H., Vip, MMY., Freemantle, J., Haan, E.A. (1992). Molecular cytogenetic and clinical studies of 42 patients with marker chromosomes, Am J Med Gellet, 43, Collins, C., Kuo, W.L., Segraves, R., Fuscoe, J., Pinkel, 0., Gray, J.W. (1991). Construction and characterization of plasmid librarics enriched in sequences front single human chromosomes, GenomÎcs, 11, de Grouchy, J., Turleau, C. {I 984). Clinical atlas ofhuman chromosomes, New York: John Wiley, Devilee, P., Cremer, T., Slagboom, P., Bakker, E., ScholI, H.P., Hager, H.O., Stevenson, A.F.G. (1986). Two subsets of human alpoid rcpelitive DNA show distinct prefcrcntial localization in the periccntric regions of chromosome 13, 18 and 21, Cylogenet Cell Genel, 41, DonJon, T.A., Lalande, M., Wyman, A., Bruns, G., LaU, S.A. (1986). Isolation ofchromosome 15-specific DNA segments that are absent in individuals with Ihe Prader WiJJi syndrome, Proe Natl Acad Sci USA, 83, Francke, U. (1972). Quinacrine mustard fluorescence of human chromosomes: characterization of unusual translocatiolls, Am. J. Med. Gene!., 24, Hulten, M.A., Gould, C.P., Goldman, A.S.H., Waters, J.J. (l991). Chromosomc in situ suppressioll hybridisation in clinical cytogcllelics, J Med Genet, 82, leshima, A., Takeshita, K., Shirasaka, Y., Nakao, Y., Kisa, T. (1985). Distal 15q lrisomy with DandYMWalker malformation in a fcmale infant, Jpn J Hum Genet, 30, Jauch, A., Daumer, C., Lichter, P., Murken, J., Schroeder-Kurth, T., Cremer, T. (1990). Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical c)10genetics, Hum Genet, 85, Klever, M., Grond-Ginsbach, C.J., Hager, H-D., Schroeder-Kurth, T.M. (1992). Chorion ie villus metaphase chromosomes and interphasc nuclei analyzed by chromosomal in situ suppression (CISS) hybridizatioll, Prenal Diagn, 12, Los, F.l. Hagenaars, A.M., Marrink, J., Cohell-Overbeek, T.E., Gaillard, J.L.J., Brandellburg, H. (1992). Maternal serum alpha-feloprotein levels and fetal outcome in early sccond trimester oligohydramnions, Prenat DiagIl, 12, Me. Demlid, H.E., Duncan, A.M.V., Higgins, M.J., Hamcrton, J.L., Rector, E., Braseh, K.R., White, B.N. (1986). Isolation and characterization of an a-satcllile repeated sequence from human chromosome 22, Chromosoma, 94,

124 Pinkel, 0., Landegent, J., Collins, C., Fuscoe, J" Segraves, R" Lucas, J"Gray, J. (1988), Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4, Prae Nar! AeadSci USA, 85, , Rosenberg, C" Blakcmore, K.J., Keams, W.G., Giraldez, R.A., Escallon, C.S" Pearson, P.L., Stetten, G. (1992), Analysis ofreciprocal translocations by chromosome painting: applications and limitations ofthe technique, Am J Hum Gel/ct, 50, Sachs, E.S., Van Hemel, J.O., Den Hollander, J.C., Jahoda, M,GJ. (1981). Marker chromosomes in a series of prenatal diagnoses. Cytogenelic and follow-up studies, PreJ/al Diagn, 7, Sachs, E.S., Van Hemel, J.O., Los, FJ. (1992). Prenalal cytogenetic and postnalal molecular studies on 46,XX male, Lal/ccI, 339, Speleman, F., Mannens, M., Redeker, B., Vercruyssen, M., Van Oostveldt, p" Lcroy, J., Slater, R. (1991). Characterization of a de novo duplication of IlpI4->p 13, using fluorescent in situ hybridization and southem hybridization, Cytogcnet Cel! Genet, 56, Speleman, F., Van Roy, N., Wiegant, J., Verschraegen-Spae, M.R., Benoit, Y., Govaeri, P., Goossens, L., Leroy, J.G. (1992). Detection of subtie reciprocal translocations by fluorescence in situ hybridization, Clin Genet, 41, Stetten, G., Blakemore, KJ., Couricr, A,M., Coss, C.A., Jabs, E,W. (1992). Prcnatal identification of small mosaic markers of different chromosomnlorigins, PreI/al DiagIl, 12, Tantravahi, U., Nicolls, R,D., Stroh, H., Ringer, S., Neve, R.L., Kaplan, L., W1tarion, R., Wurster-HiIl, 0., Graham, J.M., Canru, RS., Frias, J.L., KOllssen: B.G., Lalt, S.A. (1989). Quantitalive calibration and use of DNA probes for investigaling chromosome abnonnalities in the Prader-Willi syndrome, Am J of Med Genet, 33, Van Hemel, J.O., Eusscn, B., Wesb)'-van Swaay, E" Oostra, B.A.(1992). Molecular detcction of a translocation (Y; 11)( q 1 L2;q24) in a 45,X male with signs of Jacobsen syndrome, Hum Genet, 88, 66 I

125 Appendix publication II Am. J. Med. Genet., 66, (1996) Copyright \Viley-Liss IJlc., Reproduccd with pennission.

126

127 Short Report: Ring Chromosome 18 in a Fetus with only Facial Anomalies Frans 1. Los, Cardi van den Berg, Armando P.G. Braat, Firas K. Cha'ban, Johan M. Kros, and Diane Van Opstal Abstract A prenatally detec!ed case of dng ehrol11osome 18 [46,XX,r(18)] in amniotie fluid eells of a fetus displaying an abnol'mal faeial profile on ultra sound as thc only malformation is l'cpol'ted. Thc chromosome 18 origin of fhe ring chromosome, of a supernmncl'ary marker chrolllosome in same eells, Rnd of Illicronuclci was demollstrated by fluorescent in situ hybridization,,,i th a whole chl'omosomc 18 paint (Cambio) and 18 centromere probe L1.84. DNA investigations showed demions of 1811 as weil as 18q matcrial of 1'(18), whielt tul'llcd out fo be ofpaternal origin. Autopsy ofthe fetus aftel' tcrmination of pregnancy at 20 weeks of gestation showed no additional malfol'mations, in agreement n'ith the pl'cvious ultrasoulld findings. Inh'oduction Some cases of prenatally detected structural chromosome 18 abnormalities have been reported; isochromosome 18q [i(18q)] [Froster-Iskenius et al., 1984; Wurster-Hill et al., 1991], mosaic i(18p) [Göcke et al., 1986], 18p- [Göcke et al., 1988], mosaicism of deletion (18)(pll )/i(l8q) [Sulton & Ridler, 1986], and ring chromosome 18 [r(l8)] (Biben et al., 1992]. We describe the prenatal detection of a 46,XX,r(l8) karyotype in amuiotic fluid celis investigated with conventional cytogenetic techniques and fluorescent in situ hybridization (FISH). DNA investigations for the establishment of the parent of origin as weil as potential deletions of 18p and 18q material of the r(l8) were carried out. Furthermore, the prenatal ultrasound findings and a detailed clinical description are presented. Clinical Report A 39 year-old pregnant woman (04, P2, Abt) asked for prenatal diagnosis because of advanced maternal age. In two previous pregnancies prenatal diagnosis had also been performed with normal results alld favourable pregllancy outcollle. Her family history showed a sister with Down syndrome; the family history of her husband (40 years of age) was unremarkable. Amniocentesis was performed at a gestattonal age of 16 weeks. No abnonnalities were noted on ultrasoulld investigation. Aller the finding of a r( 18), detailed ultrasonography was perfornled at t 9 weeks of gestation; 110 stmctural malformatiol1s \Vere seen in a fetus with normal biometry (Table 1). lntracranial anatomy was normal. The only 127

128 Tablc 1. Fctal ultrasonographical biomelry at 19 weeks and body measllremenls aflcr termination of pregnancy al 20 weeks of Ihc fetus wuh 46,XX,I'(18)* Ultrasound biometry Body measurements Parameter! fetus (mm) reference values (mm) 5% 50% 95% fetus (mm) reference values (mm) mean± I.SD CHL ± 17 HC ± 12 DBP IOD/OCD II ± 1.8 OOD/OCD ± 3.4 Femur Length AC handlenght ± 2.9 middlefmger lenght ± 1.5 foot lenght 29' 35 ± 2.5 lnd ± 3.4 *Ultrasonographical reference values according to Snijders & Nicolaides, 1994, and Trout ct alo, 1994; body measurement reference values according to Chambers et al., I CHL = crown to heellcngth, HC = head circumference, DBP = distantia biparietalis, IODIlCD = inter orbital distance/inner canthal distance, OOD/OCD = outer orbital dislance/ollter cunthal distance, AC = abdominal circumference, IND = inter nipple distunce. 2 Meusurement outside mean ± 2. SD-area. Figllre 1. Ultrasonographical image of the fctal faeiat profile at 19 weeks, shoning the recedillg forehead, l'ethrognathla alld pronoullced upper Iip/philtrulll. 128

129 remarkable finding was a slightly abnormal facial profile with a reeeding forehead, pronotmeed tipper lip/philtnllll and rethrognathia (Fig. I), wbieh might fit the fetal phenotype of the 18p- syndrome [Göeke et al., 1988]. Sinee the r(l8) ulrned out to have arisen de-novo and the chanees for a norlllal phenotype were counselled low [Schinzel, 1984], the parents opted for termînation of pregnancy. At 20 weeks of gestation, labour was indueed by intravenously administered prostaglandin. A female fetus was bom of 270 gr (Mean-Ix S.D) [Chambers et al., 1993] with normal body-measurements (Tabie I). A rcccding forehead, pronotlllced convex philtrulll, lllicra- and rethrognathia, a braad neck, hypoplastic alae nasi, and dysplastic ears were nated (Fig. 2). Autopsy did not demonstrate any internal malfonnation; all argans silowed a normal weight and development far gestational age. The brain had developed normally with normalmidline structures. Figure 2. (A) Frontal and (D) latentl view of the fetus at 20 weeks. showing the rcccding forehead, hypo plastic alae nasi, pronounced convex jlhiurum alld icro/rethrognathia. Cytogenetic and DNA Studies Amniotic fluid cells were cultured by the in-situ lllethod on glass coverslips. Trypsin-Giemsa staining was tlsed. The karyotype was 46,XX,r(18) in the majority ofinvestigated clones (21 out of 27). In 5 clones a mosaicism of 45,XX,-18/46,X:X,r( 18) was encountered and one clone showcd a mosaicism of 45,XX,-18/ 46,XX,r(l8)/ 47,XX,r(18),+ marker chromosome (Fig. 3). In one ceil, a double-sized (dicentric) ring chromosome was found (Fig. 3). Figure 3. Partial karyotype of eultured amlliotie Duid eells (Trypsin-Giemsa staining). A. r(18). B. marker ehromosome (mar).c. double r(18), each accompallied by the normal ehromosome r (18) 18 lll~r 18,loubln(l8) 129

130 In fetal fibroblasts, only the 46,XX,r(l8) line was found in 16 investigated cells. Karyotypes ofthe parents were normal46,xy and 46,XX, respectively. FISH was perfonned on unstaillcd slides of cultured amniotic fluid cells with a whole chromosome 18 paint (Cambio Ltd., Cambridge, V.K.) and c1lfolllosomc 18 centromere probe L1.84 [Devilee et al., 1986]. Hybridization with the chromosollle 18 paint was perforllled according to thc procedure recolluuencled by the manufacturer. FISH with L1.84 was done as described before [Van Opstal et al., 1995]. Slides were exalllined with a Leitz aristoplan fluorescence microscope and images were capturcd by thc Genetiscan Probe Master Systcm (Perceptive Scientific Instnullents Ltd., Chester, U.K.). Hybridization with the whole chromosomc 18 paint resulted in a fluorescent staining of both ring and marker chromosome (Fig. 4A), as well as of micronuclei found in the vicinity of same iuterphase nuclei, indicating a chromosome 18 origin. Hybridization with L1.84 l'ielded strollg signals on both ring and marker chromosome (Fig. 4B), also demonstrating a chromosome 18 origin. Figurc 4. FISH signals on norm al chroll10some 18, 1'(18) and marker chromosome (mar) aftcr in situ hybridization wuh A. whole chrolllosome 18 paillt (Cambio) und n. 18 centl'omere probe L1.84 to cultul'ed ulilniotic fluid cellmctaphases. DNA isolated from cultured amniotic fluid eells and blood of bath parents was investigated bl' perfonning PCR anall'sis of various mierosatellite markers on chromasome 18 ta establish potential deletions of 18p and 18q material of r(18) and determine the parent of origin (Fig. 5). The PCR products of DI8S59 and D18S40, located on 18p, and of D18S34, D18S35, D18S42, lvlbp and D18S70, located on 18q [Le Beau et al., 1993; GeUlis van Kessel et al., 1994] showed an informative pattem. Unfortunately, 18p marker DI8S52 and 18q marker DI8S38 [Le Beau et al., 1993; Geurts van Kessel et al., 1994] turned out to be noninformative. PCR analysis demonstrated the absence of the patenml alleles in fetal eells at the loci D18S59, D18S70, MBP, and DI8S42 whilst a paternal as weil as a matemal allele were present at the loci D18S35, D18S34, and DI8S40 (Fig. 5). These DNA investigations indicated that r( 18) was of paternal origill alld displayed an 18p deletion of undetenuined size together with a large 18q deletion, at least dele 18)(q21.33). 130

131 Figure 5. Ideogram of chromosome 18 with the localization of the tested microsatellitc mm kers. per analysis of D18S59, D18S40, D18S35 and D18S42 shows the absence of patel'llal alleles Al or 1\ at loci D18S59 and D18S42 In fetal eells. p q ~D18S59 Dl8S52 I D18S40 xii _.U ~ ~ ~ I 11.1 ~I=:::.-- Al D18S DI8S59... AI ~ _,\1 IJ[, DI8S40 ~ ~ 21.2 t- Dl8S35 I ~ AI, D18S D18S U 'Sf.~ A'I MBP DI8S35 DI8S D18S70 18 := ;H Al Discussion A de-novo r(18) was estabhshed prenatally in alllniotic fluid cells with cytogenetie evidenee for lllitotic instability snch as intra-clollal mosaicism [Rocchi et al, 1984; Kosztolányi, 1987a], the presence of a snpernulllerary marker chrolllosome [Koulischer et al., 1980; MacDermot et a!., 1990], dicentric ring formation [Kosztolányi, 1987a; 1987b], and the occurrenee of micronuclei [Kosztolányi, 1987a]. FISH showed the ring and marker ehromosome to be chrolllosome 18-derived and the micronuclei to contain chromosome 18 material DNA illvestigations demonstrated a deletion of 18p and 18q material of r(18), whieh was of paternal origin. These investigations were perfonlled on DNA isolated from amniotic ±luid cells after termination of pregnancy, but they could have been perfonned prenatally. Especially in cases of de-novo ring chromosomes without any ultrasound abnormalit)', in which the fehls might on I)' be affected with the nring syndromen [Kosztolányi, 1987b; Pezzolo et al, 1993], DNA data conceming subtelomeric deletions are important for genetic counselling. In contrast ta the expected eoncomitance of 18p and 18q deletions with serious fetal malformations, the uitrasound findings were surprisingly nonnal, apart from the facial profile. This case eonfirms the importance of the interpretatian of fetal faciat profile abnormalities on ultrasound, which turned out to be in agreement with clinical obsen'atiolls of the fehls after 131

132 tennination ofpregnaney. The fems had na malfonnations of the intemal organs, but ouly faeial anomalies, whieh is in contrast with au earlier prenatal diagnosis of de-nova r(l8) [Eiben et al., 1992]. However, the facial atlomalies resembied those of a reported prenatal case with 18p- syndrome [Göeke et al., 1988], and, remarkably, also those of cases with tetrasomy i8p [Göcke et al., i986] and mosaic monosomy i8p/trisomy i8q [Sulton & Ridier, i986]. The hypopiastic aiae nasi together with some other reported anomalies ofthe i 8p-/r(i 8) phenotype as absent permanent teeth, hypothyroidism, diabetes mellitus and anorectal malfonnations [Schinzel, 1984] suggest similarities between this phenotype and the autosomal recessive inherited Johanson Blizzard syndrome (JBS)[phenotype ; McKusick i994]. Some cases of JBS might be recurrences of ehromosome 18 microdeletions, transmitted through parental germ line mosaicism, a well-knowil alternative expianatioll for autosomal recessive inheritance [Petrella et al., i993]. The phenotypic overlap between the r(i8)1l8p- syndrome and JBS ieads to the assumption that the 18p region is a candidate area for a potential JBS gene. Acknowledgemellts \Ve like to thank Mrs. J. du Paraut and Mrs. J. van Deursen for preparing the manuscript aud Professor M.F. Niermeijer for advise. Refel'ellces Chambers HM, Knowies S, Staples A, Tamblyn M, Haan EA (1993): Anthropometric measurements in Ihe second trimester felus. Earl)' Hum Dev, 33: Devilee P, Cremer T, Slagboom P, Bakker E, Scoll HP, I'lager HD, Stevenson AFG, Cornelisse Cl, Pearson PL (1986): Two subsets ofhuman alphoid rcpetitive DNA show distinet preferentiallocalization in the pericentric regions of chromosomes 13, 18 and 21. Cytogenet Cell Genet, 41: Eiben B, Unger M, Sloitenberg G, Rutt G, Goebel R, Meyer A, Gamerdinger U, Hammans W, Hansen S, Hauss 1-1, Vogel M, Schwanitz G (1992): Prenatal Diagnosis ofmonosomy 18 and ring chromosome 18 mosaicism. PrCllat Diagn, 12: Froster-Iskenius U, Coerdt W, Rehder H, Schwinger E (1984): lsochromosome 18q with karyotype 46,XX,i(18q). Cylogenetics and Pathology. CUn Genet, 26: Geurts van Kessel A, Straub RE, Silvemlan GA, Gerken S, Overhauser J (1994): Report of the second international workshop on huntan c11romosome 18 mapping. Cytogellel Cell Genet, 65: Göcke H, Muradow I, Zerres K, Hansmmm M (1986): Mosaicîsm of isochromosome 18p. Cytogenetic and morphological findings in a male fetus at 21 weeks. Prenat Diagn, 6: Göcke H, Muradow I, Stein W (I988): The fetal phenolype of the 18p- syndrome. Report of a male fetus at twenty-one wceks. AIIII Génél, 31: Kosztolányi G (1987a): Decreased cell viability of fibroblasls from two patients with a ring chromosome: an in vitro reflection of growth failure? Am J Med Genel, 28: i32

133 Kosztolányi G (l987b): Does "ring syndrome" exist? An analysis of 207 case reports on patients with a ring autosame. Hum Genet, 75: Koulischer L, GilJerot Y, Richard J (1980): Caryotype 47,XX,r(I8),+mar chez la nièce d'un trisomique 21 leucémique avec chromosome du cri du chat. AI111 Génét, 23: Le Beau MM, Overhauser J, Straub RE, Silvernwn G, Gilliam TC, Ott J, O'Conncl P, Francke U, Geurts van Kessel A (1993): Report ofthe rlist international workshop on human chromosome 18 mapping. C)'togenet Ce/l Genet, 63: MacDermot KD, Jack E, Cooke A, Turieau C, Lindenbaum RH, Pearson J, Patel C, Bames PM, Portch J, Crawfurd M d'a (1990): lnvestigation of three patients with Ihe "ring syndrome", including familial tmnsmissiojl ofriog 5, aod estimation ofreproductive risks. Hum Genet, 85: McKusick VA (1994): "Mendelian inheritance in man. A calalog of human genes and genetic disorders". Baltimore and London: Thc Johnson Hopkins University Press, pp Petrella R, Rabinowitz JG, Skinmann B, Hirschhorn K. (I993): Long-term follow-up of two sibs with Larsen syndrome possibly due to parental genn-line mosaicism. Am J Med Genet,: 47: Pezzolo A, Gimelli G, Cohen A, Lavaggetto A, Romallo C, Fogu G, Zuftàrdi 0 (1993): Presence of telorneric and subtelomeric sequcnces at the fusion points of ring chromosomes indkntes thai thc ring syndrome is caused by ring instability. Hum Genet, 92: Rocchi M, Cigui I, Archidiacono N, Pecile V, Porcelli G, Filippi G (1984): A yotlng girl with ring (18) mosaicism: cytogenetic studies and PEP A mapping. Clin Genet, 26: Schinzel A (1984): "Cataloguc ofunbalanced chromosome aberatiolls in man". Berlin: W. de Gruytcr, pp Snijders RJM, Nicolaides KH (1994): Fctal biometry at weeks' gestation. U/lrasollnd abs/et G)'lIecol, 4: Sulton SD, Ridler MAC (1985): Prenatal detection of monosol11)' 18p and trisom)' 18q mosaicism with unexpected fetal phenotype. J. Med Genet, 23: Trout T, Budorick NE, Pretorius 011, McGahan JP (1994): Significance of orbital measuremenls in thc fetus. J U1trasOlllld Me{1, 13: Van Opstal D, van dcn Berg C, Jahoda MGJ, Brandenburg H, Los FJ, In 't Veld PA (1995): Relrospective study of trisomy 18 io chorionic villi with fluorescent in situ hybridization on archival direct preparations. Prellat Diagn, 15: Wurster-Hill DH, Marin-Padilla JM, Moeschier JB, Park JP, McDennct M (1991): Trisomy 18 and isp- features in a case ofisochrotllosome 18q [46,XY,i(I8q}]: prenatai diagnosis and autopsy report. CUn Genet, 39:

134

135 Appendix publication 111 Lancet, 342, 802 (1993) Copyright The Lancet, Reproduced with pennissioll.

136

137 Fetal aneuploidy diagnosed by fluorescence in-situ hybridisation within 24 hours after amniocentesis. D. Van Opstal, J.O. Van Hemel, E.S. Saehs Sir,-Results of anmiocentesis at 16 weeks are available, at the very eartiest, after 8-12 days because of time-consuming ecu culture. Quickcr resuits would cause less anxiety in pregnant women alld alsa be an improvement in cases of serious fetal anomalies when terminatioll is considered. Thc most common fetal chromosome aneuploidies are trisomy 21 and 18. \Ve have used fluorescence in-situ hybridisation (FISH) with probes specific for chromosomes 18,21, X and Y on uncultured amlliocytes 1,2 of20 pregnancies at high genetic risk because of advanced maternal age (2: 40 years) or in cases of fetal anomaly detected on ultrasound. Gestational age varied hetween 16 and 34 weeks. Direct preparations for FISH were made from 2 mi of eaeh sample. The samples were eentrifuged for 5 min at 150 g, the eell pellet was resuspended in 2 ml 1 % sodium citrate, and kept at 37 C for 20 min. The cells were eentrifuged, resuspended in 2 ml methanol/aeetie-acid (311), kept at -20'C for 20 min, and spun. The supernatant was discarded and at least two slides were made from each cell suspension. The probes used are defined in the tabie. About 50 eells per probe were monitored for eaeh sample. Probes FISH* 2 3 X (pbamx5) 10 (96%) 9 (93%) 1(88%) Y (Amprobe RPN1305X) 10 (97%) (LI.84) 0 19 (93%) 1(70%) 21 (C BI1l ) 0 19 (86%) 1(67%) *No ofpregnancies and (mean %) of nuclei with 1-3 signais. pbamx5, Willard H.F., et al., Nucleic Acids Res 1983; 11: Amprobe RPNI305X, Amersham. L1.84, Oevilee P, et al. Cytogenet eeu Genet 1986; 41: C Bl B02134 provided by Dr. H. Lehrach, London. Results were obtained in all patients within 24 h after amniocentesis (tabie). Of20 patients, 19 had normal signal distribution. 9 female fetuses silowed two X-signals and 10 male fetus one X-signal and one Y-signal, respeetively. These 19 samples also showed two signals for the 18 and 21 probes. Trisomy for the X, 18 and 21 probes was seen in one sample from the first pregnancy of a 26-year-old woman obtained at 27 weeks because of fetal anomalies on ultrasound (spina bifida, hydrocephalus, and left rocker-bottom foot). All normal filldings and the trisomy were confirmed by karyotyping ofamniotic-fluid cell cultures. Our results show that FISH with probes speeifie for ehromosomes X, Y, 18, and 21 on 137

138 uncultured amniocytes eau rapidly deteet the most frequent ehromosome alleuploidies, and is therefore a valuable additional tooi for prenatal diagnosis, specifieally for pregnancies at high genetic risk. Refel'ences Zheng, Y.L., Ferguson-Smilh, M.A., Wamer, J.P., Ferguson-Smith, M.E., Sargenl, C.A., Carter, N.P. Analysis of chromosome 21 copy number in uncultured amniocytesby fluorescence in situ hybridizalion using a cos11lid conlig. Prellal Diagn 1992; 12: Lebo, R.V., Flandemleyer, R.R., Diukman, R., Lynch, E,D., Lepercq, J.A., Golbus,M.S. Prenalal Diagllosis wilh repetitive in situ hybridization probes. Am J Med Genet 1992; 43:

139 Appendix publication IV PrenatDiagn, 15, (1995) Copyright John Wiley & Sons. Reproduced with permission.

140

141 A chromosome 21-specific cosmid cocktail for the detection of chromosome 21 aberrations in interphase nuclei Diane Van Opstal, Jan O. Van Hemel, Bert H.J. Eussen, Annet van der Heide, Cardi van den Berg, Peter A. In rt Veld, Frans J. Los Summary Fluorescent in situ Itybridization (FISH) witlt a 21qll-spccific probe (CB21c1), collsisting of three non-overlapping cosmids, bas been applied to intcl'phasc amniocytes of pregnaucies at increased risk for fetal aneuploidy (N=78) and to interphase lyl11phocytcs, cultured nnd unculturcd, of paticnts rcfcltcd fol' DmYll syndromc (N=19, and 28, rcspcctively). In thc uncultul'cd amniocytcs six chl'omosome aberratiolls were detecfed: thrce cases of trisomy 21, a triploidy, a de-noyo 46,XX,t(21q21q), and a mosaic 46,XY/47,XY,+dic(21)(qll)/48,XY,+dic(21)(qll),+dcl(21)(qll). In 15 cuitured and 20 uncuitured blood samples, FISH corrcctly diaguoscd a trisomy 21 (full or mosaic) at the illterphasc level, wbieh was confil'med in all cases by subsequcnt karyotyping. Because of specific nnd strong signais in intcrphasc nuclei, CB21cl appcares to be a useful tooi for rapid detection of Chl'OlllOSOlllC 21 abnol'lllalities. Introduction Fluorescent iu situ hybridization (FISH) has proven to be a powerfnl method for the rapid detection of chromosomal aneuploidies in uncultured amniocytes (Klinger et al., 1992; Van Opstal et al., 1993), in uncultured mesenchymal chorionic villus cells (Bryndorf et al., 1994), in fetal cells circulating in matemal blood (Gänshirt-Ahlert et al., 1993), and in pre-embryonic blastomeres (Munné et al., 1994). The reliability of this technique depends highlyon the specificity and the hybridization efficiency of the probes. Centromeric repetitive alphoid DNA probes arc aften applied for illterphase cytagenetics as they produce strong signais. However, for the detection of the most frequently encountered cluomosome abnormality in prcnata! diagnosis, trisomy 21, only the probe LI.26 (Devilee et al., 1986) is available. This probe has been reported to be succesfull for detection of trisomy 21 by several anthors (Zahed et al., 1992; Lebo et al., 1992; Rao et al., 1993). However, others showed that this probe was not reliable, as the copy number of the sequence recognized by the probe is a highly polymorphic trait and somctimcs appears ta be too small to produce any sigllal which rcsulted in false negative outcames (Verma et al, 1992; Mizauno and Young, 1992; Seres-Santamaria et al., 1993; Cacheux et al., 1994). Moreover, LI.26 cross-hybridizes to the centromeric region of chromosome 13 which does not allaw distinction between trisomy 21 and trisomy 13. Far bath reasons, we investigated the utility ofthe present 21qll cosmid cocktail, CB21cl, for its applicability in pre- and postnatal diagllosis. 141

142 Matel'ials alld methods Cell preparaliolls Amniocentesis was perfofllled because of advanced maternai age (~:40 yrs.) (N=14), ultrasound abnormalities (N=61), and confirmation of trisomy 21 l110saicism (N=2) and of a de-novo translocation (21 q21 q) in previous direct chorionic villi. From routine amniotic fluid samples one slide was prepared directly (Van Opstal et al., 1993) for hybridization with the present 21-probe and the renminder was cultured by the in-situ method for cytogenetic analysis. Metaphase spreads of lymphocytes were prepared according to standard techlliques. Interphase nuclei ofuncultured Iymphocytes were obtained by incubating the cells in 0.075M KCL at 37"C for 18 min. Subsequently, the cells were fixed by three changes of methanolacetie acid (3:1) and dropped onto slides. Routine trypsin-giemsa banding was applied for cytogenetic analysis of the cultured anll1iotic fluid ceus and the lymphocytes. The pretreatment of the slides consisted of a RNase (Pharmacia) treatment (100 ~glml in 2*SSC) at 37"C for one hour, followed by a pepsin (Serva or Sigma) treatment (4 mglml for amniocytes and 100 ~g1ml for Iymphocytes) in O.OIN HCL at 37 C for 15 min. Uncultured amniotic fluid preparations were postfixed in 3.7 per cent formaldehyde (Merek) in PBS for 10 min. DNA probe alld labellillg The 21 cosmid cocktail, CB21c1, consists of tlrree non-overlapping cosmids, ICRFc102B02134, ICRFc102BII128, and ICRFc102C0755, which map to thc D21S13 locus at 21qll (Stinissen et al., 1990; 1991). The probes were labeled with biotin-ii-dutp hl' nick translation with the BioNick system (Gibco BRL). Probe deleclion mul signal allalysis The hybridization mixture, consisting of 50 per cent formamide (.Merck)/2*SSC, 10 per cent dextran sulphate (pharmaeia), one per cent Tween-20 (Bio Rad), 5 f-lg salmon sperm DNA (Sigma), 5 ~g trna (Gibco BRL), I ~g Cot-l DNA (Gibco BRL), and 32 ng ofeach ofthe tlrree biotinylated probes was denatured at 90 0 e for 5 min and immediately put on ice, followed bl' one hour preannealing at 3rC. Target DNA on the slides was denatured bl' immersion in 70 per cent fonnamide/2*sse for 3 min at sooe and dehydrated in an ethanol series. The hybridization reaetion was performed overnight at 37 C. After hybridization, the slides were washed three times in 50 per cent formamide/2*ssc at C [or 5 min, followed bl' tlltee changes of 2*SSC, twice at C and once at C, respectively. They were treated with alternating layers of fluoresceinated avidin and biotinylated goat anti-avidin (Vector Lab), and finally the slides were motll1ted in 0.2M Tris HCL/glycerol (1:9 vlv, ph 7.5) containing 2 per cent DABCO (Sigma) and the fluorescent counterstains propidium iodide (0.5 l.glml) and DAPI (0.25 ~glml). Coded slides were examined uuder a Leica Aristoplan epifluorescence equipped microscope and images were captured with the Genetiscan ProbeMaster system (Perceptive Scientific International Ltd. (PS!), Chester, U.K.) including a Xybion cooled CCD 24-bit color camera. One hundred 142

143 Figure I. FISH wilh Ihe 2Jqll-specific probc CB21cl. (A) Inlcrphasc Iymphocyles ora 1Il0saic Irisomy 21 case showing Iwo and thrcc signais. (B) Inlerphase Iymphocyle showing three signa Is, each consisling or Ihree spots produced by the indï\'idlial cosmids. (C) Uncliltured all1l1iocyles showing Ihree signals in a case or ruil trisomy 21 and (0) a de-no\'o unbahlllced Imnslocalion (21 q2iq). (E) Uncultured 3muiocyle and (F) metaphase showing fi\'e signa Is in a case with two norm a I chromosomes 21 and an extra del(21)(qll) and dk(21)(qll). 143

144 lymphocytes and a mean of 41 (range 10-84) amniocytes were counted in each case. Nuclei without sigllals were not illcluded in the data. Results FISH was perfomled on interphase nuclei of cultured (N~I9) and uncultured (N~28) blood samples and of uncultured amniotic Iluid samples (N~78). TIle signal distributions in cultured and uncultured lymphocytes are shown in tablc 1 and 2, respectively. More than 99 per cent of the nuclei showed compact and bright signals (Figure IA). Separate spots per signal, produced byeach of the three cosmids in the cocktail, \Vere sometîmes noticed in the largest cultured cells (Figure lb). The signal distributiolls allowed a clear discrimination between normal samples and samples with a trisol11y 21 (full as weil as mosaic). Table 1. Signal distribution afler FISH with eb21et in 19 samples of euhured Iymphocytes No. of Mean percentage (range) of nuclei showing }-4 signals Karyotype cases ,--* (2-4) (83-95) (3-12) ,--*,+21 (0 6) (94-24) (68 94) (0 6) Mos 46,XXl47,XX,+2I(21179) *-XXorXY Mos 46,XXl47,XX,+21 (82118) Table 2. Signal distribution after FISH whh e021et in 28 samples of cultured Iymphocytes No. of Mean percentage (range) of nuclei showing 1-4 signals Karyotype cases I 46,--* (t t8) (78 99) (0 8) (0 4) t ,--*,+21 (0 4) (4-35) (6t-93) (0 tol Mos 46,XXl47,XX,+21(64/36) *-XXorXY 0 2t 79 0 Mos 46,XXl4 7,XX,+ 21 (40/60) Although the number of analysable nuclei varied runong the uncultured amniotic fluid samples (mean 41; range 10-84), their distributions did suggest six chromosome

145 Tablc 3. Signa I distribution after FISH wuh CB21cl in 78 samples of uncultured amniotic "uid samples N Mean percentage (range) of nuclei showing 1 4 signals Karyotype cases Disomy21 1 (0-30) (70-100) (0-18) , 2,+21 (0-8) (10-38) (52-86) (0-4) ,XXX ,XX,I(21 q21 q) Mos 46,XY/47,XY,+dic(21)(qll)/48,XY, +dic(21)( q 11), +del(21)( q 11 )(25/21/54) I ~Aneuploidy of othcr chromosomes not mentioned, 2~ XX or XY aberralions (TabIe 3). Karyotyping ofthe cultured cells revealed full trisomy 21 in Ihrce cases (Figure IC), a triploidy, and a de-novo unbalanced translocation (2Iq2Iq) (Figure ID). In the sixth case with 23 per cent of the uncultured cells showing two signais, and 77 per cent three to six signals (Figure IE), subsequent chromosome analysis of the ceu culhlfes revealed different karyotypes with marker (mar) clrromosomes; 46,XY/47,XY,+mar1l48,XY,+marl,+mar2 (25%/21 %/54%). FISH on metaphases ofthis case identified marl as a diccntric chromosome 21 (dic(21)(pter->qll::qll->ptcr)) showing two signals with CB21 cl, and mar2 as a deleted chromosome 21 (del(21 )(q II->qter)) showing only one signal (Figure 11). Discussion Several types of chromosome 21-specific probes have been investigated for interphase cytogenetics; chromosome 21-specific!ibraries (Pinkel et al., 1988; Lichter et al., 1988; Kuo et al., 1991), plasmid clones (Lichter ct al., 1988), YAC's (Bryndorf ct al., 1992), AJu-PCR YAC's (Romana et al., 1993; Bryndorfet al., 1994), single cosmids (Lichter et al., 1989), and cosmid contigs (Klinger et al., 1992; Ried et al., 1992; Ward et al., 1993; Spathas et al., 1994). \\Te investigated the utility of the present 21-probe CB21cl, consisting of three nonoverlapping cosmids, for interphase cytogenetics. \Ve preferred to use a mixture of these probes because their joint signal was brighter than that produced bl' the individual cosmids. Separate spots per signal produced byeach of the tlrrce non-overlapping cosmids were only occasionally encountered in the largest cultured lymphocytes (Figure lb), but were never observed in uncultured cells. Zheng et al (1992), using a cosmid contig on uncultured al11niocytes. described that more than 80 per cent of the cells in amniotic fluid are degenerate squamous epitllelial celis which are unsuitable for FISH atlalysis. According to Spathas et al (1994) th is problem may be solved by coating the slides with 3'-mninopropyltricthoxysilane which results in high quality preparations with the majority of cells showing hybridization sigilais. However, they also 145

146 mention that gestation time may influence the quality of the slides. This is in agreement with our results as a large proportioll of amuiocytes in most samples of relatively late gestational age did not show any signais. However, in our experience this problem is lllainly restricted ta cosmid probes and in alesser degree to centromeric probes. \Vith CB21cl we correctly diagnosed all cases of trisomy 21. Moreover, despite its localization in 21qll instead of in the Down specific region (2 1 q22), we deteeted a Robertsonian translocation (21q21q) in uncultured amniocytes in which two of the tllfee signals were situated in close proximity in the interphase nucleus. However, these paired spots could easily be distinguised from the twin spots due to DNA replication in 02 cells. As free trisomy 21 and Rabertsonian translocations of chrolllosome 21 make up 97 per cent and 3 per cent respectively of all cases of Down syndrome investigated during alo year period in our postnatal cytogenetic laboratory, CB21cl is able ta detect all cases of Down syndrome at the interphase level. Only an exceptional case showing an unbalanced reciprocal translocation of chromosome 21 with breakpoints distal to 21qll, can not be idelltified with the present probe but oniy with a probe from the Down region. CB21 cl could also detect an extra dicentric and deleted chromosome 21 in a rare prenatalmosaic case. Both marker chromosomes lacked the Down specific region and would have golle undetected if a probe was applied lllapping to 21 q22 sueh as used by other investigators (Zheng et al., 1992; Klinger et al., 1992; Ried et al., 1993; Ward et al., 1993; Spathas et al., 1994). In order to minimize the risk of amisdiagnosis in further studies, we suggest the hybridization of both CB21cl as weil as a probe derived from the Down syndrollle region. In five cases of uncultured amniocytes most nuclei showed tluee signals indicating the presence of three copies of chromosome 21 in the fetus. In two of these cases a differential diagnosis of triploidy and of an unbalaneed transloeation (21 q21q) eould be made. In the ftrst case a triploidy was expected since in addition to CB21cl cllfomosome 18-, X-, and Y specific probes were routinely applied to all amniotic fluid samples, and tllfee signals were found in the majority ofthe eells after hybridization with CB21cl as weil as with the 18- and X-specific probes. In the second case FISH was perfornled for verification of a de-novo t(21q21q) in previous direct chorionic villi. The presence ofthree signals illmost nuclei, with two signals in close proximity, conftrmed the presenee ofthe t(21q2iq) in fetal eells. Five lllosaic cases involving trisomy 21 in Iymphocytes and tetrasomy/pentasomy of the 21qll region in uilcultured amniocytes could be identified with the present prohe. However, the level of lllosaicism in interphase nuclei did not always match that in cultured cells, whieh in some cases might be explained by selection during cel! culturing. Theoretically, detection of a Iow level of mosaicislll with FISH on interphase nuclei will be difficult because of the broad range of nuclei exhibiting two and three signais. FISH on uncultured amniocytes has the potential for a rapid prenatal diagnosis of the most common chromosome abnormalities. However, it does not allow the detection of other chromosome aberrations than those detected by the probes that are commonly used (in our laboratory for chromosomes i8, 21, X, and Y). At this moment we use the teehllique as an adjunct test in two prenatai instances: firstly, for the veriflcation in allllliotic fluid ceus of a specific chromosome aberration previously detected in chorionic villi and potentially eonfined to the placenta, and secondly, for the rapid detection of the most COIlUllon chromosome abnormalities (trisomy 21, trisomy 18, triploidy, and 45,X) in pregnancies eomplicated by fetal anomalies detected by ultrasound. A normal FISH result in these particular cases is always complementcd by a full cytogenetic analysis of the eultured cells. An abnormal FISH 146

147 result only can not be the basis for irreversible therapeutic action. However, interphase FISH can complement ultrasound findings and previollsly detected abnormalities in chorionic villi and provide help in caunselling procedures. In aur experience, probe CB21c1 which cauld detect aneuploidies as weil as same unbalanced structural rearrangements af chromasome 21, cantributes ta the detection afthe most COllllnon abnormality in clinical cytogenetics. References Bryndorf, T., Christensen, B., Philip, J., Hansen, W., Yokobata, K., Bui, N., Gaiser, C. (1992). New rapid test for prenatal detection oftrisomy 21 (Down's syndrome): preliminary report, BI' Med J, 304, Bryndorf, T., Christensen, B., Xiang, Y., Philip, J., Yokobata, K., Bui, N., Gaiser, C. (1994). Fluorescence in situ hybridization wilh a chromosome 21-specific cosmid contig: I-day delection of trisomy 21 in uncullured mesenchymal chorionic villus cells, PreI/at Diagn, 14, Cacheux, V., Tachdjian, G., Druart, L., Oury, J.F., Sérero, S., Blot, P., Nessmann, C. (1994). Evaluation of X, Y, 18, and 13/21 alpha satellite DNA probes for interphase cytogenetic analysis of uilcultured amllioc)1es by fluorescence in situ hybridization, Prenar Diagn, 14, Devilee, P., Cremer, T., Slagboom, E., Bakker, E., ScholI, H.P., Hager, H.D., Stevensoll, A.F.G., Comelisse, CJ., Pearson, P.L. (1986). Two subsets ofhuman alphoid repitilve DNA show distinct preferentiallocajization in the pericentric regions of chromosomes 13, 18, alld 21, Cytogenel Celt Genet, 41, Gänshirt-Ahlert, D., Börjesson-Stoll, R., Burschyk, M., Dohr, A., Garritscn, H.S.P., Helmer, E., Miny, P., Velasco, M., Walde, e., Patlerson, D., Teng, N., Bhat, N.M., Bieber, M.M., Holzgrcve, W. (1993). Detcctioll of feta! trisomies 21 and 18 from matemal blood using triple gradient and magnetic eell sorting, Am J Reprad ,30, Klinger, K., Landes, G., Shook, 0., Harvey, R., Lopez, L., Locke, P., Lemer, T., Osathanondh, R., Leverone, B., Houseal, T., Pavelka, K., Dackowski, W. (1992). Rapid detection of chromosome aneuploidies in uncultured amniocytcs by using fluorescence in situ hybridization (FISH), Am J Hum Gel/et, SI, Kuo, W.L., Tenjin, H., Segraves, R., Pinkel, D., Golbus, M.S. (1991). Detection of aneuploidy involving chromosomes 13, 18, or 21, by fluorescence in situ hybridization (FISH) 10 interphase and metaphase amniocyles, AmJ Hum Gel/el, 49, Lebo, R.V., Flandenneyer, R.R., Diukman, R., Lynch, E.D., Lepereq, J.A., Golbus, M.S. (1992). Prenatal diagnosis with repetitive in situ hybridization probes, Am J,\led Genet, 43, Lichter, P., Cremer, T., Chang Tang, CJ., Watkins, P.c., Manuelidis, L., Ward, D.e. (1988). Rapid detection of human chromosome 21 aberrations by in situ hybridization, Prae.Natl Acad Sci USA, 85, Lichter, P., Jauch, A., Cremer, T., Ward, O.C. (1989). Deleclion of Down syndrome by in situ hybridi7.ation with chromosome 21 specific DNA probes, in Patterson, 0., Epstein, C.J. (eds): Molecular genelics of chromosome 21 and Down syndrome, New York, Wiley-Liss, 69-78, Mizunoe, T., Young, S.R. (1992). Low fluorescence alpha satellitc region yields negative rcsult, PreI/at diagn, 12,

148 Munné, S., \Veier, H.U.G., Grifo, J., Cohen, J. (1994). Chromosome mosaicism in human embryos, Bio! Reprod, 51, Pinkel, 0., Landegent, J., Collins, C., Fuscoe, J., Segmves, R., Lucas, J., Gray, J. (1988). Fluorescence in situ hybridization with human chrolllosome-specific Iibraries: Detection of trisomy 21 and translocatiolls of chromosollle 4, Prae Nal! Aead Sci USA, 85, Rao, P.N., Hayworth, R., Cox, K., Grass, F., Pettenati, M.J. (1993). Rapid detection ofaneuploidy in uncultured ehorionic villus eells using fluoresecnce in situ hybridizatioll, PreI/at Diagn, 13, Ried, T., Laudes, G., Dackowski, W., KIillger, K., Ward, D.C. (1992). Multicolor fluoresccnce in situ hybridization for the simultaneous detection ofprobe sets for chrolllosomes 13, 18,21, X and Y in uncultured amniotic fluid ceus, Hum Mol Gellet, I, Romana, S.P., Tachdjian, G., Druart, L., Cohen, D., Berger, R., Cherif, D. (1993). A simple method for prenalal diagnosis oftrisomy 21 on uncultured alllniocytes, Eur J Hum Genet, 1, Seres-Santamaria, A., Catala, V., Cualrccasas, E., ViIlanueva, R. (1993). Fluorescent in-situ hybridizatioll and Down's syndrome, Lancet, 341, Spathas, D.H., Divane, A., Maniatis, G.M., Ferguson-Smith, M.E., Ferguson-Smilh, M.A. (1994). Prcnatal detection of trisomy 21 in uncultured amniocytes by fluorescence in situ hybridization: a prospective study, PreI/aI Diagn, 14, Stinisscn, P., Van Hul, W., Van Camp, G., Backhovens, H., Wehnert, A., Vandenberghe, A., Van Broeckhoven, C. (1990). The pericentromeric 21 DNA marker pgsm21 (D2IS13) contains an expressed HTF island, GenoIJ/fes, 7, Stinissen, p" Van Broeckhoven, C. (1991). A new (CA)n repeat polymorphism at the D2IS13E locus, Nucl Aefds Res, 19,5089. Van Opstal, D., Van Hemel, J.O., Sachs, ES. (1993). Fetal aneuploidy diagnosed by fluorescence in-situ hybridization within 24 hours after amniocentesis, Lancet, 342, 802. Verma, R., Lukc, S. (1992). Variations in alphoid DNA sequence escape detcction of aneuploidy at interphase by FISH technique, Genomics, 14, \Yard, B.E., Gersen, S.L., Carelli, M.P., McGuire, N.M., Dackowski, W.R., Weinstein, M., Sandlin, C., Warren, R., Klinger, K. (1993). Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: clinical experience with 4,500 specimens, Am J Hum Genel, 52, Zahed, L., Murer-Orlando, M., Vekemans, M. (1992). In situ hybridization studies for the detection ofcommoll aneuploidies in CVS, Prenat Diagn, 12, , Zheng, Y,L., Ferguson-Smith, M.A., Warner, J.P., Ferguson-Smith, M.E., Sargent, C.A., Carter, N.P. (1992). Analysis of chromosome 21 copy number in uncultured amniocyles by fluorescence in situ hybridization using a cosmid contig, PreI/aI Diagn, 12,

149 Appendix publication V Prenat DiagIl, 15,51-55 (1995) Copyright Joml Wiley & Sons Limited. Reproduced with penllission.

150

151 Retrospective study of trisomy 18 in chorionic villi with fluorescent in situ hybridizatioll on archival direct preparations Diane Van Opstal, Cardi van den Berg, Milena G.l. Jahoda, Helen Branclellburg, Frans J. Los, Peter A. in ft Veld Summary Trisomy 18 in direct chorionic "iilus preparatious needs furthcl' invcstigation since the chromosome abnormality ma)' be con fin cd to fhe placenta alld may not I'cprcscnt thc acturl fetai lmryotype. \Ve performcd, rctrospectively, fluorescent in situ hybl'idization (FISH) with the chromosomc 18 cclltromel'c pl'obc on intcl'phase nuclei of destaincd slides of all cases of full tl'isomy 18 (n=22) and mosaic tl'isomy 18 (n=8), detected among 7600 first trimester chorion ie villi samples during an cight yeal" pel'iod ( ). More nuclei displaying three signals were cncountcrcd in cases of ruil alld mosaic tl'isomy 18 collfil'med in fetal tissue than in nojlmconfirmed cases. FISH CRn he useful fol' the ycrification of trisomy 18 in dircct chorionic "mi preparatiolls. Introdllction Trisomy 18 in non~mosaic as weil as mosaic appearance in direct preparations of placetttal chorionic villi may not represent the chromosomal status of the fetus (Simoni et al, 1985; Wirtz et al., 1991). ConfIrmatory studies of long-term villi cultures are used as one of the means of verification. However, the culturing of chorionic villi adds significantl}' to the reporting time, while colltamillatioll of the sample with maternal tissue can illterfere with the accurate interpretation ofthe results (Vejerslev aud Mikkeiseu, 1989; Kalousek et al., 1992). Moreover, discrepancies bet ween the karyotype of cultured villi and fetal tissue have been reported (Hogge et al., 1986; Wang et al., 1993). We performed, retrospectively, fluorescent in situ hybridization (FISH) with a chromosome 18~specific probe on interphase nuclei in destained archlval direct villus preparations of30 trisomy 18 cases, to investigate whether this techllique can be used as a possible quick and accurate method ofverification oftrisomy 18 in chorionic villi direct preparations. Chorionic vi/li samples and slide preparatiolls Materials and methods Thirty cases of trisomy 18 (eight mosaic and 22 non-mosaic) were encountered in 7600 consecutive first trimester chorionic vilh samples over an eight year period ( ). 151

152 Sampling was performed transcervicauy in 10 cases alld transabdominally in the remaining cases as described previously (Jahoda et al., 1989; 1990). Indications for prenatal diagnosis \Vere advanced maternal age andlor ultrasound abnormalities. Kalyotyping was perfonned on trypsin-giemsa stained direct preparations (Simoni et al., 1983; Gibas et al., 1987). A mean of 20 metaphases (range 5-50) in the non-mosaic and 29 (range 9-50) in the mosaic cases was analysed. Preparations of long-term villi cultures (Smidt-Jensen et al., 1989) were karyotyped in some lllosaic cases. Giemsa stained arcl1îval direct villi preparations of the 30 trisomy 18 cases and of 30 contro! cases with a 110rmal karyotype, rnatched for maternal age, gestational age, fetal gender and storage-time, were destained prior to hybridization (Klever et al., 1991). DNA-probe alld labellillg The 18 centromere-probe L 1.84 (Devilee et al., 1986) was used for detection ofthe chromosome 18 copy numbcr in metaphases and interphase nuclei. The probe was labelled with biotin- 11-dUTP bl' nick translation with the J3ioNick systcm (BRL, Gaithersburg, USA). Fluorescent IJl Situ llybridizatioll (FISH) Gluiprobe detectiol1 The centromere probe L 1.84 (40 ng in I 0 ~I 60% fonnamide/2xssc) and chromosomal DNA were denatured simultaneously for tllfee min at 80 o e. Hybridization was allowed to proceed overnight at 37 e. After hybridization the slides were washed tlrree times in 50% formamide12xssc at 42"C for five min, followed bl' three changes of2xssc, twice at 42"C and once at 65 C. respectively. The probe was visualized by alternating layers of fluoresceinated avidin and biotinylated goat anti-avidin (Vector Lab, Burlingame,USA). The slides were motll1ted in anti-fade medium containing the fluorescent counterstains propidium iodide (O,06~{glml) and DAPI (0,6~g/ml). Slides \Vere examined under a Leitz Aristoplan fluorescence microscope and cells were photographed on Kodak Ektachrome 400 ASA daylight film. Samples \Vere analysed in a blind fashioll on coded slides. For each case the llumber of fluorescent spots was counted in 200 hybridized intact non-overlapping and non-clumped interphasc nuclei. The specificity of probe hybridization was checked in metaphases present on each slide. Cytogel1etic al1alysis Results Twenty-two of the 30 cases with a trisomy 18 in direct villus preparatiolls revealed a noilmosaic trisomy 18 karyotype. The pregnatlcies were terl11inated at the parents request; one pregnancy resulted in all intra-uterine fetal death within a week after sampling. The diagnosis of trisomy 18 was confirmed in the fetus bl' karyotl'ping skin fibroblasts in 15 of the 22 cases. In one early case we could not confirm the trisomy 18 in fetal cells, which showed a normal 46,XY karyotype. In the six remainillg cases cytogenetic confirmatoly studies could not be carried out or failed. Eight of the 30 cases with a trisomy 18 displal'ed a Illosaic trisomy 18 (TabIe 1). Five 152

153 pregnancics were terminated at the parents request and the trisoilly 18 was confirmed in fetal tissue in only duce instanees. In case No.26, showing a double trisomy of cluomosolllcs 18 (mosaic) and 21 (non-illosaic) in chorionic vuli, only a fuil trisomy 21 was recovered in skin fibroblasts and the cytogenetic confirmation in case NO.23 failed. Tllfce pregnancics continucd after extensive follow-up studies, including a long-term villus culture (LTC) and amniocentesis. They resulted in the birth of healthy children. In two out of five mosaic cases, in which LTC was perforllled, the culture silowed a mosaic trisomy 18 while fetal skin fibroblasts did not exhibit tlus chromosome aberration (Nos. 26 and 30). Tablc 1. Eighf cases of mosaic trisomy 18 in first-trimcstcr chorionic villl Case CVS Follow-up Outcome no. OP LTC XX/47,XX,+18(217) TOP 24 46,XX/47,XX,+ 18(3/47) F: 47,XX,+18(45) TOP 25 46,XXl47,XX,+ 18(1/31) 46,XX(7) A: 46,XX(15) Healthy ~ 3200gr 26 47,XX,+21//48,XX, 47,XX,+21//48,XX, F: 47,XX,+2I(16) TOP +18,+21(1/19) +18,+21(28/23) 27 46,XY/47,XY,+18(12/23) 47,XY,+ 1 8(32) F: 46,XY/47,XY,+18(2/36) TOP 28 46,XX/47,XX,+ 18(19/19) 46,XX(27) A: 46,XX(8) Healthy ~ 3375gr 29 46,XXl47,XX,+ 18(1/15) F: 47,XX,+18(16) TOP 30 46,XX/47,XX,+ 18(28/2) 46,XXl47,XX,+ 18(17113) A: 46,XX(19) Healthy!f! 2750gr CVS Chorionic villus sample, DP direct villus preparation, L TC long-temt villus culture, A amniocentesis, F=fetal fibroblast culture, TOP=tcrmination ofpregnancy. The numbers in parentheses denote the Ilumber of eeils analysed. FISH illielphase allalysis Fluorescent in silu hybridization (FISH) with the 18 centromere probe L1.84 was succesfully applied to destained.rchival slides of all but two of the trisomy 18 cases and to the corresponding normal controls. The mean percentage of nuclei showing one, two and three signals in (he 30 normal cases was 7%, 92% and 1%, respectively. Figure I shows the percentage of nuclei with two alld tlrree signals in individual cases of full- and 1l1osaic trisomy 18. The mean percentage of nuclei showing alle signal was 2% (range 0%-4,5%) in the non-mosaic and 3% (range O%~9%) in the mosaic trisomy 18 cases. In the series with a non-mosaic trisomy 18, the FISH results on interphase nuclei c10sely matched the cytogenetic fmdings in the direct preparations (Figure 1 A). The confirmed cases were foulld to express t1rree fluorescent signals in more than 83% oftheir nuclei (mean 87,5%). In the only non-confirmed case (No. 8), the percentage of nuclei with tluee signals was 72%, which is far outside the 95% confidence 153

154 Flgure 1. Percentage of interphase nuclei showing three (closed bars) and two (batched bars) fluorescent signals after in situ hybridization witlt a IS-spcciflc probe on direct "ilius prelmrations of (A) cases wub non-liiosaic and (B) lilosaic trisomy IS. (* = not-conflrmcd case) 100 A. Non-mosaic trisomy ao 70 "., 60 "0 " " 50 Ö -,p * case number 100 B M osalc t " nsomy ao "., 60 "0 " " 60 Ö -,p. 40 / / / / / 20 / 10 0 / I / * 27 28* 30* case number 154

155 interval of the confinlled cases. In the series with a mosaic trisomy 18, a broad range of signal distributions was found which did not alwal's match the cytogenetic anall'sis of the direct preparations (Figure IB); four mosaic cases (Nos. 24, 25, 26 and 29) with more than 90% of abnormal cehs in GTG metaphase analysis silowed 3 fluorescent signals in 76%, 66%, 44% and 77% ofthe interphase nuclei, respectively. In general, cases that were confirmed as heing trisomy 18 in amniotic fluid and/or fetal cells (Nos. 24, 27 and 29) silowed a higher percentage of 3 signal-containing nuclei (76%, 66% and 77%, respectively) than did the non-confirmed cases Nos. 25, 26, 28 and 30 (66%, 44%,18% and 13%, respectively). Discussion The diagllosis of trisomy 18 (mosaic alld non-mosaic) in direct chorionic vhli preparations in the first trimester of pregnancl' is complicated bl' the occurence of false positive (Sachs et al., 1990; Breed et al., 1990; Ledbetter ct al., 1992) and false-negative results (Leschot et al., 1988; Kalousek et al., 1989). Conftrmatory studies of long-term villi cultures have been proposed as a means of verification, as mesenchymal cells in the villus core are suggested to have a closer ontogenetic relation to the fetal ce lis than the trophoblast cells (Crane and Cheung, 1988). Our o\vn results as weil as various earlier reports argue against the use of long-term villus cultures (L TC) as sole and sufficient independent confirmation (\Virtz et al, 1991; Miny ct al., 1991; Ledbet!er et al., 1992). Cytogenetic analysis of a subsequent amniotic fluid sample seems the most reliable procedure for verification of trisomy 18 in chorionic villi. We studied the usefulness of interphase FISH as a possible quick and accurate methad of ftuther investigation of trisoml' 18 in chorionic vhli direct preparations. It was shown that FISH with a chromosome 18-specific probe, applied to interphase nuclei in direct villus preparations of non-mosaic trisomy 18 cases, has a streng predictive value for the chromosomal status of the fetus and contributes significantly to the res1.dts of the classical cytogenetic metaphase-analysis. The non-collfirmed case of full trisoml' 18 had a significantly lower lulmber of interphase nuclei displal'ing three signals than the real, confirmed cases of trisomy 18. In cases of mosaic trisomy 18 the application of F1SH adds probably also to the classical cytogenetic anall'sis. Higher levels of three signal containing nuclei were found in the dlree confirmed cases as compared to the four non-confrrmed cases. Ifthe percentage of nuclei with 3 signals turned out to be lower than than 66%, the trisomy 18 was not confirmed in fetal cells. The FISH data were better able to predict the fetal outcome than the cytogenetic data from the direct preparations. However, they yielded ambivalent results in some cases; an illtermediate level (66%-83% in our series) oftirree signal nuclei could either correspond to a true mosaic, a non-mosaic trisomy 18 in the fetus, or a false positive result. We showed ihat the application of FISH 011 chorion ie villi direct preparations with a trisomy 18 karyotype, which is frequently found to be confined to the placenta, has a predictive value for the fetal chromosome constitution and therefore can aid in the counselling procedures. However, a definite result eau ouly be achieved bl' karyotyping a subsequent amniotic fluid sample. 155

156 References Breed, A.S.P.M., Mantingh, A., Beelmuis, J.R., Kloosterman, M.D., Ten Bolscher, R, Anders, GJ.P.A. (l990). Thc predictive vallie of cytogenetic diagnosis after CVS: 1500 cases, PreI/at Diagn, 10, eraoe, J.P. Cheung, S,W. (1988), An embryogenie model 10 explain cytogenetic Înconsistencies observed in chorionic villus versus fetal tîssue, Prenat Diagn, 8, Devilee, P., Crcmcr, T., Slagboom, P., Bakker, E" Scall, H.P., Hager, H.D., Stevenson, A.F.G., Comelisse, C.l., Pearson, P,L. (1986). Two subsets ofhuman alphoid repititive DNA show distinct preferential Iocalization in the pericentric regions of chromosomes 13, 18, and 21, Cytogenet Cell Genet, 41, I. Gibas, L.M., Grujic, S., BaIT, M.A., Jackson, L.G. (1987). A simple technique for obtaining high qualîty chromosome preparations from chorionic villus samples using FdU synchronization, Prenat Diagn, 7, Hogge, W.A., Schonberg, S.A., Ooibus, M.S. (1986). Chorionic villus sampling: Experience of the first 1000 cases, Am J Obstet GJ'lIecol, 154, Jahoda, M.G.J., Pijpers, L., Reuss, A., Los, F.J., Wladimiroff, J.W., Sachs, KS. (1989). Evaluation of transcervical chorionic villus sampling with a completed follow-up of 1550 consecutive pregnancies, PreI/at Diagn, 9, Jahoda, M.G., Pijpers, L., Reuss, A., 8randenburg, H., Cohen-Overbeek, T.E., Los;F.J., Sachs, E.S., Wladimiroff, J. W. (1990). Transabdominal villus sampling in early sccond trimester: a safe sampling method for women ofadvanced age, PreI/at Diagn, 10(5), Kalousek, D.K., Barrelt, IJ., McGiIlivray, B.C. (1989). Placental mosaicism and intra-uterine survival of trisomy 13 and 18, AmJ Hum Genet, 44, Kalousek, 0., Darrett, L, Oärtner, A. (1992). Spontaneous abortion and confined chromosomal mosaicism, Hum Genet, 88, Klever, M., Grond-Ginsbach, C., Shcrthan, H., Schroeder-Kurth, T.M. (1991). Chromosomal in situ suppression hybridization after giemsa banding, Hum Genet, 86, Ledbetter, D.H., Zachary, l.m., Simpson, l.l., Golbus, M.S., Pergament, E., Jackson, 1., Mahoney, M.l., Desnick, R.J., Schulman, 1., Copeland, K.L., Verlinsky, V., Vang-Feng, T., Schonberg, S.A., Babu, A., Tharapel, A., Dorfmann, A., Lubs, H.A., Rhoads, G.G., Fowler, S.E., De la Cruz, F. (1992). Cytogenetic results from thc U.S. collaborative study on CYS, PreI/at Diagn, 12, Leschot, N.l., Wolf, H., Weenink, G.H. (1988). False negative findings at third trimesterchorionic villus sampling (C.Y.S.), Clin Genet, 34, Miny, P., Hammer, P., Gcrlach, B., Tercanli, S., Holzgreve, W., Eiben, B. (1991). Mosaicism and accuracy of prenatal cytogenetic diagnosis after chorionic villus sampling and placental biopsies, Prenaf DIagIl, 11, Sachs, E.S., Jahoda, M.GJ., Los, F.J., Pijpers, 1., Reuss, A., Wladimiroff, 1.W. (1990). Interpretation of chromosome mosaicism and discrepancies in chorionic villi studies, Am J ofmed Genet, 37, Simoni, G., Brambati, B., Danesina, c., RoselIa, F., Terzoli, G.L., Perrari, M., Fraccaro, M. (1983). Efficient direct chrolllosome analysis and enzyme determinations from chorionic vuo samples in rmt trimester of pregllancy,lllllll Genet, 63,

157 Simoni, G., GimeIIi, G., Cuoeo, C., TerzoIi, G.L., RoselIa, F., Romitti, L., Dalprà, L., Nocera, G., Tibiletti, M.G., Tenti, P., Fraccaro, M. (1985), Diseordanee belween prenalal c)10genetie diagnosis after chorionic "iili sampling and ehromosomal eonstitution of the felus, IJl: Fraeearo, M., Simoni, G., Brambati, B. (Eds.). Fust trimester fetal diagnosis, Springer-Verlag, Berlin Heidelberg, Smidt-Jensen, S., Christensen, B., Lind, A. (1989). Chorionie viiius culture ror prenatal diagnosis ofehromosome defeets: reduetion ofthe long-temt eultivation time, PreI/at Diagn, 9, Vejerslev, L., Mikkelsen, M. (1989). Tlte European collaborative study on mosaicism in chorionic villus sampling: dala from , Prenat Diagn, 9, Wang, B.B.T., Rubin, c.n., Williams, I.I.J. {I 993). Mosaicism in chorionic villus sampling: an analysis of incîdenee and chromosomes involved in 2612 consecutive cases, PreI/at Diagn, 13, Wirtz, A., GIoning, K.-P.H., Murken, J. (1991). Trisomy 18 in ChOrîOllic villus sampling: problems and consequences, Prenat. Diagn., 11,

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159 Appendix publication VI J Med Genet, 32, Copyright Journal ofmedical Genetics. Reproduced with pennission.

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161 Recun'ence of Di George syndrome: antenatal detectioll by FISH of a molecular 22q 11 deletioll lo. Van Hemel. C. Schaap, D. Van Opstal, M.P. Mulder, M.F. Nienneijer, J.H.C. Meijers Abstract \Ve report on an antcnatal diagnosis by FISH of a familial 22ql1 dclction associated witb DiGcorge syndrome (DGS). Thc deletioll was seen in thc pro band with symptoms of full DGS, in Ihe physically normal falher and in a subscquenl pregnallcy. Aftel' bil'lh this child showed hypocalcacmia, a T -eeu deficit and a rightsided atcus aortae. The DOS involves conotruncal hemt defects, hypoplastic or absent thymus and parathyroids, and facial dysmorphisms (McKusick index # ). Most likely, a deletion of a gene or a group of contiguous genes [rom 22q 11 is the cause of this disorder. This is supported by the detectioll in DOS chijdren of unbalanced chromosomal translocations l or microdeletions, mostly involving 22qll. Deletions of specit1c DNA sequences have also been described3.4. Approximately 8% of index patients with DOS show familial transmission of the 22q 11 deletion 4 Noticeably, most of the parents with deletions have only minor symptoms or just signs of learning disability or mild mental retardation. Antenatal diagnosis applying fluorescence in situ hybridisation (FISH) with 22q 11 specific probes has been performed4, with normal outcome. \Ve offered genetic counseling to a couple whose second child had died two weeks after birth. Ihis girl had the typical sympto111s of complete DOS. She had a truncus arteriosus communis type II with atrio-ventricldar septum defect and a rightsided arcus aortae. Other s)'mptol11s were neonatal hypocalcaemia and T-ceIl disturbances. At necropsy only one parathyroid gland and a small hypoplastic thymus were present. High resolution cytogenetic studies perfonned on her fibroblasts (celiline F92-31; 93RD59) showed a deletion at 22qll, which could be confirmed by applying FISH' with probe M5 I, that detects a molecular deletion in the DGS critical region of chromosome 22 (Mulder et al., submitted) (Fig.1). Both parents were physically norma!, although they bath had mild leaming disabilities; especially the làther. In childhood he had recurrent upper ainvay-infections alld was frequently hospitalized. However no data C01.lld be obtained about possible inul1unological disturbances. The ~1ther had a tendency to depressiol1 and alcohol abuse. In Iymphocyte metaphases of both parents no microdeletions were visible. After the mother became pregnant, she asked for antellatal diagnosis. FISH on lymphocyte metaphases from both parents and fetal amniotic cells was done with M51. The molecular deletion was detected in the male fetus and in its father. Cytogenetically these deletions were not visible. The parents were informed about the risk for DOS, velo-cardio-facial syndrome (VCFS), cardiac defects, and mental retardation 6,7. The parents decided to continue the pregnancy. Ultrasound studies in the 20th and 23rd week of pregnancy did not show cardiac or other 161

162 anomalies. The boy was bom at term and developed hypocalcaemia with Iow parathyroid honnone levels. T-cell fimction studies indicated a moderate T-cell deficit. Echocardiography revealed a rightsided arcus aortae without intracardial anomalies. Now at six lllonths of age, hls clinical condition is stabie. Figure 1. Part ora metaphase arter FISH wuh eosmid probe 1\151, region 22qll (arrow) and cosmid 1\169, region 22q13.3 (arrowheads), the latter used for rccognition of chromosome 22. Only one chrolllosomc 22 shows the M51 signais, pointlng to a deletion in the homologous 22. This is the first reported prenatal diagnosis of a molecular deletion in the DGS critical region of chromosome 22. Such a deletion is associated with a spectrum of malformations covered by the acronym CATCH 22 8 Thc anomalies seen in this family show the phenotypic variability ofthe M51 deletion. The anomalies ofthe youngest boy are less severe than those of the proband, with psychiatrie problems in the father. A variety of psychiatrie problems have been deseribed in vers patients', After deteetian af the 22q deletian in the [ather additional inverstigatiolls were performed. Serum calcium and inullunological screening, including IgG, IgA, IgM, IgG-subclasses and complement reactions (CH50) were all normal. Intra-cardiac abnonllalities were excluded by echocardiography; the only abnonnality was a right sided aarta deseendells deteeted by X-ray afthe ehest. In this family and others thc range of phcnotypes associated with the molecular deletions detected with probe M51 complicates genetic counseling. The classical DGS syndrome is usually sporadic but may be transmitted as an autosomal dominant trait; Shprintzen syndrome is usually autosomal dominant. \Ve propose that parents with a proven l110lecular deletion are counseled as having an increased risk for cardiac defects, DOS, immunological disturbance, and cleft palate, occurring as single abnonnalities or in syndromic farms. Furthcr studies are 162

163 needcd to detennine the potential ofprobe M5l to differentiate between isolated and [amilial farms of congenital heart defects with a very low alld a 50% recurrellee risk, respectively. For the antenatal diaguosis of microdeletions associated syndromes like DGS, FISH appears to be a rapid and reliable method'o, It lua)' aiso be reliably applied in chorionic villus mitoses whieh usually give alesser quality of thcir chromosomes. All early first trimester diagnosis is important as all option duril1g genetic counseling when there is a 50% recurrence risk of familial DGS 7, after careful explanation ta the parents of the substantial varlability of this disorder. Acknowlcdgements For skilfull lahoratory \Vork we awe ta Bert Eussen and Annet van der Heiden, alld Profs. H. Galjaard and J.C. Molenaar for their continuous support. Referenccs l. Greenberg F. (1993). DiGeorge syndrome: an historical review of clinical and cytogenetic features. J Med Genet; 30: Wilson DI, Cross IE, Goodship JA, Brown J, Scambler PI, Bain HH, Taylor JFN, Walsh K, Bankier A, Bum J, WolslenhoJme J. (1992). A prospeclive C}1ogcnctic sludy of 36 cases ofdigeorge Syndrome. Am J Hum Genet; 51: Care)' AH, Kelly D, Halford S, Wadey R, Wilson 0, Gooclship J, Bum J, Paul T, Sharkey A, Dumanski J, Nordensklold M, Williamson R, Scambler Pl. (1992). Molecular genetic stud)' of the frequenc)' of monosomy 22qll in DiGeorge syndrome. AmJ Hum Genet; 51: Driscoll DA, Salvin J, SeIlingcr B, Buclarf ML, McDonald-McGin DM, Zackai EH, Emanuel BS. (1993), Prevalence of22qi I microdeletiolls in DiGeorge and veiocardiofaciai syndromes: implications for genetic counsejling and prenatai diagnosis. J Med Genet; 30: Kievits T, DaU\verse JG, Wiegant J, Devilee P, Breuning MH, Comelisse CJ, Ommen OJB van, Pearson PI. (1990). Rapid subchromosomallocalization of cosmids by nonradioactive in-situ hybridization. Cytogene/ Cel! Genel; 53: DriscoII DA, Budarf ML, Emanuel BS. (1991). Antenatal diagnosis of DiOeorge sylldrome. Lancet; 338: Wilson DI, Goodship JA, Bum J, Cross IE, Scambler PJ. (1992). Deletions within chromosome 22ql I in familiai congenital heart disease. Lal/cel; 340: Wilson DI, Bum J, Scambler P, Goodship J. (1993). Sylldrome ofthe Illonth. DiGeorge syndrome, part of CATCH 22. J Med Genet; 30: Dunham I, Collins J, Wadey R, Scambler PJ. (1992). Possible mie for COMT in psychosis associated with velo-cardio-f..1cial s)'11drome. Lancet; 340: Desmaze C, Scambler p, Prieur M, Sidi D, Le Deist F, Aurias A. (1993). Routine diagnosis of DiGeorge syndrome by fluorescent in situ hybridization. Hum Genel; 90:

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165 Appendix publication VII Prenat Diagn, in press Copyright John \Vile)' & Sons. Reproduced with pennissioll.

166

167 Prospective prenatal investigations on potentialuniparental disomy in cases of confined placental trisomy Diane Van Opstal, Cardi van den Berg, \Vout H. Deelen, Helen Brandenburg, Titia E. Cohen Ovcrbeck, Dicky J.J. Halley, Aus ~rf.'v. van den Ouweland, Peter A. in 't Veld, Frans 1. Los Summa!'y In most rcpol'tcd cases of uuiparclltal disomy (UPD) associated n'ïth confincd placental mosaicisill (CPIV!)! a high level of lliosaicism Ol' n full tl'isomy was follud in chorionic villi (CV). At fhe time wc shntcd OUl' investigatiol1s it was not quite clear ",hethel' fetal urd also cxisted in fhe morc fl'cqucntly occul'illg low levels of lllosaicism. DuI"ing a four-year pcl'iod a follow-up amniocclltcsis was perfol'illcd in all cases of lilosaic Ol' noil-mosaic tl'isomy dctcctcd in CV semi-direct pl'cpamtiolls and suspectcd to be confillcd to the placenta. 'Vc pcl'fol'liied fluorescent in situ hybridization (FISH) on uncultured amniotic fluid cells to differentiate hefweell gencralized mosaicislll and CPi'VI. "rc found 29 cases ofcpi'\'i mul we detcrmincd the incidence ofupd iu23 of these cases. NOl'mal hiparental chl'omosomc contl'ihufions were fonnd in 22 cases. In olie case wc detecfed a matcl"ual heterodisollly fol' chromosome 16. UPD appeal'cd to hc a rare phcnolllenon in thc cases of ep.m (type I mullo)" 111) that we encountered in 3958 consecutively investigatcd CV samples! and is Ilot the cause of thc pregnanc)' colllplicatiol1s folllid in seven out of 23 cases with CPNI. In trad IIctiall In 1980, Engel introduced the concept of uniparental disomy (UPD) i.e. the presence of a chromosome pair derived from olle parent in a diploïd offspriug. Eight years later, Spence et al. (1988) reported the first example of UPD in a 16-year-old girl with short stature and eystie fibrosis; she had inherited lwo copies of the maternal chromosome 7 with a CF mutation. In 1992, the first case of UPD associatcd with confined placenta 1 mosaicisl11 (CPM) was documellted; a patient with Prader-Willi syndrome, caused by maternal UPD for chromosome 15, was barn after prenatal detectioll of a trisomy 15 in choriollic "illi (CV), and a subsequent normal karyotype in amniotic Huid (AF) cells (Purvis-Smith et al., 1992). This case supported the hypothesis that the loss of all extra chromosome from an initially trisomic zygote is one of the meehanisms that eould lead to fetal UPD (Engel aud DeLozier-Blanehet, 1991). Bennet( ct al. (1992) studied two cases oftrisomy 16 confined to the placenta and found maternal UPD in one ofthcm with a paternal chromosame 16 present in chorionic villi but being lost in the fetus, provillg that a trisolnic zygote cau indced luldergo postzygotic loss of a supernumerary chromosome rcsulting in a diploid fetus. Various cases of UPD associated with CPiv[ have been published uptill now (Kalousek and Barrelt, 1994). The type of CPM as weil as the 167

168 chromosome involvcd, and the onglll of the trisomy (meiotic or mitotic), which itself is corrclated with thc level of 1l10saicism, all seem to be associated with thc incidence of UPD (\Volstenhollllc, 1996; Robinson et al, 1997). UPD can affect human dcvelopment through imprinting or homozygosity for reccssivc traits, and may be relatcd to pregnancy and perinatal complications occasionally foulld in cases of CPM such as intra-uterine growth retardation (lugr) (Kalousek et al., 1991), [ctalloss (Goldberg ct al., 1990), or poor periuatal outcome (Johnson ct al., 1990). However, the presence of cytogenetically abnormal cells in the extracmbryonic tissues may have a direct effect on placental function with subsequcntly these complications of pregnancy (Kalousek alld Barrett, 1994). The purpose of our study, which started in 1992, was to determine the incidencc of UPD associated with CP:~vl in a consecutive series of CV samples that we recieved in our laboratory during tour years. Twenty-nine cases oftrisomy CPt."I were found, and in 23 cases wc studied extensivcly the level of mosaicism in CV with interphase fluorescent in situ hybridization (FISH), and determincd the paren tal origin of thc partieular ehromosome pair in cultured AF cclls. Additionally, wc investigated uncultured AF cells for the prcscncc of tri som ic cells to prcclude generaliscd mosaicisl11, concealed in cultured cells. PaNenls wui procedures Matcrials alld mcthods Twcnty-nine cases of confined placental (mosaic) trisomy (type 1 and/or IJl), with at least two trisomic ceils on a totalof 30 metaphases in CV semi-direct preparations, were encountered in 3958 consccutively investigatcd CV during a four-year period ( ). In all these cases follow-up amniocentesis was oftèred to thc parents for c)10genctic studies and UPD invcstigations. Por thc determination of the level and extent of thc mosaicism, we applied intcrphase FISH on CV and unctlihired AF cells. In six out of29 cases, UPD studies could not be pcrformed because blood of the parcnts was not available. Sa, thc present series compriscs the remaining 23 cases. Five to 35 mg (mean 14 mg) of CV were sampled transabdominally at 11 to 14 weeks of gestation as described prcviously (Jahoda et al, 1990). Indications tor prcnatal diagnosis were advanced maternal agc (:?: 36 years) (n=20), risk of X-linked lllcntal retardation, recurrence risk of Niemann Piek type A, and risk of X-Iinked adrenomyeloneuropathy (each n=i). At 16 weeks of gestation, 18 to 20 mi of AF was samplcd in all but one case. In two cases a fetal skin biopsy was taken after intra-uterine dcath at 15 alld 33 weeks of gestation. C)'logel1etic ill\'esagatiol1s CV were incubated overnight using fluorodeoxyuridine (FdU) synchronization (Gibas et al, 1987). Karyotyping was periormcd on Trypsin-Gicmsa stained semi-direct preparations. A mean of 32 (range 19-50) metaphases were analysed. Culhlring of CV samples was nol perfbrmed during thc stud)' pedod. AF-cells were cultured according to standard techniqucs by lhe in situ lllcthod on glass coverslips. Trypsin-Giemsa staining was routinely used. A mean of 15 clones (range 7 26) of culhlred AF cells were analysed. 168

169 Table L Chromosome spccifïc probes IJsed (or FISH Chromosome Probe Reference 3 pu3.5 Waye and Willard, pu7tl Waye et al., pul2h8 Looycnga et al., plluri95 MoyzÎs et al., LI.84 Devilee et al., M51 Mulder et al., 1995 X pbamx5 Willard et al., 1983 bltejphase FISH ((l1((lysis FISH was perfonncd on CV semi-direct preparations and on 2 mi of uncultured AF ceils as deseribed prcviausly (Van Opstal et al., 1993; 1995). The probes used in this study are Iisted in table 1. For cach case the numher of fluorescent spots was counted in a mean of 185 (range ) hybridizcd CV interphase nuclei, and a mean of 154 (range ) hybridized uneultured AF eells. For interpretation ofthe FISH results, the same probes were applied to a series of normal CV and AF cell samples. Statistical aila1ysis of data obtained from these control samples was used to determinc the 95 % confidenee interval of the one-sided upper refcrence limit (97,5%) for the proportion of cells with tl11'ee signals for each of the probes used, according to Lomax et al. (1994) (Tabie 2). This cut-off level was used to discriminate the normalnon-illosaic state from the Iowest detcetable level ofmosaicislll. DNA onolysis DNA was extracted from blood ofboth parents and in all but onc case from cultured AF cells according to standm'd techniques. In one case the tètal DNA source consisted of cultured skin fibroblasts. In t111'ce cases Trypsin-Giemsa stained CV chromosome preparations, whieh had been stored at room telllpcrature tàr two 11l0nths to two ycars, were also used for DNA extraction. Briefly, celis of one slide wcre collected in 10 lllwi NaCl/lO mivf EDTA and spun down far 15 sec at rpm. Thc eell pellet was resuspendcd in 50 mm NaOH and bailed for 20 min. Aftel' neutralization with I M Tris.HCI, the suspension was spun down to remove celi dcbris alld ~ll ofthe supernatant was used for PCR analysis. Molecular analysis of the parcntal origin of bath cjli'omosomes of a chromosome pair in AF celis (and skin fibroblasts in one case), and of the extra chromosomc in CV, was perfonned with polylllerase chain reaction (per) ampli11cation of polymorphic microsatcllite repcats. Loci that were examincd for each of the chromosolllcs involved, are Iisted in table 3. Infonnation on primer sequence and map location is available from the Genome Database. Sixty ng of fresh DNA and 0.5 ta 2 pi of arehival DNA salut ion (CV-slide extraeted) was amplitlcd in a total volmne of 15 pi containing 60 ng of each primer, 0.1 ~tl 100 mlvf dntp- 169

170 Table 2. One*sided upper reference limit (97,5 %) and corresponding 95 % confidence interval (Cl) for the proportion of CV and AF eeus with three FISH signals obtained from a series of diploid controls Probe (number of controls) p"3.5 (5 CV, 4 AF) pa7tl (12 CV, II AF) pu12h8 (4 CV) phuri95 (16 CV, JO AF) LI.84 (12 CV, 38 AF) M51 (5 CV) Upper reference (97,5 %) limit (95 % Cl) cv AF 1,84 (1,09;2,6) 1,57 (0,53;2,6) 3,78 (2,43;5,13) 5,35 (3,18;7,53) 3,27 (1,29;5,25) 6,04 (4,21;7,87) 5,44 (3,41 ;7,48) 2,37 (1,95;3,51) 7,37 (5,75;9,0) 6,69 (4,02;9,37) pbamx5 (5 CV, 16 AF) 3,36 (1,29;5,43) 4,08 (2,63;5,54) Note. CV chorionîc vîlli semi-direct prepnrations; AF unculturcd amniotic fluîd eells. Table 3. List of l1iicrosatellite loci IJsel! for parent of origill studies Chromosomc Markers * 2 02S73, ~IHCrCO, 02SI03, 02S72, CTLA4, 02S102, 02S S1270, 03S1304, DJSIOO, 03S1360, 03SII, GLUT2, 03S S531, 07S472, 07S488, 07S471, 07S473, O7S504, 07S495, 07S483, 07S S104, 09S52, 09S43, 09S51, 09S S62, 012S61, 012S43, 012S64, 012S S175, 013S220, 013S170, 013S159, 013S174, 013S158, 013S HBAPI, 016S407, 016S298, 016S285, 016S308, 016S261, 016S419, 016S301, 016S266,016S S59, 018S52, 018S452, MF080, 018S40, 018S34, 018S35, 018S42, 018S43, MBP, 018S S66,020S48,020SI02,020S S257, 022S156, 022S120, 022S315, IL2RB, CYP2D X Notc. * in order pter--> qlcr MAOA, OXSI003, OXS426, OXS453, OXS

171 mix with a Iower dctp concentration, 0.45 ~" 50 mm MgCI.6H,O (BRI., Gaithersburg, USA), 1.5 ~" 10XPCR buffer (BRI., Gaithersburg, USA), 0.3 ~I of25 mm spermidine, 0.75 ~I 1% \VI (BRI., Gaithersburg, USA), O.I~",,"P-CTP, and 0.1 ~I Taq-polymerase (5U1~") (BRI., Gaithersburg, USA). Samples were processcd through 25 cyc1es for fresh and 40 cycies for archival DNA samples, each cycle consisting of 1 min denaturation at 94 oe, 1 min annea Iing at 55 C, and 1.5 min extension at 72, The alllplit1cations \Vere performed in a Pcrkln Elmer Cetus DNA Thermul Cycler The alleles were separated by electrophorcsis on a 6 % denaturing polyacrylamide gel. Gels \Vere fixed with 10 % aeetie acid! 10 % methanol and dried. They were exposed to X-ray film overnight at room temperahlre. Conclusions about parental origin of a particular chromosome required at least two informative markers. C)ltogenetic Gnalysis alld FISH studies Resuits On a total of 3958 CV samples that \Vere invcstigatcd cytogenetically during a four-year period, 69 cases (1,7 %) of CPM (type land/or III) were detected. In 29 cases (42 %) a trisollly was involved, of which 23 cases comprises the present series. Cytogelletic results of CV arc presented in table 4; normal karyotypes were present in amniotic fluid cells or fetal skin fibroblasts (case 22). In three cases (cases 16, 17, 18) a non-lllosaic trisomy 16, and in 18 cases a mosaic trisomy involving the chromosomes 2, 3, 7, 12, 13, 16, 18, 22, alld X was found in CV. In the relllaining two cases a Jllosaic double trisomy of chromosomes 7 and 9 (case 7), and of chrolllos0111es 13 alld 20 (case 14) was present in the sample. The proportion of abnoflllal cells in the lllosaic cases varied betweell 7% and 98%. FISH results on interphase nuclei in semi-direct CV and uncultured AF ceil preparations are shown in table 4. \\'here na results are shown, appropriate probes were not available at the time of investigation or na results were obtaî.ned due to inefficient hybridization. In case 22, AF was Ilot available because ofintra-uterine dcath alld tcrmination ofpregnancy at 15 weeks of gestation. In tlnee ofthc 19 cases in which FISH results were obtained on CV sildes, a discordance was found betweell standard cytogenetic and FISH analysis: FISH did not conflnn the eytogenctic presence ofmosaicism. In one of the 14 cases, in whieh FIS I-I rcsuits were obtained on uneultured AF celis, a diserepancy was foulld between FISI-I analysis ofuneultured eells and cytogenetic analysis of the eell cultures (case 16); 26 % of the unculturcd eells showed three chromosome 16 signais, whereas the eell cultures showed a l10rmai karyotype. In all other cases, neither classical cytogenetic nor FISH analysis revealed the existence of a trisoluic cclllille in AF eeus. DNA studies Parent of Origill studies in AF cells (or skin fibroblasts in casc 22) of both homologues of the chromosoll1c that was tound to be trisoluic in CV, revealed a normal biparcntal chromosolllc contribution in 21 of thc 23 cascs, and an abnormal result in two cases (cases 13 and 17). 171

172 Table 4. Karyotypes. inte!:;ehase FISH rcsults. and pregnaney outeomes in 23 cases of CPM Case Karyotype in CY* %of Inte!".Ehase FISH results Pregnaney outeome No. (numberof eells) trisomie eells % of nuclei with 14si~s (gestational age. birth weight. pereentile) N CV AF CV AF CV AF CV AF CV AF 47.XX.+2[4]146.XX[25] wks. 4180g. P XY.+3[6]/46.XY[29] wks g. P XY.+3[7]/46.XY[22] wks. 3600g. P XY,+3[32]/46.XY[1 ] wks. 4050g. P XX.+7[3]146XX[30] wks. 2660g. P" XX.+7[3]J46,XX[26] wks. 3920g. P XY [2)/46'xY[17] wks. 3060g. PlO xX.+7[5]/46.XX[26] wks., 3500g. P ,XY,+ 7[6]J46'xY[24] wks. 4000g. P XY.+7[12]/46.XY[18] wks. 381Og. P XX.+ 12[2]/46'xx[28] wks. 2115g. <P2.3..., XX.+ 13[2)J46'xx[28] 7 41 wks g. P25-50 N XY.+ 13[2]/46.xY[28] 7 40 wks. 3850g. P XY [3]J46'xY[27] wies g. PI0-25 solutio placenta 15 47,XX.+16[2]/46.XX[28] wks. 4000g. P XX.+16[30] <;, 3 IUD at 33 wks.. 845g. <<Pl.3. MCA 17 47XX.+16[37] wks g. P 'xY.+16[32) wks. 2850g. P50. neonatal infeetion. and wet lung XX.+ 18[2]/46XX[28] wks g. PlO XX+18[2]/46.XX[28] wks g. P5-lO XX.+ 18[l9]l46.XX[19] wies. 3375g. P 'xx.+22[49]/46.XX[1] IUD at 15 wks. 14 g.<<p2.3. MCA XXX[9]146.XXI21] wks. 4lO01:::. P75-90 Note...: karyotypes in nrnniotic fluid cell cultures wen: n01'm.:l1 in all cn.~es. N== nurnberof interphase nuclei: CV: chorionic villi semi direct prejxlrotions: AF", unculrured nmniotic fluid cell~: _'" not tested or non-infol'm.:ltivc rcsujl~: "gray box" dcnoter discordnnce between cytogenetic luid FISH re.~ull~: IUD", intr:l-uterine death: MCA", multiple congenitül abnormnlities: p", percentiles of Dutch ncon:ltes (K1oo~terman. 1970)

173 Figure 1: Autoradiograms of polymorphic tlinuclcotidc repeat markcl's (UBAPI, , and 016S285) on chromosome 16 in cases 17 (A and 11) and 18 (C) (F= father, 1\1= mother, CV= chorionic "iiii, AF= amniotic "uid). (A) shows loss of the paternal allele Al in AF celjs of case 17 leading to maternal UPO. (B) shows thc presence of two different materilal alleles (AI and A2), and absence of a paternal allcle (A3), consistent with lilaternal heterodisolny, in AFC oh'ase 17. (C) dcmonstrates presence of two maternal (A2 and A4) aml olie patelïllli aljele (AI) in CV, and sllbscqucnt loss of all extra matcrnal allele (A4) in AF cells, leadil1g to biparental illheritance ofchromosomes 16 in case 18. A n (' F M CV AF _ A4 HBAPI DI6S305 DI65285 In case 17, showing a non-mosaic trisomy 16 in CV, UPD for chromosame l6 was found in AF eells, with four ofthe tested markers, two loeated on 161' (BBAPI and DI6S298), aud two on 16q (D16S261 and DI6S305), showing an informative patten!. The fetal pattem silowed a maternal heterodisomy as mother and tètus were bath heterozygous for {he same allel es of D16S285, D16S298, D16S308, and DI6S305 (Fig, I), Non-patemit)' and maternal eell contamination (MCC) were excluded by analysis of three highiy informative markers, F ABP, THOI, aud HPRT on chromosomes 4,11, and X, rcspectively. In case 13 na paternal and only one maternal allele could be detccted in AF cells for two of the tested markers (D 13S170, D I3S 174). Amplifieatioll of pol)'lllorphic markers 10cated 011 other chromosomes (D21S120 on chromosome 21, aud D9S43 on chromosome 9) also revealed thc presence of a maternal but absence of a paternal alle Ie which proved nonpaternity in this case. In the tluce non-mosaic trisomy 16 cases (cases 16, 17, and 18) we studied the parental origin of the supernumerary chromosome in CV. PCR amplit1cation of DNA extractcd from CV slides revealcd thc presence of two maternal and anc paternal allele for at least one polymorphic marker in all three cases (fig. 1). t\'icc of thc slides of cases 16 and 18 was excludcd by PCR amplification of markers on some other cluomosomes (D 1 S 158 and D21 S 156 in case 16; D20S120, D18S40, D3SII, D13S159, and Dl7S250b in case 18). Pregllancy outcome (Tabie 4) In 16 cases, the pregnmlcies were uncventfui and resulted in thc birth of illfants with birth weights beyond the 10th centije (Kloosterman, 1970). In seven cases pregnancy complicatiol1s were observed. Thcrc were two cases ofintra-uterine growth rctardation (IUGR) (cases 11 and 173

174 20), and one case of preterm delivery at 33 weeks because of solutio placenta (case 14). In case 17 maternal hypertension and deteriorating renal functioll were diagnosed al 34 weeks of gestation. The mother recovered rapidly after the delivery of a 2880 g fenmie infant at 38 weeks of gestation. Inta-uterine death and multiple congenital abnonllalities were observed in cases 16 and 22. In case 16, the abnormalities iuvolveel severe IUGR, faeial elysmorphisms, simian crease right, atria I septal defect, hypoplastic truncus pulmojmlis with valvular atresia, ventricttlar septal defect, l'ight lung with one lobe, and left lung with two lobes. In case 22, sevcl'c IUGR, faeial dysmorphisms, malrotation of the intestine, asplenia and atrial septal defect were the major malfonnations. In one njither case (case 18) transient perinatal complications wcre encountered (wet lung and illfcction). Discussioll The incidence ofupd in a diploid fetus supported bya trisomic placenta is theoretically 1/3, if the concept ion originally was trisomic and toss of anc copy of the trisojnic chromosomc in the embryonic progenitor cells during cleavage occurred randomly and resulted in CPN! (Engel and Deloizicr-Blanchet, 1991). This theoretical tïgure has achmlly been established for chromosome 16 anel 22 (Kalousck et al., 1993; \Volstenholme, 1995), since these trisomies are predominantly of meiotic origin, and a significant con-elation was fotmd between the presence of UPD and a mciotic origin of the trisomy (Robinson et al., 1997). Trisomies of many other chrolllosomes, such as 2, 3, 7, 8, and 9, seem to be primariiy thc rcsult ofsomatic duplication (Kalousck et al., 1996; Shaffer et al. 1996; Wolstenholme, 1996; Robinson ct al., 1997). Robinson et al. (1997) fouud fetal UPD iu 17 out of 94 cases of trisomy CPM, including 13 cases of UPD 16. However, their shldy poptilation lllight not be considered a random sample of CP!,,! cases founcl during prenatal diagnosis, becausc of inclusion of postnatal cases ascet1ained through IUGR noted at birth, and because of a high numbcr of trisomy 16 cases, since they were the initial focus of their research, which bath may be responsible tor an overestimation of the frequency of UPD. The purpose of our study was to determine thc incidence ofupd assoeiated with CPM in a series of consecutively investigated CV samples received during a four-year period. As a consequence of our approach to cytogenetic investigations of CVS, UPD studies \Vere only performed in cases of CPM type I (abnol'lna1 cells confined to cytotrophoblast) and undetermined CP?,,! type 111 (abnonnal cells in cytotrophoblast as weil as extraembryonic mesellchyme). Of29 cases oftdsomy CPM that we found during four years, 23 cases could be investigated, and the incidence of UPD showcd to be I iu23. This low frequency of UPD is in agreement with the correlation of fetal UPD with high levels oftrisomic cells in the trophoblast (Robinson et al., 1997). In 18 out of23 cases a low mosaic trisomy was present in semi-direct CV preparations, with less than 50% of abnormal cclls, and in half of the cases even less than 10%. The trisomies in all these cases most probably originated from a postzygotic mitotic non-disjunction (Crane and Chellng, 1988; \Voistenholme, 1996; Robinson et al., 1997). In five cases wc foll11d a high mosaic or fuu trisomy in semi-direct CV prcparations. In three cases of non-mosaic trisomy 16 we silowed that the trisomy arosc as a result of a maternalmeiotic error, as collld be expecled from carlier reports (Hassold ct al., 1995). Trisomic zygote rescue resulted in CP!v1 in these cases and causcd UPD tor chromosome 16 in one of them by eliminating thc paternal chromosome

175 Although most reported cases of UPD were found to be associated with CP1vI t)'pe lil, with high levels of trisolllic cells in. bath placental lineages, a few cases were fàund to be associated with CPM type I (Jones et al., 1995; Wilkinson et al., 1996). Robinson et al. (1997) showed that CPM type II (abnonnal cells confined to the extraembryollic lllcsenchyme) may sometimes have a meiotie origin with a risk for UPD, althaugh Wolstenholme (1996) stated, on the basis of observations and theoretical considerations, that meiotic errors are not assumed to be associated with CPM type I!. Some 21 cases of CPlvf and maternal UPD for chromosome 16 have been described previously (llennett et al., 1992; Dworniczak et al., 1992; Kalollsek et al., 1993; Sutcliffe et al., 1993; Miny et al., 1994; Vaughan et al., 1994; Kalollsek anel Barrctt, 1994; Whiteford et al., 1995; Schneider et al., 1996; O'Riordan et al., 1996; Kalollsck and Vekemans, 1996; Robinson et al., 1997). In most cases IUGR has been observed, and in some cases congenital malformations were found as well (imperforate anus, two-vcssel umbilical cord, club-foot, inguinal hernia, hypospadias, clinodactyly, and atrioventricular septal defect). However, in a few cases, including the present case, UPO 16 was làund to be associated with a 110rl11al outcome (Kalousek and Barrett, 1994; Robinson et al., 1997). This further supports the hypothesis that the impaired feta I growth in cases of UPO 16 may not he the result of the fetal UPD itself, but rather due to a malfunctioning placenta, caused by high levels of trisomic cells in thc placcnta (Kalollsck and Barrelt, 1994; Wolstenholme, 1995; Brandenburg et al., 1996), or by imprinting effects Iimited to placental tissues and in utero growth (Robinson et al., 1997). The maternal hypertension and deteriorating renal function in the present UPD 16 case further supports this hypothesis. ivioreover, there appears to be an association between CPN! for chromosome 16 and all unexplained abnormal profile of matcrnal serum markers (Vaughall et al., 1994; Zimmennan et al., 1995; Tantravahi et al., 1996), also pointing in the direction of a dysfunctional placenta. I-Iowever, this can IlOt explain the presence of fetal congenital malfonnations in some cases of UPD 16. Thcrefàre, another possible explanation lllight be th at in sylllptolllatic cases trisoilly 16 cells are in fàct not confined to the placenta, but a mosaic trisomy 16 is also present in the fetus or infant. Clinical observation ofthe fetus and FISI-I studies in case 16 of our series supports this hypo thesis. FISH revealed a raised proportion of trisomic ceus in uncultured AF, although cytogenetic as weil as PISH allalysis of the AF cell cultures onl)' revealcd disomic eells (FISH data on cell cultures not shown). These discrepant FISI-I resuits between uncultured and cultured tissue may be explained by a proliferative advantagc of nor111al cells in the ceil culhlres. Postmortem examination of the fetus after intra-uterî.ne death at 33 weeks of gestation revealed several congenital malformations which fitted a mosaic trisomy 16 phenotype (Devi et al., 1993). Postnatal cytogenetic alld FISH studies of the fetus were oniy sliceesfuil in culhlred ovary tissue, revealing disomic cells, and in fetal Iymphocytes with 5 % of interphasc nuclei showing tlrree clrromosome 16 signals with FISH (nonnal up to 3% in a series of ten control samples). In the two other cases of fuu trisomy 16 in the present series the FISH results in AF were normal. Based on these results, we believe that the detection oftrisoll1y 16 in uncuihlred AF cells with FfSH might have a predictive valne for the acttml fetal chromosomal constitution and outcome. In general, we silowed that FISH is areliabie method for rapid diflèrentiation bet ween generalizcd mosaicism and CP~II, as in IJ out of 14 cases FISH results were in agreement with cytogenetic results, and they could be achievcd within two days after sampling in all cases. The on I)' case with discrepallt results is discussed above. 175

176 In conclusion, the incidence of UPD in a series of CPM (type I alld/or lil) cases, collected over a four-year-period in our laboratory, is very low, indicating that in most cases thc trisomic eell line in CV origillatcs fiom somatic duplication, which is supported by thc low level of mosaicism in most of these cases. Furthcl111ore, it illdicatcs that the obstetrical complicatians as IUGR and IUD, found in thc present series, are not associated with UPD. Aclmowledgements \Ve wish ta thank Carola lansen for expert teclmical assistence, Armando Braat for generating figure I, and thc Rotterdam Foundation of Clinical Genetics for its tinancial support. Refel'ences Dcnnctl, P., Vaughan, J., Henderson, 0., Loughna, S., r.,'toore, G. (1992). Association between confiiled placental trisomy, fetal uniparental disomy, and earl)' intrauterine growth retardation, Lal/cet, 340, Brandenburg,H., Los, FJ., In 't Veld, P. (1996). Clinical significance of placenta-confined nonmosaic trisom)' 16, Am J Obstet Gynecol, 174 (5), Crane, J.r., Chcung, S.W. (1988). An embryogenie model to explain cytogenetic inconsistencies observed in chorion ie villus versus fetal tissue, Prenat Diagl/, 8, Devi A.S., Velinov, ]1\,1., Kamath,!'vt.V., Eisenfeld, L., Neu, R., Ciarleglio, L., Grcenstein, R., Berm, P. (1993). Variabie clinical expression ofmosaic trisomy 16 in the newborn infant, Am J Med Genet, 47, Devilee, r., Crcmcr, T., Slagboom, P., Bakker, E., Scholl, H.P., Hager, H.O., Stevenson, A.F.G., Comelisse, CJ., Pcarson, P.L. (1986). Two subsets ofhuman alphoid repetitive ONA show distinct prefercntial localization in the perkentrie regions of chromosomes 13, 18, and 21, Cytogellel Cel! Genet, 41, I. Owomiczak, B., Koppers, B., Kurlemann, G., Holzgrcvc, W., Horst, J., rvi in)', P. (1992). Uniparental disomy with normal phenotype, Lancet, 340, Engel, E. (1980). A new genetic concept: uniparental disomy and its potential cltecl, isodisomy, Am J Med Genet, 6, Engel, E., OcLoizier-Blanchet, CD. (1991). Uniparental disomy, isodisomy, and imprînting: probable effects in man and strategies lar their detcction, Am J Hum Gel/el, 40, Gibas, L.M., Gmjic, S., Barr, M.A, Jackson, L.G. (1987). A simplc technique for obtaining high quality chromosome preparations from chorionic villus samples using FdU synchronization, Prenat iagl1, 7, Goldberg, J.O., Porter, A.E., Golbus, ~:I.S. (1990). Current assessment offetallosses as a direct consequence of chorionic villus sampling, Am J Med Genet, 35, Hassold, T., Merrill, M., Adkins, K., Frccman, S., Shcrman, S. (1995). Recombination and maternal agedependent non-disjunct ion: molecular studies oftrisomy 16, Am J Hl/IJl Genet, 57, Jahoda, M.GJ., Püpers, L., Reuss, A, Brandenburg, H., Cohen-Overbeek, T.E., Los, FJ., Sachs, E.S., Wladimiroff, J. W. (1990). Transabdominal villus sampling in early second trimester: a safe sampling method for women of advanced age, Prenat DiagIl, 10,

177 Johnson, A., Wapner, RJ., Davis, G.I-I., Jackson, L.G. (J 990). Mosaicism in chorionic villus sampling: an association with poor perinatal outcome, Obslel G)'lIeco/, 75, Jones, c., Booth, c., Rita, D., Jazmines, L., Spiro, R., r., lcculloch, B., I\.JcCaskill, C., ShalTer, L.G. (1995). Identificatioll of a case of maternal uniparental disomy of chromosome 10 associated with colll1lled placental rnosaicism, Prenat Diagn, 15, Kalousek, D.K., I-Ioward-Peebles, P.N., Olson, S.B., Barrett, J.J., Dorfman, A., BJack, S.H., Schulman, J.D., WilsOIl, R.D. (J991). ConftnnatÎon of CVS Jllosaicisrn in term pjacentae and high frequency of intrauterine growth retardation association with confined placental mosaicism, Pre/lat magn, 11, Kalousek, D.K., Langlois, S., Barrelt, 1., Yam, 1., Wilson, D.R., Howard-Peebles, P.N., Johnson, I\:LP., Giorgiutti, E. (1993). Uniparental disomy for chromosome 16 in hurnalls, Am J Hum Genef, 52, Kalousek, D.K., and Barrelt, I. (1994). Genornic imprinting relaled 10 prenatal diagnosis, Prellal Diagn, 14, Kalousek, D.K., Langlois, S., Robinson, W.P., Telenius, A., Rernard, L., Barrelt, J.J., Howard-Peebles, P.N., Wilson, R.D. (1996). Trisomy 7 CVS Illosaicism: pregnanc)' outcome, placellial and DNA anal)'sis in 14 cases, Am J Met! Genel, 65, Kalousek, D.K., and Vekemans, r (1996). Confined placenta! mosaicism, J,\led Gel/cf, 33 (7), Kloostennan, G.J. (1970). On intrauterine gro\\1h, Int J G)'lIccol Obs/et, 8 (6), Lomax, B.L., Kalousek, D.K., Kuchinka, B.D., Barrett, J.J., liarrison, K.J., Safavi, H. (1994). The utilization of interphase cytogenetie analysis forthe detection ofmosaicism, HUIII Genel, 93, Looijengn, L.H.J., Smit, V.T.H.RrvL, Wessels, J.\V., rviolievanger, P., Oosterhuis, J.W., Cornelisse, C.J., Devilee, P. (1990). Localization and polymorphism of a chromosome 12-specific u satcllitc DNA sequence, Cytogenel Cell Genel, 53, 2 I r-, Iiny, P., Koppers, R, Bogdanova, N., Exeler, R., Holzgreve, W., Horst, J., Dworniczak, B (1994). Uniparental disomies after prenatal diagnosis of confined placentnl mosaicism nnd intrauterine growth retardation, 7th Int. Conf. on Early Prenatal Diagnosis, Jerusalem, Isme!, A22. tvtoyzis, R.K., Albright, K.L., Bartholdi, lv1.f., Cram, L.S., Deaven, L.L, Hildebrand, C.E., Joste, N.E., Longmire, J.L., Meyne, J., Schwarzacher-Robinson, T. (1987). "uman chromosome-specil1c repetitive DNA sequences: novel markers for genetic analysis, Chromosoma, 95, /I,'fulder, M.P., Wilke, M., Langeveld, A., Wilming, L.G., Hagemeijer, A., \'tm Drunen, E., Zwarthon~ E.C., Riegman, P.II.J., Deelen, \V.H., van den Ouweland, A.M.W., Halley, , rvleijers, c. (1995). Positional mapping of loci in the DiGeorge critical region at chromosome 22q I I usîng a new marker (D22S I 83), 111/111 Genet,96, O'Riordan, S., Grcenough, A., Moore, G.E., Bennett, P.,Nicolaides, K.H.(J996). Case report: uniparental disomy 16 in association wîth congenital heart discase, Prenal Diagn, 16, Purvis-Smith, S.G., Saville, T., Manass, S., Yip, M.-Y., Lam-Po-Tang, P.R.L., Duffy, B., Johnston, f-i., Leigh, 0., McDonald,B. (1992). Uniparental disomy 15 resulting from "correction" oran initial trisomy 15, A1II J HI/m Gellel, 50, Robinson, \V.P., Bnrrett,!.J., Bernard, 1.., Telenius, A., Bemasconi, F. Wilson, R.D., Best, R.G., Howard Pecbles, P.N., Langlois, S., Kalousek, D.K. (1997). Meiotic origin oftrisomy in confined placental mosaicism is correlated with presence of feta 1 uniparental disomy, high levels oftrisomy in trophoblast, and increased risk of fetal intrauterine growth restriction, Am J Hum Genet, 60,

178 Schneider, A.S., Bischoft~ F.Z., McCaskill, C., Luz Coady, ~t, Stopfer, J.E., Shaffer, L.G. (1996). Comprchensive 4-year follow-up on a case ofmatemal heterodisomy for chromosome 16, Am J Med Genet, 66, Shaftèr, L.G., Langlois, S., l\kcaskill, C, l... lain, O.M., Robinson, W.P., Barreu, I.J., Kalousek, O.K. (1996). Analysis ofnine pregnancies with confined placental mosaicism for trisomy 2, PreI/at Diagn, 16, Simoni, G., Sirchia, S.M. (1994). Confined placental mosaicism, Prenal Diagn, 14, Spence, J.E., Perciaccante, R.G., Grcig, G.l\t, Willard, H.F., Ledbetter, O.H., Hcjtmancik, J.F., Pollack, rvt.s., O'Bden, W.E., Beaudet, A.L. (1988). ljniparentul disomy us u mechanism for hullwn genetic disease, Am J Hum Genet, 42, Sutcliffe, rvu., ~\'tueller, O.T., Gallardo, L.A., Papenhausen, P.R., Tedesco, T.A. (1993). ]'vlaternal isodisomy 16 in a norm al 46,XX following trisolnic conception, AIII J 1ll/111 Gel/el, 53 (Suppl.) A Tuutravahi, U., l\,tatsumoto, c., Delach, J., Craflèy, A., Smeltzer, J., J3enu, P. (1996). Trisomy 16 mosaicism in amniotic!luid ccll cultures, Prenat DiagIl, 16, Van Opstal, D., Vun Hemel, J.O., Sachs, E.S. (1993). Fetul aneuploidy diagnosed by fluorescence in situ hybddization withill 24 hours afler amniocentesis, The Lal1cet, 342, 802. Van Opstal, D., van den Berg, C., Jahoda, l\tg.j., Brandenburg, 11., Los, F.J., in 't Veld, P.A. (1995). Retrospective study of trisomy 18 in chorionic villi with fluorescent in situ hybridization on archival direct preparations, PreI/at DiagIl, IS, Vaughan, J., Ali, Z., Bower, S., Bennett, P., Chard, T., Moore, G. (1994). Human matemal uniparental disomy for chromosome 16 and felal dcvclopmcnt, Prenal Diagn, 14, Waye, J.S., England, S.8., Willard, H.F. (1987). Genomic organization of alpha satellite DNA on Imman chromosomc 7: evidence for two distinct alphoid domains on a single chromosolllc, Mol Cel! Biol, 7, Waye, J.S., Willard, H.F. (1989), Chromosollle specificity ofsatellitc DNAs: short- and long-range organization ofa diverged domctric subset ofhumun alpha satellite from chromosome 3, Chromosoma, 97, Whiteford, M.L., Coutts, J., AI-Roomi, L., ~vlather, A., Lo\\1her, G., Cooke, A., Vaughan, J.I., 1\'loore, G.E., Tolmie, J.L. (1995). Uniparental isodisomy for cluomosome 16 in a growth-rctardcd infant with congenital heart disease, Prenat Diagn, 15, Wilkinson, T.A., James, R.S., Crolla, J.A., Cockwcll, A.E., Campbell, P.L., Tempie, l.k. (1996). A case of Illalemal uniparental disomy of chromosome 9 in association with confined placental mosaicism for lrisomy 9, Prenat Diagn, 16, Willard, II.F., Smilh, K.O" Sutlterland, J. (1983). Isolation and characterization of a major tandem repeat family from the human X chromosomc, Nucleic. Acids Res., 11, Wolstenholme, J. (1995). An audit oftrisomy 16 Înlllan, PreI/af Diagn, 15, Wolstcnholme, J. (1996). Confined placental mosaicism for trisomies 2, 3, 7, 8, 9, and 22: their incidence, likely origins, and mechailisills for celilineage compartmentalization, Prenat Diagn, 16, Zimmennann, R., Lauper, U., Strcicher, A.,!luch, R., Huch, A. (1995). Elevated alpha-fetoprotcin and human chorion ic gonadotropill as a marker for placental trisomy 16 in Ihe sccolld trimester, Prenaf Diagn, 15,

179 Appendix publication VIII Prenat Diagn, in press Copyright JOllll Wile)' & Sons. Reproduced with permission.

180

181 Uniparental disomy with and without confined placental mosaicism: a model for trisomic zygote rescue. Frans 1. Los, Diane Van Opstal, Cardi van den Berg, Armando P.G. Braat, SenuD Verhoef, Evelille Wesby-vall Swaay, Alls M.W. vall den Ouwelalld, Dicky J.J. Halley Summary In tbe population of chiidrcn born aftel' prcnatal cytogenetic illvcstigation in chorionic villi at our department from 1992 to 1995 (N=3940), three are knowil to us with uniparcntal disomy. Onc case of matcrnal heterodisomy 16 was prcnatally discovcrcd because of trisomy 16 in direct chol'ionic "iili with subsequently normal amniotic fluid cells. Thc afhel' two had narmal karyotypes in chorionic villi. Matcrnal heterodisomy 15 was postnatally dctcctcd in ouc of them because of Prader-'Villi syndl'ome. Maternal hetero/isodisomy 16 was accidentally cncountcred in the afhel' case in the course of prenatal DNA analysis of the tuberous sclerosis complex 2 regio!l at 16p13.3. A model is presented for the undcrstanding of thc yariolls combinations of karyotypes in direct chorionic vhh, cultured chorionic villi and thc fetus in case of successful and unsuccessful trisomic zygote reseuc. Introduction Uniparental disomy (UPD) is prenatally lllainly observed or suspected in case of (mosaic) trisomy in (semi)-direct (short term culture; STC) and cultured (long term culture; LTC) chorionic villi with subscquently normal alllniotic fluid cells (Kalousek & Barrelt, 1994; Ledbetter & Engel, 1995; Wolstenholme, 1996). The process ofthe 10ss or removal ofone of the tirrce chromosomcs from the trisomic conception, at least from the ceus that will form the fcttis proper is known as trisomic zygote rescuc, and the tlrree situations of abnormal karyotypes in STC villi, LTC villi, or both with a nonnal karyotype in the fetus are designated confmed placellfal lllosaicislll (CPM) type, I, 2 and 3, respectively (Kalousck & Barrett, 1994; Kalousek & Vekelllans, 1996; Wolstenholme, 1996). Various cases of CPM (type 1 or 3) and fetal UPD have been doclllllented (purvis-slllith et al., 1992; Cassidy et al., 1992; Bennet et al., 1992; Kalousek et al., 1993; 1996; Webb et al., 1995; 1996; Langlois et al., 1995; Jones et al., 1995; Wilkinson et al, 1996). Furtherlllore, cases of generalized mosaicislll with UPD in the disomic eell line are known (Sirchia et al. 1994; Harrison et al., 1995; Christian et al., 1995; De Pater et al., 1997; Van den Berg et al., 1997). \Ve would like to present tluce cases of UPD in which pecnatal cytogenetic investigations were carried out in chorionic villi at our department; two showed normal karyotypes in STC villi and liiaternal UPD 15 and 16, respectively, the third displayed CPM trisomy 16 in STC villi and maternal UPD 16. A model for trisolnic zygote rescue, based on the embryogenie 181

182 modeloriginalil' described bl' Crane and Cheung (1988) and modified bl' Bianchi et al. (1993), is presented for the explanation of the possible combinations of karyotypes in the various compartments in fetal UPD with or without CPM or UPD in the disomic een line in case of generalized mosaicism. Material alld Methods Duritlg the ycars , 3940 prenatal cytogenetic illvestigations were carried out in chorionic villi at Dur department. Thc majority ofinvestigations (N=3731) was perfonned for cytogenetic reasons (advaneed maternal age, ultrasound abnommlities, parental carriership of cluomosomal rearrangement, recurrenee risk for fetal aneuploidy). In the remaining cases (N~209), karyotyping was perfornled in addition to DNA or metabolic investigations. Chorionic villus sampling (CVS) was carried out transabdominally in all cases as described before (Jahoda et al., 1990). In case of anl' mosaic trisomy or lidi unusual trisomy (trisoml' athef than 13, 18 or 21) in chorionic villi, follow-up anmiocentesis and DNA analysis were offered for the differentiation between generalized chromosomal abnormality and CPM, and for the establislunent of potential UPD (Van Opstal et al., I 997a). Preparation ofstc chorionic villi slides was done according to Gibas et al (1987). We did not perform LTC villi preparations in this period. Routine Trypsin Giemsa staining was used. Normally, 16 eells were investigated, but in ease of mosaicism we investigated at least 30 metaphases. Amniotic fluid cells were cultured according to standard techniques with the in situ method on glass coverslips. Slide preparation for fluorescence in situ hybridization (FISH) on first trimester STC chorionic villi, and on STC and L TC placental villi were carried out as described before (Van Opstal et al., 1995). For the detection of the copy number of chromosome 15 or 16, the probes phuri95 for chromosome 16 (Moyzis et al., 1987) and ptra-20 for chromosome 15 (Choo et al., 1990) were used. The probes CW9D and CW23, located at 16p13.3 were used for the investigation of deletions in the tuberous sclerosis complex (TSC)2 region (European Chromosome 16 Tuberous Sclerosis Consortium, 1993). Probe labelling, detection, and visualization were carried out as described before (Van Opstal et al., 1995; Van den Berg et al., 1997). Two hundred non-overlapping and non-chuubed interphase nuclei of each sample were investigated. Metaphase analysis was carried out on 10 cells. Fetal DNA was i~olated from fresh chorionic villi, uncultured as weil as cultured amniotic fluid celis, and celis scraped from stored src villi slides (Van Opstal et al, 1997b). Postnatal DNA was isolated from peripheral blood accordillg to standard techniques. In a pregnancy at risk for TSC (McKusick phenotype ; McKusick, 1994) from a family showing linkage to the TSC2 region on 16p13.3, DNA analysis was perfonlled with the flanking markers 3'HVR, KG8, and 16AC2.5 (DI6S291) (Janssen et al. 1994). DNA analysis for Prader Willi syndrome (PWS) was carried out with probe PW71B and the microsatellite markers LS6-1 and GABRp3 (Van den Ouweland et al., 1995). Prenatal or postnatal investigations on the parent of origin of the cluomosomes 15 and 16 were carried out with analysis of various polymorphic microsateliite markers on chromosome 15 (CYPI9, DI5S87 and ACrC) and on chromosome 16 (HBAI, D16S404, D16S298, D16S261, D16S285, D16S415, Dl6S320, DI6S503, D16S515, D16S422 and Dl6S305). Data on localisation and primer sequences were derived from the Gename Data Base. 182

183 Clinical data on the course ofpregnancy, delivery and baby were couected in all ti"ee cases, Results Two cases of UPD \Vere found among the women who underwent CVS on cytogenetic indications, one with a normal karyotype (case I) and the other with a trisomy 16 (case 2) in STC villi. The third case of UPD, again with a normal karyotype in STC villi, occurred in a women who underwent CVS on a DNA indication (case 3). Tablc 1. Cytogenefic alld FISH data of fhe thl'ee UPD cases (upd 15 in case 1, UPD 16 in cases 2 and 3) Case karyotype in FISH on STC villi: nuclei with 1 4 signals(%) Karyotype in AF STCvilli [control mnge (%)]* eells or [no. of eeiis 1 Iymphocytes Probe ,XY [16J ptra-20 I ,XY [0-2] [97-100J [0-3] [O-IJ 2 47,XX,+16[37] phuri ,XX [1.5-15] [80-96J [O-IOJ [0-7J 3 46,XY[40J phuri ,XY [1.5-15] [80-96J [O-IOJ [0-7J AF ~ amniotic fluid; * The control range consisted out of3 nomlnl cases for ptra-20 and 15 for phur-195. Case 1. A boy of2735 g (25th percentiie) was bom after an uncomplicated pregnancy at 37 weeks by cesarean section due to malposition. His lllother underwent CVS on the indication of advanced maternal age (39 years) with normal cytogenetic results (Tabie I). The boy displayed congenital hypotonia, transiellt feeding difficulties, bitemporal narrowing ef the head, short palpebral fissures and hypogenitalism, suspect for PWS. Thc diagnosis of PWS was ascertained by demonstrating the absence ofa paternal fragment with probe P\V71B and maternal heterodisomy 15 with various microsatellite markers (Tabie 2). Additional retrospective FISH investigation on astored STC villi slide confinned the absence of trisomy IS ceus (TabIe I). Analysis of DNA from.nother stored STC villi slide confinned maternal heterodisomy IS in the first trimester chorionic villi (Tabie 2). Case 2. \Vith the fellow-up protocol for linusual tiïsomy in STC villi, case 2 was prenatally identified. This case has been extensively reported by Van Opstal et al. (l997a). In short: afier prenatal diagnosis becallse ofadvanced matemal age (40 years) a CPM ofnon-mosaic trisomy 16 was established with matemal heterodisomy 16 in ananiotic f1uid cells (TabIe land 2). A normal girl was hom at term with a normal birthweight. 183

184 Tablc 2. Alleles of the investigated polymorphic microsatcllite markers in the three UPD cases Case Marker Localisation Fathcr Mother CV AF cellsl UPD Iymphocytes LS6-1 15qll-ql2 2,2 1,3 1,3 Mat H OABRp3 15qll-ql2 1,2 2,3 2,3? CVPI9 15q2LI 1,2 3,3 3,3 3,3 Mat? ACTC 15ql3-qler 2,3 1,4 1,4 1,4 Mat H DI5S87 15q25-qter 1,3 1,2 1,2 1,2? 2 HBAI 16p13.3 1,3 2,2 2,2,3 2,2 Mat? DI6S298 16p12_1 1,2 3,4 3,4 Mat H DI6S261 16q12.1 2,3 1,1 1,1,2 1,1 Mat? DI6S285 16q12.1 1,3 1,2 1,2,3 1,2? DI6S308 16q12.1 1,2 2,3 2,3? D16S305 16q24.3 3,3 1,2 1,2,3 1,2 Mat H 3 3'HVR 16p13.3 1,4 2,3 3,3 Mat K08 16p13.3 1,3 2,3 2,2 2,2 Mat D16S291 16p13.3 1,2 3,4 4,4 4,4 Mat HBAI 16p13.3 1,4 2,3 2,2 Mat D16S404 16p13.1 1,4 2,3 3,3 Mat [ D16S285 16q12.1 1,4 2,3 2,3 Mat H DI6S415 16q12.1 1,3 2,4 2,4 Mat H DI6S320 16ql3 2,2 1,3 1,3 Mat H DI6S503 16q21 3,3 1,2 1,2 Mat H DI6S515 16q22.3-q23. I 1,4 2,3 2,3 Mat H DI6S422 16q24.2 2,3 1,2 1,1 Mat DI6S305 16q24.3 1,3 2,4 2,2 Mat CV = chorionic villi; AF = anmiotic fluid; Mat = matemal; H = hcterodisomy; 1= isodisomy;? - inconclusive. Case 3, In the third case, maternal iso/heterodisomy 16 was accidentally encountered durillg prenatal DNA atlalysis in a pregnancy of a TSC affected mother. Marker analysis silowed the abscence of paternal alleles and the presence of one type of maternal alleles only (Tabie 2). A deletion of the 16pl3.3 region was exc\uded with FISH by showing the presence of signals of the probes CW9D and CW23 on botll chromosomes 16. Extended chromosome 16 marker analysis showed alternate regions of maternal iso- and heterodisomy; fortunately, the isodisomic parts comprised the normal region of 16p13.3, leaving the fetus unaffected with TSC Crable 2). The katyotype in STC villi was 46,XY; additional FISH studies revealed a signal distribution compatible with disomy 16 (Tabie I). Ultrasound investigation at 19 weeks of ge station did not show fetal abnonnalities. However, the second half of pregnancy was complicated by intrauterine growth retardation and pregnancy induced hypertension. At 37 weeks, a boy was delivered by cesarean section with a birthweight of 1850 g «5th percentile) without congellital malfonnations. Af ter bîiih the placenta was received for further investigation. FISH on STC and LTC villi of 10 different placental biopsies showed signal distributions of the 16 centromere probe phur195 within the range of normal control samples. 184

185 DisclIssion UPD ean be purely heterodisomic, combined hetero/isodisomic or purely isodisomic, dependent on the meiotic division in which the non-disjunctional error occurred and the extent of cross-over hetween the holllologues of the chromosome pair involved (Engel, 1980; Engel & DeLozier-Blanchet, 1991). Heterodisomy can be without consequences, but isodisomy of (parts of) chromosomes may lead to autosomal reeessive disorders (Engel, 1993; Ledbetter & Engel, 1995). However, heterodisomy results in autsomal dominant disease when the parent colltributing bath chromosomes is affected. In Dur third case, the fetus was saved from beil1g affected with TSC because the TSC2 locus of the 110ll11aI chromosome 16 was contained in a region of isodisomy. Irrespective of the heterodisomic and isodisomic component, UPD causes developmental disturbances when imprinted regions present on some clrromosomes are involved (Bennet et al., 1992; Engel, 1993; Ledbetter & Engel, 1995). It is believed that trisomic zygote rescue rcsulting in fetal UPD is associated with CPM, espeeially CPM type land 3 involving non-mosaic trisomies (Ledbetter & Engel, 1995; \Voistenholme, 1996). In our three cases of UPD, prenatal cytogenetic investigations in chorionic villi revealed only one case with CPM and two cases whh completely normal karyotypes, suggesting that a normal karyotype in choriollic villi lllight IlOt be all exception in UPD. Prcnatal cases of generalized mosaicism ofvarious trisomies with UPD in the disomic celllines (HalTison et al., 1995; Christian et al., 1995; Van den Berg et al., 1997) as weil as postnatally established mosaic cases of autosomal and sex chromosomal aneuploidy in whieh the mosaicislll is due to toss of one of the clrromosomes (Niikawa & Kajii, 1984; Robinson et al., 1995) further suggest that trisomic zygote rescue ll1ight not always be a successful event. The very high rate of mosaic aneuploidy in 2- to 8-cell stage hunmn embryo's (Munné et al., 1994; Almeida & Bolton, 1996; Kligman et al., 1996) indieates that trisomic zygote reseue l11ight be a rather conunon phenoll1enon. The exact mechanism of trisolnic zygote rescue is not known; anaphase lagging or nondisjunction in an early postzygotic ceu division has been proposed (Kalousek et al., 1991). Since correction by anaphase lagging (AL) \Vill result in one disomic and one trisomic daughter cel!, this type of trisoinic corrcction seems not to he perfect. Correction by noildisjunetion (ND) will result in one viabie disomic and one lethal quadrisomie cell and reduce the number of eells in the embryo whieh might delay normal development (Tarin et al., 1992). Therefore, we propose an alternative correction mode, chromosome demolition (CD), as a process of deliberate fragmentation andjor removal of one of the set of tlrree clrrornosomes during metaphase or anaphase resulting in two disomic daughter cells. \Vith each of these correction modes (AL, ND and CD), we would like to present a model for the arising of the "adous combinations of karyotypes in STC villi, L TC villi, and fetus from trisomic zygote rescue. \Ve consider trisomic zygote rescue to consist out of one correction event in the first to fourth postzygotic ceil division with a subsequently unknown (random or non-random) distribution of trisomic and disomic cells among the progenitor cells of the inner eeu mass (lcm) and throphoblast eompartment untill the 16-eell stage (fig. 1). In order to show all possible combinations of karyotypes, the data are presented linder a random distribution (fig. 2 and 3). In their embryogenie model, Crane and Cheung (1988) and Bianehi et al. (1993) assume eells to lose their omnipotentiality after the 8-cell stage, and the lcm to contain 16 eells at the 64-eell (blastoe)'st) stage; four eells will form the fetus proper and 12 eells the extra-embryonic mesoderm (EEM). 185

186 Figurc l.trisomie zygote reseuc by cllromosome demolition (CD), noil-dlsjunetion (ND), and anaphase Jagging (AL). ODe example of a distribution of trisomie and disonlie eells among fetus, extra-embryonlc mesoderm (EEM), and trophoblast is shown for caeh eorreetion mode in the 1,1 anl! yd eell division. (A) CD eorreetlon, 1" eell dh'isiodj (8) ND eorrectioll, 1,1 eell di\'isionj (C) AL correeuon, lol eell di\'isiodj (D) CD eorrection, y d eell dh'isionj (E) ND correeuon, 3,d cell divisionj (F) AL eorreetioll, 3 rd eell di\'ision. Cdl dhi<ion Cd! di,i!ioo CD CD A D NO ND n E AL AL Besides these assumptions. we assume {hat within the lcm, cells Can allocate one or both daughter cells freely to the compartment of the fetus proper and/or that of the EEM after the irreversible separation of lcm and trophoblast compartment at the 16-cell stage. Furthennore we assume that in case of a reduced number of eells by ND eorrection, compensatory reallocation may oceur between the trophoblast and lcm eompartment (untill the 16-cell stage), eomparable to the situation after preimplantation diagnosis (Tarin et al, 1992). 186

187 Figurc 2. Theoretical dlsfributions of trisomie and dlsomie eells among the inner eell mass (lcm) and trophoblast compartmellt at the t6-eell stage after trisomic zygote reseue by chromosome demolition (CD) or anaphase lagging (AL). The resulting karyotypes in fetns and cuuured (LTC) "iiii, originafing from thc lcm, and in (semi-) direct (STC) "ilh, originating from the trophoblast, are shown. EEM = extraembryonic mesoderm; N = normalj Abn = abnormalj ihos = niosaicism. I = example shown in figure IA; 1 = cxample shown in figurc IC; J "" example showll in figure ID; ~ = example shown in figure IF. Cd! Corre.:tioo IDI ((~tu, + r:e),{j ReluJting TrophoN!l.11 Resulling dr..isk'n mok cdj, J...aryotypes cdjs karyotype (al 16-<:dllla;;e) Fems LTC,il!î (at 16-<:ell %Ige) STC\"Hli " CD 0000 N N 0000 N N ml88 ~Io, 0000 N,Mos ~1{l5 ~Io, mess AL Ab" N CD... Ab" NI , 00.. N,Mos,Abn Mos m888 Mo, 00.. Mo<;,Abn Mos Mos N AM Ab" Mos " '" 0000 N N mm AM 0000 N,Mos Mos mm Mos J... Ab" AL Ab" N 00.. N,Mos,Abn ~Ios mm Mos CD 00.. Mo,,Abo Mo' m~ Mos N AM... Ab" Aoo ml88 Mo, 00.. N,Mo'.Abn Mo, mm Aoo' AL '" 00.. ~lo,.abn ~Ios mm Mos IV CD N Ab" A"" ~Io;; mm.,.. 00 ~f05,abn M,,, mm Am IV AL N A"",\bn Ab" mm Mo~ I 0 disomie trucmic I TriSOlllic zygote rescue by CD and ND correction can explain all prenatally encountered combinations of karyotypes in the trophoblast, EEM, and fetal compartment in fetal UPD or UPD in the disomic cellline in case of generalized mosaicism. AL correction cannot produce the important combination of UPD with normal karyotypes in all compartments, and seems not to be the mechanism of first choice in trisomic zygote rescue. In our opinion, CD correction is the preferable mechanism at least in the first two eell divisions since it will not result in a critical diminished number of eells in the embryo. From the third cell division onwards, there is not mueh difference between the duee types of correction. Among the theoretical combinations of karyotypes in the variolis colllpartments described by Pittalis et al (1994), CPM type 2 does not occur in our model, eonfirllling otller statements with respect to the absence of an association between CPM type 2 and UPD (Bianchi et al., 1993; 187

188 Figure 3. Theoretical distributions of trisomic and disomlc cells among the inner ceu mass (lcm) and trophoblast coolpartment af the 16-cell stage afler trisomic zygote rcscue by non-disjunctlon (ND) wuh and \\ithout compensatory reallocation. The resulting karyotypes in fetus and cultureel (LTC) villi, originating from the lcm, and (semi-) direct (STC) villi, originating from the trophoblast are sbown. EEM = extra-elllbryonic mesoderm; N = normalj Abn = abnormalj Mos = mosaicism. I = example shown in figure lbj 2 = example shown in figure IE. Cel! COITl'~tion lcm (f~tus + EE~I) Resuhing TfOphobJast Resulring dr.ision m"'",dis J,.aJ)"OI)p;, rel" kaiyol)'j'.' (at 16-w!l stage) re"" LTC\;!Il (al 16-<xIl5tJ.g~) STCvilli NO 0000 N N 0000 N N o 00000o Am NI 0000 N,Mos ~fos lil!! Mos 11 NU 00.. N.Mo,,Abn Mos 1118 Mos 00" Mo,,Abn Mos ma Mos N Am... Ab" AM 1188 Mos Ab" N 000 N N /I'N, M" 000 N,Mos Mos NIfb Mos 11 NO - Ab" N OH Mos,Abn ~fo' ln9, Mos N AM Ab" AM Ni/9, Mos 00 N N l1li8 Mo, 11 No 00 N AM lm8 Mo, Mos ~Ios Ab" N.. Ab" AM M" 00" N~\los,Abn Mo,... AM' 111 NO 00.. Mos,Abn ~Ios aam Mos N Am... Ab" AM l1li8 Mos 000 N,Mos Mo> NIM AM - AM N 111 NO OH Mos,Abn Mo, I'IIIN> Mos N AM AM Ab" NN!ib Mos 00 N N man Ab" 111 NO.. 00 N AM mm Mos Mos Mos Ab" N Abo Am II1II8 Mos -OH Mo,,Abn Mos... Alm N AM Ab" AM IV NO mm Mo, 00.. Mo~,Abn Mos NIM Am... Ab" N AM I 0 disomi~ trisomic 1 AM I'IIIN> Mos i88

189 Walstenholrne, 1996). The opposite situation of abnormal karyotypes in fetus and STC villi with a normal karyotype in LTC villi, designated generalized mosaicism confmed culture normality (GMDC) does occur in Dur model. In their series, GMDC was not obscrved and considered to be very rare (Pittalis et al., 1994). Sa, the distribution of chromosomally nonnal and abllormal eells among the vadaus compartments after trisomic zygote rescue secms not to be random but directed towards a rankorder in the compartmcntalization of the fetai. EEM, and trophoblast compal1mcllts, respectively, with the maximal number of normal eells. Corrcction in the first cell division with normal karyotypes in all compartments and biparental inheritance ofall chromsomes is the best result oftrisomic zygote rescue and is ulllloticed. Acknowledgements We are grateful to Professor H. Galjaard for his support aud to Mrs. J. du Parant for preparing the mamlscript. Refercnces Almeida, P.A., Solton, V.N. (1996). 1l1C relationship betwecn chromosomal ablloffimlity in the human preimplantation embryo nnd developmcllt in vitro, Reprod FertiI Dev, 8, Bennet, p" Vaugban, J., Henderson, D., Loughna, S., Moore, G., (1992). Association between confilled placental trisomy, fetal uniparental disomy, nnd early intrauterine growth retardation, Lancet, 340, ianchi, O.W., Wilkins-Haug, L.E., Enders, A.C., Hay, E.D. (1993). Origin of exlraembryonic mesoderm in experimentai animais: relevanee to chorionic Illosaicism in humans, Am J Med Gel/el, 33, , Cassidy, S.8., Lai, L.W., Erickson, R.P., Magnuson, L" TIwmas, E., Gendron, R., Hermmnn, J, (1992). Trisomy 15 with 105S of the patemal 15 as a cause of Prader-Willi syndrome due to matemal disollly, Am J Hum Genet, 51, Choo, K.H., EarIe, E., Vissel, B., Filby, R.G. (l990). Identification oftwo distinct subfamilies ofalpha satejiite DNA that are highly specific for human chromosome 15, Gel/oll/fes,7, Christinn, S.L., Smith, A.C.M., Macha, M., Black, S.H., Elder, F.F.B., Johnson, J.M.P., Resta, R.G., Surti, U., Suslaki, L., Verp. M.S., Ledbetter, D.H. (l996). Prenatal diagnosis ofuniparentai disomy 15 following trisollly 15 mosaicislll, Prenat Dfagn, 16, Crane, J.P" Cheung, S.W. (1988), An embryogenie model 10 expiain cytogenetic inconsis-tencies observed in chorionic villi versus fetai tissue, Prenat Dfagll, 8, De Pater, J.M., Schuring-Blom, G.H" Van den Bogaard, R., Van der Sijs-Bos, C.J.M" Christiaens, a.c.m.l., Sloutenbeek, PH., Leschol, N.J. (1997). Maternal uniparental disomy ror chromosome 22 in a child with generalized mosaicism ror trisomy 22, Prenat Dfagn, 17, Engel, E. (1980). A new genetic concept: uniparental disomy and its potential effect, isodisomy, Am J Med Genet,6, Engel, E. (1993). Uniparental disomy revisited: the first twelve years, Am J Aled Genet, 46,

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