Chromosomal Microarray Analysis (CMA)

Chromosomal Microarray Analysis (CMA) Medical Genetics Laboratories Department of Molecular and Human Genetics Baylor College of Medicine

Table of Contents Overview of CMA Examples of Common Findings Examples of Mosaicism Examples of Complex Abnormalities Examples of Small Copy Number Variants CMA Comprehensive-CMA plus SNPS Resolving Variants of Uncertain Significance Prenatal CMA Considerations Prenatal CMA Case Examples Types of Cancer Arrays Conclusion

Overview of CMA

Chromosomal Microarray Analysis CMA is an array-based comparative genomic hybridization methodology that allows for analysis of the chromosomes for a large number of genetic disorders. With a single test, CMA can identify the abnormalities that are detectable by both routine chromosome analysis and FISH analysis. CMA has greater sensitivity than older methods of chromosome analysis.

Genomic Resolution Karyotype [4-5 Mb, whole genome] FISH [40 to 250 kb per probe, single site] CMA [average resolution ~30kb, whole genome]

Limits of Resolution Chromosome vs. Array CGH Analysis Chromosome 1 Limits of detection for G-banded chromosome analysis is 4-5 Mb 4 Mb region of microarray data showing a 1 Mb deletion encompassing ~70 oligos

What is included? Whole genome copy number analysis Coverage is more dense at genomic sites associated with known genetic conditions Currently 180,000 to 400,000 probes (oligos) covering the genome (depending on version) Exon coverage of 1700-4400 disease-associated genes (depending on version) Pericentromeric regions useful for detecting marker chromosomes Subtelomeric regions Backbone coverage (30kb resolution) Verification of results by FISH analysis and/or partial karyotype when indicated Parental studies to determine if observed copy number changes are inherited or de novo

CMA Process [A] Experimental Procedure [B] Laser Scanner Patient Control [D] Array Profile Hybridization of genomic DNA to the array of small DNA fragments (oligo probes) Mix [C] Actual Array Laser Scanner Duplication Deletion Shinawi, M. and Cheung, S.W. (2008) The array CGH and its clinical applications. Drug discovery today, 13, 760-70.

1. Evolution of CMA - increasing pixels! Feb 2004 July 2005 Nov 2006 Mar 2007 Feb 2008 June 2009 VERSION V4 BAC V5 BAC V6 BAC V6 OLIGO V7 OLIGO V8 OLIGO 180K Interrogating probes 366 BACs 853 BACs 1475 BACs 44K oligos 105K oligos oligos 420 genes >1700 genes Genomic disorders 40 75 >140 >140 (+61 regions *) (exons) Subtelomeric regions 41 41 41 41 41 41 # of clones / subtel ~ 4 ~10 ~10 ~10 (x20) NA NA Coverage per subtel ~ 5 Mb ~ 10 Mb ~ 10 Mb ~ 10 Mb NA NA Pericentromeric regions none 43 43 43 43 43 # clone / region ~3 ~ 3-5 ~ 3-5 ~3-5 (x20) NA NA 1 clone per 1 clone per Backbone coverage NA NA 1 band 1 band 30 kb 30 kb LCR regions 290 290 Design Improvement FISH test + subtel More Disorders + subtel + pericentr More disorders BAC clones + each chr oligo band emulation More More Disorders Disorders mito + mito + exon + LCR regions coverage Detection Rate 6.50% 9.04% ~12% ~12.5% ~15.4% TBD

N >46,000 Evolution of CMA - increasing pixels! Whole Genome Coverage June 2009 Oct 2010 July 2011 July 2011 2012 V8.0 OLIGO V 8.3 OLIGO V9.0 OLIGO VERSION V8 OLIGO +SNP Screen V 8.1.1 OLIGO +SNP Screen +SNP Screen Interrogating probes 180K Oligos 400K Oligos 180K Oligos 400K Oligos 400K Oligos >1700 genes >1700 genes 1779 genes 1936 genes 4864 genes Genomic disorders Exon coverage Exon coverage Exon coverage Exon coverage Exon coverage HG18 HG18 HG19 HG19 HG19 Subtelomeric regions 41 41 41 41 41 Pericentromeric regions 43 43 43 43 43 Backbone coverage 30 kb 30 kb 30 kb 30 kb 30 kb LCR regions 290 290 290 290 290 Design Improvement More Disorders More Disorders Mito Mito + exon + exon coverage coverage + 120K SNP More Disorders More Disorders More Disorders Mito Mito Mito + exon coverage + exon coverage + exon coverage + Additional 79 Additional 200 Additional 2500 genes genes +120K SNP genes +120K SNP 38 non-coding regulatory elements

Advantages of CMA Screens for a large number of disorders simultaneously Detects conditions that are difficult to identify clinically Atypical or mild phenotypes; e.g., VCFS/DiGeorge Conditions that lack distinctive features Detects deletions and duplications simultaneously Detects submicroscopic unbalanced chromosome rearrangements Detects mosaicism (as low as 10%) Cheung et al. (2007) Am J Med Genet A. (15):1679-86 Detects interstitial subtelomeric deletions/ duplications

Limitations of CMA Does not detect balanced translocations, inversions, low level mosaicism, point mutations Can detect copy number variants (CNVs) of unknown clinical significance Most are easily resolved by parental studies, however, clinicians should carefully evaluate parental phenotypes and developmental histories so data can be appropriately interpreted. CNVs <500 kb containing no identified genes at time of analysis are not reported CNVs >300 kb containing genes, even if clinical significance is unknown, are reported CNVs <300 kb must contain a gene known to be associated with disease in order to be reported

Examples of Common Findings

Normal Result

Trisomy 21 Chromosome 21-specific plot

DiGeorge Syndrome / VCFS Top: Whole genome view of CMA data Bottom: Left: FISH confirmation Inset: Partial karyotype of chromosome 22 (arrow points to deleted 22) Right: Chromosome 22-specific oligo plot of CMA data.

DiGeorge Syndrome / VCFS del 22q11.2

Microduplication 22q11.2 Syndrome (3 Mb ) Duplication of the DiGeorge / Velocardiofacial Syndrome region on chromosome 22q11.2

Microduplication 22q11.2 Syndrome (3 Mb ) Chromosome 22 Partial karyotype of chromosome 22 (arrow indicates duplicated 22) FISH confirmation 3 red signals indicates duplication

Example of Mosaicism

Mosaicism Example Indication Microcephaly, congenital vertical talus arr 18q21.2q23(47898780-76103255)x1.nuc ish 18q21.2(RP11-25O3x1)[45/200]dn Chromosomal Microarray Analysis revealed an approximately 28.2 Mb LOSS in copy number in the distal and subtelomeric regions of the long arm of chromosome 18 suggestive of mosaicism. This deletion includes the critical region of chromosome 18q deletion syndrome (OMIM 601808). FISH analysis and partial chromosome analysis revealed mosaicism for a deletion in the long arm of one chromosome 18 in 22% (45/200) of interphase cells examined. The remaining 78% (155/200) of cells showed a normal hybridization pattern. UPDATE: Parental FISH analysis with the above clone showed no evidence of the same LOSS in the father (KCL144199). UPDATE: Parental FISH analysis with the above clone showed no evidence of the same LOSS in the mother (KCL 146866). Therefore, this result most likely represents a de novo event. Genetic counseling is warranted.

Mosaicism (contin d) Indication Microcephaly, congenital vertical talus

Examples of Complex Abnormalities

Complex X Chromosome Abnormality

Complex X Chromosome Abnormality Chromosome X-specific plot

Complex Chromosome 1 Abnormality A Chr 1 BCM array B Nimblegen 2.1 TAR region duplication 1q41q42 microdeletion syndrome region not duplicated 3.1 Mb (dup) 1q42.12 221,378,468 224,471,629 4.4 Mb (dup) 1q42.3q43 232,592,278 236,988,147 C D van der Woude E 4.7 Mb (del) 1q32.2 205,060,076 209,804,762 F 0.3 Mb nml AKT3 not deleted G 1.8 Mb (del) del 1q43 237,337,843 239,105,025 RP11-279E18 D1Z1 RP11-339I11 D1Z1 RP11-478H16 RP5-1090A23 RP11-478H16 RP5-1090A23

Complex Chromosome 1 Abnormality A. CMA identified a complex rearrangement including two gains and two losses in chromosome 1q. B. High density array CGH using Nimblegen 2.1M array showed a 0.3 Mb single copy sequence between the distal duplication and deletion. C. Chromosome analysis showed that the gained materials (red arrow) due to the duplications were translocated into the 1q32 region. D-G. FISH analyses confirmed the deletions in 1q32.2 (D) and 1q43 (E) and the two copy number gains (F). In addition, FISH analysis on metaphase cells (G) suggested an inversion between the two regions with copy number gains and showed the duplicated segments of 1q42.12 and 1q42.3q43 were located next to each other as indicated by a white arrow. Liu et al. Cell In Press

Evaluation for a Marker chromosome

Evaluation for a Marker Chromosome Chromosome 1-specific plot marker CMA identified that the marker chromosome originated from chromosome 1.

Examples of Small Copy Number Variants (including exon deletions)

Deletion of an Exon of ERBB4 on 2q34 INTERPRETATION OF RESULTS: Chromosomal Microarray Analysis revealed a LOSS in copy number in the distal long arm of chromosome 2, spanning a minimum of 0.229 Mb and a maximum of 0.296 Mb. This deletion disrupts the ERBB4 (erythroblastic leukemia viral oncogene homolog 4) gene. A recent publication describes haploinsufficiency of ERBB4 gene in a patient with early myoclonic encephalopathy and profound psychomotor delay (Eur J Hum Genetics. 2009 Mar 17(3):378-82). Clinical correlation is recommended and genetic counseling is warranted.

Deletion of an Exon of ERBB4 on 2q34 Whole chromosome 2

Deletion of Exons of EP300 on 22q13.32 Figure 3 A. Profile of the microarray analysis showing the deleted region as indicated in the red circle [the gain on Xp as shown in green dots is also present in the mother (data not shown)]; B. The deleted oligos displayed in the UCSC genome browser corresponds to the exons of the CREBBP gene. C. The MLPA profile demonstrated copy number changes in the 2 exons of the CREBBP gene (exons 27-28). D. The deletion profile is present in the child but not in the parents indicating the deletion is de novo in origin.

Deletion of Exons of EP300 on 22q13.32 A B C CREBBP exons 27 28 deletion 27 28

CMA Comprehensive CMA +SNP

400 K CMA comprehensive showing normal CMA with a paternal UPD15 (Isodisomy) Chromosome 15 Whole genome Copy number plot Agilent-SNP In house array Agilent-SNP Genome Workbench Illumina

CMA Comprehensive (180K + SNP Screen) Normal CMA profile AOH observed in close relative mating (example : father and daughter mating)

Resolving Variants of Uncertain Significance

Examples of Clone Plots Current case Nonpolymorphic clone plot Current case Polymorphic clone plot Highly unusual Common variant

Resolving Variants of Uncertain Significance Mother Father Fetus Chr 12 CNV (paternal) Trisomy 21

Incidental finding [from the father while absent from the fetus] Proband Mother Father

Prenatal CMA Considerations

Why Consider Prenatal CMA? Combined incidence of known microdeletion/ duplication syndromes is at least ~1/1000 Many are not detected on standard chromosome analysis, especially in prenatal samples with lower resolution Many have moderate to severe phenotypes after birth, but no prenatal signs that would raise suspicion and trigger specific FISH testing Women of all ages are equally likely to have affected pregnancies No screening tools have been developed for microdeletion / microduplication disorders

Baylor Prenatal CMA Clinical Protocol Parental samples required for testing Informed consent strongly recommended Entire sample can be sent to Baylor (30+cc amniotic fluid {>16 wk gestation} or 30+ CVS) for routine cytogenetics and CMA OR direct sample may be split (15cc amniotic fluid or 15 mg dcvs) can be sent and remainder sent to another lab for routine cytogenetics. Direct CMA testing on direct CVS or direct amniotic fluid with (collected >16 weeks gestation) with culture in reserve. Maternal cell contamination studies Reporting in 7-10 days from direct sample

Pre-test discussion A pre-test discussion should include: How the testing works What is being tested for Possible test results Benefits of testing Limitations/risks of testing Assessment of the individual s understanding of the testing Assessment of parental clinical and developmental history

Possible Test Results No abnormality detected No gain or loss of chromosomal material was detected in the regions tested A gain or loss was detected that is known / expected to be benign (i.e. does not cause disease) Abnormality detected A gain or loss of chromosomal material known to result in a defined genetic condition has been detected Results of uncertain significance A gain or loss of chromosomal material not known to result in a defined genetic condition has been detected This means that a change was found, but there is little or no medical knowledge about the particular change. Whether the change may lead to medical problems and what types of problems it may cause is uncertain. In this case additional testing is performed, including analysis of DNA from the parents.

Benefits of CMA testing CMA testing may discover an abnormality that may not have been detected by routine chromosome testing. The information gained from CMA testing may be important for making decisions about the pregnancy or for making medical decisions about the baby s care after delivery.

Limitations of CMA testing Detection rates It is possible that the baby could have one of the medical conditions included in the CMA test, but the CMA test was unable to detect the condition. For some conditions included in the CMA test, 99% of cases can be detected. For others, the detection rate may be lower because they can have multiple underlying causes. Findings of uncertain significance It is possible that the test will detect an abnormality for which there is very little medical information available to predict the type of problems that may develop in the baby. Expectant parents may be left with ambiguity and this may increase their anxiety about the pregnancy Need for further testing and impact on family members As with any genetic test, results may indicate a need for further testing and may also impact other family members.

Assessment of Patient Understanding Patients should understand that: CMA DOES NOT test for ALL genetic conditions Detection rates are not 100% Even if the results are normal, the baby could still have a birth defect(s) or mental retardation from causes not detected by the CMA testing

Prenatal CMA Case Examples

Case 1 CVS (13 weeks) Indication: AMA, Abnormal ultrasound (nuchal thickening) and Normal chromosome analysis referred to us for CMA 8.7 Mb loss in 4q21.23q22.1 Copy number loss of this region is associated with CNS overgrowth, facial anomalies, hypotonia and developmental delay [PMID 9098490]

Case 1 CVS (13 weeks) Confirmation of the deletion by FISH. In retrospect, perhaps karyotype shows deletion. Deleted chromosome Deleted chromosome

Case 2 Indication: AMA, parental concern Duplication detected at 17p11.2 involving RAI1. Duplication of RAI1 has been associated with Potocki-Lupski syndrome. FISH using the probe FLI in green used in the clinical laboratory confirmed an additional copy of RAI1. The control probe PMP22 in red shows the expected 2 signals.

Case 3: Origin of Marker Chromosome Indication: Karyotype analysis performed at another laboratory showed a supernumerary small marker chromosome in 5 of 18 cells (28%). FISH studies with probes specific for chromosomes 13, 14, 15, 18, 21, 22, X and Y were unable to identify the origin of the marker. CMA detected a 10 Mb (maximum 19 Mb) gain in copy number on the short arm of chromosome 20. Metaphase FISH analysis confirmed that the marker chromosome was an isochromosome 20p resulting in tetrasomy for 20p, and was observed in 20% (4/20) of the cells examined.

Case 3: Origin of Marker Chromosome Mar 20 20

Types of Cancer arrays HEME-ONC Array(44K) 180K CGH/SNP Cancer Array (CCMC Design) BCM 400K CGH/SNP Cancer Array

Utility of Heme-Onc CMA First cancer gene targeted oligonucleotide microarray for genome profiling of hematological malignancies at a high resolution. Heme-Onc CMA is for: Acute leukemias CLL Multiple myeloma Lymphomas Myelodysplastic syndrome (MDS) Higher sensitivity for detection of abnormalities in CLL than the current FISH panel

Heme-Onc Array Design Guideline Even Distribution Target Genes Implicated in Cancer V.1.0 10X X Targeted Regions: Selected Genes (494) Regions Implicated in Leukemia Excluding Regions: Repetitive Elements Low Copy Repeats Assembly Gaps Copy Number Polymorphism (TCAG V1+UCSC)

Heme-Onc Array Design Example Gene 10X Backbone Regions X

Resolution of the Heme-Onc Array 494 genes 1 OLIGO per 7.5 kb Backbone region 1 OLIGO per 78 kb

Case 1: Indication CLL 1. The initial chromosome study showed an abnormal clone with deleted chromosome 11q and deleted chromosome 13q in 10% of the cells examined. 46,XY,del(11)(q13q23),del(13)(q14q22)[3]/46,XY[27] 2. These findings were consistent with the CMA results (blue circles) 3. FISH confirmation showed that the deletions are present in >40% of cells. 4. CMA also detected additional findings as indicated in the pink box.

Case 1: Indication CLL 2p16 gain 42% deleted by FISH 45% deleted by FISH

Case 2: Indication CLL 1. Initial FISH results using CLL FISH panel nuc ish(p53x1)[27/500] 5% nuc ish(d13s319x0)[113/500] 27% nuc ish(cep12x3)[119/500] 27% 2. These findings were consistent with the CMA results (blue circles) 3. CMA also detected additional findings as indicated in the pink boxes

Case 2: Indication CLL dup15q dup 11q del 9p

Case 3: Indication MDS 1. Initial chromosome analysis detected two abnormal clones 47,XX,inv(3)(q21q26),+mar[11]/46,idem,-7[9] 2. CMA detected a loss of chromosome 7 (red circle) except for the pericentromeric region (blue arrow). 3. Subsequent FISH analysis using a centromere probe for chromosome 7 confirmed the marker chromosome is derived from chromosome 7. 4. CMA is able to detect gain or loss of genomic material but not balanced rearrangements such as the inverted chromosome 3 present in this case.

Case 3: Origin of the marker chromosome inv(3) marker 298857 56131915 Chr 7 95893139 mar derived from chr 7 158767840

Case 4: Indication MDS Right Panel: Initial chromosome analysis detected an abnormal clone: 46,XY,-7,+mar[11]/46,XY[10] Bottom Panel: CMA detected a gain of chromosome 3q and a loss and gain of chromosome 7q. chr 3 nl 7 marker

Case 4: CMA Results continued Chr 3 Chr 3 Chr 7 Marker chromosome is an isoderivative 7 ider(7)(q22)t(3;7)(q25.3;q22)

Case 5: Indication-CLL- Homozygous loss on 13q14 A. CMA detected a LOSS of copy number (deletion) on chromosome 13. B. The chromosome 13-specific plot shows the coverage and the boundaries of the deleted segment. C. The size of the deleted segment and genes involved. D. The size of the segment within the deletion showing a homozygous LOSS and the genes involved.

Case 5: Indication-CLL- Homozygous loss on 13q14 A. B. C. Copy number LOSS on chromosome 13 D. Homozygous loss

Case 5: Indication-CLL- Homozygous loss on 13q14 Confirmation FISH analysis using the CLL FISH panel from Vysis E. E. 63% (313/500) cells had one signal for D13S319 (red) probe. F. F. 27% (146/500) cells had no signals (homozygous loss) for D13S319 probe localized to chromosome 13q14 consistent with the results from array CGH. 13q14 Red Signal 13q34 Aqua Signal 12cen Green Signal

180K CGH/SNP Cancer Array (CCMC Design)

180K CGH/SNP Cancer Array (CCMC Design) 44K 60K 105K 180K MLL DDX6 CBL2 Backbone Backbone 512 cancer genes or cancer-related genes Average of 2 probes per exon. Average resolutions <10 Kb (large exons) to <10 Kb (cancer genomic regions) in targeted regions. ~30 Kb in backbone regions.

Myelodysplastic Syndrome (180K CGH/SNP Array) 4 copies 3 copies 1 copy 0 copy 4 3 2 1 0 BBBB ABBB AAAB AAAA Myelodysplastic Syndrome Partial Chromosome 6p Amplification to 4 copies SNP data show 0, 1, 3, 4 copies

Unbalanced Balance Translocation Acute Myeloid Leukemia (180K CGH Array, CCMC Design) Red: D7S486 Green: D7Z1 3p21.3 1 2.7 Mb 3q21.3 1.6 Mb 3q26.2 87 Kb 12p13.3 1 2.7 Mb 57 Mb 36 Mb Red arrow: deletions; green arrow: duplication

BCM 400K CGH/SNP Cancer Array

BCM 400K CGH/SNP Cancer Array 400K MLL DDX6 Backbone Backbone 2,300 cancer genes or cancer-related genes 235 cancer associated-mirnas Average of 6 probes per exon. Average resolutions <1 Kb (large exons) to <10 Kb (cancer genomic regions) in targeted regions. ~12 Kb in backbone regions.

Myelodysplastic Syndrome (BCM 400K CGH/SNP Array) 3 copies Copy Gain LOH Triplication of one allele 1 copy Copy Loss LOH Homozygous deletion 3 copies Copy Gain LOH Triplication of one allele SNP CGH

Conclusion CMA is a powerful molecular cytogenetic tool for detecting genomic imbalances both in constitutional as well as in cancer diagnostics CMA is a high resolution technology capable of detecting chromosomal DNA copy number changes throughout the genome Baylor designed CMA detects chromosomal abnormalities that would go undetected by older techniques such as karyotype or non-exon focused arrays