Genetics, genomics and gene discovery in the auditory system

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1 # 2002 Oxford University Press Human Molecular Genetics, 2002, Vol. 11, No Genetics, genomics and gene discovery in the auditory system Cynthia C. Morton * Departments of Obstetrics, Gynecology, and Reproductive Biology and Pathology, Brigham and Women s Hospital and Harvard Medical School, Boston, MA, USA Received April 5, 2002; Accepted April 10, 2002 The sounds of silence have forever been broken as genetics and genomics approaches in human and model organisms have provided a powerful and rapid entry into gene discovery in the auditory system. An understanding of the complexities and beauty of the biological process of hearing itself is unfolding as genes underlying hereditary hearing impairment are identified. Genes involved in modifying hearing are also being found, and will be critical to a full comprehension of genotype phenotype relationships. Investigations in the auditory system will provide important insight into how the nervous system decodes molecular information. Deafness represents a common sensory disorder that can interfere dramatically in the acquisition of speech and language in children, and in the quality of life for a growing aged population. As newborn screening for hearing impairment is being implemented in many birth hospitals, the prospects for precise clinical diagnosis, appropriate genetic counseling and proper medical management for auditory disorders has never been at a more exciting crossroad. INTRODUCTION It has long been recognized that heredity plays a major role in hearing impairment. Despite the fact that understanding the genetic basis of hearing loss has fascinated human and medical geneticists for decades, only within the past few years have the genes and molecular mechanisms underlying deafness begun to be discovered. In part, this results from various obstacles to investigation of the auditory system including: inaccessibility of the sensory end organ for hearing, the cochlea, within the dense temporal bone; length of time to direct pathologic observation of the deaf ear due to an otherwise normal lifespan of individuals with hearing loss; unparalleled genetic heterogeneity; and assortative mating. The history of the genetics of deafness has had a sordid past of blatant discrimination of the deaf (1), and there is mistrust about genetics among some members of the deaf population. Nevertheless, the past few years have witnessed an explosion of discoveries that are providing fundamental insight into the biology of hearing. The frequency of hearing loss is estimated at one per thousand newborns and half of all cases are attributed to genetic causes. In addition to being a common etiology of congenital deafness, mutations in genes are responsible for progressive hearing loss, and no doubt will be found to play an important role in progressive hearing loss with ageing (presbycusis). Environmental etiologies of hearing loss are likely to represent a declining proportion of cases as better therapies for bacterial and viral infections (e.g. vaccines) are implemented, acoustic trauma in the workplace is recognized and prevented, and ototoxic drugs (e.g. aminoglycosides) are avoided in genetically susceptible individuals. GENETICS OF DEAFNESS The study of the genetics of deafness is unique among inherited disorders for several reasons and illustrates well various concepts in human genetics. Notably, there is incomparable genetic heterogeneity with over 90% of matings among the deaf resulting in all hearing offspring. This reflects matings among the deaf with mutations in different genes as well as matings of couples in which one individual is deaf due to a genetic mechanism and the other due to an environmental etiology. Matings among the deaf are well recognized and, with the exception of assortative mating for stature, may represent one of the most common genetic traits on which an altered mating structure occurs in human populations. Furthermore, the hearing offspring of deaf couples who themselves can be native signers are more likely than random members of the population to have a partner who is deaf due to a shared language and culture. Both genetic heterogeneity and assortative mating confound gene discovery using traditional methods of genetic linkage analysis. *To whom correspondence should be addressed at: Department of Pathology, Brigham and Women s Hospital, 75 Francis Street, Boston, MA 02115, USA. Tel: ; Fax: ; cmorton@partners.org

2 1230 Human Molecular Genetics, 2002, Vol. 11, No. 10 Table 1. Gene discovery in the auditory system No. Gene Protein or RNA Map position Syndromic or Disorders Reference nonsyndromic hearing loss 1 ATP6B1 2cen q13 SHL Distal renal tubular acidosis (76) associated with sensorineural deafness 2 BSND barttin 1p32.3 SHL Bartter syndrome (77) 3 CDH23 cadherin 23 10q21 q22 NSHL þ SHL Usher syndrome type 1D (10) (USH1D) Usher syndrome type 1D (11) (USH1D) þ DFNB12 4 CLDN14 claudin 14 21q22 NSHL DFNB29 (78) 5 COCH cochlin 14q12 q13 NSHL DFNA9 (22) 6 COL1A2 collagen type 1 a2 7q22.1 SHL Osteogenesis imperfecta (79) 7 COL2A1 collagen type 2 a1 12q13.1 q13.2 SHL Stickler syndrome (STL1) (80) 8 COL4A3 collagen type 4 a3 2q36 q37 SHL Alport syndrome (23) 9 COL4A4 collagen type 4 a4 2q36 q37 SHL Alport syndrome (23) 10 COL4A5 collagen type 4 a5 Xq22 SHL Alport syndrome (20) 11 COL11A1 collagen type 11 a1 1p21 SHL Stickler syndrome (STL2) (81) 12 COL11A2 collagen type 11 a2 6p21.3 NSHL þ SHL Stickler syndrome (STL3) (82) DFNA13 (83) 13 DDP Xq22 SHL Mohr-Tranebjaerg syndrome (16) 14 DFNA5 7p15 NSHL DFNA5 (19) 15 DIAPH1 diaphanous 5q31 NSHL DFNA1 (84) 16 DSPP dentin sialophosphoprotein 4q21.3 SHL Dentinogenesis imperfecta 1 (85) (DGI1), DFNA39 17 EDN3 endothelin 3 20q13.2 q13.3 SHL Waardenburg Shah syndrome (86) (WS4) 18 EDNRB endothelin receptor B 13q22 SHL Waardenburg Shah syndrome (87) (WS4) 19 EYA1 8q13.3 SHL Branchio-oto-renal syndrome (88) 20 EYA4 6q22 q23 NSHL DFNA10 (89) 21 GJA1 connexin 43 6q21 q23.2 NSHL Autosomal recessive deafness (67) 22 GJB2 connexin 26 13q12 NSHL DFNB1 (24) DFNA3 (25) 23 GJB3 connexin 31 1p34 NSHL DFNA2 (26) Autosomal recessive deafness (90) 24 GJB6 connexin 30 13q12 NSHL DFNA3 (66) 25 KCNE1 21q22.1 q22.2 SHL Jervell and Lange Nielsen (91,92) syndrome (JLNS2) 26 KCNQ4 1p34 NSHL DFNA2 (93) 27 KVLQT1 11p15.5 SHL Jervell and Lange-Nielsen (94) syndrome (JLNS1) 28 MITF 3p12.3 p14.1 SHL Waardenburg syndrome type II (28) (WS2) Tietz syndrome (95) 29 MYH9 myosin heavy chain 9 22q13 NSHL þ SHL May Hegglin and Fechtner (96) syndromes DFNA17 (97) 30 MYO6 myosin 6 6q13 NSHL DFNA22 (49) 31 MYO7A myosin 7A 11q12.3 q21 NSHL þ SHL Usher syndrome type 1B (7) (USH1B) DFNA11 (6) DFNB2 (8,9) Atypical Usher syndrome (98) 32 MYO15A myosin 15A 17p11.2 NSHL DFNB3 (99) 33 NDP norrin Xp11.3 SHL Norrie disease (14,100) 34 OTOF otoferlin 2p22 p23 NSHL DFNB9 (29) 35 PAX3 2q35 SHL Waardenburg syndrome type I (27) (WS1) Waardenburg syndrome types (101) I þ III (WS1 þ WS3) Craniofacial dysmorphism, (102) hand abnormalities, profound sensorineural deafness (CDHS) 36 PCDH15 protocadherin 15 10q21 q22 SHL Usher syndrome type 1F (103,104) (USH1F) 37 POU3F4 Xq21.1 NSHL DFN3 (105)

3 Human Molecular Genetics, 2002, Vol. 11, No Table 1. (Continued) 38 POU4F3 5q31 NSHL DFNA15 (69) 39 PMP22 peripheral myelin 17p11.2 SHL Charcot Marie Tooth disease (106) 40 12S rrna ribosomal RNA mitochondrial NSHL Sensorineural deafness (74) 41 SLC19A2 1q23.3 SHL Thiamine-responsive megaloblastic (107,108) anemia with diabetes mellitus and deafness 42 SLC26A4 pendrin 7q31; 7q21 q34 NSHL þ SHL Pendred syndrome (PDS) (17) DFNB4 (109) 43 SOX9 17q24.3 q25.1 SHL Campomelic dysplasia (110) 44 SOX10 22q13 SHL Waardenburg Shah syndrome (WS4) (111) 45 STRC stereocilin 15q21 q22 NSHL DFNB16 (31) 46 TCOF1 treacle 5q32 q33.1 SHL Treacher Collins syndrome (15) 47 TECTA alpha tectorin 11q22 q24 NSHL DFNA8, DFNA12 (21) DFNB21 (112) 48 TMC1 9q13 q21 NSHL DFNA36 (12) DFNB7/B11 49 TMPRSS3 21q22.3 NSHL DFNB8, DFNB10 (113) 50 trna-glu transfer RNA mitochondrial SHL Diabetes and deafness (114) 51 trna-leu transfer RNA mitochondrial SHL Myopathy, encephalopathy, lactic (115) acidosis and stroke-like episodes (MELAS) Diabetes mellitis and deafness (116) 52 trna-lys transfer RNA mitochondrial SHL Myoclonic epilepsy and ragged-red fiber (117) disease (MERRF) 53 trna-ser transfer RNA mitochondrial SHL Retinitis pigmentosum and progressive (118) sensorineural hearing loss 54 trna-ser(unc) transfer RNA mitochondrial NSHL þ SHL Sensorineural deafness (119) Progressive myoclonic epilepsy, ataxia (120) and hearing loss Palmoplantar keratoderma and deafness (121) 55 USH1C harmonin 11p15.1 SHL Usher syndrome type 1C (USH1C) (30) (122) 56 USH2A usherin 1q41 SHL Usher syndrome type 2A (USH2A) (18) 57 USH3 3q21 q25 SHL Usher syndrome type 3 (USH3) (123) 58 WFS1 wolframin 4p16 SHL Wolfram syndrome (124,125) NSHL DFNA6/A14 (126) DFNA38 (127) Hundreds of syndromic forms of deafness have been described (2 4) and the underlying genetic mutation identified for many of the more common forms, but only 30% of genetic cases are estimated to be part of a heritable syndrome. Thus, the vast majority of genetic deafness is designated as nonsyndromic and to date over 65 loci have been mapped. Nonsyndromic hearing impairment is categorized further by mode of inheritance: approximately 77% of cases are autosomal recessive; 22% autosomal dominant; 1% X-linked; and < 1% due to mitochondrial inheritance (5). Dominant loci are designated with the prefix DFNA, recessive loci DFNB, X-linked loci DFN and modifying loci with DFNM. Generally, patients with autosomal recessive hearing impairment have prelingual and congenital deafness and patients with autosomal dominant hearing impairment have postlingual and progressive hearing impairment. This observation may be explained by the complete absence of functional protein in recessive disorders, while in autosomal dominant disorders, dominant mutations may be consistent with initial function and subsequent hearing impairment due to a cumulative, degenerative process. A recent tally of nonsyndromic hearing loss disorders reveals 32 autosomal dominant, 27 autosomal recessive, and 4 X-linked forms (4). It remains to be shown whether each of these loci will correspond to a unique gene. In fact, various deafness disorders have been found already to be the result of the same genetic etiology (e.g. DFNA8 and A12; DFNA6, A14 and A38). In addition, at least 58 auditory genes have been identified: 16 for autosomal dominant and 11 for autosomal recessive loci, and 1 for an X-linked locus; 6 mitochondrial genes and at least 38 genes for syndromic hearing loss have also been discovered (n.b. some genes cause multiple forms of deafness) (Table 1). Although this magnitude of progress is remarkable and significant advances have been made, it is clear that many more genes for hearing await detection. Mutations within the same gene have been found to result in a variety of clinical phenotypes with different modes of inheritance. For example, mutations in MYO7A are pathogenetic in the autosomal recessive deafness and blindness disorder Usher syndrome type 1B (USH1B), and in two nonsyndromic hearing disorders, DFNB2 and DFNA11, displaying autosomal recessive and dominant segregation, respectively. It has been suggested that the mutation in DFNA11, a 9 bp deletion in the coiled-coil domain of MYO7A which is involved in dimerization, could have a dominant negative effect (6) as compared to splicing and missense mutations observed in recessive USH1B and DFNB2 (7 9). Another example of phenotypic heterogeneity also involving Usher syndrome is seen in mutations in CDH23 detected in USH1D and DFNB12 (10,11). Mutations in PDS cause Pendred syndrome and nonsyndromic autosomal recessive

4 1232 Human Molecular Genetics, 2002, Vol. 11, No. 10 hearing loss, DFNB4. Similarly, mutations in WFS1 cause Wolfram syndrome and also account for an autosomal dominant nonsyndromic deafness, DFNA6/A14/A38. Most recently, DFNA36 and DFNB7/B11 were determined to be the result of mutations in TMC1 (12). GENOMIC APPROACHES TO GENE DISCOVERY IN THE AUDITORY SYSTEM Traditional methods for mapping disease genes, such as genetic linkage analysis, have a less than totally optimal use in gene discovery efforts for hearing disorders, mainly because of the complex genetic nature of deafness. Successful use of genetic linkage for mapping hearing disorders, especially for autosomal recessive nonsyndromic loci, has been restricted largely to consanguineous kindreds or populations in which there has been limited immigration. Even in families in which a heritable hearing disorder is successfully mapped, there is often an insufficient number of recombination events to narrow a chromosomal interval, resulting in a candidate region consisting of megabases of genomic DNA. Positional cloning has been productive for a modest number of human deafness genes including NDP (13,14), TCOF1 (15), DDP (16), SLC26A4 (17), USH2A (18) and DFNA5 (19). Positional candidate genes from human (e.g. COL4A5 (20), TECTA (21), COCH (22), COL4A and 4A4 (23), GJB2 (24,25), GJB3 (26)) and mouse (e.g. PAX3 (27), MITF (28), OTOF (29), USH1C (30), STRC (31)) among others have been the primary method for gene identification. Organ and tissue-specific methods for auditory candidate genes A complementary method to genetic linkage analysis for gene identification is one that utilizes tissue or organ-specific cdna libraries to provide candidate genes (32 34). A transcript map of the inner ear provides a ready source of positional candidate genes for mutation screens in gene discovery efforts. To this end, cochlear cdna libraries constructed from human (35,36) and mouse (37) have provided precious biological tools for gene discovery in the mammalian cochlea. A cdna library from an analogous organ in chicken, the basilar papillae, has also been of great value (38). Almost human (Morton fetal cochlear cdna library) and 1600 mouse (Soares mouse NMIE cdna library) inner ear ESTs are currently available in GenBank and the sequences of an additional 9000 mouse ESTs will soon be deposited there. ESTs derived from two sequencing projects from human cochlear cdna clones (39,40) have elucidated thousands of potential positional candidate genes for hearing disorders (41) in addition to providing a snapshot of gene expression in the week gestational age fetal cochlea. BLAST analysis of 8153 human ESTs revealed that about 50% (n ¼ 4040) had sequence similarity to a total of 1449 known human genes. The most abundantly expressed gene was COL1A2, and two other collagens, COL3A1 and COL1A1, were among the most highly represented transcripts. In total, 10 different collagen genes were present in the cochlear ESTs. Forty-three human homologs of nonhuman mammalian genes were also identified, and among them are ESTs for membrane proteins, extracellular proteins and trafficking proteins. Of the remaining 4055 ESTs, Table 2. Web resources for genetic and genomic studies of the auditory system Web page name Summary of contents URL Connexin-deafness homepage Information on connexin mutations Corey lab Microarray expression data from mouse inner ear coreylab/index.html Harvard Medical School Center Resources for families, clinicians and hearing.harvard.edu for Hereditary Deafness researchers Hereditary hearing impairment in mice Results from a large scale mouse screening program Hereditary hearing loss homepage Syndromic and nonsyndromic disorders, mitochondrial disorders, otosclerosis Morton hearing research group Human cochlear ESTs summary and map hearing.bwh.harvard.edu locations MRC Institute of Hearing Research inner ear mutant table Mouse mutants with various inner ear defects MutantsTable.shtml MRC Institute of Hearing Research inner ear developmental gene expression table Developmental gene expression in the inner ear in a variety of species genetable/index.shtml National Institute on Deafness and Other Research from the Laboratory of Molecular Communication Disorders Genetics and other resources OMIM Human heritable disorders query.fcgi?db ¼ OMIM Otobase Repository of candidate genes for inner ear Hudspeth-sgi.rockefeller.edu disorders in human, mouse and zebrafish Tour de l oreille, Université Montpellier Anatomy, physiology and pathophysiology of the auditory system audition/english/start.htm Univ. of WI Dept. of Neurophysiology Hearing Educational and research resources and Balance Washington Univ. inner ear protein database Catalog of biochemical characterization of tissues and fluids of the inner ear oto.wustl.edu/thc/innerear2d.htm

5 Human Molecular Genetics, 2002, Vol. 11, No Table 3. Mouse hearing loss mutants and their human homologs Mouse mutant Gene Human disorder(s) References Ames waltzer (av) Pcdh15 Usher syndrome type 1F (USH1F) (103,104,128) Beethoven (Bth) and deafness (dn) Tmc1 DFNA36, DFNB7/B11 (12,50) chondrodysplasia(cho) Col11a1 Stickler syndrome type 2 (STL2) (81,129,130) Col1a1 transgene disruption Col1a1 Osteogenesis imperfecta (OI) ( ) Col11a2 targeted null Col11a2 Stickler syndrome type 3 (STL3), DFNA13 (82,83) Col4a3 targeted null Col4a3 Alport syndrome (23,135) Disproportionate micromelia (Dmm) Col2a1 Stickler syndrome type 1 (STL1) (80,136,137) and mutant transgenes Dominant megacolon (Dom) Sox10 Waardenburg Shah syndrome (WS4) (111,138,139) dominant spotting (W) Kit Piebald trait (PBT) ( ) Eya1 bor and targeted null Eya1 Branchio-oto-renal syndrome (BOR) (88,143,144) Fgfr3 targeted null Fgfr3 Craniosynostosis (145,146) Gata targeted null Gata3 Hypoparathyroidism, sensorineural deafness (147,148) and renal dysplasia syndrome (HDR) Kcne1 targeted null and Kcne1 pkr Kcne1 Jervell and Lange Nielsen syndrome (JLNS2) (91,92,149,150) Kcnq1 targeted null Kcnq1 Jervell and Lange Nielsen syndrome (JLNS1) (94,151,152) lethal spotting (ls) Edn3 Waardenburg Shah syndrome (WS4) (86,153) microphthalmia (mi) Mitf Waardenburg syndrome type 2 (WS2), (28,95,154,155) Tietz syndrome Ndph targeted null Ndph Norrie disease (ND) (13,14,156) Pax2 targeted null Pax2 Renal-coloboma syndrome (157,158) piebald (s) Ednrb Waardenburg Shah syndrome (WS4) (87,159) Pou3f4 targeted null and sex-linked fidget (slf) Pou3f4 DFN3 (105, ) Pou4f3 targeted null and dreidel (ddl) Pou4f3 DFNA15 (69,163,164) quivering (qv) Spnb4 Charcot-Marie-Tooth disease type 4F (CMT4F) (165) shaker-1 (sh1) Myo7a Usher syndrome type 1B (USH1B), DFNB2, (7,9,98,166,167) DFNA11, atypical Usher syndrome shaker-2 (sh2) Myo15a DFNB3 (99,168) Slc26a4 targeted null Slc26a4 Pendred syndrome (PDS), DFNB4 (17,109,169) Snell s waltzer (sv) Myo6 DFNA22 (48,49) splotch (Sp) Pax3 Waardenburg syndromes types 1 and 3 (WS1, WS3), (27,101,102,170,171) Craniofacial dysmorphism, hand abnormalities, profound sensorineural deafness (CDHS) Tecta targeted null Tecta DFNA8/A12, DFNB21 (21,112,172) Thrd targeted null Thrb Thyroid hormone resistance (173,174) tremble (Tr) Pmp22 Charcot-Marie-Tooth disease type 1A (CMT1A) (106,175,176) waltzer (v) Cdh23 Usher syndrome type 1D (USH1D), DFNB12 (10,11,177) 3277 had sequence similarity to other ESTs representing 2266 unique clusters; 778 categorized into 700 clusters had no sequence similarity to known genes or ESTs and can be considered to be cochlear-specific. Identification of additional known genes, ESTs and cochlear-specific ESTs provides new candidate genes for both syndromic and nonsyndromic deafness disorders. A variety of web resources have been developed for genetic and genomic studies of the auditory system that facilitate candidate gene approaches and are listed in Table 2. In addition to sequence-based approaches, gene expression chips provide an important means to explore the repertoire of cochlear messages in the normal and diseased state. Data from gene chip assays are available on the web for normal mouse cochlea for ages P2 and P32 (42). Several preferentially expressed cochlear genes, namely COCH (43) and OTOR (44,45), have been identified from the human fetal cochlear cdna library; COCH was further shown to be responsible for a sensorineural deafness and vestibular disorder, DFNA9 (22). Various genes have been identified from a similar approach using mouse inner ear transcripts and include Otog (37), Ocn95 (46), Ush1c (30), Fdp (mouse homolog of OTOR) (47) and Strc (31); the human homolog of Ush1c was found to underlie mutations in USH1C and of Strc to be etiologic in DFNB16. Mouse models for discovery of hearing genes Identification of mouse models of specific forms of deafness is of great interest as the mouse is clearly the model organism of choice for the study of hearing loss in humans. The mouse cochlea is highly similar in structure to that of the human. Studies of mouse mutants from fetal to adult ages makes possible investigation into the pathology at developmental time points simply not possible in humans. Of particular relevance to understanding the pathobiology and underlying molecular mechanisms of genetic mutations is the ability to evaluate early developmental stages in mouse mutants because hair cell defects may result from degenerative processes secondary to a primary abnormality in another cell type. Although there has been tremendous progress in identifying genes underlying deafness, there are still relatively few mouse models (Table 3). In some instances, identification of the mouse mutation has greatly preceded detection of the human disorder (48,49),

6 1234 Human Molecular Genetics, 2002, Vol. 11, No. 10 whereas in other cases discovery of the genetic basis for deafness has occurred concurrently (12,50). Positional cloning of deafness genes in the mouse is facilitated by the ability to breed large numbers of mice with the same mutation to narrow the interval for study. A large screen of inbred mouse strains by ABR threshold analyses at The Jackson Laboratory is currently underway, and likely to identify mutants that will lead to discovery of new genes and modifying genes for deafness (51). In addition, large numbers of new mouse mutants for investigating mammalian gene function including deafness are being generated rapidly through ENU mutagenesis (52). This chemical mutagenesis program also makes possible genedriven approaches to mouse mutants and using this approach, two missense and one stop mutation were recently identified in Gjb2, the most common cause of nonsyndromic deafness in the human population (53). Besides the spontaneous deaf mouse mutants and those generated from mutagenesis programs, a number of gene targeting experiments have been performed following identification of the human gene, and have provided valuable mouse mutants for investigation. As the human and mouse DNA sequencing projects are finished, the mouse human synteny maps will also become better defined and it will become increasingly easier to locate potential mouse mutants for mapped human deafness disorders. GENE DISCOVERY IN THE AUDITORY SYSTEM: THE PATH TO IMPROVED DIAGNOSIS AND CLINICAL CARE Inspection of the genes identified in hearing disorders and those among the gene lists from the EST sequencing projects reveals a great diversity of transcripts, perhaps not surprising due to the large variety of cells and complexity of the inner ear. The finding of a large percentage of cochlear ESTs not identified in any other tissue may indicate the existence of genes that are unique to the cochlea. Certainly, the great degree of genetic heterogeneity reflected in the many different syndromes involving hearing loss and mapped loci is indicative of a large number of genes orchestrating the hearing process. Grouping the genes discovered to be etiologic in deafness disorders into functional categories begins the process of understanding their role in hearing. Knowledge of the pathways in which many of these genes function will be an exciting journey in hearing science; no doubt pathways exist that are not yet imagined. Another important aspect of gene discovery for deafness disorders is that it makes possible the development of diagnostic tests and accurate genetic counseling. Appropriate medical management and therapeutic options may be based on an understanding of the specific disorder. Gap junction proteins: the connexins Prominent among the group of genes are those encoding gap junctions. A somewhat surprising finding in the field has been the prevalence of mutations in a single gene encoding the gapjunction protein connexin 26, GJB2, accounting for up to 50% of all cases of autosomal recessive prelingual deafness in tested populations (54 65). Connexin 26 gap junctions are believed to play a critical role in the recycling of potassium ions from their entry into hair cells during sensory transduction from the endolymph through to the stria vascularis, where other potassium channels pump potassium back into the endolymph. The gap junction itself, the connexon, is formed from six monomers of connexin and forms a pore between cells by binding with a connexon on an adjacent cell. Several recurrent mutations have been found in GJB2 (e.g. 35delG, 167delT, and 235delC), some with ethnic predilections. In addition to the recurrent mutations, the gene is small (681 bp) making it especially amenable for genetic testing. Screening for mutations in GJB2 has already emerged as the cornerstone of genetic testing for hearing loss and has been incorporated in some centers into the clinical work-up of infants who fail newborn hearing tests. The connexin-deafness homepage provides information on connexin mutations in deafness (Table 2). Besides GJB2, genes for other gap junction proteins have been found to be associated with hearing loss including GJB3 (26), GJB6 (66) and GJA1 (67). A curious finding in genetic testing of the deaf for GJB2 has been the frequent observation of heterozygosity for a mutation. Various explanations have been proposed including the possibility that the deafness was due to another gene or that there was a mutation in a non-coding region of GJB2 not evaluated in the test. Recent identification of a 342 kb deletion in the GJB6 gene (D(GJB6 D13S1830)) as the second most frequent mutation causing prelingual deafness in the Spanish population may provide the sought after answer in many cases (68). GJB6 maps within the same chromosomal region as GJB2, and these recently published data suggest that mutations in the DFNB1 locus can result in a monogenic or digenic pattern of inheritance. Of note, the typical mutationdetection assays commonly in use may miss such large deletions. Genes for maintenance of hair cell function Another group of genes of intense interest are those required for survival of sensory hair cells. The POU domain transcription factor gene POU4F3 is required for terminal differentiation and maintenance of inner hair cells and an 8 bp deletion in the POU homeodomain results in progressive hearing loss in DFNA15 (69). Studies of such genes may result in valuable insight into the molecular triggers for hair cell degeneration. Loss of hair cells is presumed to be a fundamental cause of progressive age-related hearing loss (presbycusis) and an understanding of this degenerative process might provide the basis for therapeutic intervention. The recent finding of the transmembrane cochlear-expressed gene TMC1 uncovers a common cause of nonsyndromic recessive deafness in Pakistan and India at the DFNB7/B11 locus on chromosome 9 in bands q13 q21; mutations in TMC1 account for the deafness phenotype in % of 230 families screened (12). Cloning of TMC1 resulted from an interesting genomicsbased approach that first involved identification of a predicted gene (subsequently designated TMC2) on chromosome 20 with sequence similarity to query sequences in a tblastx analysis of a BAC from the linked genetic interval. TMC1 mutations were also found to be etiologic in DFNA36 (12) and in the mouse mutants deafness (dn) (12) and Beethoven (Bth) (50). It is predicted that TMC1 protein may mediate an ion-transport or channel function required for the normal function of hair cells.

7 Human Molecular Genetics, 2002, Vol. 11, No The recessive dn mutant displays no auditory response and has secondary hair cell degeneration and the dominant Bth mutant appears to have normally functioning hair cells prior to degeneration. Modifier genes Molecular analyses of the auditory system have already yielded a number of genes in mice and humans that influence the expression or function of other genes. Studies of these genes are certain to provide insight into the interaction of their gene products. Notable among the mouse genes are tub (tubby) and moth1 (modifier of tubby hearing) (70), and dfw (deafwaddler) and mdfw (modifier of deaf waddler) (71). Moth1 can worsen or prevent the hearing impairment in tubby, depending upon the type of moth1 allele and whether one or both copies of the allele are functional. One allele of Mdfw can protect dfw heterozygotes from hearing loss, whereas another is permissive for hearing loss in dfw heterozygotes. In humans, the locus for a modifier gene (DFNM1) for the deafness haplotype in DFNB26 has been identified on chromosome 1 in band q24 (72). Mitochondrial genes A variety of mitochondrial disorders have been found to involve hearing loss, perhaps reflecting the highly metabolic state of the hearing process (73). Of particular interest has been the A1555!G mutation in 12S rrna that is recognized as the most frequent cause of aminoglycoside-induced deafness and as the etiology of a nonsyndromic deafness (74). In a recent study a nearly identical degree of mitochondrial dysfunction was observed in enucleated lymphoblastoid cells derived from both symptomatic and asymptomatic individuals from the same kindred (75) supporting the possibility of a nuclear gene in modifying the effect of the mutation. Investigations in hereditary deafness have revealed many lessons in genetics, foremost among them a sensory system with profound genotypic and phenotypic heterogeneity. 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