SACKLER SCHOOL OF MEDICINE DEPARTMENT OF HUMAN GENETICS AND MOLECULAR MEDICINE

IDENTIFICATION AND CHARACTERIZATION

OF GENES FOR NON-SYNDROMIC IN THE ISRAELI POPULATION, INCLUDING GENES FOR

THESIS SUBMITTED FOR THE DEGREE “DOCTOR OF PHILOSOPHY” BY ZIPPORA BROWNSTEIN I.D. 054075189

UNDER THE SUPERVISION OF PROF. KAREN B. AVRAHAM

SUBMITTED TO THE SENATE OF TEL AVIV UNIVERSITY SEPTEMBER 2005

THIS WORK WAS CARRIED OUT UNDER THE

SUPERVISION OF

PROFESSOR KAREN B. AVRAHAM ACKNOWLEDGMENTS

To my supervisor, Prof. Karen B. Avraham, for her guidance, continuous support and endless trust. I am fortunate for being a member of her team.

To Prof. Moshe Frydman for dedicating so many days of his free time for collecting blood from my study population all over the country, for teaching me patiently all I needed for the statistical analysis and for sharing his extensive knowledge with me.

To Hashem Shahin for helping me with the radioactive labeling and for his endless willingness to help in anything needed.

To Ronna Herzano for regarding even the silliest questions as the most clever ones, for guiding my first steps in molecular biology and for sharing her remarkable knowledge with me.

To Orit Ben David for spending many hours helping me to prepare my first poster and first lecture. I owe her all the compliments I have received for my presentations since then.

To Irit Gottfried for helping me “create” the first Excel audiogram, which is still the template for every audiogram drawn in the lab.

To Rami Khosravi for accompanying me on many evenings to collect blood from the deaf and their families. Even the most frightened stubborn child could not resist Rami, since he is so kind, gentle and professional.

To Dr. Avi Goldfarb for driving with me, as far as it was needed, to draw blood from members of Family Z and Family O, and for his endless patience to answer all the questions presented to him by the individuals we met.

To Tama Sobe for her advice and tips that make laboratory life (and protocols) easier.

To Tzlil Ofir for his extreme kindness and never ending patience.

To Helen Berman for her help, efficiency and companionship.

To Orit Dagan for her companionship, sharing mapping worries and walking together along the doctoral pathway.

To Prof. Elon Pras and to Dr. Dafna Benayahu for supervising my doctoral research.

To all the members of the lab, in the past and present, for making these years so exciting and joyful.

To my parents, Menachem and Bella Krol, for their endless support and love, and for always believing I can achieve anything in the entire world.

To my husband Shmuel and to my children Vered, Reuven, Revital and Aharon, for their continuous love and support and for never stopping to believe in me. Without them I would not have reached this far.

TABLE OF CONTENTS

I. INTRODUCTION...... 1

I.1. Anatomy and physiology of the ear...... 2 I.1.1 The outer ear...... 3 I.1.2. The middle ear...... 3 I.1.3. The inner ear...... 5 I.2. Function of the ear...... 10 I.2.1. The mechanism of ...... 10 I.2.2. Vestibular maintenance ...... 13 I.3. ...... 15 I.3.1. Basic and audiological terms...... 16 I.3.1.1. Pure-tone audiometry ...... 16 I.3.1.2. Types of HL...... 17 I.3.1.3. Types of audiograms ...... 18 I.3.2. Further auditory evaluation ...... 19 I.3.2.1. Impedance audiometry ...... 19 I.3.2.2. Speech audiometry ...... 19 I.3.2.3. Auditory brainstem response (ABR) ...... 21 I.4. Genes for NSHL in the Israeli population...... 21 I.4.1. Cx26 and Cx30 ...... 23 I.4.2. Correlation between connexin-associated deafness and outcome of cochlear implants...... 26 I.5. ...... 27 I.5.1. Genes involved in Usher Syndrome in the Israeli population...... 28 I.5.2. PCDH15 ...... 28 I.6. Otosclerosis ...... 29

II. RESEARCH GOALS...... 33

III. MATERIALS AND METHODS...... 34

III.1. Ascertainment of probands and families...... 34 III.1.1. Letters, informed consent and questionnaires...... 34

124 III.1.2. Clinical assessment ...... 34 III.1.3 Audiometry ...... 34 III.1.4. Blood collection...... 35 III.2. Molecular analysis ...... 36 III.2.1. Materials ...... 36 III.2.1.1. Protocols for buffers, solutions and gels...... 36 III.2.1.2. Reagents...... 38 III.2.1.3. Instruments...... 39 III.2.1.4. Kits...... 40 III.2.2. General methods ...... 40 III.2.2.1. DNA extraction...... 40 III.2.2.1.1. Isolation of DNA by kit ...... 40 III.2.2.1.2 Isolation of DNA by salting-out technique...... 40 III.2.2.2. Establishment of cell lines ...... 41 III.2.2.3. RNA extraction from tissues and cell culture...... 41 III.2.2.4. Reverse transcriptase-PCR...... 42 III.2.2.5. Basic PCR touchdown protocol...... 42 III.2.2.5.1. Reaction protocol...... 42 III.2.2.5.2. PCR program ...... 42 III.2.2.6. Electrophoresis of PCR products using agarose gels...... 43 III.2.2.7. DNA sequencing...... 43 III.2.2.7.1. Cleaning DNA following PCR ...... 43 III.2.2.7.2. Extraction of DNA fragments from agarose gels ...... 43 III.2.2.7.3. Sequencing of the purified PCR Product...... 44 III.2.2.8. SDS-PAGE ...... 44 III.2.2.8.1. DNA radioactive labeling ...... 44 III.2.2.8.2. DNA silver staining ...... 44 III.2.3. GJB2 (Cx26) mutation analysis...... 46 III.2.3.1. Identification of GJB2 mutations by restriction enzyme digestions...... 46 III.2.3.1.1. Detection of 35delG mutation by BslI restriction enzyme digest...... 46 III.2.3.1.1. 1. Amplification protocol...... 46 III.2.3.1.1. 2. PCR program ...... 46 III.2.2.3.1. 3. Restriction enzyme digestion...... 47 III.2.3.1.2 Detection of 167delT mutation by MwoI restriction enzyme digest...... 47

125 III.2.3.1.2.1. Amplification protocol...... 47 III.2.3.1.2.2. PCR program ...... 47 III.2.3.1.2.3. Restriction enzyme digestion...... 48 III.2.3.1.3. Identification of the GJB2, exon 1 mutation, IVS1+1(G->A), by restriction digest...... 48 III.2.3.1.3.1. Restriction enzyme digestion...... 49 III.2.3.2. Detection of GJB2 mutations in the open reading frame by direct...... 49 sequencing...... 49 III.2.3.2.1. Amplification protocol...... 49 III.2.3.2.2. PCR program ...... 50 III.2.3.2.3. Cleaning and sequencing of PCR products...... 50 III.2.3.2.4. Detecting GJB2 mutations by bioinformatics...... 50 III.2.4. Identification of the ∆(GJB6-D13S1830) mutation (Cx30)...... 51 III.2.5. Identification of the R245X mutation in the PCDH15 gene...... 52 III.2.5.1. Allele-specific PCR (ASPCR) ...... 52 III.2.5.2. Detection of the R245X mutation by restriction enzyme digestion...... 53 III.2.5.3. Detection of the R245X mutation by sequencing exon 8 of PCDH15...... 54 III.2.6. Otosclerosis...... 54 III.2.6.1. Family O ...... 54 III.2.6.2. Linkage exclusion ...... 55 III.2.6.3. Linkage analysis...... 56 III.2.6.3.1. Genome scan...... 56 III.2.6.3.2. Determining the location of the OTSC4 locus ...... 56 III.2.6.3.3. Haplotype analysis...... 57 III.2.6.4. Candidate genes ...... 58 III.2.6.4.1. Mutation analysis...... 58

IV. RESULTS...... 60

IV.1. Family O ...... 60 IV.1.1. Ascertainment...... 60 IV.1.2. Audiological evaluation...... 61 IV.1.2.1. Age of onset...... 61 IV.1.2.2. Symmetry of HL...... 61

126 IV.1.2.3. Severity and type of HL...... 61 IV.1.2.4. Audiometric configuration...... 66 IV.1.2.5. Progression and operation outcome...... 66 IV.1.2.6. Immittance testing ...... 69 IV.1.3. Linkage exclusion...... 70 IV.1.4. Linkage analysis ...... 70 IV.1.5. Candidate genes...... 75 IV.2. Cx26 and Cx30 ...... 76 IV.2.1. Prevalence of the del(GJB6-D13S1830) mutation in the Israeli population ...... 76 IV.2.2. A multicenter study of the prevalence of the del(GJB6-D13S1830) mutation in the DFNB1 locus in hearing-impaired subjects...... 77 IV.2.3. A multicenter study of the prevalence of the del(GJB6- D13S1854) mutation in the DFNB1 locus in hearing-impaired subjects...... 77 IV.2.4. GJB2 and GJB6 mutations in the Israeli population ...... 77 IV.2.5. Genotype-phenotype correlation of connexin mutations...... 78 IV.2.6. Correlation between connexin-associated deafness and outcome of cochlear implants...... 79 IV.3. Evaluation of PCDH15 mutations in the Israeli deaf population...... 80 IV.3.1. R245X mutation detection...... 80 IV.3.2. R245X carrier frequency ...... 82 IV.4. Ascertainment of additional large families with HL ...... 83 IV. 4.1. Family Z...... 83

V. DISCUSSION ...... 85

V.1. Family O ...... 85 V.1.1. Phenotypic characterization of hereditary otosclerosis locus OTSC4 ...... 85 V.I.2. Mapping of hereditary otosclerosis linked to OTSC4...... 88 V.2. Cx26 and Cx30...... 94 V.2.1. GJB2 and GJB6 mutations in the Israeli population...... 94 V.2.2. Prevalence of del(GJB6-D13S1830)...... 94 V.2.3. Genotype-phenotype correlation of connexin mutaions ...... 96 V.2.4. Correlation between connexin-associated deafness and outcome of cochlear implants...... 98

127 V.3. PCDH15 mutations in the Israeli deaf population...... 99 V.4. Family Z...... 103

VI. FUTURE STUDIES…………………………………………………………………….104

VII. REFERENCES...... 107

VIII. APPENDIX…………………………………………………………………………...124

128 LIST OF FIGURES

Figure I-1. Overview of the outer, middle and inner ear...... 2 Figure I-2. The tendons of the tensor tympani and the stapedius muscles...... 4 Figure I-3. Diagram of the inner ear in cross-section of the human temporal ...... 5 Figure I-4. The ...... 7 Figure I-5. The tip links of hair cells...... 8 Figure I-6. An electron micrograph image shows a single "hair cell bundle" located in the inner ear...... 9 Figure I-7. Frequency distribution along the human cochlea basilar membrane: passive tonotopy...... 11 Figure I-8. Traveling wave along the basilar membrane...... 11 Figure I-9. Linear acceleration detection...... 14 Figure I-10. Angular acceleration detection...... 14 Figure I-11. Heterogeneity of hereditary HL...... 16 Figure I-12. Normal hearing audiogram...... 17 Figure I-13. Diagram of cross-section of cochlear duct indicating the localization of the different connexins...... 23 Figure I-14. Connexin expression in the mature organ of Corti...... 26 Figure I-15. Otosclerosis of otic capsule: erosion into normal otic capsule...... 30 Figure I-16. A focus of otosclerosis, shown on a temporal bone section...... 30 Figure III-1. Schematic location of the 3 primers designed to detect the ∆(GJB6-D13S1830) mutation in one PCR reaction...... 51 Figure III-2. Mutation detection assays for the R245X mutation of the PCDH15 gene...... 52 Figure IV-1. Pedigree of Family O...... 60 Figure IV-2. Pure tone audiograms of affected Family O members...... 65 Figure IV-3. Audiograms of individual IV:14, female, born in 1963...... 67 Figure IV-4. Post-operation progression of otosclerosis of individual IV:4, female, born in 1939...... 69 Figure IV-5. Haplotypes of the OTSC4 linked region on chromosome 16q22.3-23.1...... 74 Figure IV-6. Physical map of the OTSC4 region on the Genethon genetic map (Dib et al. 1996)...... 75 Figure IV-7. Pedigrees of families with children homozygote for the R245X mutation in PCDH15...... 82

129 Figure IV-8. Pedigree of Family Z...... 83 Figure IV-9. Haplotypes of Family Z...... 84 Figure V-1. A diagnostic algorithm for Ashkenazi Jewish children presenting with SNHL.102

130 LIST OF TABLES

Table IV-1. Analysis of Audiograms in Individuals with Otosclerosis of Family O*...... 63 Table IV-2. Analysis of in Individuals of Family O with Otosclerosis...... 64 Table IV-3. Comparison of Female/Male Degree of HL...... 65 Table IV-4. Evolution of HL of individual IV:14 and post-operative outcome...... 68 Table IV-5. Exclusion of OTSC1, OTSC2 and OTSC3 loci by genotyping with markers from the respective chromosomal regions...... 70 Table IV-6. Linkage analysis of the three chromosomal loci with LOD scores >1.5 (in italics), obtained by the genome scan...... 71 Table IV-7. Two point LOD Scores between the OTSC4 locus and chromosome 16q22.1-23.1 markers with penetrance set at 80%...... 72 Table IV-8. Comparison between highest LOD Scores for each marker considering individual IV:6 affected vs. not affected, with penetrance set at 80%...... 73 Table IV-9. Cx30 deletions found in the Israeli population...... 76 Table IV-10. Cx26 and Cx30 mutations in the Jewish Israeli population...... 78 Table IV-11. Connexin mutations in CI children...... 80 Table IV-12. R245X Mutation Frequencies among NSHL Askenazi Jews in Israel and Carrier Rate of Mutation in a Control Group...... 81

131 ABBREVIATIONS

AARS: alanyl-tRNA synthetase ABR: auditory brainstem response ADAT1: adenosine deaminase, tRNA-specific 1 AMPA: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (glutR 1- 4) AP1G1: adaptor-related protein complex 1, gamma 1 ASPCR: allele specific PCR ATBF1: AT-binding 1 ATP: adenosine triphosphate BCAR1: breast cancer anti-estrogen resistance 1 BERA: brainstem electric response audiometry bp: base pairs Bsl1: Bacillus species BspMI: Bacillus species M CALB2: calbindin 2 CDH1: cadherin 1, type 1, E-cadherin (epithelial) CDH3: cadherin 3, type 1, P-cadherin cDNA: complementary deoxyribonucleic acid CFDP1: craniofacial development protein 1 CGI-37: comparative gene identification transcript 37 CHST4: carbohydrate sulfotransferase 4 CHST5: carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5 CHST6: carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 6 CI: cochlear implant CIDR: center for inherited disease research CIRH1A: cirrhosis, autosomal recessive 1A (cirhin) cM: centimorgan CMV: cytomegalo virus CNS: central nervous system CNTNAP4: contactin associated protein-like 4 COG4: component of oligomeric golgi complex 4 COG8: component of oligomeric golgi complex 8 COR: correlation between paired samples

CTRL: chymotrypsin-like Cx: connexin CYB5-M: cytochrome b5 outer mitochondrial membrane dB: decibel DC: Deiters’ cells

Ddw/ddH2O: double distilled water DDX19: DEAD (Asp-Glu-Ala-As) box polypeptide 19 DDX19L: DEAD (Asp-Glu-Ala-As) box polypeptide 19-like DDX28: DEAD (Asp-Glu-Ala-Asp) box polypeptide 28 DEPC: diethylpyrocarbonate DERPC: decreased expression in renal and prostate DFNA: deafness, autosomal dominant DFNB: deafness, autosomal recessive DHODH: dihydroorotate dehydrogenase DHX38: DEAH (Asp-Glu-Ala-His) box polypeptide 38 DMSO: dimethyl sulfoxide DNA: deoxy ribonucleic acid dNTP: deoxynucleotide triphosphate DPEP2: dipeptidase 2 DPEP3: dipeptidase 3 EDTA: ethylene diamine tetraacetic acid EEG: electroencephalogram ENU: N-ethyl-N-nitrosourea ERG: electroretinogram EST: expressed sequence tag EXs: exons EXOSC6: exosome component 6 F: female FA: familial FA2H: fatty acid 2-hydroxylase FF: free field FLJ: "full-length long Japan" collection of sequenced human cDNAs, ID of hypothetical gene FS: frame shift FUK: fucokinase

GABARAPL2: GABA(A) (gamma-aminobutyric acid type-A) receptor-associated protein- like 2 GLG1: golgi apparatus protein 1 HAS3: hyaluronan synthase 3 HC: Hensen’s cells HL: hearing loss HP: haptoglobin HPR: haptoglobin-related protein HYDIN: Hydrocephalus inducing Hz: Hertz IHC: inner hair cells ID: identification number KARS: lysyl-tRNA synthetase Kb: killobases kHz: kilo Hertz KIAA: hypothetical gene characterized in the “Kazusa cDNA sequencing project” LCAT: lecithin-cholesterol acyltransferase precursor LDHD: lactate dehydrogenase D LOC: LocusLink ID of hypothetical protein LOD: logarithm of the odds LT: left LYPLA3: lysophospholipase 3 (lysosomal phospholipase A2) M: male MARVELD3: MARVEL [MAL (myelin and lymphocyte protein) and Related proteins for VEsicle trafficking and membrane Link] domain containing 3 µg: microgram MGC: mammalian gene collection id of hypothetical protein µl: microliter mM: millimolar MON1B: MON1 homolog B mRNA: messenger ribonucleic acid Mwo1: Methanobacterium wolfeil NB: narrow band

NCBI: National Center for Biotechnology Information NFAT5: nuclear factor of activated T-cells 5 NFATC3: Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3 NM: name of gene NOB1P: nin one binding protein NSHL: non-syndromic hearing loss NQO1: NAD(P)H dehydrogenase [quinone] 1 NUTF2: nuclear transport factor 2 OHC: outer hair cells OMIM: Online Mendelian Inheritance in Man ORF: open reading frame OTSC: otosclerosis PBS: phosphate buffered saline PC: pillar cells PCDH15: protocadherin 15 PCR: polymerase chain reaction PDF: peptide deformylase-like protein PDPR: pyruvate dehydrogenase phosphatase regulatory subunit PKD1L3: Polycystic kidney disease 1-like 3 PMFBP1: polyamine modulated factor 1 binding protein 1 PolyA: Polyadenylation signal POU: Pit1/ghf1, Oct1/Oct2 and Unc86 Pou3f4: POU domain, class 3, transcription factor 4 Pou4f3: POU domain, class 4, transcription factor 3 PRMT7: protein arginine N-methyltransferase 7 PSDM: PCR-mediated site-directed mutagenesis PSKH1: protein serine kinase H1 PSMB10: Proteasome (prosome, macropain) subunit, beta type, 10 PSMD7: proteasome (prosome, macropain) 26S subunit, non-ATPase, 7 PTA: pure tone average RCD-8: Autoantigen RE: restriction enzyme RNA: ribonucleic acid rpm: rounds per minute

RT: right RT-PCR: reverse transcription PCR S: sporadic SDS: sodium dodecylsulfate SEQ: sequence SF3B3: splicing factor 3b, subunit 3 SG: spiral ganglion cells SHL: syndromic hearing loss SIAT4B: ST3 beta-galactoside alpha-2,3-sialyltransferase 2 SLC7A6: 7 (cationic amino acid transporter, y+ system), member 6 SLC12A4: Solute carrier family 12 (potassium/chloride transporters), member 4 SMPD3: Sphingomyelin phosphodiesterase 3, neutral membrane (neutral sphingomyelinase II) SNP: Single nucleotide polymorphism SSCP: Single-strand conformation polymorphism SNTB2: basic beta 2 syntrophin SPL: Sound Pressure Level SSLP: Simple Sequence Length Polymorphism TAE: Tris Acetic acid EDTA TAT: tyrosine aminotransferase TERF2: telomeric repeat binding factor 2 TERF2IP: Telomeric repeat binding factor 2, interacting protein THAP11: THAP (mammalian Thanatos-associated protein) domain containing 11 TEX292: testis expressed gene 292

Tm: melting temperature

TM: tectorial membrane TM domain: transmembrane domain TSNAXIP1: Translin-associated factor X interacting protein 1 TXNL4B: thioredoxin-like 4B UCSC: the University of California Santa Cruz UNQ: “Universidad Nacional de Quimes” hypothetical protein UTR: untranslated region UV: ultra violet VOR: vestibulo-ocular reflex

VPS4A: vacuolar protein sorting 4a WWP2: WW domain containing E3 ubiquitin protein ligase 2 ZFP1: zinc finger protein 1 homolog ZNF19: zinc finger protein 19 ZNF23: zinc finger protein 23 ZNRF1: zinc and ring finger protein 1

ABSTRACT

Hearing impairment affects approximately one in 1000 newborns. Progressive hearing loss (HL) affects up to 10% of the population by the age of 65 and ~50% by age 80.

Inherited HL accounts for at least 60% of deafness, including 30% syndromic HL (SHL) and

70% non-syndromic HL (NSHL). The most common form of NSHL is autosomal recessive, which accounts for about 80% of cases. Over 100 genes are thought to be involved in HL; thus far, about 96 loci have been mapped and almost 43 genes cloned.

In the Jewish Israeli population, I analyzed statistically (in the 1980s) the number of genes for autosomal recessive, prelingual, nonsyndromic deafness in the general deaf population, and in the various ethnic groups in Israel derived from Europe (Ashkenazim),

Asia (Eastern) and Africa (Sephardim). I estimated that there are 8-9 loci in the general deaf population. Intraethnic and interethnic matings, within and between the mentioned groups, gave an estimate of 7 and 22 loci, respectively, indicating that in different ethnic groups

different loci may exist. During the past eight years, we came to a stage where we can

actually determine how many genes there are and what proteins they encode. In my doctoral

thesis, I tried to interpret my statistical evaluation of genes for HL in the Israeli population to

molecular identification and characterization of the genes.

Thus far, mutations in four genes are known to lead to NSHL in the Israeli

population. These include connexin 26 (GJB2), connexin 30 (GJB6), IIIA (MYO3A),

and POU4F3. The latter two genes are associated with deafness that was identified in two

extended families. GJB2 mutations, however, are associated with 39% of children born with

HL in both familial and non-familial cases. The two most prevalent mutations are 35delG and

167delT, which count for 72% of the DFNB1 mutations in Israel, and have a 2.9-7.5% carrier

frequency in Ashkenazi Jews. The del(GJB6-D13S1830) mutation is most often associated with a heterozygote

GJB2 mutation in deaf individuals, though I identified one person homozygous for the

deletion in Israel. Our GJB6 data was published as part of a multicenter study in the

American Journal of Human Genetics (2003). In this multicenter study, it was shown that the

del(GJB6-D13S1830) mutation is most frequent in Spain, France, the United Kingdom, Israel

and Brazil (5.9%-9.7% of all DFNB1 alleles), is less frequent in the United States, Belgium

and Australia (1.3%-4.5% of all DFNB1 alleles), and is very rare in Southern Italy (Del

Castillo et al. 2003).

Another multicenter study was conducted to investigate the prevalence of the

second novel deletion involving the gene encoding connexin-30, del(GJB6-D13S1854) (del

Castillo et al. 2005) in different countries. I screened 159 Jewish probands, participating in

the study, with non-syndromic and non-connexin hearing impairment, and the novel deletion was not found in any of them. A Letter to the Editor was accepted to the Journal of Medical

Genetics.

Out of 222 Israeli probands screened for mutations in connexin 26 and connexin 30,

56 were homozygotes or compound heterozygotes for mutations in connexin 26 and 21 were heterozygotes. Seven out of the 21 Cx26 heterozygotes were double heterozygotes

Cx26/∆30 and 1 out of the 222 probands was homozygous for the del(GJB6-D13S1830). I

screened for Cx26 and Cx30 mutations in 79 probands and analysed all the data.

Our 64 probands with connexin mutations participated in a worldwide multicenter

study comprising 1531 genetically and audiometrically documented individuals with

connexin related autosomal recessive HL (Snoeckx et al. 2005). The goal was to develop a

detailed genotype-phenotype correlation for this frequent form of hereditary HL. In this study

sample, the degree of HL associated with biallelic truncating mutations is significantly more severe than the HL associated with biallelic non-truncating mutations. Since the most

prevalent mutations in Israel are of the truncating type, the HL they cause is mainly profound.

Most of the profound hearing impaired children today are undergoing cochlear

implantation and so did most of the deaf children participating in our research, which lead us

to another study regarding the correlation between connexin-associated deafness and

outcome of cochlear implants. Thirty out of the 50 CI children that underwent genetic screening in our laboratory were selected for a study evaluating speech perception after implantation, 17 with connexin 26 or 30 mutations and 13 without connexin mutations. Each child in the connexin group was carefully matched with a non-connexin implanted child according to age of implantation, duration of implant use and mode of communication. There was no evidence for additional disabilities or handicaps in either group. I performed the genetic analysis and speech perception was evaluated at the Speech and Hearing Center,

Chaim Sheba Medical Center, Tel-Hashomer. A retrospective analysis was made on their performance at 6, 12, 24, 36 and 48 months post-implantation. Test material was selected according to the child's age, cognitive and language abilities. Both groups showed significant improvement in speech perception results after implantation and no significant differences in

speech perception results were detected between the groups. Our results in the current study

support the findings obtained in earlier connexin studies. However, there were other results

from other studies that found better results in the connexin group. These differences can be

attributed to the small number of subjects, criteria for selecting the control group and other

confounding factors that affected the results. In addition, our findings provide evidence that

when we carefully select very “clean” groups that differ only in the etiology of deafness, the

performance with the implant is similar. In our control group, although the cause of deafness

is unknown, it is likely that many of these cases have a genetic etiology, particularly in those

cases in which more than one individual in the family suffers from HL. Furthermore, we would expect cochlear implantation outcome to be similar for deafness with non-genetic

etiology that involves sensory HL but not central auditory pathways.

For SHL, mutations causing USH1, USH2, and USH3 have been found in Israel.

Novel mutations in the USH2A gene have been found in Jews of Iranian and Moroccan

origins. The N48K USH3A mutation has been identified in Jewish Ashkenazi individuals. I

characterized the R245X mutation of the PCDH15 gene causing USH1F in the Ashkenazi

Jewish population in Israel. I detected this mutation in Ashkenazi Jewish children diagnosed

with NSHL below the age of ten, prior to the onset of retinitis pigmentosa, Ten percent (2/20)

were homozygous for the R245X mutation. The carrier rate of the R245X mutation among

the normal hearing Ashkenazi population in Israel was estimated at 1% (Brownstein et al.

2004). Early screening for this mutation in Ashkenazi Jewish children thought to have NSHL

is essential. We suggest testing for R245X in children under the age of approximately 10 of

Ashkenazi Jewish descent with no family history of NSHL who test negative for a GJB2 or a

GJB6 mutation. This molecular test may identify impending RP prior to detection by ERG, and permit timely rehabilitation and genetic counseling for the parents.

We also studied the molecular basis for deafness in an Israeli family with otosclerosis (OTSC4) and progressive NSHL (DFNA51). Otoscelerosis differs from the HL

described above since it belongs to the late onset, progressive, conductive category of HL.

Otosclerosis is a common bone disorder of the otic capsule that leads to a progressive hearing

impairment, with a prevalence of 0.2%-1% among Caucasian adults. The age of onset is

usually 20-40 years. The mode of inheritance is autosomal dominant, with reduced penetrance.

Thus far, the chromosomal locations of four loci for otosclerosis have been reported on

chromosomes 3q22-24 (OTSC5), 6p21.3-22.3 (OTSC3), 7q34-36 (OTSC2) and 15q25-q26

(OTSC1). I mapped and clinically analyzed hereditary otosclerosis linked to the OTSC4 locus

in Israeli Family O. I performed an audiological analysis of the hearing impaired members of Family O in order to evaluate the clinical characteristics of HL in this family and a whole

genome scan was carried out using microsatellite markers in order to identify the

chromosomal region of the mutant locus. Twenty four individuals of Family O, both affected

and unaffected, were ascertained and a pedigree was constructed. Otosclerosis was surgically

confirmed in three affected individuals and in the other nine affected members of the family,

the hearing impairment was diagnosed as otosclerosis based on audiological evaluation,

medical history and family history. A large variability exists among the affected members of

the family in all aspects of HL. The different stages of otosclerosis are directly correlated with

type and severity of HL and audiogram configuration. Our investigation is unique in that it shows considerable inter-subject and inter-aural variability in the same family, and activity of the otosclerotic lesion could provide convincing explanations for all these differences.

Linkage to the 16q21-23.2 interval was identified and confirmed with a LOD score

of 3.973 at θ= 0. The new locus for otosclerosis was designated OTSC4. The OTSC4 interval of 9-10 Mb included 93 genes, of which several are involved in the immune system and bone homeostasis that might have been good candidates for genes causing otosclerosis. Sequencing all of the genes in the OTSC4 locus revealed no mutations in any of them. The failure to detect mutations by sequencing all the genes in the OTSC4 region could be attributed to a combination of factors, namely i) intrinsic limitations in the techniques used for mutation screening; ii) hypothetical existence of mutations in regulatory regions in introns or anywhere on the chromosome. Functional assays providing accumulation vs. deficiency data may lead us to the target gene. Since there is a large range of hypotheses regarding the etiology of otoscelerosis, almost all gene in the region might be a candidate for otosclerosis., Therefore it is not realistic to characterize all 93 genes in the region in terms of expression and function. A combination of advanced studies of regulatory regions, more data regarding gene interaction, and development of more sophisticated techniques may lead us to the OTSC4 gene. Identification of the gene will help to reveal the etiology of the disorder and the functional and structural analysis of the protein, opening new options for diagnosis, treatment, and prevention of otosclerosis.

1. Brownstein, Z., T. Ben-Yosef, O. Dagan, M. Frydman, D. Abeliovich, M. Sagi, F. A. Abraham, R. Taitelbaum-Swead, M. Shohat, M. Hildesheimer, T. B. Friedman and K. B. Avraham (2004). The R245X mutation of PCDH15 in Ashkenazi Jewish children diagnosed with nonsyndromic hearing loss foreshadows retinitis pigmentosa. Pediatr Res, 55, 995-1000. 2. del Castillo, F. J., M. Rodriguez-Ballesteros, A. Alvarez, T. Hutchin, E. Leonardi, C. A. de Oliveira, H. Azaiez, Z. Brownstein, M. R. Avenarius, S. Marlin, A. Pandya, H. Shahin, K. R. Siemering, D. Weil, W. Wuyts, L. A. Aguirre, Y. Martin, M. A. Moreno-Pelayo, M. Villamar, K. B. Avraham, H. H. Dahl, M. Kanaan, W. E. Nance, C. Petit, R. J. Smith, G. Van Camp, E. L. Sartorato, A. Murgia, F. Moreno and I. del Castillo (2005). A novel deletion involving the connexin-30 gene, del(GJB6- d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment. J Med Genet, 42, 588-594. 3. Del Castillo, I., M. A. Moreno-Pelayo, F. J. Del Castillo, Z. Brownstein, S. Marlin, Q. Adina, D. J. Cockburn, A. Pandya, K. R. Siemering, G. P. Chamberlin, E. Ballana, W. Wuyts, A. T. Maciel-Guerra, A. Alvarez, M. Villamar, M. Shohat, D. Abeliovich, H. H. Dahl, X. Estivill, P. Gasparini, T. Hutchin, W. E. Nance, E. L. Sartorato, R. J. Smith, G. Van Camp, K. B. Avraham, C. Petit and F. Moreno (2003). Prevalence and evolutionary origins of the del(GJB6-D13S1830) mutation in the DFNB1 locus in hearing-impaired subjects: a multicenter study. Am J Hum Genet, 73, 1452-1458.

I. INTRODUCTION

Hearing impairment affects approximately one in 1000 newborns and 4% of people aged younger than 45 years (Estivill 1998). Progressive hearing loss affects a greater proportion of the population; in the U.S.A. ~10% of persons suffer hearing loss (HL) by age

65 and ~50% of persons by age 80 (Nadol 1993). Inerited HL accounts for at least 60% of deafness [reviewed in (Gorlin et al. 1995)], including 30% syndromic hearing loss (SHL) and

70% non-syndromic hearing loss (NSHL). The most common form NSHL is autosomal recessive, which accounts for about 80% of cases (Van Camp et al. 1997).

Over 100 genes are thought to be involved in HL; thus far, about 90 loci have been mapped (Hereditary Hearing Loss Homepage, http://webhost.ua.ac.be/hhh/) and almost 40 cloned [(Friedman and Griffith 2003); http://hearing.harvard.edu/db/genelist.htm].

In the Jewish Israeli population, I analyzed statistically (in the 1980s) the number of genes for autosomal recessive, prelingual, nonsyndromic deafness in the general deaf population, and with regards to the various ethnic groups in Israel derived from Europe

(Ashkenazim), Asia (Eastern) and Africa (Sephardim). I estimated that there are 8-9 loci in the general deaf population. Intraethnic and interethnic matings, within and between the mentioned groups, gave an estimate of 7 and 22 loci respectively, indicating that in different ethnic groups, different loci may exist (Brownstein et al. 1991). The two statistical methods used assumed equal frequency of alleles for deafness at each locus, and this assumption led to a minimum estimate of the number of loci involved. One method was based on the ratio of the number of deaf couples yielding all deaf offspring to the total number of fertile deaf couples (Stevenson and Cheeseman 1956). The second method was based on the ratio of the number of deaf couples yielding all deaf offspring to those who have only hearing children

(Chung and Brown 1970). During the past eight years, thanks to the rapid advancement of

1 techniques in molecular biology, the publication of the human and mouse genomes, and the development of functional genomics, we are able to actually determine how many genes there are and what proteins they encode. In my doctoral thesis, I tried to prove my statistical evaluation of genes for HL in the Israeli population by means of molecular identification and characterization of the genes.

I.1. Anatomy and physiology of the ear

The ear is located within the temporal bone and consists of three main structural components (Schuknecht 1993): the outer ear, the middle ear, and the inner ear (Figure I-1).

The ear’s main functions are amplification, transduction and encoding of the external mechanical input (sound) to an electrochemical output traveling to the brainstem nuclei and cortex. The outer and middle ears comprise the sound conductive system and are both gas- filled compartments.

Figure I-1. Overview of the outer, middle and inner ear. (www.emsee.ca/ GoodImpression.htm l).

2 I.1.1 The outer ear

The outer ear is comprised of the pinna that acts to focus and aid in the localization of sound, and the external auditory meatus which terminates as a canal, within the temporal bone, at the tympanic membrane (Figure I-1). The tympanic membrane emulates an irregular cone, the apex of which is formed by the umbo (at the tip of the manubrium, the handle of the malleus). The adult tympanic membrane is about 9 mm in diameter.

I.1.2. The middle ear

The middle ear is composed of three major components that lie inside the tympanic cavity. These include the tympanic membrane, the auditory ossicles, and two small auditory muscles, the tensor tympany and stapedius. The middle ear is connected to the nasopharynx via the Eustachian tube, thereby allowing pressure to equalize from both sides of the tympanic membrane.

The ossicular chain, made up of the malleus, incus, and stapes (Figure I-1), serves to conduct sound from the tympanic membrane to the cochlea. These ossicles transfer sound waves across the tympanic cavity and serve as an amplifier of the sound wave in the transition from air to fluid movement in the inner ear. The malleus, the most lateral of the ossicles, has a head (caput), manubrium (handle), neck, and anterior and lateral processes.

The anterior ligament of the malleus, extending from the anterior process, creates the axis of ossicular rotation. The incus, the largest of the three ossicles, is immediately medial to the malleus. The incus has a body and three processes: a long, a short, and a lenticular. The body of the incus articulates with the head of the malleus. The lenticular process, at the terminus of the long process, articulates with the stapes. The stapes is the smallest and most medial of the ossicles. Its head articulates with the lenticular process of the incus, whereas its footplate sits in the oval window, surrounded by the stapediovestibular ligament. The arch of the stapes,

3 composed of an anterior and a posterior crus, links the head and the footplate (Glasscock and

Gulya 2003).

The tensor tympani muscle inserts in the pharyngotympanic tube, attaches to the manubrium (handle) of the malleus, and is innervated by a branch of the mandibular nerve.

The stapedius muscle extends from the wall of the tympanic cavity to the neck of the stapes and is innervated by a branch of the facial nerve. Contraction of these muscles occurs as a reflex to high volume sounds and results in the tympanic membrane becoming tenser, thus reducing the effectiveness of sound transmission and protecting the inner ear from loud noise

(Figure I-2).

A B

Figure I-2. The tendons of the tensor tympani and the stapedius muscles. A. The tensor tympani tendon attaches the muscle to the manubrium of the malleus. Contraction of the tensor tympani displaces the malleus (and consequently the tympanic membrane) medially, thus adding tension to the tympanic membrane. The muscle body of the tensor tympani is enclosed in its own temporal bone cavity and lies essentially in an anterior- posterior plane (perpendicular to its tendon) along the medial aspect of the eustachian tube. B. The stapedius tendon attaches the muscle most commonly to the head of the stapes. Contraction of the stapedius displaces the stapes posteriorly. The body of the stapedius muscle is also enclosed in its own temporal bone cavity and lies in a superior-inferior plane (perpendicular to its tendon). (audilab.bmed.mcgill.ca/ ~daren/media/ossicles).

4 I.1.3. The inner ear

The inner ear, which resides in a bony cavity, is a fluid-filled organ composed of two major regions, the cochlea, which processes the auditory signal and the vestibular apparatus

(utricle, saccule, and ampluae of the semicircular canals) which helps maintain balance by responding to gravity and acceleration (Figure I-3).

Figure I-3. Diagram of the inner ear in cross-section of the human temporal bone. (Forge and Wright 2002).

The processing of auditory signals takes place in the cochlea, which is a bony canal

that coils around its’ central core called the modiolus, having 2.75 turns in humans. The

inside of the cochlea is separated in the middle by the osseous lamina that consists of thin

bony ridges and plates. It subdivides the cochlea into two large cavities, the scala vestibuli

and the scala tympani. Both of these cavities contain a liquid called perilymph that is similar

in composition to cerebrospinal fluid. A third cavity, the scala media (cochlear duct), lies between the scala vestibuli and the scala tympani (Figure I-3). The scala media, is a

triangular membranous canal that is limited by the Reissner's membrane (top), the basilar

5 membrane (bottom) and stria vascularis (strial – abneural side), and contains the endolymph fluid.

The molecular as well as physical and biological properties of the cochlear duct vary throughout its length and are reflected in differences and gradients of gene expression (Davis

2003).

The stria vascularis is an ion transporting epithelium composed of three cell types: marginal, intermediate, and basal cells. Together, they generate the high endolymphatic potassium ion concentration and positive endocochlear potential. The basilar membrane, the bottom of the scala media, stretches between the spiral lamina on the modiolar side, and the spiral ligament on the strial side. The organ of Corti, which is the sensory transduction organ of the cochlea, rests just on top of the basilar membrane throughout the cochlear duct. The organ of Corti is comprised of hair cells, supporting cells, neurons, blood vessels and the tectorial membrane (Figure I-4) (Kessel and Kardon 1979; Schuknecht 1993).

6

AA B

Figure I-4. The cochlea.

A. The cochlea, shown “unrolled”. (emusician.com/mag/Can-you-Fig1.jpg). B. Cross section of the cochlea. The cochlear duct is inserted in the perilymph. It is filled with endolymph and contains the organ of Corti between the tectorial and the basilar membranes. The relative movement of the two membranes leads to deflection of the stereocillia of the inner hair cells (one row) and the outer hair cells (three rows), which generates the influx of potassium ions through channels at the tip links of the stereocillia. The influx of potassium ions from the endolymph activates the hair cells, which leads to the stimulation of the underlying nerve cells that convey the auditory signal to the auditory cortex. Epithelial supporting cells are shown in red. (Willems 2000).

The supporting cells include several different types of cells, most of which have a poorly understood function. It has been shown that the supporting cells function to recycle the potassium ions (Figure I-4), form a sealed barrier between the endolymph and perilymph filled spaces, maintain the hair cell function, and structurally support the organ of Corti, specifically as the hair cells are in contact with supporting cells. The supporting cells are coupled to each other by gap junctions (comprised of connexins 26, 30, 31 and 43, in the cochlea, and 26, 30 and 43, in the vestibular system), allowing them to act as a 'functional unit' (Forge et al. 2003). These ion

7 channels enable the passage of ions, second messengers and small metabolites between adjacent cells. The importance of these gap junctions is demonstrated by their roles in human hereditary

NSHL (http://www.uia.ac.be/dnalab/hhh/). Notably, there are no gap junctions between hair cells and supporting cells, delineating their separate functions.

The hair cells of the cochlea are the sensory cells. At their apical surface lie bundles of stiff- filled microvilli, named stereocillia. Thirty to 300 stereocilia form the hair bundle, which is the mechanoreceptive structure of the hair cell. The stereocilia are linked to each other by extracellular filaments, called tip links (Pickles et al. 1984; Furness and Hackney 1985) (Figure I-

5).

Figure I-5. The tip links of hair cells. Schematic representation of an outer hair cell crowned with an array of stereocilia connected by tip links. The vibrations of the basilar membrane caused by the oscillations of the perilymph induce shearing of the tectorial membrane, that leads to bending of the stereocilia, which stretches the filaments that link neighboring stereocilia, thereby opening unidentified potassium channels in the membrane of the stereocilia. (Willems 2000).

The hair cells of the cochlea are divided into inner and outer hair cells (IHCs and OHCs) that differ in their structure, pattern of innervation and function. IHCs are pear shaped and their apical bundles are arranged in a straight, crescent-like pattern. The IHCs receive most of the

8 afferent innervation of the organ of Corti and very little efferent innervation. IHCs are therefore considered the 'hearing' cells of the cochlea. OHCs are more cylindrical in shape, and unlike the

IHCs, both the cells and their stereocilia increase in length from base to apex and from modiolar to strialar ends of the cochlea. OHCs stereociliary bundles form a V or W shape (Figure I-6), with the open end directed towards the modiolar side of the cochlea. In the mature inner ear, all hair cell stereociliary bundles are uniformly oriented. OHCs are directly innervated by efferent neurons, mainly from the contralateral superior olive nucleus in the brainstem.

Figure I-6. An electron micrograph image shows a single "hair cell bundle" located in the inner ear. There are 17,000 of these hair cells in each ear that detect and amplify sound waves. (http://www.npr.org/templates/story/story.php?storyId=4 107255).

OHCs feature a unique lateral membrane composed of densely packed proteins,

on top of a lattice of helical actin filaments crosslinked by (Holley and Ashmore 1990;

Zheng et al. 2000). OHCs can contract in a frequency dependent manner and it has been shown

that prestin is the OHC molecular motor (Ashmore 1991). OHCs probably function to increase

the sensitivity of the cochlea to sound, as a loss of OHC results in an increase of 60-80 dB in

the threshold of hearing.

The tectorial membrane, which covers the organ of Corti and touches mainly the OHC

stereocilia (via Kimura's membrane), is an extracellular aqueous matrix synthesized by the

interdental cells and is fixed to Hensen's cells in its lateral (strial) border by a marginal net. The

9 tectorial membrane consists of fine cross-linked fibrils with several ear specific proteins (e.g. Otogelin and α- and β- tectorins) (reviewd in (Lim 1986; Forge and Wright 2002).

Nerves, ganglions and blood vessels are located between the bony plates of the osseous spiral lamina. Their dendrites extend into the organ of Corti to the base of the hair cells. Each outer hair cell is usually connected to a number of nerve cells and each inner hair cell to a single nerve cell.

I.2. Function of the ear

I.2.1. The mechanism of hearing

The sound waves are funneled through the outer ear canal and reflect on the tympanic membrane resulting in its vibration, which is conveyed to the inner ear by the three middle ear interconnected ossicles, the malleus, incus and stapes. The malleus is attached to the tympanic membrane and the footplate of the stapes is attached to the oval window at the base of the cochlea, near the scala vestibuli. The three ossicles of the middle ear function like a lever to increase the energy of the sound waves, mainly through the difference in surface area between the malleus and the stapes footplate, resulting in a final gain of about 18-fold (~30 dB SPL)

(Posner 1985). This will compensate for the loss in intensity that these sound waves will suffer when transferred to a fluid environment in the cochlea. Movement of the stapes in and out of the fluid filled cochlea results in a fluid wave in the scala vestibuli that will cause a vibration of the basilar membrane of the cochlear duct, as the two compartments are separated by only a thin membrane. The fluid wave then reaches the helicotrema and travels toward the membrane sealed round window. The structure of the basilar membrane is reminiscent of a harp, with short and densely packed fibers at the base and long and more sparce fibers towards the apex. Thus the traveling wave peaks at a frequency dependent location, with higher frequencies close to the base and lower frequencies close to the apex (reviewed in Petit et al., 2001) (Figure I-7).

10

Figure I-7. Frequency distribution along the human cochlea basilar membrane: passive tonotopy. From base (20 kHz) to apex (20 Hz), some characteristic frequencies are indicated. Note the progressive enlargement of the basilar membrane. (Pujol:http://www.iurc.montp.inserm.fr/cric/audition /english/index.htm).

The traveling wave moves the tectorial membrane relative to the stereocilia of the inner and outer hair cells, upon which a deflection of the stereocilia bundles occur, which triggers the opening and closing of mechanosensitive ion channels located along the stereociliary tips (Figure I-8).

Figure I-8. Traveling wave along the basilar membrane. This wave reaches a maximal amplitude at a certain area of the organ of Corti, directly related to the frequency of the incoming sound (characteristic frequency for this location). In this area, the traveling wave introduces a shearing force between the tectorial membrane and the stereocilia of the inner and outer hair cells, upon which a deflection of the stereocilia bundles triggers the opening of mechanosensitive ion channels located along the stereociliary tips.

11 These mechanotransducer channels are nonspecific cation channels and when a force is delivered to them, the open probability of the channel is altered upon which a transducer current flows into the cell that is largely carried by potassium ions that enter the cells along the electrical gradient that exists across its cell membrane. The transducer current produces a voltage drop (receptor potential) across the membrane of the hair cell. This is thought to occur at the place of maximum amplitude of the traveling wave exclusively and as such only a limited number of hair cells are thus activated, depending on the frequency of the sound.

The OHCs then translate the resulting changes in membrane potential into macroscopic changes in the length of their cell bodies, which is mediated by Prestin, a transmembrane protein located in the cell membrane of the outer hair cells (Zheng et al.

2000). This generates the mechanical energy that is required for amplifying the sound- induced vibrations in the cochlea, which are largely responsible for hearing sensitivity and frequency-resolving capability of the ear (Dallos and Fakler 2002).

By contrast, IHCs function as the sensory receptors of the hearing organ and convey essentially all auditory information to the brain. Indeed, the receptor potential induces a release of neurotransmitter and it appears that the excitatory transmitter glutamate mediates fast signalling in the first synapse of the auditory and vestibular pathways, i.e. the contact between the hair cells and afferent dendrites of spiral and vestibular ganglion cells. After release of glutamate, postsynaptic receptors, primarily of the AMPA type, are activated, which may subsequently induce an action potential (Ottersen et al. 1998). In addition, IHCs are equipped with a small pool of presynaptic AMPA receptors (Matsubara et al. 1996).

Besides its fast excitatory properties, glutamate is known to have neurotoxic properties when released in large amounts or when incompletely recycled. This so-called excitotoxicity may occur during noise trauma and anoxia and may be responsible for subsequent hearing loss

(Pujol and Puel 1999). Therefore it is imperative that glutamate be effectively removed from

12 the synaptic cleft after its release and subsequent activation of the postsynaptic receptors, and it is the supporting cells that have been shown to be responsible for this process (Ottersen et al. 1998). K+ ions that pass through the sensory cells during mechanosensory transduction can be recycled back to the endolymphatic space, via two independent gap junction systems, the epithelial cell gap junction system and the connective tissue cell gap junction system.

Indeed, influx of K+ ions into activated hair cells is released basolaterally to the extracellular space of the organ of Corti, from which they enter the cochlear supporting cells. From here they move via the epithelial cell gap junction system laterally to the lower part of the spiral ligament, from which they enter the connective tissue gap junction system, and are eventually released back into the endolymph through the tight junctional barrier of the stria vascularis

(Kikuchi et al. 2000).

I.2.2. Vestibular maintenance

The vestibular system is one of the most ancient sensory systems in evolution and is thought to be a derivation of the lateral line organ of primitive fish. It monitors the position and motion of the head in space both by linear and angular acceleration to the brain. The utricle and saccule detect linear acceleration, while the semicircular canals detect angular acceleration.

The utricle and saccule detect linear acceleration through the hair cells in their macula

(Kessel and Kardon 1979; Schuknecht 1993). The utricle macula lies on a horizontal plane and the saccule macula on a vertical one. In both, the hair cells are polarized in different directions and thus each cell can be either stimulated or inhibited while the adjacent cell may have the opposite outcome. The hair cells protrude into a fibrogelatinous structure, similar to the tectorial membrane in the cochlea, called the otoconial membrane. The otoconial membrane is composed of the otoliths (also called otoconia) that are calcium carbonate based

13 crystals with a certain mass. Due to this mass the otoliths lag behind the macula when the skull moves due to acceleration causing a stimulation of the hair cells in the macula (Figure I-

9). Deceleration causes the otoconia to move ahead of the macula, causing the reverse effect.

Figure I-9. Linear acceleration detection. A diagram showing the detection of linear acceleration ithe saccule and utricle. (http:// www.nsbri.org/HumanPhysSpace/fo cus7/ep_structure.html).

The three semicircular canals, corresponding to the three dimensions in which we move, allow the detection of angular acceleration. The semicircular canals can be looked at in a very simplified fashion as endolymph-filled hoops, with a swelling at its base called the ampulla. The hair cells of the semicircular canals lie in the ampulla on the crista and are separated from the cupula by a narrow endolymph-filled space. All the hair cells have the exact same polarity so that deflection in one particular direction produces either stimulation or inhibition in the hair cell bundle of a specific ampulla. The flow of the endolymph in the ampulla causes either stimulation or inhibition of the sensory hair cells, depending on which semicircular canal it is and the direction of the angular movement itself (Figure I-10).

Figure I-10. Angular acceleration detection. A diagram showing the detection of angular acceleration by a semicircular canal. (http://thalamus wustl.edu/course/audvest.html).

14 I.3. Hearing loss

HL can be caused by environmental and/or genetic factors, including exposure to ototoxic drugs, rubella during pregnancy, trauma, excessive noise, and/or mutations in one of the 30,000 genes that define our genome. Even when environmental factors are involved, modifying genes may influence the onset or severity of the hearing impairment. HL is classified according to cause (genetic or non-genetic), association (syndromic or nonsyndromic), onset (before or after language acquisition – pre-lingual or post-lingual, respectively), type (sensorineural, conductive or mixed), severity (mild 21-40 dB, moderate

41-60 dB, moderately severe 61-80 dB, severe 81-100 dB and profound, more than 100 dB) and frequencies (low – lower than 500Hz, middle 500-2000Hz and high – over 2000Hz)

(Willems 2000). About 70% of all genetically based HL is nonsyndromic (NSHL) where the only symptom observed is hearing loss, and 50% of prelingual NSHL is considered to be the result of monogenic disorders. Thirty percent is in the form of SHL, where HL is associated with other symptoms. NSHL is inherited in a recessive mode in approximately 80% of the cases, in a dominant mode of inheritance in approximately 20%, and is either X-linked or mitochondrial in 2-3% of this group (Figure I-11). Approximately 36% of people over the age of 75 suffer from , a high-tone hearing impairment that presents with advanced age, much of which is thought to be due to genetic etiology (Nadol and Merchant

2001).

15

Autosomal X-linked Recessive (1-2%) (~ 80%) Autosomal Dominant (20%) Non syndromic (70%)

Syndromic (30%) Mitochondrial (1-2%)

Figure I-11. Heterogeneity of hereditary HL. A pie diagram showing the distribution of hereditary HL throughout the population.

I.3.1. Basic audiometry and audiological terms

I.3.1.1. Pure-tone audiometry

Pure tones are generated by an audiometer within a frequency range of 250 to 8000

Hz. The hearing threshold is measured for each ear separately, for both bone and air conduction, in decibel steps. Hearing responses are recorded on an audiogram.

Audiogram – A graph showing hearing loss as a function of frequency as measured by an audiometer. 0 dB on an audiogram denotes the hearing threshold level regarded as the normal audiometric standard at that frequency (Figure I-12).

16

Figure I-12. Normal hearing audiogram. Grey area indicates normal hearing range. (http://www.tchain.com/otoneurology/testing/hearing_test.htm#audiometry).

Air conduction (AC) - normal conduction of sound to the inner ear via the sound-conducting apparatus. AC is measured using earphones.

Bone conduction (BC) - Sound conducted via the of the skull to the inner ear. BC is measured using a vibrator placed on the mastoid bone behind the ear.

Air-bone gap (ABGap) - The difference between the threshold for hearing acuity by BC and by AC. ABGap is indicative of impairment of hearing due to interference with the acoustic transmission (AC) of sound through the outer and middle ear.

I.3.1.2. Types of HL

Conductive HL – thresholds obtained by AC are elevated, whereas thresholds measured by

BC are within normal. Conductive HL occurs when sound waves are prevented from passing

17 from the air to the fluid-filled inner ear. This may be caused by a variety of problems in outer or middle ear, including buildup of earwax (cerumen), infection, fluid in the middle ear, a punctured eardrum, or fixation of the ossicles, as in otosclerosis. Other causes include scarring, narrowing of the ear canal, tumors in the middle ear, and perforation of the tympanic membrane. Once the cause is found and removed or treated, hearing usually is restored.

Sensorineural HL (SNHL) – both AC and BC thresholds are similarly elevated. SNHL develops when the auditory nerve or hair cells in the inner ear are damaged. The source may be located in the inner ear, the nerve from the inner ear to the brain, or in the brain. SNHL, commonly referred to as "nerve deafness," frequently occurs as a result of the aging process in the form of presbycusis, which is a gradual loss occurring in both ears. Tumors such as acoustic neuromas can lead to SNHL, as can viral infections, Meniere’s disease, meningitis, and cochlear otosclerosis. SNHL can also be the result of repeated, continuous loud noise exposure, certain toxic medications, or an inherited condition. Generally, it is non-reversible.

Uncovering the genes responsible for deafness, may in time lead to therapies for some forms of SNHL.

Mixed HL – a combination of both conductive and SNHL. Both AC and BC thresholds are elevated, but AC more than BC, i.e. ABGap is present.

I.3.1.3. Types of audiograms

Sloping – thresholds occur at successively higher levels from 250-8000 Hz. The difference between thresholds at 250 and 8000 Hz is >20dB (corner audiograms with thresholds indicating no response at the limit of the audiometer were not considered in the algorithm).

Rising - thresholds occur at successively lower levels from 250-8000 Hz. The difference between thresholds at 250 and 8000 Hz is >20dB.

18 Flat – thresholds across frequencies do not vary more than 20 dB from each other.

U-shaped – one or more adjacent thresholds between 500 and 4000 Hz are ≥20 dB relative to the better threshold at 250 and 8000 Hz.

Tent-shaped - one or more adjacent thresholds between 500 and 4000 Hz are ≤20 dB relative to the poorer threshold at 250 and 8000 Hz.

Other – configurations that do not meet the criteria for inclusion in any other category.

Definitions of audiometric configurations are according to Pittman and Stelmachowicz

(Pittman and Stelmachowicz 2003).

I.3.2. Further auditory evaluation

I.3.2.1. Impedance audiometry

This technique forms part of the functional diagnosis of the sound conduction apparatus, and it includes the following two methods of investigation:

Tympanometry - measures the impedance or acoustic resistance at the tympanic membrane and is an indirect test of tubal function.

Measurement of the stapedius reflex - The change in impedance at the tympanic membrane caused by the acoustic stapedius reflex is measured. The stapedius reflex is absent in different pathologies including retrocochlear sensorineural deafness, otosclerosis and other middle ear diseases, in facial nerve lesions and in brainstem lesions.

I.3.2.2. Speech audiometry

Speech-reception threshold (SRT) - obtained by measuring the lowest level at which speech can be identified at least half the time. Two-syllable test words are usually used to obtain the

SRT (taken from http://www.emedicine.com/ent/).

19 In addition to determining softest levels at which patients can hear and repeat words, the SRT is also used to validate pure-tone thresholds because of high correlation between the

SRT and the average of pure-tone thresholds at 500, 1000, and 2000 Hz. In clinical practice, the SRT and 3-frequency average should be within 6 dB. This correlation holds true if hearing loss in the 3 measured frequencies is relatively similar. If one threshold within the 3 frequencies is significantly higher than the others, the SRT will usually be considerably better than the 3-frequency average.

Speech-discrimination testing - also referred to as suprathreshold word-recognition test. The primary purpose of the discrimination test is to estimate the ability to understand and repeat single-syllable words presented at conversational or another suprathreshold level.

Initial word lists compiled for word-recognition testing were phonetically balanced

(PB). This term indicated that phonetic composition of the lists was equivalent and representative of the tested language. The PB word lists each contain 50 single-syllable words that are presented at specified sensation levels. Different PB lists are presented to a patient if more than one PB list is needed for testing. Words can be presented via tape, CD, or monitored live voice. Patients are asked to repeat words to the audiologist. Each word repeated correctly is valued at 2%, and scores are tallied as a percent-correct value. Varying the presentation level of monosyllabic words reveals a variety of performance-intensity functions for these word lists. In general, presenting words at 25-40 dB sensation level (refer to the SRT) allows patients to achieve maximum scores. Lowering the level results in lower scores. For individuals with HL, words can be presented at a comfortable loudness level or at the highest reasonable level before discomfort occurs. When words are presented at the highest reasonable level and the word-recognition score is 80% or better, testing can be discontinued. If the score is lower than 80%, further testing at lower presentation levels is recommended. If scores at lower levels are better than those obtained at higher presentation

20 levels, "roll over" has occurred, and these scores indicate a possible retrocochlear (or higher) site of lesion.

Another use of discrimination testing is to verify speech-recognition improvements achieved with hearing aids. Testing can be completed at conversational levels in the sound field without the use of hearing aids and then again with hearing aids fitted to the patient.

Score differences can be used as a method to assess hearing with hearing aids and can be used as a pretest and posttest to provide a percent-improvement score.

I.3.2.3. Auditory brainstem response (ABR)

The principle of ABR, or brainstem electric response audiometry (BERA), is that the subject is exposed to an acoustic stimulus repeatedly, and an electroencephalogram (EEG) assesses changes in brain activity. The method is important in the differentiation of cochlear from retrocochlear hearing disorders, and in the investigation of hearing in infants and young children.

I.4. Genes for NSHL in the Israeli population

Thus far, mutations in four genes are known to lead to NSHL in the Israeli population. These include connexin 26 (Cx26) (GJB2) (Sobe et al. 2000), connexin 30

(Cx30) (GJB6) (Del Castillo et al. 2003), myosin IIIA (MYO3A) (Walsh et al. 2002), and

POU4F3 (Vahava et al. 1998). The latter two genes are associated with deafness that was identified in one extended family each. Three different mutations in MYO3A are responsible

for progressive late onset sensorineural high tone hearing loss (HTHL), inherited in a

recessive mode in one Israeli family of Iraqi (Mosul) descent. Correlation between onset and

severity of HL and mutation was observed (Walsh et al. 2002). A POU4F3 mutation causes

late onset progressive HL with a sloping moderate to severe audiogram, inherited in a

21 dominant mode in one Israeli family of Libyan descent (Vahava et al. 1998). The autosomal recessive GJB2 mutations, however, are associated with 39% of children born with HL in both familial and non-familial cases. The two most prevalent mutations are 35delG and

167delT, with a 2.9-7.5% carrier frequency in Ashkenazi Jews (Lerer et al. 2000; Sobe et al.

2000). The del(GJB6-D13S1830) mutation is most often associated with a heterozygote

GJB2 mutation in deaf individuals, though one person homozygous for the deletion has been identified in Israel (Del Castillo et al. 2003).

For SHL, mutations causing USH1, USH2, and USH3 have been found in Israel, all involving combined deafness and blindness due to retinitis pigmentosa (RP), with a variable age of onset. Mutations in the USH2A gene found in Iranian and Moroccan Jews cause

moderate SNHL in the first decade of life (Adato et al. 2000). The N48K USH3A mutation

has been identified in Ashkenazi Jews with postlingual progressive HL (Adato et al. 2002;

Ness et al. 2003). The R245X mutation of the PCDH15 gene causing USH1F was identified

in congenitally deaf Ashkenazi Jews (Ben-Yosef et al. 2003). I charaterized this mutation in

Ashkenazi Jewish children in Israel diagnosed with NSHL below the age of ten, prior to the

onset of RP. Ten percent (2/20) were homozygous for the R245X mutation. The carrier rate

of the R245X mutation among the normal hearing Ashkenazi population in Israel was

estimated at 1% (Ben-Yosef et al. 2003; Brownstein et al. 2004). Early screening for this

mutation in Ashkenazi Jewish children thought to have NSHL is essential, since the earlier the diagnosis is made, the better chance these children have for early habilitation and being able to communicate optimally in society, even after they have lost a portion or all of their vision.

Genes involved in otosclerosis are also being studied in Israel. I mapped the fourth

locus for otoscelerosis (OTSC4) in an Israeli family of Yemenite descent, with progressive

conductive to mixed HL.

22 I.4.1. Cx26 and Cx30

GJB2 was the first autosomal recessive gene causing NSHL, discovered in 1997. It is now known to be the single gene harboring mutations for up to half of all cases of human

recessive NSHL (Kelsell et al. 1997). GJB2 encodes Cx26, a gap junction protein. Cx26

belongs to a family of more than 20 members that share a common structure of four

transmembrane segments. Most cell types express more than one connexin species, which

may form homomeric or heteromeric connexons. In the auditory system, intercellular

channels are formed predominantly by Cx26 but also by Cx30, Cx31 and Cx43 (Kelley et al.

1999; Forge et al. 2003) (Figure I-13).

Figure I-13. Diagram of cross-section of cochlear duct indicating the localization of the different connexins. (Forge et al. 2003).

Cx26 seems to be involved in maintaining a high extracellular electrical potential in

the cochlea by facilitating the circulation of K+ ions (Forge et al. 1999). A surprising finding

was that despite the extreme genetic heterogeneity of deafness, this gene is responsible for a

high proportion of NSHL. Mutations in GJB2 are responsible for up to 50% of severe to

profound prelingual recessive deafness in several worldwide populations (Denoyelle et al.

1999), for 38.7% in the general deaf Israeli population (Sobe et al. 2000), and for 70.4%

23 among Ashkenazi Jews (Lerer et al. 2000). However, the screenings for mutations in GJB2 in subjects with autosomal recessive hearing impairment revealed an unexpected problem; a high number of patients carried only one mutant allele. Exhaustive screening of the coding region (fully contained in exon 2), of exon 1 and of splice sites, did not reveal any mutation in the second allele. These cases accounted for 10 to 42% of all subjects with mutations in

GJB2 in distinct populations (Del Castillo et al. 2003). This could be attributable to intrinsic drawbacks in the techniques for mutation detection, to the high frequency of carriers for some

GJB2 mutations, or to the hypothetical existence of mutations in other non-coding parts of the gene. However, it was also suspected that other mutations may be found in the DFNB1 locus but not in the GJB2 gene, which would explain those large fractions of heterozygous subjects. Recently, this hypothesis received experimental support by the finding of a novel mutation in the DFNB1 locus. In 2001, Lerer et al discovered a GJB6 deletion in the

Ashkenazi Jewish population, found on one allele in conjunction with a GJB2 mutation

(Lerer et al. 2001). A few months later, Del Castillo reported a 342 kb deletion in GJB6 in

Spain. The GJB6 deletions in Israel and Spain were subsequently found to be the same (Del

Castillo et al. 2001). The deletion was not affecting GJB2, but truncating the adjacent GJB6 gene, which encodes connexin 30 (Lerer et al. 2001; Del Castillo 2002; Pallares-Ruiz et al.

2002). This deletion was found accompanying in trans the only GJB2 mutant allele in heterozygous affected subjects (double heterozygosity), and was also found in homozygosity in several cases (Del Castillo 2002; Pallares-Ruiz et al. 2002). Double heterozygotes for

Cx26 and Cx30 mutations manifest the same phenotypes as homozygotes for Cx26 as well as homozygotes for Cx30 (Lerer et al. 2001; Del Castillo 2002; Del Castillo et al. 2003). The deletion breakpoint junction was isolated and sequenced, revealing the loss of a DNA segment of about 342 kb, with one breakpoint inside the GJB6 coding region. This deletion,

24 named del(GJB6-D13S1830), was the accompanying mutation in up to 50% of the heterozygotes with only one GJB2 mutant allele (Del Castillo 2002).

Another novel deletion involving the gene encoding Cx30, del(GJB6-D13S1854), was found in trans with mutations in the GJB2 gene in subjects with autosomal recessive non- syndromic hearing impairment. This second deletion is smaller than the previous and spans

232 kb (del Castillo et al. 2005).

These findings can be interpreted on the basis of a digenic pattern of inheritance of mutations in GJB2 and GJB6. This hypothesis is supported by several facts: i) Both Cx26 and

Cx30 colocalise in the same inner ear structures: in the supporting cells of the organ of Corti, in the basal cell region of the stria vascularis, and in type I fibrocytes of the spiral ligament

(Forge et al. 2003) (Figures I-13, I-14); ii) Connexons composed of Cx26 can bind connexons composed of Cx30 to form heterotypic gap-junction channels (Dahl et al. 1996); iii) Cx30- deficient mice exhibit a severe constitutive hearing impairment, and lack an endocochlear potential (Teubner et al. 2003). Nevertheless, the fact that no point mutations in GJB6 have been found up to now in cases of autosomal recessive hearing impairment argues against this hypothesis. An alternative explanation is that the deletion eliminates a regulatory element located far upstream GJB2, which would be essential for the expression of this gene in the inner ear. So far, such an element has not been isolated.

25

A Figure I-14. Connexin expression in the mature organ of Corti. A. Cx26 antibody labeling in a section of guinea pig organ of Corti (basal turn). Extensive labeling at borders of all non-sensory cells. Numerous individual large puncta are present at borders between Deiters’ cells below OHC, which are not labeled, and between Hensen’s cells, indicating each cell forms several gap B C junctions with each of its neighbors in both the radial and longitudinal directions. Scale bar: 10 µm. B. Cx26 antibody labeling (green) in a whole mount of gerbil organ of Corti counterstained with propidium iodine (red) to show nuclei. Arrow indicates single large gap junction between adjacent inner pillar cells in the longitudinal direction along the organ of Corti. On the outer edge, each Hensen’s cell (Hc) shows several labeled puncta delimiting the cell border, indicating that each cell forms gap junctions with each of its neighbors. Scale bar: 20 µm. C. Cx30 antibody labeling in a whole mount preparation of mouse organ of Corti. The distribution of Cx30 labeling coincides with that of Cx26. Image at a level below the outer hair cells, to show labeled puncta around each Deiters’cell (Dc). Arrow indicates large gap junctions between inner pillars cells, and Hc indicates Hensen’s cells with labeled puncta around the borders of each one. Scale bar: 20 µm. (Forge et al. 2002).

I.4.2. Correlation between connexin-associated deafness and outcome of cochlear implants

One of the most common rehabilitation options for the severe to profound HL population are cochlear implants. The performance with cochlear implants is highly variable and depends on many factors such as age of implantation, residual hearing, and mode of communication. While the contribution of these factors to speech perception abilities have been documented, they were found to explain less then 50% of the variance in the results

(Clark 1997). Thus, there are clearly additional factors involved that account for the remaining variability.

26 One possible factor that can affect the results of a cochlear implant is the etiology of deafness. It is assumed that speech perception performance with the cochlear implant might be poorer in etiologies which are known to cause neural and or central damage to the auditory system (e.g., cytomegalovirus, meningitis, auditory neuropathy) (Pyman et al. 2000;

Francis et al. 2004), compared to those which are known to primarily affect the hair cells

(e.g., hereditary NSHL). One such etiology, which is common and known to affect the cochlea, is genetic mutations in the GJB2 or GJB6 genes, leading to NSHL.

Several studies assessed speech perception of implanted children with GJB2 mutations compared to a control group, but overall, the results were inconclusive. In one study, better results were found in the Cx26 group at six months post-implantation

(Matsushiro et al. 2002). In another study, there was a tendency toward better results in the

Cx26 group but this was not statistically significant (Fukushima et al. 2002). Recently,

Sinnathury et al. found better speech intelligibility and better auditory perception results

(Sinnathuray et al. 2004) in the Cx26 group, while other studies found similar results in the two groups (Dahl et al. 2003; Cullen et al. 2004).

I.5. Usher syndrome

Usher syndrome accounts for more than 50% of the deaf-blind population

(Boughman et al. 1983), and approaches a prevalence of 1/10000 between the ages of 30-49

(Hope et al. 1997). It is defined by congenital SNHL, progressive RP leading to blindness in the second to fourth decades of life and vestibular disfunction. Three clinical subtypes are described, based on the degree of deafness, the age of onset of RP and the presence of vestibular disfunction. Usher syndrome type 1 (USH1) is the most common and most severe, characterized by congenital profound deafness, onset of RP in the beginning of the second decade of life and constant vestibular dysfunction. USH2 is distinguished from USH1 by a

27 mild to severe high tone HL (HTHL), and by higher variability of the progression of RP, even though the age of onset overlaps in the two subtypes. USH3 has low prevalence, and is distinct from the others because of the occasional presence of vestibular dysfunction, the progressiveness of the HL, and the later onset of RP (Petit 2001).

I.5.1. Genes involved in Usher Syndrome in the Israeli population

Genes for seven USH1 loci have been mapped (Petit 2001; Mustapha et al. 2002) and four have been cloned (Weil et al. 1997; Bork et al. 2001; Ahmed et al. 2002; Boeda et al. 2002). Myosin VIIA (MYO7A) underlies USH1B, as well as non-syndromic forms of HL,

DFNB2 and DFNA11 (Liu et al. 1997; Liu et al. 1997; Weil et al. 1997). Mutations in cadherin 23 (CDH23) (Bork et al. 2001; Astuto et al. 2002), are responsible for both USH1D and non-syndromic DFNB12, and USH1C and DFNA18 are allelic mutations of the gene encoding harmonin (Ahmed et al. 2002). Defects in protocadherin 15 (PCDH15) cause

USH1F (Ahmed et al. 2001; Alagramam et al. 2001). In the Ashkenazi Jewish population, the

R245X mutation of PCDH15 accounts for more than half (58.3 %) of USH1 cases (Ben-

Yosef et al. 2003).

I.5.2. PCDH15

PCDH15 is located on chromosome 10q11.2-q21. Protocadherins are thought to be involved in neural development, neural circuit formation and formation of the synapse

(Suzuki 2000). Mice carrying a mutation in Pcdh15 show disorganization in the placement of the stereocilia. It was suggested that PCDH15 plays a role in regulation of planar polarity in the sensory neuroepithelium of the inner ear. Members of the cadherin superfamily are also required in the eye and inner ear for maintenance of normal function (Alagramam et al.

2001).

28 The Israeli population is genetically heterogeneous and divides into two main ethnic groups, the Ashkenazi Jews originating from Europe, and the Sephardic/Oriental community who came from the Mediterranean Sea area and the Middle East. In the present thesis we report the estimated prevalence of the R245X mutation among Ashkenazi Jewish deaf probands in Israel, and carrier rates I found in the population for this mutation.

Since the R245X mutation is known to be involved in USH1F, some children incompletely diagnosed with NSHL excluded for CX26/Cx30 mutations, may have this mutation. This would indicate that they have USH1 and hence will develop RP during late childhood.

I.6. Otosclerosis

Otosclerosis is a common disorder of the otic capsule of the human temporal bone.

An unknown trigger initiates bone remodeling, characterized by an active phase of resorption of mature bone and deposition of a spongy vascularized bone, that eventually results in an inactive phase of a dense sclerotic mass (Chole and McKenna 2001; Zhao et al. 2002; Gros et al. 2003; Menger and Tange 2003) (Figure I-15).

29

Figure I-15. Otosclerosis of otic capsule: erosion into normal otic capsule. An active focus of otosclerosis is seen invading into the normal otic capsule bone. The process appears to be an initial osteolysis followed by new bone deposition. (Chole and McKenna 2001).

Invasion of otosclerotic foci into the stapedio-vestibular joint leads to fixation of the stapes in the oval window, resulting in clinical otosclerosis that occurs in about 0.2 to 1% of

Caucasians (Gordon 1989) (Figure I-16).

Figure I-16. A focus of otosclerosis, shown on a temporal bone section. This horizontal section demonstrates the two phases of otosclerosis, a spongiotic or active phase and a relatively inactive sclerotic phase. The footplate of the stapes is replaced by otosclerosis, as seen lateral to the vestibule. The focus contacts the spiral ligament in this case; the involvement of the otosclerotic focus near the spiral ligament may lead to sensorineural hearing loss. (Chole and McKenna 2001).

30 Otosclerosis is characterized by progressive conductive hearing impairment ranging up to 60 dB, which might develop into mixed or even SNHL. The HL first affects the low frequencies. This first stage is thought to be caused by the presence of highly cellular fibrous tissue that characterizes the spongiotic phase. As the pathological changes progress to a stage of localized bony fixation of the anterior part of the footplate, it is thought to result in a moderate conductive HL spanning all frequencies. The HL increases to moderately severe when the diffuse bony ankylosis involves the entire circumference of the annular ligament, completely preventing the motion of the stapes in the oval window (Cherukupally et al. 1998;

Zhao et al. 2002). Cochlear otosclerotic foci adjacent to the basilar membrane might constrict the cochlear lumen, with distortion of the basilar membrane, leading to inhibition of the traveling sound wave and therefore adding a sensorineural component to the HL (Linthicum and Lalani 1975). Another possible explanation is that the SNHL is caused by hydrolytic enzymes secreted into the perilymph by an impaired blood supply to the stria vascularis, due to vascular shunts (Causse et al. 1989; Ramsay and Linthicum 1994). A sensorineural component may also be a result of the Carhart notch effect that is characteristic of otoscelerosis (Lopponen and Laitakari 2001). The Carhart effect is an elevation in BC threshold, mainly in the middle frequencies (500-2000 Hz). This is caused by the fixation of the stapes that may dampen the resonance of the middle ear that is normally around 1000 Hz.

The BC threshold elevation can be restored after a successful operation.

Controversy exists as to whether or not pure SNHL can be caused by isolated cochlear otosclerosis without stapedial involvement, since histological evidence of pure cochlear otosclerosis is very rare (Youssef et al. 1998). Nevertheless, it is quite common for SNHL to develop as the disease progresses. Long-term follow up reveals that approximately 10% of individuals with otosclerosis ultimately develop severe to profound SNHL (Ramsay and

Linthicum 1994).

31 The age of onset for otosclerosis is usually 20-40 years and in most cases both ears are involved, but HL is often asymmetric (Goh et al. 2002). A definitive diagnosis of otosclerosis can be made only by surgery, but existence of family history of the disease, as well as some other clinical characterizations, are considered sufficient to diagnose otosclerosis in a family where only some members were surgically confirmed (Sakihara and

Parving 1999; Lopez-Gonzalez and Delgado 2000; Zhao et al. 2002). Both environmental and genetic factors have been implicated in otosclerosis. A 2:1 female-to-male predominance suggests hormonal involvement as well (Vartiainen 1999). An association between mutations in the collagen gene COL1A1 that underlies , and otosclerosis was found in a small percentage of cases (McKenna et al. 2002). In some other cases of otosclerosis, a collagen autoimmune mechanism was suggested since antibodies against collagen II and IX were present (Niedermeyer and Arnold 2002).

Otosclerosis is inherited in an autosomal dominant fashion with reduced penetrance, estimated at 40% (Morrison 1967; Menger and Tange 2003). Thus far, four locations have been reported for otosclerosis genes, chromosomes 15q25-q26 (OTSC1) (Tomek et al. 1998),

7q34-36 (OTSC2) (Van Den Bogaert et al. 2001), 6p21.3-22.3 (OTSC3) (Chen et al. 2002) and 3q22-24 (OTSC5) (Van Den Bogaert et al. 2004), although none have been cloned. I mapped the fifth locus (OTSC4) to chromosome 16q22.1-23.1.

32 II. RESEARCH GOALS

1. Ascertainment of large families with deafness, Family O and Family Z, and audiologica evaluation of hearing impaired members in the families.

2. Identification of new genes for deafness in large families, including the following steps:

a) Exclusion of mutations in known genes for deafness in the Israeli population.

b) Linkage exclusion of known deafness loci for genes associated with the same

type of HL (i.e. otosclerosis).

c) Genome-wide scan.

d) Statistical evaluation of linkage.

e) Fine mapping of linked regions with additional markers.

f) Identifying genes by the positional-candidate gene approach.

g) Evaluation of candidate genes by sequencing DNA.

3. If new genes are identified:

a) Determining prevalence of the mutations and correlation to ethnic group, and

analyzing genotype-phenotype correlations.

b) Characterizing the genes identified in terms of expression and function by RT-

PCR, in situ hybridization methods, and immunohistochemistry.

4. Characterization of newly discovered deafness genes in the Israeli population, Cx30 and

PCDH15, i.e. determining prevalence, carrier rate, correlation to ethnic group and genotype- phenotype correlations.

5. Correlation of Cx26 mutations and efficacy of cochlear implants.

33 III. MATERIALS AND METHODS

III.1. Ascertainment of probands and families

III.1.1. Letters, informed consent and questionnaires

Letters addressing deaf organizations, centers and individuals were prepared, explaining the genetic etiology of deafness, the study being performed in our laboratory and its advantages (Appendix VII.1.1). The deaf, or parents of deaf children, were asked to sign consent forms (Appendix VII.1.2). When first contacted, a questionnaire was filled in providing details about HL, presence of other symptoms, information about other deaf members in the family and ethnic origin (Appendix VII.1.3). An appointment was arranged for the entire family (deaf and hearing), for drawing blood, testing hearing and for further clinical assessment. The study was approved by the Tel Aviv University Helsinki Committee.

III.1.2. Clinical assessment

I collected all the clinical data from available family members. Particular emphasis was given to medical history regarding subjective degree of HL, age of onset, evolution of

HL, symmetry of hearing impairment, hearing aids, presence of , medication, noise exposure, pathologic changes in the ear, and other relevant clinical manifestations that might point to a syndrome, such as condition of eyes, kidney, heart, thyroid or any other results of clinical tests performed in the past. Information about deceased family members was obtained from their living family members. After clinical histories ensured that the HL was not a result of non-genetic causes (e.g., trauma, infection, drugs), a family tree was drawn.

III.1.3 Audiometry

Some of the affected individuals had audiograms obtained in hearing centers. In case of congenital non-syndromic, non-progressive HL, there was no need to repeat the hearing

34 test. When progressive HL was involved, we asked for all the audiograms available and if there was not an updated audiogram, we performed audiometry with a portable audiometer

(Maico MA-40) in the person's home, in a quiet but not soundproof room.

Pure tone audiometry (air and bone conduction levels) was performed. AC was performed at 250, 500, 1000, 2000, 4000 and 8000 Hz, and BC at 500, 1000, 2000 and 4000

Hz.

When audiometry was performed in professional hearing centers, patients underwent speech audiometry as well, and some of the hearing impaired that were tested further had also immittance testing results (tympanometry and acoustic reflexes) and/or ABR results available.

Severity of HL was classified according to ASHA as follows: <16 dB, normal hearing;

16 dB to 25 dB, slight HL; 26 dB to 40 dB, mild HL; 41 dB to 55 dB, moderate HL; 56 dB to

70 dB, moderately severe HL; 71 dB to 90 dB, severe HL; and >90 dB, profound HL (Clarke

1981). Definitions of audiometric configurations are according to Pittman and Stelmachowicz

(Pittman and Stelmachowicz 2003).

III.1.4. Blood collection

Blood samples were collected from each participating family member by venipuncture after obtaining informed consent in accordance with the guidelines of the Tel Aviv University

Helsinki Committee. Up to 10ml blood was mixed with EDTA (0.1ml 0.5M EDTA for 20ml blood) to prevent clotting (Miller 1988). For establishment of cell lines, blood was also collected into heparin vacutainer tubes.

35 III.2. Molecular analysis

III.2.1. Materials

III.2.1.1. Protocols for buffers, solutions and gels

Use Working Protocol (per 1L) Final Concentration Material Protocol Isolation of 8.28gr NH4Cl 0.15M NH4Cl Red Blood Cell 0.79gr NH HCO 1mM NH HCO Lysis Buffer DNA by 4 3 4 3 0.2ml of EDTA 0.5M, pH=7.4 0.1mM EDTA (pH=7.4) Salting-Out Complete to 1L with ddH2O Store at 4°c Technique Isolation of 50ml Tris-HCl 1M, pH=7.5 25mM Tris-HCl (pH=7.5) 1X Lysis Buffer

DNA by 33.4ml NaCl 3M 0.1M NaCl 1mM EDTA Salting-Out 2ml EDTA 0.5M

Technique Complete to 1L with ddH2O Store at room temperature Isolation of Dissolve Proteinase K in Proteinase K

DNA by ddH2O to 5mg/ml final Salting-Out concentration Technique Agarose gel Dissolve Ethidium Bromide Ethidium in ddH O to 1mg/ml final 2 Bromide concentration

Agarose gel 242gr Tris base, pH 8.0 2M Tris base, pH 8.0 50x TAE 57.1ml Acetic Acid 1M 57.1 mM Acetic Acid Buffer 100ml EDTA 0.5M pH 8.0 Complete 0.05M EDTA, pH 8.0 to 1L with ddH2O Store at room temperature Running PCR 1%,1.5%, 2%, 4% Agarose Agarose Gel products (FMC) 1x TBE buffer 0.01% Ethidium Bromide Running PCR 0.25% BromoPhenol Blue 5x Loading products XyleneCyanole FF Buffer on gel 30% glycerol in water Radioactive Dissolve Ammonium 10% APS

36 labeling Persulfate in ddH2O to (Ammonium Silver staining Persulfate) 0.1g/ml final concentration Radioactive 108gr Tris-HCl 0.9M Tris-HCl 10x TBE Buffer labeling 55gr Boric Acid 0.9M Boric Acid 5x TBE Buffer . Silver staining 40ml EDTA 0.5M, pH 8.0 0.02M Na2EDTA 2H20, pH For 10X complete with ddH2O to 1L 8.0 For 5X complete with ddH2O to 2L Store at room temperature Radioactive 157.5ml 40% Poly Acrylamide Gel 6% Poly Acrylamide Gel Urea Solution labeling SolutionTM (Boehringer Mannheim) Solution Silver staining 52.5ml 10x TBE 46mM Tris-HCl 441gr Urea 46mM Boric Acid . Complete with ddH2O to 1L 1mM Na2EDTA 2H20, pH 8.0 7.35M Urea Radioactive 9.5ml Formamide 95% Formamide Acrylamide labeling 0.4ml EDTA 0.5M, pH 8.0 0.02M EDTA Stop Silver staining 5mg Bromophenol Blue 0.05% Bromophenol Blue Solution 5mg Xylene 0.05% Xylene (Loading Solution) 25µl of 4N NaOH 0.01M NaOH Silver staining 500 µl ethanol 99.2% ethanol Bind Silane 1.5µl bind silane 0.3% bind silane 2.5µl acetic acid 0.5% glacial acetic acid Radioactive 80ml Urea Solution 100% Urea Solution 6% Denaturing labeling 0.05% TEMED 400µl 10% APS Polyacrylamide 40µl TEMED 0.05% APS. Gel Silver staining 75ml Urea Solution 100% Urea Solution 6% Denaturing 0.07% APS 500µl 10% APS Polyacrylamide 50µl TEMED 0.07% TEMED Gel Silver staining 200ml Glacial Acetic Acid 10% Glacial Acetic Acid Fix Solution 1800ml ddH2O 90% ddH2O Silver staining 2gr Silver Nitrate AgNO3 6mM Silver Nitrate AgNO3 Staining 3ml 37% formaldehyde 0.06% formaldehyde Solution Complete to 2L with ddH2O Silver staining 60gr Sodium Carbonate Na2CO3 0.29M Sodium Carbonate Developer 3ml 37% Formaldehyde Na2CO3 Solution 400µl Sodium Thiosulfate 0.06% formaldehyde Complete to 2L with ddH2O 0.02% Sodium Thiosulfate

37 III.2.1.2. Reagents

Reagent Supplier Product Specifications Use ReddyMix PCR ABgene Cat# AB-0575-DC-LD PCR Master Mix r-Taq Polymerase Takara Biomedical Buffer included Radioactive labeling Reverse Transcriptase Gibco BRL SuperScript II RNase H RT PCR Reverse Transcriptase enzyme MwoI New England Cat# 573S Restriction assay– Biolabs® Inc. 167delT of Cx26 BslI New England Cat# 555S Restriction assay– Biolabs® Inc. 35delG of Cx26 BspMI New England Cat# 502S Restriction assay– Biolabs® Inc. EX1 of Cx26 HphI New England Cat# 158S Restriction assay– Biolabs® Inc. R245X of PCDH15 ∝-dCTP Amersham Redivue 10µCi/µl Radioactive Pharmacia Biotech ~3000Ci/mmol labeling UK Polyacrylamide Boehringer Acrylamide/ Methylene Gels for Mannheim Bis-Acrylamide radioactive mixture 30% 19:1w/v. labeling and silver Cat# 1871 749 staining Oligonucleotide Gibco BRL Desalted; Unpurified RT PCR Primers Oligo (dT) Primer Invitrogen RT PCR Random Primer Invitrogen RT PCR Agarose Hispanagar Type: D1-250 Gels Deoxynucleoside- Boehringer Cat# 1 277 049 Radioactive triphosphate Set Mannheim Working mixture: labeling 1mM each Autoradiographic Film Fuji Photo Film New RX (ref. 03E010) Radioactive Co. LTD labeling TEMED AMRESCO Ultra pure grade Radioactive labeling and silver staining Silanizing Agent FMC Gel SlickTM Solution Radioactive labeling and silver staining

38 III.2.1.3. Instruments

Reagent Supplier Product Use Specifications PTC 200 MJ Research PCR Thermal Cycler Inc. Polyacrylamide Bio-Rad Sequi-Gen GT cat# Radioactive labeling Gel 165-3860-63 and silver staining Electrophoresis Apparatus Polyacrylamide Bio-Rad Power Pac 3000 Radioactive labeling Gel Cat# 165-5056-60 and silver staining Electrophoresis Power Supply Agarose Gel EC Apparatus MiniCell EC370M Running PCR Electrophoresis Corporation products Apparatus Agarose Gel EC Apparatus EC-103 Running PCR products Electrophoresis Corporation Power Supply PAA Gel Drier Savant SGD 4050 Radioactive labeling Instruments, Inc Spectroscopic Pharmacia Gene Quant II DNA concentration DNA Biotech Concentration Measurement Automated Applied Big Dye Terminator Sequencing Sequencing- Biosystems Inc. Cycle sequencing kit Reactions Automated Applied ABI Prism 377 DNA Sequencing Sequencing- Biosystems Inc sequencing unit Electrophoresis

39 III.2.1.4. Kits

Kit Supplier Product Use Specifications PCR Purification Boehringer High Pure PCR Sequencing Kit Mannheim Product Purification Kit cat# 1732676 Gel Extraction Kit Qiagen Cat# 28704 Sequencing Wizard SV Gel and Promega Cat# A9281 Sequencing PCR Clean-Up System Advantage-GC Clontech Cat# K1908-1 PCR of GC-rich Genomic Kit regions Taq DNA Qiagen Cat# 201203 PCR of GC-rich Polymerase with Q- regions solution DNA Isolation Kit Boehringer Cat# 1667 327 DNA extraction For Mammalian Mannheim Blood RNeasy® Mini Kit Qiagen Cat# 74104 RNA extraction Expand Reverse Roche Cat# 1 785 834 RT PCR Transcriptase Silver Staining Kit Amersham PlusOne DNA Silver Silver Staining Pharmacia Biotech Staining Kit AB

III.2.2. General methods

III.2.2.1. DNA extraction

III.2.2.1.1. Isolation of DNA by kit

Genomic DNA was extracted from whole blood (Miller 1988) or buccal smears

(Richards et al. 1993) using the DNA Isolation Kit for Mammalian Blood manufactured by

Boehringer Manheim.

III.2.2.1.2 Isolation of DNA by salting-out technique

Five to ten ml of blood was collected in a sterile EDTA vacutainer. Red Blood Cell

lysis buffer (see section II.2.1.1) was added 3 times the volume of the blood and mixed

gently. Tubes were kept on ice for 20 minutes and then centrifuged at 2000 rounds per minute

(rpm) for 10 minutes at 4°C. The supernatant was carefully removed and the pellet was

40 resuspended in 3 ml Red Blood Cell lysis buffer and centrifugation was repeated. After breaking the pellet, it was suspended in a mix of 3ml of 1X Lysis buffer (see section II.2.1.1),

100ul of 20% SDS and 50µl of 5mg/ml Proteinase K (see section II.2.1.1), followed by incubation at 55°C for 3 hours or 37°C overnight. After incubated, 1ml of 6M NaCl was added to the lysate and vigorously vortexed. Then it was centrifuged at 3000 rpm for 20 minutes at room temperature. The supernatant (the upper phase) was transferred gently into a

15ml (or 50ml, depending on volume) tube, avoiding the salt protein deposit. Twice the volume of the tube of 100% cold ethanol (EtOH) was added and gently mixed by inverting the tube. DNA was removed with a glass Pasteur pipette, washed in 70% EtOH (in an eppendorf tube) and air-dried for a few minutes on the Pasteur pipette. DNA was then dissolved in ddH2O (200-1000ml depending on the amount of DNA) and left at room

temperature overnight.

III.2.2.2. Establishment of cell lines

Blood collected into heparin vacutainer tubes was sent to the National Laboratory for

the Genetics of Israeli Populations at Tel Aviv University for cell line establishment using

Epstein-Barr-Virus (EBV) transformation (Neizel 1986).

III.2.2.3. RNA extraction from tissues and cell culture

RNA was isolated using RNeasy® Mini Kit (Qiagen) according to the manufacturer’s

protocol.

41 III.2.2.4. Reverse transcriptase-PCR

The RNA extracted was used as a template for reverse transcription, using the Expand

Reverse Transcriptase Kit manufactured by Roche according to the manufacturer’s protocol.

III.2.2.5. Basic PCR touchdown protocol

III.2.2.5.1. Reaction protocol

• PCR ReddyMix (Abgene) 15µl • Primer F 1µl1 • Primer R 1µl1 • Sample DNA 1µl2 • DDH2O 12µl

1Primer concentration: 10pM. 2Optimal DNA concentration: 100-300ng per reaction.

We multiply each portion by the number of samples that we are doing +1 (for a control).

III.2.2.5.2. PCR program

1. 95ºC – 5’ 2. 95° - 30’’ 3. 60º* - 30’’ 4. 72° - 1’** 5. 95º - 30’’ 6. 59° - 30’’ 7. 72º - 1’ 8. 95º - 30’’ 9. 58° - 30’’ 10. 72° - 1’ 11. 95° - 30’’ 12. 57º - 30’’ 13. 72° - 1’ 14. 95° - 30’’ 15. 56º - 30’’ 16. 72° - 1’ 17. 95º - 30’’ 18. 55° - 30’’ 19. 72° - 1’ 20. Go to 17 – 30 cycles 21. 72º - 10’

42 22. 4º - 10’ 23. end

*Annealing temperature was changed in correlation with primers Tm. ** Extension time according to size of fragment - 30’’ for each 500bp.

Run 3 µl of the reaction on a 1% agarose gel to check PCR product.

III.2.2.6. Electrophoresis of PCR products using agarose gels

The concentration of the agarose gel depends on the sizes of the DNA fragments being run. One percent agarose gels containing 0.01% ethidium bromide were usually used, prepared using 1X TAE running buffer (see section II.2.1.1). Three µl of PCR product were loaded onto the gel and run in 1X TAE running buffer at 80V for 30-45 minutes, depending on the fragment size. DNA fragments were observed using ultraviolet light and photographed using the FUJI thermal imaging system FTI-500.

III.2.2.7. DNA sequencing

III.2.2.7.1. Cleaning DNA following PCR

If the PCR product showed a clear band on an agarose gel, it was cleaned using the

Wizard® SV Gel and PCR Clean-Up System (Promega).

III.2.2.7.2. Extraction of DNA fragments from agarose gels

When more than one band of PCR products were seen on an agarose gel, the DNA fragment of the desired size was cut from the agarose gel, using a UV lamp, and then DNA was cleaned using the Wizard® SV Gel and PCR Clean-Up System (Promega).

43 III.2.2.7.3. Sequencing of the purified PCR Product

The DNA concentration of the purified product solution was determined using the

Gene QuantII (Pharmacia Biotech). DNA samples were prepared for sequencing using 10ng per 100bp of PCR DNA with 1ul 10mM primer and adding ddH20 to a total of 16ul.

Automated sequencing was performed at the Tel Aviv University Sequencing Department

(Faculty of Life Sciences) using the ABI Prism BigDye Terminator Cycle Sequencing Ready

Reaction Kit and the ABI 377 DNA sequencer (PE Biosystems).

III.2.2.8. SDS-PAGE

III.2.2.8.1. DNA radioactive labeling

The urea solution (see section III.2.1.1) was slowly poured, using a syringe, between

the two precleaned glass plates that are separated by 0.4 mm thick spacers. The longer glass

plate was coated with silanizing agent. A 0.4 mm thick shark tooth was inserted into the gel’s

edge between the two plates before the gel polymerized. The polymerized gel was placed in

the sequencing apparatus and 1X TBE Running Buffer was added.

Five µl from each PCR amplified product were mixed with 5µl of Acrylamide Stop

Solution (see section III.2.1.1). The mixture was boiled for 5 minutes to denature the PCR

products and then transferred immediately to ice. Eight µl of each sample was loaded in each

well and electrophoresed at 60W for 2-3 hours depending on the size of the marker’s

fragments. After the run was completed, the gel was transferred onto 3 mm Whatman paper, dried under vacuum at 80°C for 30 minutes and then exposed to X-ray film.

III.2.2.8.2. DNA silver staining

Electrophoresis of PCR products was done using a six percent denaturing

polyacrylamide gel (see section III.2.1.1). The gel was slowly poured, using a syringe,

44 between the two precleaned glass plates that were separated by 0.4 mm thick spacers. The longer glass plate was coated with silanizing agent to prevent the gel from sticking to the glass plate. The short glass was coated with Bind Silane (section III.2.1.1) to enable the gel to bind to the glass. A 0.4 mm thick shark tooth was inserted into the gel’s edge between the two plates before the gel polymerized. The polymerized gel was placed in the sequencing apparatus and 0.5X TBE Running Buffer was added and preheated to 50°C to prepare it for running.

Four µl from each PCR amplified product was mixed with 4µl of loading solution

(section III.2.1.1). The mixture was boiled for 3 min. to denature the PCR products and then

transferred immediately to ice. Six µl of each sample was loaded in each well and

electrophoresed at 70W for 2-3 hours depending on the size of the marker’s fragments.

After the run was completed, the glasses were carefully separated and the gel stayed

attached to the short glass. The gel was now washed in several different solutions, in plastic

baths, using Promega's SILVER SEQUENCE-DNA Staining Reagents (#Q4132). The first

wash, fix solution (section III.2.1.1), was done for 20 min. followed by 3 rinses with DDW (2

min. each). The gel was then immersed in staining solution (section III.2.1.1) and agitated for

30 min. The gel was dipped for 10 sec. in DDW and immediately placed in developing

solution (section III.2.1.1). The gel was agitated until the bands were visible and then

transferred into the fix solution for 5 min. This stage was followed by two rinses with DDW

(2 min each) and then dried at room temperature.

Alleles for each marker were numbered according to their relative mobility on the gel.

45 III.2.3. GJB2 (Cx26) mutation analysis

III.2.3.1. Identification of GJB2 mutations by restriction enzyme digestions

III.2.3.1.1. Detection of 35delG mutation by BslI restriction enzyme digest

This method was based on the principle of PCR-mediated site-directed mutagenesis

(PSDM), followed by Bsl1 digestion (Storm et al. 1999). Using a primer set that consisted of a wildtype forward primer and a modified reverse primer that was modified at nucleotide

268, the amplified fragment of 207bp had a newly created restriction site for the Bsl1 enzyme. The following primers were used for amplification of this fragment.

Cx26-35delG-F 5’-GGT GAG GTT GTGTAA GAG TTG G –3’ Cx26-35delG-R 5’-CTG GTG GAG TGT TTG TTC CCA-3’

III.2.3.1.1. 1. Amplification protocol

• PCR ReddyMix (Abgene) 15µl • Primer F 1µl1 • Primer R 1µl1 • Sample DNA 1µl2 • ddH2O 12µl

1Primer concentration 10pM. 2Optimal DNA concentration 100-300ng per reaction.

III.2.3.1.1. 2. PCR program

Thermal cycling amplification was done using the following program:

1. 94ºC 3’ 2. 94º 1’ 3. 60º 1’ 4. 72º 1’ 5. go to 2 35 cycles 6. 72º 10’ 7. 4º 10’ 8. end

Run 3 µl of the PCR product on a 1% agarose gel to check PCR product.

46 III.2.2.3.1. 3. Restriction enzyme digestion

BslI 0.5µl (0.25 units/µll) 10X Buffers 3 2.0µl ddH2O 7.5µl PCR product 10.0µl

Multiply each portion by number of samples.

„ Add a known heterozygote to the digestion reaction, for a control. „ Tubes are incubated at 55oC for 16 hours. „ Load 10 µl of digested reaction on a 4% agarose gel.

Expected Fragments (bp):

Wildtype 207 Heterozygote 207 181 26 35delG Homozygote 181 26

III.2.3.1.2 Detection of 167delT mutation by MwoI restriction enzyme digest

A fragment of 322bp of the open reading frame (ORF) was amplified using Cx26-6F

and Cx26-5R primers:

Cx26-6F 5’- GAT TGG GGC ACG CTG CA –3’ Cx26-5R 5’- CCC TTG ATG AAC TTC CTC TTC TTC -3’

III.2.3.1.2.1. Amplification protocol

• PCR ReddyMix (Abgene) 15µl • Primer F 1µl1 • Primer R 1µl1 • Sample DNA 1µl2 • d.d.H2O 12µl

1Primer concentration 10pM. 2Optimal DNA concentration 100-300ng per reaction.

III.2.3.1.2.2. PCR program

Thermal cycling amplification was done using the following program:

1. 94ºC 3’ 2. 94º 30’’ 3. 62º 30’’

47 4. 72º 1’ 5. go to 2 35 cycles 6. 72º 10’ 7. 4º 10’ 8. end

Run 3 µl of the PCR product on a 1% agarose gel to check PCR product.

III.2.3.1.2.3. Restriction enzyme digestion

The detection of this mutation is possible, as the Mwo1 restriction site is created as a result of the deletion of T at position 167, while the site does not exist in the wild type sequences.

MwoI 0.5µl (0.25 units/µl) Buffer MwoI ...... 2.0µl

ddH2O 7.5µl PCR product 10.0µl

Multiply each portion by number of samples. „ Add a known heterozygote to the digestion reaction, for a control. „ Tubes are incubated at 60oC for 6 hours. „ Load 10 µl of digested reaction on a 1% agarose gel.

Expected Fragments:

Wildtype 322 Heterozygote 322 161 167delT Homozygote 161

III.2.3.1.3. Identification of the GJB2, exon 1 mutation, IVS1+1(G->A), by restriction

digest

A fragment of 540 bp of the upstream region of GJB2, including the non-coding exon

1 and part of the intron, was amplified using the following primers:

Cx26-Ex1F 5'- GGC GAC ACC ACA AAC CTC -3' Cx26-Ex1R 5'- CCT CCG TAA CTT TCC CAG TC -3'

PCR amplification was performed according to the basic PCR touchdown (60º->55º) protocol

as described in section III.2.2.5.

48 III.2.3.1.3.1. Restriction enzyme digestion

The detection of this mutation is possible, as the BspMI restriction site that exists in the wild type at position +1 of exon 1 is eliminated as a result of the substitution of G->A in this splicing site of exon 1.

BspMI 1.5µl 10X Buffers 3 ...... 2.0µl

ddH2O: 12.5µl PCR product 4.0µl

Multiply each portion by number of samples.

„ Add a known heterozygote to the digestion reaction, for control. „ Tubes are incubated at 37oC for 8 hours. „ Load 5 µl of digested reaction on a 2% agarose gel.

Expected Fragments:

Wildtype 309 231 Heterozygote 540 309 231 IVS1+1(G->A) Homozygote 540

III.2.3.2. Detection of GJB2 mutations in the open reading frame by direct

sequencing

A 722bp PCR fragment was amplified using a primer pair that spans the ORF of

connexin 26 (Kelsell et al. 1997).

Cx26-1F 5’- TCT TTT CCA GAG CAA ACC GC - 3’ Cx26-2R 5’- GGG CAA TGC GTT AAA CTG GC -3’

III.2.3.2.1. Amplification protocol

• PCR ReddyMix (Abgene) 25µl • Primer F 3µl1 • Primer R 3µl1 • Sample DNA 1µl2 3 • ddH2O 18µl

49

1Primer concentration 10pM. 2Optimal DNA concentration 100-300ng per reaction. 3If PCR product did not show a clear band on electrophoresis gel, 5µl of DMSO (10% of the volume) was added.

III.2.3.2.2. PCR program

Thermal cycling amplification was done using the following program:

1. 94ºC 3’ 2. 94º 30’’ 3. 62º 30’’ 4. 72º 1’ 5. go to 2 35 cycles 6. 72º 10’ 7. 4º 10’ 8. end

III.2.3.2.3. Cleaning and sequencing of PCR products

Three µl of the PCR products were run on 1% agarose gel (prepared as described in

section II.2.2.6.) to ensure that the amplification worked and that the desired band was

obtained, free from other artifact bands that might be amplified along with the desired band.

In most cases one clear band was obtained and the PCR product was purified using the

Wizard® SV Gel and PCR Clean-Up System (Promega). In some cases, when more than one

band was observed, only the right size band was cut from the gel using a UV lamp, and then

DNA was cleaned using the Wizard® SV Gel and PCR Clean-Up System (Promega).

Concentration of the DNA samples was measured and then the samples were sequenced as

described in section II.2.2.6.3.

III.2.3.2.4. Detecting GJB2 mutations by bioinformatics

Sequences were compared to the Cx26 sequence taken from NCBI

(http://www.ncbi.nlm.nih.gov) (i.e. GI:42558282) using BLAST

(http://www.ncbi.nlm.nih.gov/BLAST/) to determine if there were any mutations in the

50 obtained sequence. Samples that showed mutations were sequenced in both forward and reverse directions.

III.2.4. Identification of the ∆(GJB6-D13S1830) mutation (Cx30)

The Cx30 mutation consists of a 342kb deletion (Del Castillo 2002). I designed a 3- primer protocol for detecting the ∆(GJB6-D13S1830) mutation in one PCR reaction. Two

primers (F and R1) were designed to detect the deletion. In addition, I designed a third primer

(R2) for the same PCR reaction to serve as a positive control should there be no deletion. F

was designed 244bp upstream of the proximal breakpoint of the deletion, R1 is localized

216bp downstream the distal breakpoint of the deletion, and R2 is localized 681bp from F

(Figure III-1).

F R2 R

681bp

244bp 216bp centromeric telomeric

GJB2 ~35kb GJB6

Ex2 Ex1(ncd) Ex2 Ex1(ncd) 5’ 5’

342Kb deletion Figure III-1. Schematic location of the 3 primers designed to detect the ∆(GJB6- D13S1830) mutation in one PCR reaction.

The 3 primers designed:

GJB6-F 5'- TTT AGG GCA TGA TTG GGG TGA TTT -3' GJB6-R 5'- CAC CAT GCG TAG CCT TAA CCA TTT T -3' GJB6-R2 5’-TCA TCG GGG GTG TCA ACA AAC A-3’

51 PCR amplification yields products of:

Wt 681bp Homozygote for the deletion 460bp Heterozygotes 681bp 460bp

III.2.5. Identification of the R245X mutation in the PCDH15 gene

Three alternative assays were used. One is the allele-specific PCR (ASPCR) assay

(Ben-Yosef et al. 2003). Another method is a restriction digestion assay, using HphI, which is usually confirmed by the third method, sequencing of exon 8 (Figure III-2).

Figure III-2. Mutation detection assays for the R245X mutation of the PCDH15 gene. A. ASPCR assay, B. restriction enzyme assay and C. sequence analysis.

III.2.5.1. Allele-specific PCR (ASPCR)

To detect the R245X mutation by allele-specific PCR (ASPCR), two PCR reactions

were performed for each DNA sample. One amplified only the wild type allele and the other

amplified only the mutant allele. Band sizes for both reactions were 350 bp (Figure III-2).

52 One forward common primer was used for both reactions. The primers used (Ben-Yosef et al.

2003):

Common 5’- CTT TGT GTT AAA AAT GTA TTC ATA CTC CCT G -3’ Primer: CP-F WT-R 5’- AGG ACC GTG CCC AAA ATC TGA ATG AGA GCC -3’ MUT-R 5’- AGG ACC GTG CCC AAA ATC TGA ATG AGA GCT- 3’

PCR reactions were performed in a total of 30µl, using PCR ReddyMix, including 1µl of genomic DNA with an average concentration of 200ηg/µl. PCR cycling conditions were

95°C for 5 minutes, followed by a touchdown program of 61°C to 56°C and 30 cycles of

95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds. Two last steps followed of 72°C and 4°C for 10 minutes each. 20µl of reaction was separated by electrophoresis on a

2% gel.

III.2.5.2. Detection of the R245X mutation by restriction enzyme digestion

To detect the R245X mutation by restriction enzyme digestion assay, a 553bp segment of exon 8 was PCR-amplified by the primers:

361F 5’-ATA ACC ATG TTG GAC TGT TGT TTC-3’ 914R 5’-ATG TTT GCC AGG CTG GTA TCA AAC-3’

PCR conditions were as described above, except that touchdown annealing temperatures are 60°C to 56°C and time of denaturation, annealing and elongation in each cycle is 45 seconds instead of 30 seconds.

An HphI site is inserted due to the mutation. The PCR product is digested by HphI,

37°C for 10 hours and run on a 2% gel electrophoresis.

53 Expected Fragments (Figure III-2):

Wildtype 553bp Heterozygote 553bp 372bp 181bp R245X Homozygote 372bp 181bp

III.2.5.3. Detection of the R245X mutation by sequencing exon 8 of PCDH15

For sequence analysis, a 367bp segment of exon 8 was amplified by PCR using primers:

F 5’-TGC CTA ATT TCT ATA AAC TAC CTG TTG-3’ R 5’-CCC TGA AAA TAA TTT CGG ACA-3’

The PCR products were purified using the Wizard® SV Gel and PCR Clean-Up

System (Promega). Sequence was performed using the same primers as those used for PCR, the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer

Applied Biosystems) and an ABI 377 DNA sequencer. Sequencing results for WT and for homozygotes for the R245X mutation are shown in Figure III-2.

Mutations were confirmed by sequencing in both directions or by using at least two assays of the described above.

III.2.6. Otosclerosis

III.2.6.1. Family O

Twenty four individuals of Family O, both affected and unaffected, were ascertained as described above, in sections III.1.1 and III.1.2, giving particular emphasis to medical history. All family members underwent audiological evaluation performed by a portable audiometer (Maico MA-40) in the person's home, in a closed but not soundproof room, as described in section III.1.3. Two affected (IV:6 and IV:10) and one unaffected individual

54 (V:1) underwent pure tone and speech audiometry at the Speech and Hearing Center,

Hadassah Hebrew University Hospital and Medical School, Jerusalem, Israel, in a sound

treated room. Very similar audiometric curves were obtained from the Hearing Center when

compared to the results of the portable audiometer. Blood was drawn upon signing Helsinki

Committee approved consent forms, both for DNA extraction and establishment of

lymphoblastoid cell lines, and genomic DNA was extracted from the 24 family members.

III.2.6.2. Linkage exclusion

To determine the chromosomal location of the otosclerosis locus in Family O, linkage

to the known otosclerosis loci was first examined on chromosomes 15q25-q26 (OTSC1)

(Tomek et al. 1998), 7q34-36 (OTSC2) (Van Den Bogaert et al. 2001) and 6p21.3-22.3

(OTSC3) (Van Den Bogaert et al. 2002) using the following linked markers:

Locus Marker Band Location* Primers Length

OTSC1 D15S1004 15q26.2 92132022- F: GGCAAGACTCCATCTCAAAA 247-271 bps 92132356 R: GAATAAAAAGCCTGTAAACCACC OTSC1 D15S652 15q26.1 90318339- F: GCAGCACTTGGCAAATACTC 284-309 bps 90318669 R: CATCACTCAAGGCTCAAGGT OTSC2 D7S684 7q34 138000941- F: GCTTGCAGTGAGCCGAC 169-187 bps 138001294 R: GATGTTGATGTAAGACTTTCCAGCC OTSC2 D7S2513 7q34 140860602- F: GCAGCATTATCCTCAACAGC 157-181 bps 140861006 R: CACAAATGGCAGCCTTTC OTSC3 D6S291 6p21.31 36373492- F: CTCAGAGGATGCCATGTCTAAAATA 198-210 bps 36373700 R; GGGGATGACGAATTATTCACTAACT * according to http://genome.ucsc.edu, May 2004.

The radioactive labeling system (see section III.2.2.8.1) was used for band detection. Two-

point LOD Scores were calculated using LIPED v1.21 (1995).

55 III.2.6.3. Linkage analysis

III.2.6.3.1. Genome scan

A genome-wide search for linkage was performed by the Laboratory of DNA

Analysis at the Institute of Life Sciences, Hebrew University of Jerusalem. A total of 400

microsatellites spaced at 10cM intervals were analyzed. PCR product electrophoresis and

detection was performed using a 3700 Automated DNA Analyzer (Applied Biosystems).

Sizing and genotyping was performed using GENESCAN and GENOTYPER softwares

(Applied Biosystems). Linkage was evaluated using LIPED v1.21 (1995). Autosomal

dominant inheritance with 80% penetrance was assumed (Chole and McKenna 2001;

Niedermeyer and Arnold 2002; Menger and Tange 2003). Gene frequency was set at 0.0001.

Equal male and female recombination frequency was assumed. Marker frequencies were set

arbitrarily at 0.2.

III.2.6.3.2. Determining the location of the OTSC4 locus

Three regions on chromosomes 2, 16 and 16, with LOD scores above 1.5, were

further genotyped using additional microsatellite markers, which were chosen from the

UCSC Genome Browser (http://genome.ucsc.edu/).

The microsatellite markers used from the 3 regions:

Marker Band Location* Primers Length

D2S362 2q36.3 229563147- F: CTAAAAAACAACTCTGAATGTAGTC 102-116 bps 229563479 R: TATTACGGTTCCTAACAGCA D2S345 2q37.3 237583956- F: GGAAGCCACCATGAAT 249-259 bps 237584341 R: AGATCAACAGACATAACCCA D20S107 20q12 38315925- F: CTACATGATGCCTCTTGGGA 250-278 bps 38316258 R: TCAGACAATGGCAAATTCCT D20S891 20q13.12 45362995- F: GCAAGCATCTACAAGGCTCTTCAT 190-216 bps 45363372 R: CTACAGGTGAGCGCCACCAT D16S3106 16Q22.2 70745120- F: GAGACCTACAGTCTTTTGCATTTAC 166-206 70745465 R: TTTTGAAGCTGAGCAGAAGG BPS D16S507 16Q23.2 78633932- F: GCAGGGGCTAGAAGGTG 175-195 78634283 R: TGTTCGCCTCTTGCAGT BPS *according to http://genome.ucsc.edu, May 2004.

56 The DNA silver staining system SILVER SEQUENCETM Staining reagents was used

for band detection (see section III.2.2.8.2).

The OTSC4 gene symbol was approved by the HUGO Gene Nomenclature

Committee (http://www.gene.ucl.ac.uk/nomenclature/).

III.2.6.3.3. Haplotype analysis

In order to define the OTSC4 locus, additional microsatellite markers in the region,

which were chosen from the UCSC Genome Browser (http://genome.ucsc.edu/), were used.

Haplotypes were constructed using genotyping results of all the polymorphic markers

spanning the linked region. PCR amplification of these markers was performed using pairs of

specific primers and then alleles of each marker were numbered according to their relative

mobility on a denaturing polyacrylamide gel.

Table of markers: Marker Band Location* Primers Length D16S3043 16q22.1 64049633- F: CATTAATATGGAGCCTTATAGATTG 118-150 bps 64050004 R: AAATGTTGAGCACTTGAATAAAAT D16S3019 16q22.1 64686655- F: CAACTCATTCCCTGTGTGAC 206-254 bps 64687009 R: CTTACCTGGCTGTATGTAGACC D16S3107 16q22.1 66215417- F: CCAGAGTGATGGGGAATA 234-304 bps 66215744 R: TGAGCACTGTCTCAAAAAA D16S3025 16q22.1 67123897- F: TCCATTGGACTTATAACCATG 90-110 bps 67124211 R: AGCTGAGAGACATCTGGG D16S3067 16q22.1 67666164- F: GCCACCTCACACTAGCCTG 138-152 bps 67666478 R: TCACTCAAAATGGAGTCACTCTG D16S3095 16q22.1 68503729- F: TCAGTTGGAAGATGAGTTGG 134-162 bps 68504049 R: TATAGTTTGTGTCCCCCGAC D16S512 16q22.3 72625070- F: TGAGAGCCAAATAAATAAATGG 201-211 bps 72625430 R: TCACGTTGTGAATGCAAGT D16S3018 16q22.3 72730112- F: GGATAAACATAGAGCGACAGTTC 244-270 bps 72730469 R: AGACAGAGTCCCAGGCATT D16S3115 16q22.3 73087860- F: GGAGAATGGCTTTCTTGC 242-252 bps 73088205 R: CAACTCTATGATGGGGTTTTATTAC D16S3051 16q23.1 73825415- F: ACCAGGTGGTGCTGCGTGT 231-283 bps 73825708 R: AAGGGGCTGCCAGGGTTG D16S515 16q23.1 75074529- F: CATTCTGAAATTAGACAGCGATAGG 222-244 bps 75074903 R: TGTGACCAGAGGCTTGC D16S3097 16q23.1 75946559- F: TGATAGCCAAAGAAGTTGGT 194-214 bps 75946895 R: CTTGTGGGTCAATATAGATTAAAAA

57 D16S516 16q23.1 77681517- F: CCTCCAGAAACCGTGAGAT 164-176 bps 77681818 R: GGTGCCATCCTGACAGA D16S3040 16q23.1 78208997- F: TACTCCGGCAAGGACG 109-129 bps 78209301 R: GCTGCCTAGCACATGG D16S507 16q23.2 78633932- F: GCAGGGGCTAGAAGGTG 175-195 bps 78634283 R: TGTTCGCCTCTTGCAGT D16S511 16q23.2 80258655- F: CCCCGGAGCAAGTTCA 182-222 bps 80259024 R: CAGCCCAAAGCCAGATTA D16S3091 16q23.3 81537951- F: GGGAGATAGCCTTAAACTTTCTTAC 115-129 bps 81538275 R: TGTTGCTAATAACACTAGGCCA *according to http://genome.ucsc.edu, May 2004.

The DNA silver staining system SILVER SEQUENCETM Staining reagents was used

for band detection (see section III.2.2.8.2). If controversial results were obtained, band

detection was confirmed by the radioactive labeling system (see section III.2.2.8.1).

III.2.6.4. Candidate genes

The list of genes in the linked interval was taken from the UCSC Genome Browser

(NCBI Build 35; http://genome.ucsc.edu/). In order to sequence the genes, primers were

designed using Primer 3 Input, v 0.2 (http://frodo.wi.mit.edu/cgi-

bin/primer3/primer3_www.cgi). Primers were designed for cDNA for genes expressed in

lymphoblasts, covering the whole coding region of each of these genes in overlapping pieces.

Since most of the genes in the interval were not expressed in lymphoblasts, genomic primers

were designed for all coding exons.

The basic PCR touchdown protocol was used for amplification (see section III.2.2.5)

and sequencing was performed as described above (III.2.2.7).Candidate genes, primers and

expected fragments are specified in appendix VII.2.1.

III.2.6.4.1. Mutation analysis

Sequences obtained from affected family members were compared to the sequences

taken from the NCBI (http://www.ncbi.nlm.nih.gov) using BLAST

58 (http://www.ncbi.nlm.nih.gov/BLAST/) to determine if there were any mutations in the obtained sequence.

In case a sample of an affected member showed a change when compared to the sequence in the database, segregation of the change with the otoscelerosis was tested by sequencing additional DNA samples of the family, including samples of unaffected members.

If the change segregated in the family, an unaffected control group, of at least 100 samples was screened in order to differentiate between mutation and polymorphism.

59 IV. RESULTS

IV.1. Family O

IV.1.1. Ascertainment

A five generation pedigree of Israeli Family O, of Yemenite Jewish origin, was constructed (Figure IV-1). The disorder was transmitted from generation to generation and an autosomal dominant age-dependent inheritance with reduced penetrance was suggested.

Twelve members of Family O manifested different forms of hearing loss characteristic of otosclerosis (Figure IV-1), with a 2:1 female-to-male predominance (8 females and 4 males), that is concordant with the incidences published elsewhere (Pearson et al. 1974; Vartiainen

1999). Three individuals (IV:4, IV:8, IV:14) that were surgically confirmed as having otosclerosis, and a complete clinical history of each of the other affected individuals, in addition to the family history, led to the same diagnosis in patients that did not undergo surgery.

1 2 I

1 2 3 4 5 II

1 2 3 4 5 6 7 8 9 III

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 IV

1 2 3 4 V

Unaffected Unilateral Bilateral SNHL conductive or conductive or mixed HL mixed HL

Figure IV-1. Pedigree of Family O.

60

IV.1.2. Audiological evaluation

All the otosclerotic members of Family O have experienced progressive hearing loss, beginning in their late twenties to their forties. There was a large variability between affected individuals in age of onset, type of HL, shape of audiogram and symmetry of HL. There were no reports of tinnitus.

IV.1.2.1. Age of onset

No member of Family O could point to the exact age of onset of HL. Individual III:7 reported unilateral left HL since a young age, but HL was not present in childhood. Most of the women in the family started to face difficulties in hearing in their late twenties to early thirties, and the men claim to have noticed their HL in the late thirties to early forties.

Individual III:3, at 93 years of age, reported normal hearing despite having a sloping audiogram of mild to severe HL, as did individual V:2, age 26, who had a mild HL in low frequencies.

IV.1.2.2. Symmetry of HL

Out of the 12 affected members of Family O, nine had bilateral HL and three, two females and one male, had unilateral HL. No differences in severity or shape of audiogram were observed between individuals with bilateral vs. unilateral HL.

IV.1.2.3. Severity and type of HL

The severity and type of HL of the affected members of Family O are summarized in

Table IV-1, and pure tone averages across frequencies 500-4000 Hz are presented in Table

61 IV-2. Severity of HL ranges from mild to profound. Even though the youngest affected individual (individual V:2 at 26 yrs old) has a mild HL and one of the oldest (individual III:3 at 79 yrs old) has the most severe HL in the family, in between, there was little correlation between age and severity (Figure IV-2). Comparison between female and male severity of

HL showed higher degrees of HL for females in all age ranges, even though considerable variability was seen within the genders (Table IV-3, Figure IV-2).

Considerable inter-subject and inter-aural variability in type of audiogram is seen in

Family O. Conductive HL is observed in individuals IV:8, IV:9, IV:14 and V:2. Mixed HL, in individuals IV:4, IV:5, IV:10, III:3, III:8 and III:9, and individuals IV:6, III:3 and III:8 have SNHL at least in one ear. Individuals IV:4, IV:8 and IV:10 show elevations in BC thresholds characteristic of Carhart’s effect.

62 Table IV-1. Analysis of Audiograms in Individuals with Otosclerosis of Family O*.

Individual Gender Age Right ear severity Right ear Left ear severity Left ear Symmetry Carhart Surgery No. Audiogram Type Audiogram Type of HL notch III:7 Female 79 Normal Profound Flat unilateral - - Mixed to SN IV:4 Female 64 Moderately severe Flat Moderately severe Flat Bilateral + Rt ear Mixed Conductive IV:5 Male 62 Moderate to Sloping Mild to severe Sloping Rt>Lt - - profound Conductive to Conductive to SN mixed IV:6 Male 58 Normal to severe Sloping Normal to moderate Sloping Bilateral - - Mixed to SN SN IV:8 Female 51 Mild to normal to Tent shaped Moderately severe Flat Lt>Rt + Rt ear moderate Conductive Conductive IV:9 Male 50 Moderate to mild Tent shaped Normal Unilateral - - to severe Conductive IV:10 Female 49 Normal Moderately severe Flat Unilateral + - Conductive to mixed IV:14 Female 40 Moderately severe Rising Moderately severe Rising Bilateral - Lt ear to moderate Conductive to moderate Conductive III:8 Male 76 Mild to severe Sloping Profound to severe Rising Lt>Rt - - SN Mixed III:9 Female 68 Profound Flat Moderate to Sloping Rt>Lt - - Mixed profound Mixed III:3 Female 93 Mild to severe Sloping Mild to severe Sloping Bilateral - - Mixed to SN SN V:2 Female 26 Mild to normal Rising Mild to normal Rising Bilateral - - Conductive Conductive *Definition of audiometric configurations according to Pittman and Stelmachowics (Pittman and Stelmachowicz 2003).

63 Table IV-2. Analysis of Pure Tone Audiometry in Individuals of Family O with Otosclerosis.

Individual Gender Age Ear PTA-AC (dB) Range (dB) PTA-BC Range ABGap (0.25-8kHz) (dB) (dB) (dB) (0.5-4kHz) III:7 Female 79 Rt 22.5 20-45 20 15-25 2.5 Lt NR (110-120) 60 (0.5-1kHz) 55-NR NR (2-4kHz) IV:4 Female 64 Rt 60 55-65 32.5 25-40 27.5 Lt 58.75 55-65 12.5 0-30 46.25 IV:5 Male 62 Rt 66.25 45-100 33.75 5-60 32.5 Lt 37.5 25-70 21.25 0-45 16.25 IV:6 Male 58 Rt 31.25 10-70 21.25 0-50 10 Lt 26.25 10-55 17.5 0-45 8.75 IV:8 Female 51 Rt 18.75 15-20 5 0-10 13.75 Lt 65 60-85 13.75 5-25 51.25 IV:9 Male 50 Rt 50 35-70 5 5 45 Lt 13.75 10-30 1.25 0-5 12.5 IV:10 Female 49 Rt 18.75 15-40 12.5 10-15 6.25 Lt 73.75 65-80 33.75 25-40 40 IV:14 Female 40 Rt 51.25 35-70 8.75 5-10 42.25 Lt 57.5 45-70 7.5 5-10 50 III:8 Male 76 Rt 37.5 30-70 33.75 30-40 3.75 Lt 86.25 75-100 51.25 45-60 35 III:9 Female 68 Rt 88.75 80-100 43.75 35-50 45 Lt 61.25 45-100 32.5 25-35 28.75 III:3 Female 93 Rt 56.25 40-80 43.75 30-65 12.5 Lt 51.25 40-80 47.5 30-70 3.75 V:2 Female 26 Rt 22.5 10-30 11.25 0-15 11.25 Lt 20 15-30 15 15 5 Abbreviations: PTA, pure tone average across 500-4000Hz; AC, air conduction; BC, bone conduction; ABGap, air bone gap; NR, no response (at maximum output of audiometer).

64 A B

0 0

58 20 20 58 26 26 62 40 93 40 76 93 68 62 48 60 40 64 60 64 49 40 dB dB 80 80 51 68 76 100 100 79 120 120

140 140 125 250 500 1000 2000 4000 8000 125 250 500 1000 2000 4000 8000 Hz Hz

Figure IV-2. Pure tone audiograms of affected Family O members. All sclerotic ears are presented. Colors distinguish between different members. A. Female otosclerotic ears in Family O. There are six females with bilateral and two with unilateral otosclerosis but only 13 ears are presented since the female of age 51 had one ear operated successfully. Age range 26-93. B. Male otosclerotic ears in Family O. Age range 50-76. Note that there are more female than male otosclerotic ears, HL is more severe in females compared to males at certain age ranges, and females’ age range begins at a younger age.

Table IV-3. Comparison of Female/Male Degree of HL.

Females Males Age PTA1-AC (dB) Age PTA1-AC (dB) 79 100 76 38 68 89 76 88 68 58 Average 65-79 82.3 63 64 60 62 62 64 60 62 36 51 69 58 30 58 24 50 50 Average 50-64 63 40.4 93 53 93 50 49 73 40 53 40 60 26 24 26 22 Total average 59.3 42.1 Abbreviations: PTA, pure tone average across 250-4000Hz; AC, air conduction.

65 IV.1.2.4. Audiometric configuration

A large variability exists concerning shape of audiogram as well as the other aspects of HL. Individuals III:7, IV:4, IV:8, IV:10 and III:9 have flat audiograms. Individuals IV:5,

IV:6, III:3, III:8 and III:9 have sloping audiograms, tent shape audiograms are seen for individuals IV:8 and IV:9, and individuals IV:14, III:8 and V:2 have rising audiograms

(Table IV-1). In general, most of the sloping audiograms are seen in males and most of the women have flat or rising audiograms (Figure IV-2).

IV.1.2.5. Progression and operation outcome

Otosclerosis was confirmed by operation in three members of Family O. Severity of

HL pre- and post-operation of individual IV:14 is presented in Table IV-4 and Figure IV-3.

When hearing was first tested at the age of 29, an asymmetric mixed HL, Lt>Rt, was obtained. At the age of 34, after the birth of her first child, HL in the right ear progressed and by the age of 39, after having another child, a bilateral mixed HL was seen. By this time individual IV:14 was using a hearing aid alternatively in both ears. She was operated on her right ear at this age of 39, and operative findings included a fixed stapes with normal movement of the malleus and incus, consistent with the diagnosis of otosclerosis. There was no other abnormality in the middle ear and no signs of tympanosclerosis. A small fenestra stapedotomy was performed and a 0.6mm diameter 4.75mm length Platinum-Teflon prostheses (Xomed) was placed. Her results three weeks after operation revealed an improvement of 10 dB across all frequencies but 8000 Hz. A small improvement of 8.75 dB on average was observed in BC. This is consistent with the Carhart effect, where BC is often improved after surgery (Lopponen and Laitakari 2001).

66 IV:14 - 24.5.92 IV:14 - 13.5.97 Frequency in Hertz Frequency in Hertz 125 250 500 1000 2000 4000 8000 125 250 500 1000 2000 4000 8000 -10 -10 0 0 10 < 10 20 < < 20 30 < 30 40 40 50 50 Hearing 60 Hearing 60 Level in 70 Level in 70 80 80 Decibels 90 Decibels 90 (dB HL) 100 (dB HL) 100 110 110 120 120 130 130 140 140

IV:14 - 22.12.02 IV:14 - 22.1.03 Frequency in Hertz Frequency in Hertz

125 250 500 1000 2000 4000 8000 125 250 500 1000 2000 4000 8000 -10 -10 0 0 10 > 10 20 > > 20 30 30 40 Hearing 40 Hearing 50 50 Level in 60 Level in 60 70 Decibels 70 Decibels 80 (dB HL) 80 (dB HL) 90 90 100 100 110 110 120 120 130 130 140 140

Figure IV-3. Audiograms of individual IV:14, female, born in 1963. A. Audiogram at age 29, when she first had her hearing tested. B. At age 34 HL progressed and AC in Rt almost similar to Lt AC. C. At age 39, symetric bilateral mixed HL was evident prior to the operation of the right ear. D. Post operation audiogram less than one month after surgery. Only a slight improvement of hearing in the Rt operated ear was seen. A small improvement (8.75dB in average) is seen in Rt-BC as well.

67 Table IV-4. Evolution of HL of individual IV:14 and post-operative outcome.

A. Right ear.

Frequency Age 29 AC Age 34 AC Age 39 AC Age 40 AC (dB) Improvemen (Hz) (dB) (dB) (dB) (post operation) t in dB (post operation) 250 45 55 65 55 10 500 55 60 60 50 10 1000 50 55 55 45 10 2000 30 30 40 30 10 4000 30 40 45 35 10 8000 20 35 25 35 -10 PTA-AC 41.25 46.25 50 40 10 PTA-BC 22.5 22.5 22.5 13.75 8.75 ABGap 18.75 23.75 27.5 26.25 1.25 Abbreviation: PTA, pure tone average across 500-4000Hz; AC, air conduction; BC, bone conduction; ABGap, air bone gap.

B. Left ear.

Frequency Age 29 AC Age 34 AC (dB) Age 39 AC (dB) Age 40 AC (dB) (Hz) (dB) 250 80 70 70 75 500 85 70 55 70 1000 75 65 55 60 2000 65 35 40 50 4000 65 50 35 45 8000 65 50 50 45 PTA-AC 72.5 55 46.25 56.25 PTA-BC 22.5 16.25 15 20 ABGap 50 38.75 31.25 36.25 Abbreviation: PTA, pure tone average across 500-4000Hz; AC, air conduction; BC, bone conduction; ABGap, air bone gap.

Individual IV:4, a mother of four children, was operated on her right ear in 1981 at the

age of 42, with a finding of stapes fixation, confirming the diagnosis of otosclerosis. There are no audiograms available from before or immediately after the operation. She reported symmetric HL before the operation and complete restoration of hearing in the right ear after the operation. Five years later, at age 47, she faced difficulties in hearing again and her audiogram (Figure IV-4) showed a mild to moderate HL in the right operated ear and a rising

configuration of moderate to moderately severe HL in the left ear. She then began to use a

68 hearing aid. By the age of 62, her audiogram revealed bilateral symmetric mixed moderately severe HL. It is suspected that her prosthesis is dislocated or that there is an erosion of the lenticular process of the incus on the operated side. Nineteen years after surgery, BC in the operated ear deteriorated more than BC in the non-operated ear (Figure IV-4). This finding is compatible with a long term follow up post otosclerosis surgery report, noting a certain percent of post operative ears developing SNHL (Ramsay and Linthicum 1994).

IV:4 - 1.4.86 IV:4 - 7.5.01 Frequency in Hertz Frequency in Hertz 125 250 500 1000 2000 4000 8000 -10 125 250 500 1000 2000 4000 8000 -10 0 0 10 > > < > > 10 20 < 20 30 30 40 40 Hearing 50 Hearing 50 60 Level in Level in 60 70 70 Decibels 80 Decibels 80 (dB HL) 90 (dB HL) 90 100 100 110 110 120 120 130 130 140 140

Figure IV-4. Post-operation progression of otosclerosis of individual IV:4, female, born in 1939. A. Audiogram at age 47, 5 years after Rt ear was operated on and hearing was restored. Before surgery she had bilateral conductive HL, according to her report. The present audiogram shows deterioration in the hearing of the Rt operated ear. B. Audiogram at the age of 62, 19 years after surgery. The operated Rt ear deteriorated to the same degree of HL as the non-operated ear. Note that BC in the operated ear is worse than in the other ear.

IV.1.2.6. Immittance testing

Normal compliance of the tympanic membrane (type A tympanometry) was obtained bilaterally in individuals IV:14 and V:1, while individual IV:4 had normal compliance in the right ear and high compliance (AD) in the left ear. Tympanometry of IV:6 showed bilateral high compliance. Since otosclerosis causes fixation of the stapes, low compliance of the middle ear is expected. AD tympanometry, which reflects low stiffness, seems to be a paradoxical result in otosclerosis. Nevertheless, similar findings were reported in a small percent of confirmed otosclerosis in other populations as well, and an overlap of compliance

69 results was noted between otosclerotic and normal ears (Browning et al. 1985; Muchnik et al.

1989; Zhao et al. 2002). Acoustic reflexes were present bilaterally in all frequencies tested in

individual V:1, that at the age of 30 has normal hearing, and in individual IV:6 acoustic

reflexes were obtained in frequencies 500-2000Hz but not in 4000Hz, which is compatible

with his high tone SNHL. Acoustic reflexes were absent in individuals IV:4 and IV:14, as was expected according to their conductive HL.

IV.1.3. Linkage exclusion

Close linkage to the known otosclerosis loci on chromosomes 15q25-q26, 7q34-36

and 6p21.3-22.3 was excluded with negative two-point LOD Scores (Table IV-5).

Table IV-5. Exclusion of OTSC1, OTSC2 and OTSC3 loci by genotyping with markers from the respective chromosomal regions. (Dominant model, penetrance set at 80%, frequencies of alleles and of mutant allele set arbitrary at 0.2 and 0.0001 respectively.)

Two point LOD Scores at θ = Locus/Markers 0 0.001 0.05 0.1 0.2 0.3 0.4 OTSC1/ D15S1004 -4.285 -1.829 -0.176 0.062 0.191 0.152 0.054 D15S652 -4.353 -1.433 0.148 0.323 0.354 0.239 0.081 OTSC2/ D7S684 -4.411 -4.197 -1.249 -0.703 -0.249 -0.075 -0.013 D7S2513 -4.411 -4.055 -0.971 -0.451 -0.059 0.039 0.023 OTSC3/ D6S291 -4.411 -1.086 0.511 0.688 0.684 0.487 0.191

IV.1.4. Linkage analysis

Fifteen members of Family O underwent a whole genome scan. Four hundred

polymorphic markers were evaluated. Three possible regions of linkage were identified, on

chromosomes 2q37.1 (marker D2S206 - LOD score 1.918 at θ = 0.1), 16q22.3-23.1 (marker

D16S515 - LOD score 1.777 at θ = 0.1) and 20q13.12 (marker D20S119 - LOD score 1.692

at θ = 0). Further genotyping of the family members with two additional microsatellite

70 markers in each region did not support linkage to chromosomes 2 and 20 and gave an

additional two-point LOD score of 1.915 at θ = 0 in the 16q21-23.2 interval (Table IV-6),

leading to further genotyping of the region with DNA derived from all 24 family members.

Two point LOD scores were calculated for 17 markers in the chromosomal region 16q21-

16q24.1 (Table IV-7). The highest two point LOD score of 3.973 at θ = 0 was obtained for

the haplotype and Lod scores of 3.751 at θ = 0 and 3.723 at θ = 0 were obtained for markers

D16S515 and D16S3025, respectively.

Table IV-6. Linkage analysis of the three chromosomal loci with LOD scores >1.5 (in italics), obtained by the genome scan. (Dominant model, penetrance set at 80%, frequencies of alleles and of mutant allele set arbitrary at 0.2 and 0.0001 respectively.)

Two point LOD Score at θ = Marker 0 0.001 0.05 0.1 0.2 0.3 0.4 D2S362 -4.436 -4.358 -1.731 -1.105 -0.501 -0.199 -0.047 D2S2061 -99.990 0.493 1.912 1.918 1.601 1.115 0.526 D2S345 1.188 1.185 1.034 0.885 0.598 0.328 0.100 D20S107 -0.030 -0.028 0.049 0.086 0.092 0.058 0.017 D20S119 1.692 1.689 1.518 1.341 0.978 0.609 0.250 D20S891 -4.142 -2.423 -0.727 -0.435 -0.176 -0.064 -0.014 D16S3106 -0.176 -0.175 -0.148 -0.119 -0.068 -0.030 -0.008 D16S515 -99.990 0.330 1.759 1.777 1.486 1.036 0.500 D16S507 1.915 1.912 1.724 1.527 1.114 0.678 0.241 1For each chromosomal region, the middle marker was genotyped in the genome scan, revealing a LOD score ~2 (in italics), and the flanking markers were chosen to confirm or reject linkage to the locus. Note that linkage was confirmed only on chromosome 16 (in italics).

71 Table IV-7. Two point LOD Scores between the OTSC4 locus and chromosome 16q22.1- 23.1 markers with penetrance set at 80%.

Two point LOD Score at θ = Marker Distance 0 0.001 0.05 0.10 0.20 0.30 0.40 Zmax θmax (Mb)1 Haplotyoe2 3.973 3.966 3.623 3.261 2.494 1.671 .791 3.973 0 D16S3043 65.2 .370 .370 .336 .298 .216 .132 .057 .370 0 D16S3019 65.8 -1.790 .479 1.908 1.9251.625 1.153 .582 1.925 0.1 D16S3107 67.3 -2.029 -.225 1.230 1.281 1.056 .678 .251 1.281 0.1 D16S3025 68.2 3.723 3.716 3.375 3.015 2.259 1.456 .609 3.723 0 D16S3067 68.8 .418 .418 .385 .347 .261 .170 .081 .418 0 D16S3095 69.6 1.734 1.731 1.561 1.381 1.003 .603 .208 1.734 0 D16S512 73.7 3.179 3.173 2.876 2.561 1.894 1.175 .410 3.179 0 D16S3018 73.9 3.522 3.516 3.223 2.911 2.244 1.516 .718 3.522 0 D16S3115 74.2 1.832 1.828 1.631 1.424 .989 .536 .115 1.832 0 D16S3051 75.0 2.558 2.554 2.329 2.090 1.578 1.017 .416 2.558 0 D16S515 76.2 3.751 3.745 3.423 3.082 2.362 1.587 .752 3.751 0 D16S3097 77.1 -1.587 .018 1.466 1.513 1.282 .891 .412 1.513 0.1 D16S516 78.8 1.723 1.720 1.526 1.322 .894 .445 .041 1.723 0 D16S3040 79.3 2.305 2.303 2.164 1.991 1.566 1.046 .441 2.305 0 D16S507 79.8 -1.892 -.356 1.179 1.292 1.139 .771 .274 1.292 0.1 D16S511 81.4 -2.131 -0.028 1.442 1.504 1.298 0.930 0.474 1.504 0.1 D16S3091 82.7 0.204 0.203 0.172 0.141 0.085 0.040 0.010 0.204 0 1Distances between markers are taken from the UCSC Genome Browser, July 2003 version. 2Two point Lod score between the OTSC4 locus and the haplotype.

Since individual IV:6 has a high tone SNHL that is not common in otosclerosis, LOD

scores were calculated twice, once considering him affected and once as unaffected. LOD

scores obtained were 3.751 and 2.973 at marker D16S515 respectively (Table IV-8).

72 Table IV-8. Comparison between highest LOD Scores for each marker considering individual IV:6 affected vs. not affected, with penetrance set at 80%.

Marker Zmax (IV:6 aff) θ (IV:6 aff) Zmax (IV:6 n.a.) θ (IV:6 n.a.) Haplotype 3.973  3.195  D16S3043 0.370 0 0.370 0 D16S3019 1.925 0.1 1.339 0.1 D16S3107 1.281 0.1 0.695 0.1 D16S3025 3.723 0 2.945 0 D16S3067 0.418 0 0.418 0 D16S3095 1.734 0 1.734 0 D16S512 3.179 0 2.401 0 D16S3018 3.522 0 2.744 0 D16S3115 1.832 0 1.833 0 D16S3051 2.558 0 1.780 0 D16S5151 3.751 0 2.973 0 D16S3097 1.513 0.1 1.513 0.1 D16S516 1.723 0 1.723 0 D16S3040 2.305 0 1.527 0 D16S507 1.292 0.1 0.750 0.15 D16S511 1.504 0.1 0.928 0.15 D16S3091 0.204 0 0.204 0 1Marker from genome scan that identified chromosome 16 linked region.

Haplotypes were constructed as demonstrated in Figure IV-5. Flanking markers for genotyping were defined by recombination in individuals IV:9 (D16S3107) and V:2

(D16S3097). A genetic map of the candidate region showing a portion of the markers genotyped and distances between markers is presented in Figure IV-6. Distances and information regarding genes in the region were taken from the UCSC Genome Browser May

2004 Assembly.

73 1 2 I Unilateral Bilateral SNHL conductive or conductive or mixed HL mixed HL 1 2 3 4 5 II

1 2 3 4 5 6 7 8 9 III D16S3043 1 2 1 2 2 1 1 2 1 2 D16S3019 4 3 2 2 3 2 2 3 2 3 D16S3107 2 5 2 2 5 2 2 5 4 5 D16S3025 3 1 4 2 1 2 3 1 2 1 D16S3067 2 2 1 2 2 2 2 2 2 2 D16S3095 1 2 2 1 2 1 1 2 1 2 D16S3066 3 1 1 1 1 1 2 1 2 1 D16S512 3 1 2 3 1 3 3 1 2 1 D16S3018 2 3 2 1 3 2 2 3 2 3 D16S3115 2 3 3 2 3 2 2 3 1 3 D16S3051 3 2 3 3 2 3 3 2 3 2 D16S515 1 3 1 6 3 6 1 3 2 3 D16S3097 3 4 3 2 4 2 D16S516 3 2 2 3 2 3 2 2 2 2 S16S3040 3 2 2 1 2 3 2 2 2 2 D16S507 2 1 3 2 1 2 1 1 1 1 D16S511 2 2 4 4 2 3 3 2 5 2 D16S3091 2 3 3 3 3 3 1 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 IV 14 4 D16S3043 2 2 1 2 1 2 1 2 1 1 1 2 2 1 1 2 1 1 2 1 1 2 2 1 D16S3019 4 1 2 3 2 3 2 3 2 3 2 2 2 3 2 2 2 2 2 2 2 3 2 3 3 4 D16S3107 3 3 2 2 2 5 2 5 2 2 2 5 2 2 2 5 2 2 2 2 2 2 2 2 D16S3025 2 3 3 3 4 1 4 1 2 1 4 2 4 1 2 1 4 1 4 2 2 2 2 2 2 1 3 1 2 2 D16S3067 2 2 2 1 1 2 1 2 2 2 1 2 1 2 2 2 1 2 1 2 2 2 2 2 2 2 2 2 3 1 D16 S3095 1 2 1 1 2 2 2 2 1 2 2 1 2 2 1 2 2 2 2 1 1 1 1 1 1 2 1 2 1 1 D16S3066 1 3 3 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 3 3 D16S512 4 3 3 1 2 1 2 1 3 1 2 3 2 1 3 1 2 1 2 3 3 3 3 3 3 1 3 1 2 2 D16S3018 3 4 2 4 2 3 2 3 1 3 2 2 2 3 1 3 2 3 2 2 1 2 1 2 1 3 2 3 4 4 D16S3115 1 2 2 3 3 3 3 3 2 3 3 2 3 3 2 3 3 3 3 2 2 2 2 2 2 3 2 3 3 1 D16S3051 2 3 3 2 3 2 3 2 3 2 3 3 3 2 3 3 3 3 3 3 3 2 3 2 3 2 D16S515 2 2 1 3 1 3 1 3 6 3 1 6 1 3 6 3 1 3 1 6 6 6 6 6 6 3 1 3 1 1 D16S3097 1 2 3 2 3 4 3 4 3 2 3 4 3 4 3 2 2 2 2 2 2 4 3 3 D16S516 3 3 3 2 2 2 2 2 3 2 3 3 2 2 3 2 2 2 2 3 3 3 3 3 3 2 2 2 2 2 D16S3040 2 2 2 2 2 2 2 2 1 2 1 3 2 2 1 2 2 2 2 3 1 3 1 3 1 2 2 2 2 2 D16S507 1 2 1 3 3 1 3 1 2 1 2 2 3 1 2 1 3 1 3 2 2 2 2 2 2 1 1 1 3 3 D16S511 4 3 4 2 4 2 4 3 4 2 4 2 4 2 4 3 4 3 4 3 D16S3091 2 3 2 3 3 3 3 3 2 3 2 3 2 3 3 3 3 3 3 3 1 2 3 4 V D16S3043 1 2 1 2 2 1 D16S3019 4 3 4 3 2 3 3 4 D16S3107 2 5 2 5 2 2 D16S3025 2 1 3 1 3 2 1 2 D16S3067 4 2 2 2 2 3 2 1 D16S3095 3 2 1 2 1 1 2 1 D16S3066 1 1 2 1 2 3 1 3 D16S512 2 1 2 1 3 2 1 2 D16S3018 4 3 3 3 2 4 3 4 D16S3115 2 3 1 3 2 3 3 1 D16S3051 3 2 2 2 3 3 2 2 D16S515 2 3 3 1 D16S3097 3 4 3 3 D16S516 3 2 3 2 2 2 2 2 D16S3040 1 2 2 2 2 2 2 2 D16S507 2 1 2 3 1 3 1 3 D16S511 D16S3091

Figure IV-5. Haplotypes of the OTSC4 linked region on chromosome 16q22.3-23.1. The yellow haplotype segregates with the mutation. Arrows at the haplotypes of IV:9 and V:2 indicate proximal and distal recombinations, respectively, flanking the linked region. Note hearing individuals IV:15 and V:4 demonstrating incomplete penetrance.

74 Chromosome band Marker Nucleotide number Distance in Mb on chromosome from reference marker

~-12.9 Mb ~-11.5 Mb ~-11.2 Mb ~-11.0 Mb ~-10.4 Mb ~-8.9 Mb ~-8.0 Mb D16S503 63274000 ~-7.4 Mb D16S3050 64742148 ~-6.6Mb D16S3021 64960301 ~3.2Mb D16S3043 65168450 ~-2.4 Mb 65805472 D16S3019 0 ~-2.3 Mb D16S3107 67334151 ~-2.0 Mb D163025 68242631 ~-1.2 Mb D16S3067 68784850 Reference D16S3095 69622415 ~0.9 Mb D16S3066 73009352 D16S512 73747131 ~2.6 Mb D16S3018 73852173 ~3.1Mb D16S3115 74209921 ~3.6 Mb D16S3051 74947466 ~5.2 Mb D16S515 76196554 ~6.5 Mb D16S3097 77068584 ~10 Mb D16S516 78803542 D16S3040 79331022 D16S507 79755957 D16S511 81380684 D16S3091 82660134 D16S520 86197591

Figure IV-6. Physical map of the OTSC4 region on the Genethon genetic map (Dib et al. 1996). Distances between markers are taken from the UCSC Genome Browser, May 2004 version (http://genome.ucsc.edu/). Red indicates the reference marker that generated an initial Lod score of 1.777 in the genome scan and it happens to be one of the flanking markers of the region. Blue indicates the other recombination flanking the interval.

IV.1.5. Candidate genes

The region contains 79 known genes and 14 predicted genes. Primers were designed for the coding regions of all the candidate genes (Appendix VII.2.1). cDNA was screened if gene was expressed in lymphoblasts, and genomic DNA was sequenced for the other genes in the region. In some cases, sequencing of cDNA was not completely clear and genomic DNA was sequenced for the respective exons (i.e. PSMD7, GLG1 etc.). All the 93 genes in the linked region were completely sequenced and no mutations were detected. Appendix VII.2.1 presents all the genes sequenced in the linked region and the primers used. Appendix VII.2.2 lists the SNPs (including SNPs not reported in the database) detected in the genes that were screened in the OTSC4 region.

75 IV.2. Cx26 and Cx30

IV.2.1. Prevalence of the del(GJB6-D13S1830) mutation in the Israeli population

In 2001, Lerer et al discovered the del(GJB6-D13S1830) deletion in the Ashkenazi

Jewish population, found on one allele in conjunction with a GJB2 mutation (Lerer et al.

2001). Almost simultaneously, del Castillo reported a 342 kb deletion in GJB6 in Spain that was subsequently found to be the same as in Israel (del Castillo et al. 2001). I evaluated the prevalence of GJB6 mutations in the Israeli population. In particular, we were interested in determining whether the relatively large number of deaf GJB2 heterozygotes had GJB6 mutations in trans. Nineteen GJB2 heterozygotes were screened. Seven out of 19 were found to be GJB6 heterozygotes as well. 191 deaf individuals, in which GJB2 mutations were excluded, were screened for the GJB6 deletion. One individual was found to be homozygote for the deletion and 190 were found to be wild type. Table IV-9 lists the results found. All hearing impaired associated with ∆30 suffered profound HL.

Table IV-9. Cx30 deletions found in the Israeli population.

Family Proband Ethnic Group Genotype Deafness History Ashkenazi (Poland D37C + 35delG/∆30 Profound mainly) D59C Ashkenazi - 35delG/∆30 Profound D62E Ashkenazi (Poland) + 167delT/∆30 Profound Ashkenazi D84 - ∆30/∆30 Profound (Poland/Romania) ¾ Ashkenazi, D125C - 167delT/∆30 Profound ¼ Syria T450 Ashkenazi - 167delT/∆30 Profound Ashkenazi Z434 - 167delT/∆30 Profound (Romania) Ashkenazi Z453 + 35delG/∆30 Profound (Romania)

76 IV.2.2. A multicenter study of the prevalence of the del(GJB6-D13S1830) mutation in the DFNB1 locus in hearing-impaired subjects

The connexin 30 data reported above (section IV.2.1) was published, as part of a multicenter study, in the American Journal of Human Genetics (del Castillo et al. 2003). In

this multicenter study, it was shown that the del(GJB6-D13S1830) mutation is most frequent

in Spain, France, the United Kingdom, Israel and Brazil (5.9%-9.7% of all DFNB1 alleles), it is less frequent in the United States, Belgium and Australia (1.3%-4.5% of all DFNB1

alleles), and it is very rare in Southern Italy.

IV.2.3. A multicenter study of the prevalence of the del(GJB6- D13S1854) mutation in

the DFNB1 locus in hearing-impaired subjects

Another multicenter study was conducted to investigate the prevalence of a second

novel deletion involving the gene encoding connexin-30, del(GJB6-D13S1854), found in the

del Castillo laboratory. The del(GJB6-D13S1854) mutation accounts for 25.5 % of the

affected GJB2 heterozygotes which remained unelucidated after screening for del(GJB6-

D13S1830) in Spain, 22.2% in the United Kingdom, and 1.9% in Northern Italy. It was not

found in screenings performed in samples from France, Belgium, Israel, the United

States,Australia, and the Palestinian Authority. I screened159 Jewish probands participating

in the study, with non-syndromic and non-connexin hearing impairment, and the novel

deletion was not found in any of them. A letter to the editor was recently published in the

Journal of Medical Genetics (del Castillo et al. 2005).

IV.2.4. GJB2 and GJB6 mutations in the Israeli population

Out of 222 Israeli probands screened for mutations in connexin 26 and connexin 30,

56 were homozygotes or compound heterozygotes for mutations in connexin 26 and 22 were

77 heterozygotes. Eight out of the 22 Cx26 heterozygotes, were double heterozygotes Cx26/∆30.

I screened for Cx26 and Cx30 mutations in 79 probands and analyzed all the data. Table IV-

10 sumarizes the connexin 26 and connexin 30 mutaions in the Israeli population.

Table IV-10. Cx26 and Cx30 mutations in the Jewish Israeli population.

Mutations Number of Ethnic group Frequency Frequency individuals among among Cx26/Cx30 NSHL deaf Homozygotes 35delG 13 All 20% 6% 167delT 19 Ashkenazi 30% 9% 51del12insA 3 Buchari 5% 1% Compound 35delG/167delT 14 Ashkenazi, 22% 6% heterozygotes Ashkenazi- other, Syrian 35delG/L90P 3 Iraqi-other 5% 1% L90P(TÆC269)/ 1 Iraqi 2% 0. 5% IVS1+1(G->A) 51del12insA/ 1 Buchari 2% 0. 5% W24X 35delG/R32C 1 Ashkenazi 2% 0. 5% V37I/35delG 1 Ashkenazi 2% 0. 5% Heterozygotes 35delG 6 (+ 3 all 9% 3% 35delG/∆30) 167delT 6 (+ 5 Ashkenazi 9% 3% 167delT/∆30) V37I 1 Ashkenazi 2% 0. 5% M34T 1 2% 0. 5% ∆30 and ∆30 1 Ashkenazi 2% 0. 5% 35delG 3 Ashkenazi 5% 1% 167delT 4 Ashkenazi 6% 2%

IV.2.5. Genotype-phenotype correlation of connexin mutations

Our 64 probands with connexin mutations participated in a worldwide multicenter study comprising 1531 genetically and audiometrically documented individuals with connexion-related autosomal recessive HL (Snoeckx et al. 2005). The goal was to develop a detailed genotype-phenotype correlation for this frequent form of hereditary HL. The data

78 regarding all 64 probands with connexin mutations participating in the multicenter study is presented in Appendix VII.2.3.

A total of 177 different genotypes was found, of which 64 were homozygous truncating (T/T), 42 were homozygous non-truncating (NT/NT) and 71 were compound heterozygous truncating / non-truncating (T/NT). In this study sample, the degree of HL associated with biallelic truncating mutations is significantly more severe than the HL associated with biallelic non-truncating mutations. Forty-seven different genotypes showed less severe HL compared to 35delG homozygotes. Several common mutations (M34T, V37I,

L90P) were associated with mild-to-moderate HL. Two genotypes (35delG/R143W and

35delG/del(GJB6-D13S1830) had significantly more severe HL compared to 35delG homozygotes. In the Israeli population, in which T/T mutations (35delG and 167delT) are the most common (72% of connexion mutations), the degree of HL of most of the connexion hearing impaired is severe to profound, and all the deafness in which del(GJB6-D13S1830) is involved is profound. The few cases in which V37I and L90P mutations are associated moderate to severe Hl is involved. All our findings are compatible with those of the multicenter study.

IV.2.6. Correlation between connexin-associated deafness and outcome of cochlear implants

A total of 50 cochlear implanted (CI) children underwent genetic analysis to detect known mutations in the Israeli population. Connexin mutations were detected in 18 of them

(Table IV-11). Thirty out of the 50 implanted children were selected for a study evaluating speech perception after implantation, 17 with connexin 26 or 30 mutations and 13 without connexin mutations. Each child in the connexin group was carefully matched with a non- connexin implanted child according to age of implantation, duration of implant use and mode

79 of communication. There was no evidence for additional disabilities or handicaps in either

group. Speech perception was evaluated at the Speech and Hearing Center, Chaim Sheba

Medical Center, Tel-Hashomer. A retrospective analysis was made on their performance at 6,

12, 24, 36 and 48 months post-implantation. Test material was selected according to the

child's age, cognitive and language abilities. Both groups showed significant improvement in

speech perception results after implantation and no significant differences in speech

perception results were detected between the groups.

Table IV-11. Connexin mutations in CI children.

Cx26 mutation No. of CI children 35delG Hom. 3 167delT Hom. 5 35delG/167delT 7 35delG/∆30 3 None 32

IV.3. Evaluation of PCDH15 mutations in the Israeli deaf population

IV.3.1. R245X mutation detection

The PCDH15 gene contains 33 exons and is predicted to encode a protein of 1955

amino acids. The mutation R245X is caused by a C to T transition at position 733 in exon 8

of the PCDH15 gene, and leads to the substitution of an arginine by a stop codon. Since this mutation was reported only among Ashkenazim, and was not detected in other Jewish ethnic groups (Ben-Yosef et al. 2003), we selected only Ashkenazi deaf probands for examination of this mutation.

Ashkenazi Jewish individuals (n=59) diagnosed with NSHL were screened for the

R245X mutation. All the affected individuals were diagnosed with congenital NSHL, and mutations in connexin 26 and in connexin 30 were excluded in all probands. Twenty of the probands were under the age of 10, and 39 were over 10 years old. We chose this age

80 distinction since this is typically the age that RP is diagnosed after (Petit 2001). In the first

group of children under 10, 18/20 were found to be wild type for the mutation and 2/20

(10%) were homozygous for R245X. In the older group, no homozygotes were detected;

38/39 were wild type and 1/39 (2.6%) was heterozygous for the R245X mutation (Table IV-

11).

Table IV-12. R245X Mutation Frequencies among NSHL Askenazi Jews in Israel and Carrier Rate of Mutation in a Control Group.

Probands Number Wild type Heterozygous Homozygous Tested Congenital deaf, 20 18 - 2 (10%) under 10 years old Congenital deaf, 39 38 1 (2.6%) - 10 years old and above Hearing individuals 505 500 5 (0.99%) -

All deaf children were screened by the ASPCR assay. Since this assay is based on two

distinct PCR amplifications and no bands are expected depending on certain genotypes, one

cannot be sure if a band is not visualized because of the respective genotype, or since the

PCR did not work on a certain DNA sample. As screening children for the R245X mutation

is extremely important for early diagnosis of USH1F, results were confirmed by sequencing.

In the older group, results of ASPCR assays were confirmed by either sequencing or

restriction enzyme assays.

The family members of the two R245X homozygous young probands were screened

for the mutation. Family 1 (Figure IV-7) includes parents, two deaf children, 7 and 4 years of

age, both with cochlear implants, and two hearing children. The older deaf child manifested a

delay in motor development and started to walk at two years. This is characteristic for

vestibular dysfunction and is consistent with what was described as clinical features of USH

(Smith et al. 1994). Anomalies were also found in both fundoscopy and electroretinogram

81 examinations in this 7 year old child, but were not seen in his 4 years old brother. Both

parents were heterozygous for the R245X mutation, the two deaf children were homozygous

for the mutation, one hearing child was heterozygous and in the other hearing child the

mutation was not detected.

In Family 2 (Figure IV-7), two out of three children were deaf. The deaf children

were 4 and 2 years old and both underwent cochlear implantation. The two deaf children had

delays in motor development and were reported by the parents to be ‘clumsy.’ Both deaf children were homozygous for R245X.

Family 1 Family 2

+/- +/-

+/- -/- +/+ +/+ +/+ +/+ 12 9 73 4 3 1

Figure IV-7. Pedigrees of families with children homozygote for the R245X mutation in PCDH15. + and – refer to presence or absence of mutation on each allele, and numbers represent age.

IV.3.2. R245X carrier frequency

Anonymous DNA samples (n=505) of hearing Ashkenazi Jews were screened for the

R245X mutation by the ASPCR assay and if the mutation was detected on one of the alleles,

it was confirmed by sequencing of exon 8. Five out of 505 (0.99%) were heterozygous for the

mutation (Table IV-11). Since the R245X mutation was not detected among 293 non-

Ashkenazi Jews screened elsewhere (Ben-Yosef et al. 2003), and the mutation is assumed to

82 be a founder effect in the Ashkenazi ethnic group, no other ethnic Jewish groups were screened.

The PCDH15 data that was detected in Ashkenazi Israeli children diagnosed as having nonsyndromic hearing loss, and indicates that these children would develop RP, was published in Pediatric Research (Brownstein et al. 2004).

IV.4. Ascertainment of additional large families with HL

IV. 4.1. Family Z

One member of Family Z was approached by the Shema Organization for the Deaf and agreed to participate and involve her family in our study. The HL in the family is characterized by severe to profound, prelingual deafness, and most deaf individuals only use sign language for communication. The family is of Algierian origin. Once obtaining initial data a pedigree was drawn (Figure IV-8).

Z685 Z684 Z759 Z760 Z762 Z764 Z765 1943 1938 1940 1948 1945 1943 1943

Z686 Z687 Z688 Z658 Z761 Z763 1971 1972 1975 1980 1986 1978

Figure IV-8. Pedigree of Family Z. Top numbers indicate DNA samples of subjects; bottom numbers indicate year of birth.

83 Mutations in GJB2 were excluded in the proband (Z658). The family underwent a

genom scan in Mary-Claire King’s laboratory at the University of Washington, Seattle, USA.

The CIDR genotypes were searched for the longest chromosomal regions homozygous in

Z685. Her longest regions of homozygosity were on 7p, 8q, 10q, 13q (below Cx26), 15q, and

all of 19. Some markers were uninformative so this introduced a bias in favor of those

regions. Each region of apparent homozygosity was at least 40 MB. Additional markers were

genotyped in each of these 6 regions. The entire family was genotyped because the goals

were to test whether Z685 was truly homozygous across an entire candidate region and to

determine which region involved the fewest haplotypes in the deaf in the rest of the family.

The shared haplotype was detected on chromosome 10q21-q23. This region had a partially

shared haplotype across all deaf except the original deaf father (Figure IV-9), and within this

shared haplotype lies CDH23. Two markers were genotyped within introns of CDH23 and could not exclude this gene out of the region. CDH23 consists of 69 exons spanning 420 kb.

2 2 2 2 1 1 1 2 2 1 1 3 2 1 1 1 1 3 3 1 2 1 1 3 1 1 1 2 2 4 4 4

Z685 Z684 Z759 Z760 Z762 Z764 Z765 1943- 1938- 1940- 1948- 1945- 1943- 1943- 1 1 2 2 2 2 2 3 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 2 2 3 1 1 1 1 1 1 1 2 2 2 3 3 2 1 1 1 1 1 1 1 1 2 2 1 1 2 3 3 3 3 3 3 3 3 1 1 1 1 3 1 1 1 1 1 1 1 1 2 2 3 3 1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 4 4 4 4 5 4 2 2 4 4 4

Z686 Z687 Z688 Z658 Z761 Z763 1971- 1972- 1975- 1980- 1986- 1978- chr10 MB 1 2 1 2 1 2 2 3 2 2 D10s1221 56.9 1 1 1 1 1 1 1 1 1 1 D10s1225 64.1 1 1 1 1 1 1 1 2 2 1 gata121a08 69.9 1 1 1 1 1 1 1 1 2 1 D10s606 72.7 CDH23 intron 7 3 3 3 3 3 3 3 3 1 2 D10s1694 72.8 CDH23 intron 12 1 1 1 1 1 1 1 1 2 1 D10s1432 74.0 1 1 1 1 1 1 1 1 1 1 D10s2327 80.1 1 4 1 4 1 4 4 4 2 3 D10s2470 92.0

Figure IV-9. Haplotypes of Family Z. Shared haplotype is marked yellow. Two markers in this region lie within the CDH23 gene.

84 V. DISCUSSION

V.1. Family O

V.1.1. Phenotypic characterization of hereditary otosclerosis locus OTSC4

The hearing impairment that occurred in most members of Family O was diagnosed as otosclerosis based on several of the following criteria: family history of otosclerosis, age of onset, normal otoscopy, progressive conductive to mixed HL and audiograms showing a characteristic Carhart effect. The disease has also been implicated in certain cases of pure

SNHL, and even though such a diagnosis cannot be made safely without exploratory tympanotomy, we thought we could assume that individual IV:6 is included in this category due to family history and a similar audiogram configuration to that of some of his brothers. In addition, a large variability exists among the affected members of the family in all aspects of

HL so it is not inevitable that cochlear otosclerosis is one of the various forms of the disease in the family. However, since this diagnosis cannot be confirmed, and histological evidence of pure cochlear otosclerosis is very rare even though several confirmed cases were reported

(Youssef et al. 1998), we analyzed the data in both ways, considering IV:6 both unaffected and affected. The statistical calculation in both ways led to the same conclusions and did not affect the results of the linkage.

Sex differences in distribution of the disease, age of onset and audiogram configuration were observed in Family O. The 2:1 female to male predominance obtained is concordant with the incidences published elsewhere (Pearson et al. 1974; Vartiainen 1999).

Age of onset was higher in males as compared to the females in the family. All women became aware of hearing problems before the age of forty and in some cases before the age of thirty, whereas the men noticed loss in hearing only after the age of forty. These findings may correlate to the female:male ratio, as the differences in sex distribution might be

85 explained by endocrinologic factors and endocrine activity, such as puberty or pregnancy that

might cause otosclerosis to increase rapidly (Vartiainen 1999). Individual IV:14 first came

for a hearing test after her first child was born, at the age of 29. Individual IV:4 had her right

ear operated on, in her early forties, after having four children, when she felt a deterioration in hearing. In both cases the HL progression might be correlated to the pregnancies.

Individual V:2, who was not aware of hearing problems and revealed a mild HL when she had her hearing tested, is 26 years old and is pregnant with her fourth child. The males might have a similar age of onset as the females but without the hormonal factors, the progression is slower and the HL is noticed at an older age. Furthermore, higher degrees of HL were observed in the females of the family when compared to the males in all age ranges. This was

reported in other populations as well (Browning and Gatehouse 1992) and the explanation

might also be correlated to the maturational history of women that lead to more severe

expression.

Other factors may influence the type of HL and audiogram configuration of the

affected men in the family. Three out of the four men assumed to have otosclerosis have a

sloping audiogram in at least one ear, with mixed to SNHL in the high frequencies.

Individuals IV:5, IV:6 and III:8 were exposed to noise daily in their occupations, and this

might explain the HL in the high frequencies with the SN component that is characteristic of

NIHL. Individual IV:6 shows high tone SNHL with no ABG. Since he did not have his

hearing tested before, we do not know if there was a conductive component before or the HL

began as SN. It is possible that his HL is due to noise exposure and not otosclerosis, but it is

widely accepted that otosclerosis can progress into SNHL or in some cases cause pure

cochlear otoscelerosis (Youssef et al. 1998).

The variability of audiogram configuration and severity of HL in the family may

represent different stages of otosclerosis. A mild conductive rising audiogram, exhibited by

86 member V:2, is characteristic of the first stage and is thought to be caused by the presence of cellular fibrous tissue. The second stage, with localized bony fixation of the anterior part of the footplate, is thought to result in moderate conductive HL, as is the case with individual

IV:9 and IV:14. The final stage, fixation of the entire circumference of the annular ligament, is considered to result in a moderately severe conductive to mixed HL (Zhao et al. 2002).

Individuals IV:4, IV:8 and IV:10 have this kind of audiogram. Histological studies have shown that in some patients with stapes fixation, a second focus of otosclerosis can be found in the cochlea that is probably responsible for the SN component. A cochlear focus might cause profound mixed to SNHL as is the case with individuals III:7, III:8 and III:9. In rare cases there was evidence of isolated cochlear otoscelerosis (Youssef et al. 1998) that may begin with a HT loss, depending on the otosclerotic locus in the cochlea. Individual IV-6 is thought to have this form of otosclerosis. Individuals IV:4, IV:8 and IV:10 have a characteristic elevation in BC with a notch in 2000 Hz, characterized as a Carhart effect.

Otosclerosis was confirmed histologically in three members of the family, IV:4, IV:8 and IV:14. In individual IV:14, only a slight improvement was obtained in hearing of the right operated ear. However, subjectively, she reports an improvement in hearing. Hearing was tested three weeks after the operation and this might be not enough time for clearance of fluid and gelfoam from the middle ear cavity, thus affecting the recovery. Individual IV:4 appeared to have a successful operation in her right ear, but five years later, she began to lose her hearing again, and 20 years later the HL in her right ear deteriorated to the same degree of

HL as in the unoperated ear, suggesting dislocation of the prosthesis or erosion of the lenticular process of the incus. Individual IV:8 had a bilateral symmetric HL at the age of 34 and had her right ear operated on successfully. Fifteen years later, at the age of 49, her hearing in the operated ear is still normal except for frequencies 6000-8000 Hz, where she has a characteristic post operation moderate HL.

87 In conclusion, the different stages of otosclerosis are directly correlated with type and severity of HL and audiogram configuration. The present investigation is unique in that it

shows considerable inter-subject and inter-aural variability in the same family, and activity of

the otosclerotic lesion could provide convincing explanations for all these differences.

V.I.2. Mapping of hereditary otosclerosis linked to OTSC4

In the present study, we have mapped the fourth otosclerosis locus, OTSC4, to the

long arm of chromosome 16 in an extended Israeli family. All twelve affected members of

the family inherited the otosclerosis haplotype. Individual IV-6, diagnosed with SNHL,

inherited the same haplotype pattern as family members with otosclerosis, suggesting that his

HL may be due to mutations in the same gene. Nevertheless, we calculated LOD scores, labeling him as affected or unaffected. The calculated LOD scores confirmed linkage to

chromosome 16q22.1-23.1 for both options. Unaffected individual IV-15 inherited the

disease haplotype. At the age of 50, after having five children, it is extremely unlikely that

individual IV-15 will develop otosclerosis, as the onset of the disease in females in this

family was in the late twenties to early thirties, and was reported to be first noticed or deteriorating during or after pregnancies. Therefore in view of the prevalence of reduced penetrance associated with otosclerosis, we predict that individual IV:15 is bearing the mutation but that the phenotype is not expressed due to reduced penetrance. In the families in which the three OTSC loci, OTSC1-3, were mapped, there were 3, 2 and 1 individuals, respectively, without hearing loss but with the otosclerosis haplotypes. They were considered to reflect reduced gene penetrance as well (Tomek et al. 1998; Van Den Bogaert et al. 2001;

Chen et al. 2002).

Since there is a large diversity of hypotheses regarding the cause of otosclerosis, many genes in the region could fit into at least one of the theories suggested. Among the

88 proteins encoded by these genes are a few members of the cadherin superfamily, cadherin 1

(CDH1) and cadherin 3 (CDH3). Cadherins are mostly transmembrane proteins that mediate cell recognition and cell-cell adhesion, with unique cadherin domains or ectodomains (EC) consisting of negatively charged sequence motifs (Yagi and Takeichi 2000). The perturbed bone homeostasis in otosclerosis might involve a disturbance in cell signaling and/or tissue cohesion involving a cadherin. Furthermore, cadherin proteins are expressed in connective tissues, in bone and in the cochlea (Okazaki et al. 1994; Murcia and Woychik 2001; Diehn et

al. 2003; Kelley 2003) (SOURCE, http://source.stanford.edu). Both cadherins in the region

were found to be wild type by sequencing.

Conserved oligomeric Golgi (COG) 8 and COG 4 are two genes in the 16q21.1-23.1

region that belong to the COG group of multiprotein complexes. These complexes are key

determinants of the Golgi apparatus structure and are actively involved in intracellular

membrane trafficking (Ungar et al. 2002). COG complexes are expressed in the immune system (Diehn et al. 2003) and mutations in COG genes might be compatible with the

morphologic findings of an inflammatory process within the temporal bone at distinct stages

of otoscelerosis (Chole and McKenna 2001). COG 8 and COG 4 in the region were sequenced and found to be wild type in sequence in the affected individuals.

Several members of the DEAD (Asp-Glu-Ala-Asp) box proteins, DEAD (Asp-Glu-

Ala-As) box polypeptide (DDX) 19, DDX28 and DEAH (Asp-Glu-Ala-His) box polypeptide

(DHX) 38, are encoded by genes included in the OTSC4 linked region. These proteins are implicated in a number of cellular processes, including alteration of RNA secondary structure such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly (Hamm and Lamond 1998; Tanner and Linder 2001). Some members of the family are involved in cellular growth and division. Several of the DEAD box proteins are reported to be expressed in the immune system, cartilage and fibrous tissue (Grundhoff et

89 al. 1999; Allcock et al. 2001; Diehn et al. 2003; van't Wout et al. 2003), all with possible

relation to otosclerosis. All the DEAD box genes in the intervals are wild type.

A number of the genes coding for zinc finger (ZNF) proteins, ZNF19, ZNF23, zinc and ring finger protein (ZNRF) 1 and zinc finger protein 1 homolog (ZFP1), are also included in the OTSC4-linked region. Zinc finger proteins are multifunctional proteins with both transcriptional and post transcriptional functions (Ladomery and Dellaire 2002). Their broad expression pattern includes tissues involved in different stages of otosclerosis, such as the inner ear, the immune system, and fibrous tissue (Diehn et al. 2003). All the zinc finger genes in the region are wild type as well.

All the other known genes, as well as the predicted genes in the OTSC4 interval, were completely sequenced and no mutation was revealed, even though a nonsense mutation was identified in the polycystic kidney disease like 3 gene (PKDL3). I identified a 2365c->t,

R789X, change in exon 15 of the PKDL3 (Li et al. 2003), leading to a nonsense mutation segregating with affected members of Family O. Eight unrelated individuals from different ethnic origins that were surgically confirmed as having otosclerosis, were screened for the

R789X change and it was detected in one of these Ashkenazi individuals. A stop codon is formed as a result of this change, but this is not the causative mutation, as we also identified this change in a heterozygous form in 11/42 Yemenite controls, 12/45 Ashkenazi controls and one Ashkenazi individual in the control group was homozygous for R789X. This change was also found in 3 patients with polycystic kidney disease (as well as in 5 controls) (S. Somlo;

Department of Medicine, Division of Nephrology, Mayo Clinic, Rochester, MN, USA.; personal communication). No similar sequences were detected when blasted against all organisms (http://www.ncbi.nlm.nih.gov/BLAST/), which suggests that this is not a pseudogene. On the other hand, many SNPs where detected in Family O, as well as in the population screened by Somlo while sequencing the gene. Seven missense nonpathogenic

90 changes were identified in Family O and 13 in the Somlo’s population, where an ins/del

(V86Xfs), in a homozygous form, was also reported. All the changes detected in Family O were reported by Somlo as well (personal communication). Another possible explanation for the nonpathogenic stop codon is that the PKD1L3 gene consists of two transcript variants encoding distinct isoforms, as is known in other genes such as WWP2 that has 3 isoforms of

10, 14 and 25 exons, CNTNAP4 with 2 isoforms of 14 and 23 exons (http://genome.ucsc.edu)

and the 2(XI) procollagen gene, in which an in-frame termination codon was reported, suggesting that some of the splice variants encode a truncated pro- chain (Lui et al. 1996).

The possible synthesis of truncated proteins raises questions about possible differing functional roles for these polypeptides at the translational level.

Sequencing of all the 93 genes incorporated in the OTSC4 locus and not finding any mutation does not mean that the gene involved in otoscelerosis is not linked to this interval.

What it actually means is that there is no mutation in the coding regions of the genes. Most of the genes were sequenced on a genomic level and for these genes we can conclude that there are no mutations in the immediate splice sites as well, since primers were designed to include at least 50 bp flanking the exons. However, we cannot exclude mutations in intronic domains involved in splice sites that are far from the exons. Concerning the genes that were sequenced on the cDNA level, we can assume that no mutations in splice sites are involved since skipping exons, elongated exons or additional exons can be detected easily on this level.

There are many examples for splice site mutations inserting or deleting an exon or part of an exon. One example for an intronic mutation affecting the transcript is a cytidine phosphate guanosine dinucleotide C-to-T point mutation in intron 19 of the CFTR gene, termed

3849+10 kb C to T. The mutation leads to the creation of a partially active splice site in intron

19 and to the insertion into CFTR transcripts of a new 84-base-pair "exon," containing an in-

frame stop codon between exons 19 and 20 (Highsmith et al. 1994). Another intronic

91 mutation affecting splicing was identified in the COL5A1 gene involved in Ehlers-Danlos syndrome (EDS), a connective-tissue disorder characterized by skin fragility, joint laxity, and skeletal deformities. A point mutation in intron 32 (IVS32:T-25G) causes the 45-bp exon 33 to be lost from the mRNA of transcripts from the mutant gene. This mutation lies only 2 bp upstream of a highly conserved adenosine in the consensus branch-site sequence, which is required for lariat formation. This is the first description of a mutation at the lariat branch site, which plays a pivotal role in the splicing mechanism, resulting in an in-frame exon skip

(Burrows et al. 1998). Both transcript alterations described could be identified by cDNA sequencing.

Other mutations that could not have been detected by sequencing of coding regions, either on a genomic level or on a cDNA level, are intronic mutations that do not affect transcription. Such an example is that of the G to A base substitution at 39 base pairs upstream to exon 7 of the p53 gene, detected in Li-Fraumeni syndrome tumors. Sequencing of all exons of this gene, including splice sites, revealed no mutation. It was suggested that this mutation in intron 6 of the p53 gene stabilizes the wild type p53 protein, resulting in its abnormal accumulation (Avigad et al. 1997). Another mutation in a p53 intron, the p53

13964GC mutation, represents a single noncoding base change. mRNA analysis did not demonstrate aberrant transcript production but it was present at elevated frequencies in familial breast cancer patients compared to a control group. Functional activity was also detected, as prolonged cell survival was observed as well as inhibition of chemotherapy- induced apoptosis (Lehman et al. 2000). The conclusion of these findings was that mutations in the noncoding regions should be further studied

Another kind of mutations that our sequencing techniques failed to detect is promoter mutations. The promoter regions are not known in most of the genes in the OTSC4 region.

Trying to define the promoter for a gene is a project within itself and often does not lead to

92 the desired results. It is impossible in a limited time to perform this work on 93 genes.

Nevertheless, mutations in promoters were identified in several genes, among which is the

peroxisome proliferator-activated receptor (PPARG, PPAR ) gene, involved in Familial

Partial Lipodystrophy (FPLD). An A>G mutation at position –14 of intron B upstream of

PPARG exon 1 within the promoter of the PPAR 4 isoform was detected. This mutation was absent among 600 alleles from normal Caucasians. A minimal promoter sequence bearing the

mutation had significantly reduced promoter activity when used to drive reporter expression

in in vitro expression in two cell lines, compared with the wild-type sequence (Al-Shali et al.

2004). Another study also reported a mutation in a regulatory region of the CIITA gene involved in the coordination of the MHC II expression. The major histocompatibility complex (MHC) class II deficiency [bare lymphocyte syndrome (BLS)] is a rare primary

immunodeficiency disorder. Mutations in the CIITA are involved in this disorder. In some

affected individuals dysfunction of the CIITA was observed but no mutation was identified in

either coding or noncoding regions of the CIITA cDNA sequence. No mutations were

detected in promoters III and IV as well, and normal promoter function was assessed by a

luciferase assay. It was suggested that the data might indicate the existence of a region

involved in the stabilization of CIITA transcripts or a regulatory sequence required for the full

activity of each CIITA promoter (Dziembowska et al. 2002). Identification of such sequences

may elucidate genes involved in diseases, including otoscelerosis.

In conclusion, the failure to detect mutations by sequencing all the genes in the OTSC4

region could be attributed to a combination of factors, namely i) intrinsic limitations in the

techniques used for mutation screening; ii) hypothetical existence of mutations in regulatory

regions in introns or anywere on the chromosome; or iii) hypothetical existence of mutations

in other genes interacting with a specific gene in the linked region. Functional assays

providing accumulation vs. deficiency data coud lead us to the target gene, but in order to

93 take the functional direction, one has to have one or two very good candidate genes to

analyze. Nevertheless, as was discussed previously in this thesis, there is a large range of

hypotheses regarding the etiology of otoscelerosis, and if we follow all the therories suggested, any gene in the region might be a candidate for otosclerosis. It is impossible to characterize all the 93 genes in the region in terms of expression and function.

V.2. Cx26 and Cx30

V.2.1. GJB2 and GJB6 mutations in the Israeli population

A prevalence of 29% of deafness due to Cx26 and Cx30 mutations was found among the Israeli population we screened. The most prevalent are the 167delT and the 35delG mutations (72% of the connexin mutations). In total, 21% of the congenital NSHL in Israel is due to homozygotes for 167delT (9%), homozygotes for 35delG (6%) and compound heterozygotes 35delG/167delT (6%). While the 167delT mutation was detected only among

Ashkenazi Jews, the 35delG mutation was identified in all ethnic groups in Israel. The Cx30

deletion was also detected only in the Ashkenazi Jewish population. Several low probability

mutations were detected in certain ethnic groups; the 51del12insA mutation was identified

only among Buchari Jews (~1%), and the L90P mutation was detected in Jews originating in

Iraq (~1%) and not in any other ethnic group. Our results have implications for routine

genetic diagnosis of DFNB1, directing which mutations should be examined for in distinct ethnic groups.

V.2.2. Prevalence of del(GJB6-D13S1830)

The deletion truncating the GJB6 gene (Cx30), close to GJB2 on 13q12, was shown to be the accompanying mutation in about 50% of these cases in a cohort of Spanish patients

94 (Del Castillo 2002). Among the Israeli deaf population in which Cx26 was excluded as the

cause for HL, we found 9% (19/210) deaf GJB2 heterozygotes. We were interested in determining whether the relatively large number of deaf GJB2 heterozygotes had the del(GJB6-D13S1830) mutation in trans. Thirty seven percent of deaf GJB2 heterozygotes (7 out of 19) were found to be GJB6 heterozygotes as well. These findings reduced the prevalence of the carriers of Cx26 mutations among the deaf population in Israel to 6%, which is closer to the rates reported by other; ~3% of 35delG in Europe and North America

(Estivill 1998; Kelley et al. 1998; Green et al. 1999; Storm et al. 1999) and ~8.5% of 167delT

(7.5%) and 35delG (1.1%) mutations among Ashkenazi Jews (Lerer et al. 2000). Among 191

deaf individuals in which GJB2 mutations were excluded, only one was homozygote for the

deletion (0.5%) and no carriers were detected.

The data was analyzed as part of a multicenter study in which nine countries participated and was published in the American Journal of Human Genetics (Del Castillo et al. 2003). The deletion was present in most of the screened populations, with higher frequencies in France, Spain and Israel, whose percentages of unelucidated GJB2 heterozygotes dropped after deletion screening.

The effect of the deletion mutation could be due to a digenic mode of inheritance of

GJB2 and GJB6 genes that encode two different connexins; connexin 26 and connexin 30.

However, pure digenic inheritance seems unlikely as compound heterozygosity with a GJB2 mutation has not been found for other GJB6 mutations. Alternatively, it may abolish control

elements that are important in the expression of the GJB2 gene in the cochlea. Regardless of which of the options is valid, it is apparent that the deletion mutation provides new insight

into connexin function in the auditory system.

95 Analysis of haplotypes associated with the deletion revealed a founder effect in

Ashkenazi Jews (Lerer et al. 2001), and also suggested a common founder for countries in

Western Europe .

V.2.3. Genotype-phenotype correlation of connexin mutaions

Our 64 probands with connexin mutations participated in a cross-sectional analyses of GJB2 genotype and audiometric data from 1531 persons from 16 different countries with autosomal recessive, mild-to-profound, nonsyndromic hearing impairment. The goal was to develop a detailed genotype-phenotype correlation for this frequent form of hereditary HL.

In this study of persons segregating GJB2-related deafness, it was found that truncating mutations of GJB2 are associated with a greater degree of HL than non-truncating mutations. The truncating mutations 35delG and 167delT are involved in 72% of the connexin associated deafness in the Israeli population, and in concordance with the findings of the study most of them are profoundly deaf. Several of the common genotypes in this study group were also associated with mild-to-moderate HL, suggesting that complete GJB2 mutation screening, including IVS1+1G>A and del(GJB6-D13S1830), should be offered to all children with non-syndromic HL regardless of severity. The pathogenicity of missense mutations depends on many factors, including the position of the mutation in the protein and the nature of the substitution. For example, a change in an amino acid that is positioned in a functional domain or that is conserved in related genes or species is likely to be pathogenic.

However, given the complex structure and function of gap junctions, it is extremely difficult to predict the possible pathogenic effect of some missense mutations. This limitation reflects our incomplete understanding of the molecular basis for gap junction function, and it is for this reason that data from animal models and recombinant expression systems, while valuable for the investigation of mutations, should be extrapolated to humans with caution.

96 Of importance is the fact that some functional studies seem to contradict the findings of the study. Mutations that are included in this list of apparent discrepancies between gating properties and degree of HL are IVS1+1G>A, V37I and L90P. Expression studies have demonstrated a complete loss of channel activity for V37I and L90P (Bruzzone et al. 2003;

Skerrett et al. 2004), although we found these mutations to be associated with mild-to- moderate HL. Functional studies also have shown that the 35delG and IVS1+1G>A mutations do not yield any detectable Cx26 protein and mRNA, respectively (D'Andrea et al.

2002; Shahin et al. 2002), a result inconsistent with our observation that 35delG/IVS1+1G>A compound heterozygotes had significantly less severe HL compared to 35delG homozygotes.

Although most of the 35delG homozygotes manifest profound deafness, there are still some that show moderate to severe HL.

Most of the individuals with the 35delG/L90P genotype had significantly less HL compared to the reference group. The generally mild character of the L90P mutation was corroborated by additional compound heterozygous L90P combinations that all had significantly less HL compared to the 35delG homozygotes reference group. Among them was the combination involving IVS1+1G>A detected in the Israeli population.

The pathogenicity of the V37I and M34T mutations remains unresolved. The M34T variant was first described as an autosomal dominant mutation (Kelsell et al. 1997), consistent with the study of White et al. where it was reported to have a dominant-negative effect over wild-type Cx26 in Xenopus oöcytes (White et al. 1998). These dominant effects were later attributed to an artefact in the expression levels of mutant and wild-type RNA that were not controlled in the exogenous system (Skerrett et al. 2004). Other reports list the

M34T allele as an autosomal recessive mutation in the presence of other GJB2 mutations or in the homozygous condition (Wilcox 2000; Houseman et al. 2001; Kenneson et al. 2002; Wu et al. 2002), while other studies have stated that this variant is not pathogenic (Griffith et al.

97 2000; Feldmann et al. 2004). Persons with a 35delG/M34T genotype had mild-to-moderate

HL.

The pathogenicity of the V37I mutation is also debatable. While some studies have found V37I not pathogenic (Kelley et al. 1998; Kudo et al. 2000; Hwa et al. 2003;

Wattanasirichaigoon et al. 2004), in our multicenter study an association with mild HL was observed in combinations involving V37I. This result is consistent with other studies of this allele (Abe et al. 2000; Wilcox et al. 2000; Kenna et al. 2001; Lin 2001; Marlin et al. 2001;

Marlin et al. 2005). A definite conclusion about the impact of M34T and V37I on hearing cannot be made currently. Additional large scale studies, also including random individual populations with a high frequency of these variants, are needed to reach these conclusions.

Despite the genotype-phenotype correlations observed, significant phenotypic variability within genotypes does remain. This variability may reflect the effect of modifier genes and/or environmental factors leading to incomplete penetrance and variable expression

(Nadeau 2001). If modifier genes are involved, their characterization will be very important to refine genotype-phenotype correlations and improve the accuracy of phenotype prediction.

V.2.4. Correlation between connexin-associated deafness and outcome of cochlear implants

The main purpose of the study was to compare retrospectively speech perception results of children with and without connexin mutations. Our results indicate that when the two groups of implanted children are carefully matched by many variables, there are no apparent differences in speech perception scores between the two groups. Both groups show improvement in speech perception abilities post-implantation. These results differ from that obtained in other studies (Fukushima et al. 2002; Matsushiro et al. 2002; Sinnathuray et al.

2004), which found better results in the connexin group. These differences can be attributed

98 to the small number of subjects, our strict criteria for selecting the control group, i.e. age of implantation, duration of implant use and mode of communication, and other confounding factors that affected the results. In addition, in the control group, the cause of deafness is unknown and probably most of the cases are attributed to other genes, particularly in those cases in which more than one individual in the family suffers from hearing loss. The results of the study indicate that the central auditory pathway is substantially preserved in the two groups we selected for the study. One prevailing question is whether cochlear implant efficacy would be different if the patients have a connexin mutation or a mutation in another gene. However, it appears that whether hearing loss is caused from a mutation in GJB2 or a different gene has minor effects on the preservation of the auditory pathway. Indeed, this observation does fit with our knowledge regarding the pathophysiology of neuronal death in the ears of mice with mutations in different genes underlying NSHL. Some of these genes,

Cdh23, Kcnq4, Myo15, Myo3A, Myo6, Myo7a, Otof, Pres, and Strc, are expressed primarily in hair cells and Tecta, Otoa and Col11A2 are expressed in other cell types in the cochlea

(http://webhost.ua.ac.be/hhh/). Mutations in all the genes mentioned affect the sensory organ of hearing while neuronal patterning is expected to be intact. It is therefore likely that mutations in most deafness genes will result in similar results post cochlear-implantation.

Our results have implications and prognostic value regarding counseling for cochlear implant candidates with connexin mutations, as well as for other NSHL cases with no additional complications. Overall, the results of these two groups are at least at the mean level of the results with implants or even better.

V.3. PCDH15 mutations in the Israeli deaf population

Our results confirmed the prevalence of the R245X mutation, associated with Usher syndrome type 1 in the Ashkenazi Jewish population, as reported by Ben-Yosef et al. (Ben-

99 Yosef et al. 2003). The R245X mutation generates a protein translation stop codon in exon 8 of PCDH15, the gene encoding protocadherin 15, and presumably results in a truncated form of protocadherin 15. Alternatively, there may be no protein formed due to nonsense-mediated decay (NMD) (Maquat 1996). Most significantly, we report that this mutation was found in children under ten years of age who were inadvertently diagnosed with NSHL, where HL is the only symptom, prior to the age that RP develops. We tested a cohort of 59 individuals that included 39 over the age of 10 in order to exclude involvement of the R245X mutation in

NSHL.

Among the twenty probands under the age of ten years having no mutations in

GJB2 and GJB6 that were screened for R245X, we identified two (10%) that were homozygous for the R245X mutation. Both probands manifested delays in motor development, which is characteristic of vestibular dysfunction. One seven year old proband who underwent ophthalmological consultation showed an abnormal fundus and ERG, including diffuse retinal dystrophy and visual field constriction, while his four year old

brother’s results were within normal limits. Since onset of RP in USH1F is prepubertal,

visual anomalies in the younger child may develop later.

One deaf individual who was heterozygous for the R245X mutation did not carry

any other PCDH15 mutations in the reported 33 exons of PCDH15, either suggesting that we

have not yet found the partnering mutant allele of PCDH15 or more likely, this person is a

coincidental carrier of R245X.

When 90 non-Ashkenazi deaf probands were screened for the M1853L mutation, two

adult probands were heterozygous for M1853L. While examining the other family members

of the two heterozugous for M1853L, one unaffected brother in one of the families was

homozygous for M1853L. Based on our data, M1853L appears to be a benign polymorphism

(i.e. not a pathogenic mutation of PCDH15).

100 In addition to the prevalence of the R245X mutation among children diagnosed with

NSHL, we found a carrier rate of 1% among the Ashkenazi Jewish population examined in

Israel [comparable to 0.79-2.47% among Ashkenazi Jews, reported by Ben-Yosef et al.(Ben-

Yosef et al. 2003)]. This has led us to propose that all Ashkenazi probands with NSHL under the age of ten in whom GJB2 and/or GJB6 mutations have been excluded as the reason for the HL should be screened for the R245X mutation to enable presymptomatic diagnosis of

USH1. Since a GJB2 mutant allele, 167delT, is carried by approximately 4% of the

Ashkenazi Jewish population and another allele, 35delG, is carried by 0.21-1.1% of this population (Morell et al. 1998; Sobe et al. 1999; Lerer et al. 2000; Sobe et al. 2000), individuals homozygous for the R245X mutation of PCDH15 might also be coincidental carriers of one of these GJB2 mutations. The presence of only one GJB2 mutant allele may be coincidental and should not preclude screening for R245X in a deaf Ashkenazi Jewish child.

Early diagnosis is especially crucial when deaf children will develop blindness as well. In such cases, oral language may be the optimal mode of communication since sign language and lip reading are both visual. There is a need to provide these children with the best hearing amplification available, with a preference for cochlear implantation if possible, accompanied by intensive training and habilitation. A recent study of Usher syndrome suggests that auditory-oral communication is more successful if cochlear implants are implemented prior to the onset of retinal degeneration, in conjunction with speech therapy

(Loundon et al. 2003). The earlier the diagnosis is made, the better chance these children have for being able to communicate optimally in society, even after they have lost a portion or all of their vision as well.

101 In addition, early diagnosis of Usher syndrome might allow for an enhanced

social and emotional adjustment for the family, recurrence risk counseling of at risk couples and the option for prenatal diagnosis for at risk couples.

We suggest a diagnostic algorithm for Ashkenazi Jewish children presenting with

SNHL (Figure V-1). The routine implementation of comprehensive tests, such as brain CTs, etc. is cost-ineffective and may be circumvented in many cases by less costly molecular diagnostic tests (Greinwald and Hartnick 2002). We suggest testing for R245X in children under the age of approximately 10 of Ashkenazi Jewish descent with no family history of

NSHL who test negative for a GJB2 or a GJB6 mutation. This molecular test may identify impending RP prior to detection by ERG, and permit timely rehabilitation and genetic counseling for the parents.

History/Physical Examination*/Audiological Tests

Diagnosis Urinalysis, SMAC Uncertain CBC, ESR Thyroid Tests Bilateral CMV Unilateral ECG GJB2/GJB6

Computed Tomography (CT) Diagnosis readily apparent. Serology (Syndromic SNHL, Autosomal No Mutation Abnormal Dominant SNHL, Infection, Trauma)

PCDH15

No Mutation Abnormal

Lab. Tests (Dashed box above) ERG CT Genetic Counseling, Treatment, Periodic Follow-up

*Fundoscopy included

Figure V-1. A diagnostic algorithm for Ashkenazi Jewish children presenting with SNHL.

102 V.4. Family Z

There is almost no information for classical linkage in Family Z, because most of the DNA samples we have are of deaf children who are the result of deaf marrying deaf.

Such marriages provide no segregation data (the maximum Lod score possible in the family is 0.60). Therefore homozygosity mapping was performed in Mary-Claire King’s laboratory at the University of Washington, Seattle, USA. Since the parents of #685 were believed to be closely related to each other, it could be assumed that the genomic region of homozygosity in

#685 at the critical allele would be quite large (i.e. few recombination events would have occurred since the shared common ancestor of the parents of #685). A shared haplotype was detected on chromosome 10q21-q23. The CDH23 gene, which lies within this haplotype, consists of 69 exons spanning 420 kb. Mutations in CDH23 are known to cause USH1D as well as non-syndromic HL associated with DFNB12 (Pennings et al. 2004). Another

DFNB12 mutation in Family Z is possible and the CDH23 gene is being sequenced in Prof.

King’s laboratory.

Identification of the causative gene for deafness in Family Z, besides adding to the growing list of cloned genes for HL, will probably reveal another “ethnic gene” in Israel related to Jewish Algerian origin. Genes involved in HL of distinct ethnic groups have implications for routine genetic diagnosis that could be directed by the mutation-ethnic group correlation.

103 VI. FUTURE STUDIES

VI.1. Family O – Otosclerosis

All 93 genes in the OTSC4 locus were completely sequenced but mutations were

neither detected in the coding sequences nor in the splice sites. Naturally, the next step should

be to search for functional non-coding elements in the region. There are 481 ultraconserved

elements of the human genome, which are segments longer than 200 bp that are fully

conserved between orthologous regions of the human, rat, and mouse genomes and nearly all

of these segments are also significantly conserved in chicken, dog and fish genomes. In

addition, there are 5000 sequences of over 100 bp that are absolutely conserved among

human, rat and mouse. These conserved elements of the human genome, which are more

highly conserved than proteins, are most often located in introns or nearby genes involved in

the regulation of transcription and development (Bejerano et al. 2004). Recently, Bejerano et

al. published a protocol for “Computational screening of conserved genomic DNA in search

of functional non-coding elements” (Bejerano et al. 2005). Following this protocol, and

starting with what seems to be the best candidate genes in the region, might lead to cloning of

the gene for otoscelerosis in Family O.

VI.2. GJB2 and GJB6 mutations

Since mutations in GJB2 and GJB6 are responsible for a large HI population in Israel,

this study could be expanded in several directions, including searching for novel mutations in a larger deaf population. Very recently, a novel splice site mutation, IVS2 -24 A>G, was detected in the GJB2 gene in an Israeli deaf proband (personal communication). This is most

probably not the last mutation to be found. Other directions include investigating the adjacent

regions of the genes for functional non-coding elements (following the protocol described in

104 VI.1.) and extending the study of the correlation between connexin-associated deafness and outcome of cochlear implants. It is not inevitable that a larger study population and additional parameters tested might result in a different outcome.

VI.3. Family Z

Immediately after submitting this doctorial thesis, a missense mutation was detected

in the CDH23 gene in Family Z, in Prof. King’s laboratory. The mutation V2635P is a G>T

transversion at chr10:73.235.599 nucleotide, according to http://genome.ucsc.edu, May 2004.

Screening for the mutation in all the deaf probands in the lab, with particular attention

to probands belonging to the same ethnic group, will reveal the prevalence of the mutation

among the Israeli deaf population, and the carrier rate in Israel will be determined by

screening a large hearing control group.

Since the involvement of the CDH23 gene in deafness is known, expression experiments had been conducted previously. In situ hybridization analyses of the mouse inner ear revealed Cdh23 mRNA in sensory hair cells and Reissner's membrane (Wilson et al.

2001). Immunohistochemical analysis showed Cdh23 in stereocilia of immature mouse

cochlear and vestibular hair bundles (Boeda et al. 2002; Siemens et al. 2004). Recently,

Cdh23 was shown to localize along the length of growing stereocilia and to the tips of mature stereocilia (Siemens et al. 2004).Although expression studies on CDH23 have been carried out, more defined studies on the correlation between localization, cellular impairment and phenotype are needed in order to decipher the role of CDH23 in the distinct phenotypes in which the gene is involved.

105 VI.4. More Genes

The aim of this doctorial thesis was to actually determine how many genes there are

and what proteins they encode, based on the statistical evaluation of genes for HL in the

Israeli population I made in the eighties. Apparently, this goal was not completely achieved

and the study should go on. Shortly after submitting this thesis, I identified a novel insertion

mutation in the SLC26A4 gene in a proband with an enlarged vestibular aqueduct. This

mutation should be analyzed in terms of prevalence, correlation to distinct ethnic groups and

genotype-phenotype correlation as well as characterized in terms of expression and function.

Another Israeli family with dominant HL was very recently mapped to the same locus of DFNA13 on 6p21, where the COL11A2 gene is located. The family’s phenotype resembles the non-syndromic dominant mutations phenotype of this gene (Chen et al. 2005).

Sequencing of the COL11A2 gene should be the next step, following characterization of all

aspects of the gene in case a mutation is detected, or proceeding with sequencing other

candidate genes in the region if no mutation is identified in the COL11A2 gene.

Including these three newly detected genes for NSHL in the Israeli population and the

genes discussed above (GJB2, GJB6, MYO3A, POU4F3), it seems we are getting closer to

the estimated number of 8-9 genes I concluded statistically before the molecular genetics era

began.

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123 VII. APENDIXES

VII.1. Forms VII.1.1. Request to participate in a clinical research study VII.1.2. Consent to participate in a clinical research study VII.1.3. Questionnaire VII.2. Tables VII.2.1. All the Genes in the OTSC4 Region (according to http://genome.ucsc.edu, May 2004) and the Primers Used for Sequencing (Capital letters indicate sequence of primer in exon). VII.2.2. SNPs in the Candidate Genes for Otosclerosis in the OTSC4 Region on Chromosome 16q22.1-23.1 VII.2.3. Genotypic, Phenotypic and Familial Data of Connexin 26 and/or Connexin 30 Probands VII.3. Manuscripts Brownstein, Z., T. Ben-Yosef, O. Dagan, M. Frydman, D. Abeliovich, M. Sagi, F. A. Abraham, R. Taitelbaum-Swead, M. Shohat, M. Hildesheimer, T. B. Friedman and K. B. Avraham (2004). The R245X mutation of PCDH15 in Ashkenazi Jewish children diagnosed with nonsyndromic hearing loss foreshadows retinitis pigmentosa. Pediatr Res, 55, 995-1000. del Castillo, F. J., M. Rodriguez-Ballesteros, A. Alvarez, T. Hutchin, E. Leonardi, C. A. de Oliveira, H. Azaiez, Z. Brownstein, M. R. Avenarius, S. Marlin, A. Pandya, H. Shahin, K. R. Siemering, D. Weil, W. Wuyts, L. A. Aguirre, Y. Martin, M. A. Moreno-Pelayo, M. Villamar, K. B. Avraham, H. H. Dahl, M. Kanaan, W. E. Nance, C. Petit, R. J. Smith, G. Van Camp, E. L. Sartorato, A. Murgia, F. Moreno and I. del Castillo (2005). A novel deletion involving the connexin-30 gene, del(GJB6- d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment. J Med Genet, 42, 588-594. Del Castillo, I., M. A. Moreno-Pelayo, F. J. Del Castillo, Z. Brownstein, S. Marlin, Q. Adina, D. J. Cockburn, A. Pandya, K. R. Siemering, G. P. Chamberlin, E. Ballana, W. Wuyts, A. T. Maciel-Guerra, A. Alvarez, M. Villamar, M. Shohat, D. Abeliovich, H. H. Dahl, X. Estivill, P. Gasparini, T. Hutchin, W. E. Nance, E. L. Sartorato, R. J. Smith, G. Van Camp, K. B. Avraham, C. Petit and F. Moreno (2003). Prevalence and

evolutionary origins of the del(GJB6-D13S1830) mutation in the DFNB1 locus in hearing-impaired subjects: a multicenter study. Am J Hum Genet, 73, 1452-1458. Snoeckx, R. L., P. L. Huygen, D. Feldmann, S. Marlin, F. Denoyelle, J. Waligora, M. Mueller-Malesinska, A. Pollak, R. Ploski, A. Murgia, E. Orzan, P. Castorina, U. Ambrosetti, E. Nowakowska-Szyrwinska, J. Bal, W. Wiszniewski, A. R. Janecke, D. Nekahm-Heis, P. Seeman, O. Bendova, M. A. Kenna, A. Frangulov, H. L. Rehm, M. Tekin, A. Incesulu, H. H. Dahl, D. du Sart, L. Jenkins, D. Lucas, M. Bitner-Glindzicz, K. B. Avraham, Z. Brownstein, I. del Castillo, F. Moreno, N. Blin, M. Pfister, I. Sziklai, T. Toth, P. M. Kelley, E. S. Cohn, L. Van Maldergem, P. Hilbert, A. F. Roux, M. Mondain, L. H. Hoefsloot, C. W. Cremers, T. Lopponen, H. Lopponen, A. Parving, K. Gronskov, I. Schrijver, J. Roberson, F. Gualandi, A. Martini, G. Lina- Granade, N. Pallares-Ruiz, C. Correia, G. Fialho, K. Cryns, N. Hilgert, P. Van de Heyning, C. J. Nishimura, R. J. Smith and G. Van Camp (2005). GJB2 mutations and degree of hearing loss: a multicenter study. Am J Hum Genet, 77, 945-957. Brownstein Z., A. Goldfarb, H. Levi, M. Frydman, and K. B.Avraham. Mapping and Characterization of Hereditary Otosclerosis Linked to OTSC4. Arch Otolaryngol Head Neck surg., In Press. Taitelbaum-Swead R*, Z. Brownstein*, C. Muchnik, L. Kishon-Rabin, J. Kronenberg, L. Megirov, M. Hildesheimer,K.B. Avraham. Connexin-Associated Deafness and Speech Perception Outcome of Cochlear Implantation. Arch Otolaryngol Head Neck surg., In Press.

* These authors contributed equally to this study.

VII.1. Forms

VII.1.1. Request to participate in a clinical research study

REQUEST TO PARTICIPATE IN A CLINICAL RESEARCH STUDY

Department of Human Genetics and Molecular Medicine Sackler School of Medicine Tel Aviv University

We invite you to take part in a research study that takes place in the laboratory of

Prof. Karen B. Avraham at Tel Aviv University. The goal of the study is to discover

the genes that cause hearing impairment in the Israeli population. To date, mutations

in a total of four genes associated with hearing loss have been identified in the Israeli

Jewish population, out of eight to nine genes that were estimated. Connexin 26 is one

of the genes involved in 30-40% of recessive deafness among the Israeli population.

The studies on clinical and molecular characterization, probability and genotype-

phenotype correlation of connexin 26 are being carried out in the laboratory of Prof.

Karen B. Avraham, as well as searching for additional genes involved in deafness.

The research study is being conducted in collaboration with organizations for the

hearing impaired in Israel such as MICHA, SHEMA and the HELEN KELLER

organization, as well as with the medical centers in Israel: the Sheba Medical Center,

the Rabin Medical Center, the Hadassah Medical Center and others.

Finding the genes requires DNA samples and results of hearing tests from hearing

impaired individuals and their family members.

Your participation in this study would therefore involve the following:

Obtaining a Blood Sample from You

We will draw a 20-ml sample of your blood in a test tube. The procedure will be performed by a trained person using sterile techniques. DNA will be extracted from these blood samples and cells will be grown in a laboratory culture.

Hearing Tests

We need an updated audiogram of all individuals participating in the study. In case

you do not have one, we can perform the audiological evaluation with a portable

audiometer at your house or at any other place of your convenience.

Confidentiality and Privacy

All the information you give is strictly confidential. Your privacy will be preserved under all circumstances, including in scientific publications resulting from this work.

Your records will not be released to anyone without your written request or consent.

Benefits of Taking Part in this Study

The results of the hearing tests, and any results we obtain from the DNA analysis, will

be made available to you at your request. Using this information, you will be advised

to seek genetic counseling in a Genetic Center.

Studying your family may lead to an increase in understanding of hereditary hearing

impairment and may also benefit others, as we begin to understand more about how

the auditory system works and how deafness can be prevented.

For more information and/or if you agree to participate in the study, please contact

Zippi Brownstein at tel.: 03-6406417, fax: 03-6409360, or email address:

[email protected], or fill in this form and send it back in the attached envelope.

We will contact you to set up an appointment with you and your family.

Name:______

Address:______

Tel.:______Fax:______

E-mail address:______

Signature:______

We will appreciate very much your participation,

Sincerely,

Prof. Karen B. Avraham Zippi Brownstein

Tel Aviv University

VII.1.2. Consent to participate in a clinical research study

Sackler School of Medicine Tel Aviv University Ramat Aviv, Tel Aviv 69978 ISRAEL Department of Human Genetics & Molecular Medicine Investigators: Prof. Karen B. Avraham

CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY • Adult patient

• Parent, for minor patient

Patient’s name: ______I.D. number: ______

We invite you to take part in a research study that takes place in the laboratory of

Prof. Karen B. Avraham in Tel Aviv University. The goal of the study is to discover

the genes that cause hearing impairment. Finding the genes requires DNA samples

and results of hearing tests. Your participation in this study would therefore involve

the following:

Obtaining a Blood Sample from You

We will draw a 20-ml sample of your blood in a test tube. The procedure will be

performed by a trained person using sterile techniques. From these blood samples

DNA will be extracted and cells will be grown in a laboratory culture.

When blood is drawn you may experience some discomfort for a few minutes at the

site of the needle entry in a vein in your arm. Occasionally there is a small bruise at

the site of the needle stick. This will resolve and cause no problem. There is a remote

risk of fainting or local infection. In such an unlikely event you will receive

appropriate medical treatment.

Confidentiality and Privacy

All the information you give is strictly confidential. Your privacy will be preserved under all circumstances including in scientific publications resulting from this work.

Your records will not be released to anyone without your written request or consent.

Benefits of Taking Part in this Study

Your hearing status will not be changed by your participation, and there is no direct

benefit to you if we find the genetic cause of deafness in your family. However you

may derive some indirect benefits from this research. For instance, the results of the

hearing tests, and any results we obtain from the DNA analysis can be made available

to you at your request. Using this information you will be recommended to seek

genetic counseling in a Genetic Center.

Studying your family may lead to an increase in understanding of hereditary hearing

impairment and may also benefit others as we begin to understand more about how

the auditory system works and how deafness can be prevented.

Withdrawing from Participation in this Study

You may withdraw your participation in this study at any time. You will not be

penalized in any way if you withdraw and none of your rights and benefits as a

participant and/or a patient will be taken away.

I have read the explanation about this study and I hereby consent to take part in

it.

Signature of patient or parent: ______

Signature of investigator: ______

Date: ______

VII.1.3. Questionnaire

QUESTIONNAIRE

Family Number ______Family Name ______Address:______Tel.: ______Fax.: ______E-mail address: ______Husband Wife

Name ______

Year of birth ______

Land of birth ______

Hearing loss yes / no yes / no

Occupation ______

Father’s name ______

Land of birth ______

Hearing loss yes / no yes / no

Mother’s name ______

Land of birth ______

Hearing loss yes / no yes / no

Parents are relatives yes / no yes / no

Husband & wife are relatives yes / no

If yes, how? ______

Hearing impaired relatives yes / no

If yes, list:

Name Relation ______

______

Offspring

Number of children: ______

Hearing impaired children:

Name Year Age Cause Type Severity Hearing Cochlear Other of of of HL of HL Aid Implant symptoms birth onset

Remarks: ______

______

Pedigree:

VII.2. Tables

VII.2.1. All the Genes in the OTSC4 Region (according to http://genome.ucsc.edu, May 2004) and the Primers Used for Sequencing

(Capital letters indicate sequence of primer in exon) (The list is in the order the genes were sequences. Genes that were assumed better candidates were sequenced before others)

Gene DNA Primers Product size (nt) LOC64174 cDNA 3F- gcagagagctccgctaagg 847 =DPEP2 3R- gggtgtctgtggcttgcta (11 exs) NM_022355 genomic g2F- gaatcgagggtcgggact 384 g2R- ccccttgcagcaattacg genomic g3F- gctaagaggcccatctgtct 700 g4R- aggtcaggtgattactttccag genomic g5F- gttgatgggaagcgttagga 284 g5R- cacagtgacctcaggcagat genomic g6F- tagactcttgaggccgagga 472 g7R- tcctggtttgaacctcttgc genomic g10F- gaccctggactgtggttctg 694 g11R- cccattgcagagaaggaaac ` CDH1 genomic 1F- gtgaaccctcagccaatcag 371 (16 exons) 1R- tcaggacccgaactttcttg NM_004360 genomic 2F- aggtcttgagggggtgactc 426 2R- ggtgtgggagtgcaatttct genomic 3F- cgctctttggagaaggaatg 393 3R- atccagcccaaaatgtcaac genomic 4F- gctgtctggctaggttggac 297 4R- ttttccctttctctccttgg genomic 5F- agggaaaagacccagtgttg 295 5R- ggatccagcatgggttgac genomic 6F- ccccttctcccatgttttct 294 6R- tacacaacctttgggcttgg genomic 7F- attgacccagtcccaaagtg 299 7R- tgtccacgggattgagcta genomic 8F- attctggttccatgtgttgg 300 8R- acttcgcccatgagcagtg genomic 9F- aatcctttagccccctgaga 382 9R- tctgggaaagtcaccctgtc genomic 10aF- ccaaaagcaacagttaaggattt 374 10aR- aaccagttgctgcaagtcag genomic 11F- aagcgcttaagccgttttc 285 11R- gaggggcaaggaactgaact genomic 12F- aaggcaatggggattcatta 400 12R- caatggaaggggtgacatct

genomic 13F- ggcttgcgggtgtctttagt 391 13R- ccaggaaataaacctcctcca genomic 14F- taactgccccctgtctggta 299 14R- ctaatcattgcttcttccgaat genomic 15F- tgaacatagccctgtgtgtatg 278 15R- tgacacaactcctcctgagc genomic 16F- agatgacaggtgtgcccttc 357 16R- atctcaagggaagggagctg TEX292 = cDNA 1F-CAGATTGGCTGTTTCACG 208 CIRH1A 1R-CAGTTCAAGGAAGGACAC (17 exs) NM_032830 cDNA 2F-ACACAGATTGAAACTGAGC 456 2R-AGGGAGATGTTGTTGGAG genomic Ex1-F- ACTATGCGCCAGGAGCACTT 257 Ex1- R-CTCTCCCCGAGTCTACAGAGTA genomic g1F- ATGTTGTTGGAGCCTTCCAG 319 g2R-TCAGAAGAAATTAACATCAAAACACC cDNA 3F- CTTGTCCGATGGCACTATCA 998 3R- GGGCAATAGCCATAGCAGTC cDNA 4F- accagtgctggagtccatgt 592 4R- gggtagacagttcaaggaagga cDNA 5F- agtcattgccccttccaaat 284 5R- aagcaacagggtagacagttca NFAT5 cDNA 1aF- cctctgaagcagggagtgtc 819 (14 exons) 1aR- tggccttccagctttactgt mRNA NM_006599 cDNA 2aF- tgtacaacctgagacacagca 600 2aR- tcatcagaaacattttcttggaa cDNA 3aF- ctgcaaacaccctcttctcc 834 3aR- ctgcaatagtgcatcgctgt cDNA 4aF- aggacatctcacagcctggt 848 4aR- tccagaaactgcaggaggag cDNA 5aF- caactgcttcagcaaatgga 812 5aR- tattggagcttgctgttgga cDNA 6aF- tttttgcagcaccgaactc 677 6aR- ctgcaaggcagacatggat cDNA 7aF- gctctcctcagattcagttgg 583 7aR- tcaggacatcttggaatcagg TERF2 genomic 1cF- tcttcaatccgcgacagac 588 (10 ex) 1aR- gctcggaacgctgtttctat NM_005652 genomic 2F- aaaacaaaaccctacgcaagt 294 2R- ggtacggggacttcagacag genomic 3F- tctgccccacacagtaaaga 394 3R- tgaatttggacccttatcgtg genomic 4F- ttccctatttccccacaaaa 291 4R- gcgcctggctttataattgtt genomic 5F- ttcaatgtctttttcacacatcaa 393

5R- gggcttcccttcctgtatgt genomic 6F- cctggcctaaggaggagact 295 6R- ttggatttgtaagtgaacttgtttg genomic 7F- ccactcctgcgtcaagttct 593 7R- tggatgagcatcatcagtgg genomic 8F- aacatgacagtaacacgcatctg 246 8R- gtacagggccaagagtgagc genomic 9F- gagacagggcgcgtatttta 206 9R- gttttccctttggggacatt genomic 10aF- GGAACCATGCTCCTGTGAAT 248 10aR- aagtggagaggaaaggtggt COG8 genomic g1F- aacctggaacgtggtaaacg 587 (6 exs) g1R- gccggatgagaaagcataga NM_032382 cDNA 2F- ctgtttggcgacgtggag 681 2R- cacctgcaggaattgtgaga genomic g3F- atgtcagcagacaccgtcaa 683 g3R- CCCGTGTCCATCTCTTTGAT genomic g4F- cactgacttacacaaatggatgaa 289 g4R- ggctgtcccagtaattgacg genomic g5F- gcttagactggcagtgagca 488 g5R- ccggcctcccttaaatttct WWP2 cDNA 1F- aaggctaaagagggctggag 471 (25 exs) 1aR- gtcagttttccgcctgagac NM_007014 cDNA 2F- gtcaagaactcaggccaca 850 2R- gaaacggaactggtgatacttc cDNA 2aF- aagaacaatgggggcaaaat 492 2aR- agaagtcgtgttgggattcg cDNA 3F- gtttgagtcggggacgaag 674 3R- ttccttgttctcctccgtga genomic g16F- tgctggatattgggccttag 241 g16R- atgctgtagcctggggaag cDNA 4F- atactgggcaaggtgacgac 696 4R- acacagggccacatccac genomic g24F- ctagggctgactgtcgtgct 249 g24R- ACACAGGGCCACATCCAC AP1G1 genomic g1F- tggatttggcaagtcttgaa 398 (23 exs) g1R- ccagaaaaagatggacaatcc NM_001128 genomic g2F- tggggactctgggttaaaaa 300 g2R- tgggcaaaagagcaagactc genomic g3F- ctgcttcttggggagcac 260 g3R- aatctgccttctcatctcca genomic g4F- gttgccctggcataattgaa 247 g4R- cactgttctgagagacacaggact genomic g5F- ttttggaattgaccttcagga 276 g5R- tcagatccatagcttgctttca genomic g6F- cttggactgggacccttaca 288 g6R- tcaacacaaacttaagaggaagga

genomic g7F- ccaagtgagatttctttgtgtga 584 g8R- accaaaaatccagcattcca genomic g9F- ctgtgaagcaccagaggtca 227 g9R- tcttgagcagaatcgctctt genomic g10F- ggagaaaaactatcctgaattttaca 250 g10R- cggcatagagctatcaataatctg genomic g11F- tcacaagatcgtgcaaggac 388 g11R- cctcccaaactgctgagact genomic g12F- tgcttgggcaggattttcta 231 g12R- ttgtgaatttcttacctccctga genomic g13aF- catcatttctgtgtgtttctaggg 574 g14aR- aatcaacctcaactccccatt genomic g15F- cctgacttgatatcagatttagatgc 299 g15R- gcttgaaattttccatgataacaa genomic g16F- tctggcttattaaacattatctctgc 278 g16R- aaagcacaaggaatgaatcttct genomic g17F- gatggggaggggagatagat 674 g18R- ttcacgtgctggaaagtctg genomic g19F- tttgtcatgctgcctaaggtt 526 g20R- tcctttaccaaacacatgaaaa genomic g21F- tggtatctctgacctagcgttg 250 g21R-gaaaaacctaaagaaacaaagcaga genomic g22F- cagctttgctttaagttttgacc 285 g22R- CCAGCAATCACAACCTCCTT PDF genomic 1F – gtatgaaccgcgaccacttc 791 (2 EXs) 1R – tgccagtctaagcaggaagg NM_022341 genomic 2F- ATATGCGAGCCATCCAAGTT 371 2R- gctgttgcctcttgtcaatg HSPCO31= genomic 1F- gagaggaaggctttgggatt 693 CGI-37 3R- gtcagggactctgtgggaga (5 Exs) NM_016101 genomic 4F- gcttgtctgcatggatagaaat 600 5R- CACAAACACAGGAAACAAAGC ART4 genomic 1F- gcccagacttggacatagga 476 =NOB1P 2R- aaggcagctagaccccaact (9 EXs) NM_014062 genomic 3F- ccattggttatccaaggtca 247 3R- tgacagttcccataatacaaagc ` genomic 4F- tacaggcatgagccatcgt 849 6R- gccgatgacgtgtgtgac genomic 4bF- ctgggattacaggcatgagc 250 4bR- cactgtgaagagatgacttagagaga genomic 6F- TCGATCATGAACTGCAGGAG 376 6aR- gtaggaccactgccgatgac genomic 7F- caaggcagggtttgtgaagt 298 7R- taccactgcactccagccta genomic 8F- gtttgctgcctgtggcttt 291

8R- cccatcttgcagaaacgact genomic 9F- ctgcaccaggatctgcaag 477 9R- TGTTATTTCCATCGGGTGGT MTR3 = genomic 1F- attctcgacgcaaactggag 980 EXOSC6 1R- aacggaagaaccagtggatg (1 EX) NM_058219 genomic 2F- CTGATTCAGCACAGGCATGA 684 2R- gttcgcacgccaagaacc NQO1 genomic 1F- gtgcactacacacgcgactc 210 (6 EXs) 1R- ccctacaacctcctccacag NM_000903 genomic 2F- gcaactcccctgtagctgaa 599 3R- cagagaatgcagtaaagtgggtaa genomic 4F- gaggaatgggaaaggtgtga 300 4R- atcaggacagaccacccaga genomic 5F- tcttgtcaccaaggggatgt 240 5R- tgtcactgcactccagccta genomic 6F- gaagcccagaccaacttctg 458 6R- AAGTCAGGGAAGCCTGGAAA MGC34647 genomic 1F- agaatgccctggttgctatg 285 (6 EXs) 1R- TTGGGGCACCTGCTCACT NM_152456 genomic 2F- cagaggcctatgccaggtg 245 2R- gagatggagggtgctcatgt genomic 4F- tttccctcactagcctttgg 689 3R- aacatcatttgggaggttgg genomic 6F- ACTCCTGTGGGAGCTGGTC 695 5R- acagccctcacggctctc CHST4 genomic 1F- gccagaaggggaatagaagg 697 (2 EXs) 1R- AGGTGCACGATATGCAGGTT NM_005769 genomic 2F- AGGTGCGCTTCTTCAACCT 677 2R- AGTGACTGAGGCTGACACCA FLJ10079 = genomic 3F- ggggcagtatctctgaaagg 399 PDPR 3R- ctcattaatcccatgcactga (19 EXs) NM_017990 genomic 4F- cctggcagaggaccaaatac 286 4R- aatgagaaaccctgacctcgt genomic 6F- tggtgtcacttcctattgtgaga 490 5R- ggattttccactctcctgtcc genomic 7F- ggaggaaggacaaggtcaca 397 7R- tcttctgggtggagaacctg genomic 8F- ggacaggatactggcaaagaa 245 8R- tctcaagaggctgaacttgc genomic 9F- caggcaaactcaccctctgt 398 9R- tgggtgtttttgtttggttg genomic 10F- agggaagagacccagcaagt 355 10R- gaggttttctgaaaggggaga

genomic 11F- ggccaccgagaagatacctg 294 11R- tcagctggtctatggcaattt genomic 13F- tgaaaaacaaaaggcattcaa 594 12R- gtggacatgggctactcagg LCAT genomic 1F- cccactcccacaccagataa 334 (6 EXs) 1R- ttatgtcggggcttatgcag NM_000229 genomic 2F- tacctgggggttgagggtat 581 3R- gtgtgtgcaggtaccctgtg genomic 4F- ggcaggtttgtgtcagagg 599 5R- ctagaggccactgtgagcag genomic 6F- ctgtcccaccttgctcca 694 6R- gctggtgaggagtgaaacct FLJ20400 = genomic 1F- tggtgacagcactcatcaaag 682 DERPC 1R- cctaggattgggaggaccat (4 EXs) NM_017804 genomic 2aF- caggtcaggtgtgtttccag 224 2aR- tgtttccagaagcctgtgag genomic 3F- ccaaactcgggtcctagctc 686 3R- gccaagaaccacagtgccta MGC23911 genomic 1F- agcggttgtctgaacgactt 361 (4 EXs) 1R- ggcctaaaacctgacccact NM_144676 genomic 2F- cgtgatgtagcaggggttct 241 2R- tcctcattatttctgtaatttcatagg genomic 3F- aaaggggaggagaccaaaga 233 3R- ccacagatgtttctcttccaca genomic 4F- gctcaggtgaggctagatgg 343 4R- TCGACTAAGCCCCAGAAGAA cDNA 1aF- ctatgaggggcctgagactg 451 1aR- tgattttattgccattttgcttt FUK genomic 4F- ctgctcaactccacgaactg 245 (24 EXs) 4R- tttgaccttgccttttgtcc NM_145059 genomic 5F- aacagtgggagggaagtgg 249 5R- ctgatgaggtggccttgg genomic 7F- acggaggaaactccttggat 828 6R- tggaggcccttgttacttca genomic 8F- cctccctgtcccataaggtc 242 8R- tcctcagctttcctttggag genomic 10F- attcttggggtcctcactcc 640 9R- atggcactcagcttcatgtg genomic 11F- ccacctgaccccagacaac 249 11R- cccatgtgtctctgctgct genomic 13F- ctcagagaaccccgcagtg 578 12R- cagtaggcctggacgcttt genomic 14F- ggagggacacacctcaaaga 208 14R- ctggccgagtgtctctaacc genomic 15F- gatggcccagaggacagg 500

15R- ggggaagagactggagcttt genomic 16F- ctctgggtttcctccatcag 383 16R- ggcgcatttagggactgtt genomic 18F- actggcctgcagtctctagc 685 17R- ggaggaaacccagagagagg genomic 20F- gcccttgggaaagtgtcagt 595 19R- cagtactgctgctgggcttt genomic 22F- cccacaagtgacagaatactcg 500 21R- aggctgggagagtgaagctg genomic 23F- aggaccagggggacttacag 389 23R- atctccagatgggtgctgag genomic 24F- GGGAGTAGGAGGTGGAGGTT 250 24R- agcctccagaaggcctgag CALB2 genomic 1F- ctgagggcaaacgtacacct 440 (11 EXs) 1R- gtctcagcgcagaggtaagg NM_001740 genomic 2F- gctcaaaggcataggtcagg 235 2R- ccagctgggagaagacattc genomic 3F- atgctcatgcctgggaga 246 3R- tgatttccaagcttcctgct genomic 4F- gcaggtcccagtgcttagaa 245 4R- agaaaacgcaattcccacac genomic 6F- cttggggcacaagtcctg 845 5R- aatggaagggaggtgggtag genomic 9aF- gatgaagctgagcccttctg 226 9R- catgcacacaccacacacat genomic 8aF- caggctcagagggatgagg 492 7aR- caaataacccaggcaccttt genomic 10F- tgcagaagcctctcctgtg 245 10R- cccccttttgcacagagtaa genomic 11F- CAGGTGTGTGCAGGCTGTAG 332 11R- ggtgtgggtgtttgtgtttg HAS3 genomic 2F- cacaacccaagggacctaga 794 (4 EXs) 2R- actggaaatgctgcctcct NM_005329 genomic 3F- catttcccaggcatatccaa 234 3R- acagggtgtgcaggtctga genomic 4F- TGCAGGTCCCAGTTCACAT 227 4R- tcaagcagaaactgcattcg SNTB2 genomic 1F- ggcctcatgaataatgaatcg 457 (7 EXs) 1aR- CGCCTCTTGCTTCACCAC NM_006750 genomic 1cF- GGGGAGAGCCTGAGCCTGAC 605 1cR- AAGCATCCGTTTTTCCACACTG genomic 2F- cccagctctttcctccactt 390 2R- ctgcaacatgtccttaagtttg genomic 3F- tggcaggtttggtgacttct 372 3R- ccaggtcctaactgcatggt genomic 3aF- caaatttatttgaagaatgcaaaa 384 3aR- gaaccatcctggcactgaat

genomic 4F- ggccatcattgaaggaactt 400 4R- ctgagctaggagctgcaaaa genomic 5F- tcaatgagcacaaattcctga 392 5R- tctgaggggactgctgtcat genomic 6F- tcagaagattcccttttcacttg 357 6R- ggaatgtcagtccatgactttgt genomic 7F- ttgcatcttcgtttattaagttgtc 250 7R- gcaaagagtttctttttgtgctt CFDP1 cDNA 1F- ctgcggtcttgtgagtttga 999 (7 EXs) 1R- cgcacaaaaagctcacattg NM_006324 VPS4A genomic 1F- gagcgcgtggaaacagac 482 (11 EXs) 1R- gtcagtcctgcctctcgaac NM_013245 genomic 3F- atgagcgccaggaatgatag 490 2R- cagggactttccacctctcc genomic 6F- ccgctgggtcactagcag 996 4R- aggctgaagggccagctt genomic 7F- ggctgagaacaaccaaatcc 336 7R- tgtgggaagggtgagaagag genomic 9F- caggaggcctcagagactgt 810 8R- ggcttcaattgctgacacac genomic 10F- ccaaaccctcaattttctgg 298 10R- cagagcattctctttgggactt genomic 11F- AGCCCTGTCTGGCTGTTG 244 11R- gaggttggggacctggag DDX19 cDNA 1F- cctcgaatccaccagcac 779 (12 EXs) 1R- catcagcctcatccagaaca NM_007242 cDNA 2F- ggactgtgctggactggtg 850 2R- tgaacgcacttgtctcctgt ZNF23 cDNA 1F- AAGTCGGTGACCTTTGAGGA 840 (6 EXs) 1R- CTCCCCACTGTGGATTGTCT NM_145911 cDNA 2F- ACAGTGGGGAGAAACCCTTC 660 2R- CTGCCGAAATTGGGAGTTAC cDNA 3F- ATTCACACAGGCGAGAAACC 800 3R- CTGCCATATTCATGCCCTCT COG4 genomic 1F- gttcattgggcacttctgtg 300 (20 EXs) 1R- ggctcccactctccctagat NM_145818 cDNA 2F- cgaggagaaagtggtggaga 745 2R- gagtctccctggcccataat cDNA 3F- ctggggacagacatgagtga 844 3R- ctcgatgccttttgtgtcaa cDNA 4F- atgcacagcagcctccag 843 4R- ggcctgcaagtgtgatgag FLJ20511= cDNA 1F- ccacaaagtgaaagggagtga 600 TXNL4B 1R- aacacagcacagctggattc (4 EXs)

NM_017853 FLJ11171 genomic 1F- ttgctgctcatgagatttcg 812 (3 EXs) 1R- TCCTTGGCAATCAAAACTCC NM_018348 genomic 2F- TTGCAAATACCTTGCACTGG 839 2R- ACCATGTTCTTCAGCCCAAC genomic 3F- ACAAAATGGTTTGGGCAGAG 692 3R- CAAAGATCAAACCAGCCATAAA genomic 4F- GACTGCCTTCTACATTCATTGC 499 4R- GCAACAGGATGCAAATACTGG FLJ10520 genomic 3F- catcttcttcaagattgattttacatt 500 (removed 4R- tttcagatgtgcctacaatgaac from region) (11 EXs) NM_018124 genomic 5F- cttaactctgccacctggatg 250 5R- ttagcttcctcttccattcct genomic 6F- aaggaattgtatttgaggggaat 374 6R- ttttccaaaaggcaaggaaa genomic 7F- caagaaaggtggacgaagga 397 7R- cagagcgagactccatctca genomic 8F- ttgggagtatgagtgtgttctca 384 8R- tgtggaacacccactgaaga genomic 9F- gtgggaatgtgcttgaggtc 373 9R- tcaaaccctccttccctttt genomic 10F- gcgtgtgaggtttgggtact 365 10R- aaagttcatgttttcccttacca genomic 11F- gacttgttagatgatgaagaatggaa 299 11R- ATCTTGCTTGGGGCTGCT DDX28 cDNA 1F- ctccacacaccgtactgagc 847 (1 EX) 1R- cagctgcagcctgatcctac NM_018380

cDNA 2F- gagaattggcccaacaggt 600 2R- gccagttcacagtgctggag cDNA 3F- gcatcgtgacagagcagaaa 550 3R- tcccactgacaagtgtggtc LYPLA3 cDNA 3F- ggcttcaggagacaacaacc 574 (6 EXs) 3R- cagaggccaacagcaggt NM_012320 genomic g1F- AGAGAGCTGAACCTGCATCC 265 g1R- tcgtgactggaacagaagca genomic g2F- ggctgagggctcacacat 246 g2R- ctctcaggcactgttggtga genomic g3F- gatgcactgttgggacacac 593 g4R- ccatgatcacctcaccccta genomic 5F- gtgtctggcctgagaaaagc 366 5R- aagggaagagagggcagaat genomic 6F- aaccagctggcattcctaag 696 6R- GCAGATGCTTCACAGTCCAA

SLC7A6 cDNA 3F- gatgttttccagcttatcaactact 500 (11 EXs) 3R- tcaaaaggctcctttaattcag NM_003983 genomic g3F- atggtgggaactgagacagg 813 g3R- ctcccaaagtgctgggatta genomic 3aF- ataggaagtgggcagggaaa 395 3aR- AACAGAGAAGAGCCCACCAA genomic 3bF- GAGATCTCCCTGCTGAATGG 494 3bR- tgctgggattataggcatga genomic 4F- tgctctctgtaaagaggggttg 297 4R- tagaaacagccgagcagtga genomic 5F- cctcccatctcctcttcctt 382 5R- cactcccattctagccctca genomic 6aF- aactttgttcccaaggagagg 272 6R- gctgaggagggtgcagtaag genomic 7F- gcagtgattgcatttgttcc 299 7aR- cacaccctatatgcccatga genomic 8F- gctctgagctgagtttgtcca 248 8R- gcacaaatcagaaggagcaa genomic 9F- ggtgaagtggcatcagatctagta 600 10R- tgcgcatgcacacatagc genomic 11F- cagccttgttcaggttaggg 246 11R- GAGAGTTGGGCCCACAGAG ZNF19 genomic g3F- tgaatgctgcttgctattgg 246 (6 EXs) g3R- tggctctaagacttgccagaa NM_006961 genomic 4F- cctcacaggactttccctca 959 5R- ctgcccagcatgattcattt genomic 6F- gcctgtgtgatatttggcact 998 6R- CCTGAGCACTGAAGGCTTTT cDNA 3F- ggggaaaagccttattcttg 498 3R- ggatggatcagaggctgagt FLJ12331 cDNA 1F- ggaggttactactgccatcca 594 (1 EX) 1R- gccgaagagctatagccaga NM_024986 FLJ22593 cDNA 1F- cagagtccagagccaggaag 579 (removed 1R- tgagatccagcaatggttca from region) (1 EX) NM_024703 SLC12A4 genomic 1F- aagtgagcgcagcgggga 333 (24 EXs) 1R- caaggtcccgggataggtg NM_005072 cDNA 2F- ggctcagttgggtggactac 686 2R- attgttcaaagtggcattcg cDNA 3F- ccatcgagatcttgctgacc 696 3R- ggatagacttctgggcgtca cDNA 4F- cccttctgtaacaggcatca 693 4R- ttctcagccccttggtactc cDNA 5F- tggcccttatgtttgtctcc 675

5R- cgatccaccacacgtctatg cDNA 6F- cccaagaacatcgccttcta 849 6R- ctgttaccccagaccacagc DPEP3 genomic 1F- cagagtcgcgtgacttcaac 479 (10 EXs) 1R- gcatgctcagaacctcctct NM_022357 genomic 2F- aggagggtagacccaagcac 268 2R- ggtttctgtgaccccaacat genomic 3F- aggcctagggggttgaagac 497 4R- gcttggtcacacccatgc genomic 5F- ctgtgatggcccaaggtg 497 6R- gtgaaacctgggctggtg genomic 7F- acaggaggcatgttctcagg 789 8R- acgtcaggatgggaatgaag genomic 9F- ctgtgtgactagggctggtg 684 10R- cgctagggagaaggaaggac ATBF1 genomic 2aF- gccctcctaacctctttcctt 850 (10 EXs) 2aR- TCCTTGTTGCTTTTGTGTCG NM_006885 genomic 2bF- TACCCGCAGATCATCAACAC 839 2bR- TCCTCCTCCTCTTCCTCCTC genomic 2cF- GCAGAAGGAGAGAAGCAGGA 808 2cR- CCGCTTTTGCAGTAGACACA genomic 2dF- GACACTCAAGTGCCCCAAGT 825 2dR- ccactcgagaagaccagagtc genomic 3F- ttctccttttggaccccagt 688 3R- ctccccagtgctccctaact genomic 4F- tgtgaccaggatggtgtgg 400 4R- cctcaaacagaagtcctctgga genomic 5F- ctgtcgttggcagttgtttg 246 5R- ttccagggaaggaaagatga genomic 6F- acaaaccctctgggagctg 595 7R- cttgcccatgttactgcatc genomic 8F- ctggtgaaggctaattttgct 279 8R- cctgaaaacctttctccatga genomic 9aF- gtcattcaggttgggcactt 809 9aR- CACTTGTAAGGGCGAGAAGG genomic 9bF- ACAAGAAGACTCGGGCTCAG 828 9bR- CAGCAGGGTCTCAGTTGTCA genomic 9cF- GGCTCAAGTTCAAGCTCACC 805 9cR- TCTCGAGCTGTTTGAAAGGAA genomic 9dF- CTGACCAGGGAGAGAACCTG 814 9dR- AGACCTCTTGCTTCCCCAGT genomic 9dF2- ATGGAGCTGCCCATCTTCT 9dR2- 578 CGGGTTGGAAGGTTCAGTAA genomic 9eF- AGCCTGGAGGAGCTCAAGAT 835 9eR- GGTAAGGGGCACTGTGGAG genomic 9fF- CCGAGCAGAAGACCAACACT 816 9fR- CGGATATGAGCCTCAAGAGC genomic 9gF- GAGATGCCCTTTTTGCAGAG 679

9gR- ATTTGCCTGCAGATCCACTC genomic 9hF- TGTCATCTGGTCTGGTCAGC 848 9hR- ctcagagggtttgggtggta genomic 10aF- tcttgagcttcagccctacc 821 10aR- CTGCTGCTGCTGCACTTTT genomic 10bF- GAGTCTGCAGGAGGCAATTC 582 10bR- TGCTGCTCTCGTGATTGTTC genomic 10dF- GAAGCTCTGAGTCAACATCTCG 250 10dR- AAGCCCGGGAGACCACTT genomic 10fF- AGTCTGCCGCTCACTCAAAC 499 10fR- caacccacgctttttctttt SF3B3 cDNA 1F- ttggtggtggcttaagttttg 846 (26 EXs) 1R- taaggaagttgccgtgttcc NM_012426 cDNA 2F- cagcagctaatacccagcag 846 2R- ggcattcacgaaagacacaa cDNA 3F- gtatgtggcctgtggtaggg 842 3R- tcggaagagcttcacaggac cDNA 4F- tgtgctgctgaggactgtct 832 4R- gggacctcttccacaggagt cDNA 5F- aacaatggggaaaaactgga 915 5R- gtgggggaaacaaaacacac genomic 21F- tcctgtggaagtaggcatttct 300 21R- taaaactcaatgcccaccac genomic 22F- ttgctgttctgctgacccta 395 22R- ccttggctttacccacaaga LOC124491 genomic 1F- ttgtgattggaggaggcaac 469 (3 EXs) 1R- cgaagaagtgatggggaaag NM_145254 genomic 2F- gggtatgatgagtaccctctgc 397 2R- gccttggtgacatagggaga genomic 3F- aaatgttgtattttcgtgagtttg 264 3R- TGTATTCTTTTGGAGGATTCCAT CHST6 genomic 3F- tcgggtctggtggtagaatc 794 (4 EXs) 3R- GCACGATGCCGTTGTCAC NM_021615 genomic 4F- GCTCAACCTACGCATCGTG 668 4R- CTGCACCATGCACTCTCCT DHX38 genomic 2F- gcatttattgtgggatatagtagagc 999 (27 EXs) 3R- aaaaaggctgacacatttattcg NM_014003 genomic 4F- ggcagcctgtcctttctcta 287 4R- ctgactcaatgcctcagcaa genomic 5F- tttggggaattgacttttgg 807 7R- atcaaagcccagtcctgcta genomic 8F- ccttcttctgttggccatgt 997 9R- ggcccacatcaggtctctc genomic 10F- gggtagcagtccctttcaca 660 11R- ctttggagcaaggctgatgt genomic 12F- ccaatcaggaaggacagcat 841

13R- tttagcaaaaatgggtggaa genomic 14F- tgctaaagggtgtgatctgct 843 15R- ggcttcgcatcagagaagtg genomic 16F- caaagtccatggctccattc 849 18R- aatgtgagcctcccaccac genomic 19F- ctgccatgtgtagcaaccag 393 19R- gaaagccactccttccttcc genomic 20F- tccctgtgggatttcatctg 597 21R- attcccctccccaacactt genomic 22F- aacctctgggatgtgctcag 850 24R- atgccagaacgcagtgaag genomic 22R- ggagccctcctttcctctc 290 (with 22F) genomic 25F- tctggggtttttcctttcct 394 25R- tggtggggaatcagacagac genomic 26F- gatgcagcacctggtttgt 296 26R- tgcatcagggcctggtct genomic 27F- ctcctggctgtgggtgag 250 27R- CACAGATGAAAGTCCTCAGATG LOC55565 cDNA 1F- accagagctggaggttagca 700 (6 EXs) 1R- tacggtaggcagcacagttg NM_017530 genomic 2F- tgagttttatatgctcaggagctg 298 2R- ccctaaattcaggttactcttcca genomic 3F- tttctattgtaactgccaactctagg 300 3R- catcagggcaggcagaag genomic 4F- ccatcagtctgggttccttt 294 4R- gagagacaaaggccaaacca genomic 5F- aaggggacatttcccaaaag 367 5R- catcagctgcctttgcatta genomic 6F- ccactggcctcataggaaaa 788 6R- GGGTAGGTAGGTGGGAAGGA cDNA 2F- tgctgaggggaaggatattg 669 2R- agcagcactggtcctcgt SIAT4B cDNA 1F- tgccagaataggcaggctac 690 (7 EXs) 1R- acatgaaatggtgggtggtt NM_006927 cDNA 2F- gacgggcacaacttcatcat 642 2R- ggctgcagcatgattggt LOC91862 = genomic 1F- cacctgcccaagaaacttgt 394 MARVELD3 1aR- TTTTCCCAAACTCCGTGTTC (3 EXs)

NM_052858 genomic 1bF- GGGAGAGGGAGAGAGAGAGG 374 (with 1R) genomic 2F- tcctgaacgctattgaatgaga 249 2R- agaccatcccttccagaacc genomic 3aF- gaagccttcaggggtctgag 480 3aR- GAAGTACAAGGCCGGGATGT

genomic 3bF- GTTGCCATGGGTGTCCTG 400 3bR- TGGCTCAGACTACCTTCCAC TAT genomic 2F- cgctatgttatcaacatcacagg 600 (12 EXs) 3R- agcactaaagatttggcagtca NM_000353 genomic 4F- tgtcactgcactccaagtcc 250 4R- ccctctgacacccgtacttc genomic 5F- acaattgctcccaactcacc 697 6R- accccaggcttggaacttag genomic 7F- cgggatagggaaattacatga 247 7R- tttgctgtaatgaaacgattagga genomic 8F- aatcccagtcatgcttcagg 365 8R- ccccactgtgctcatgaaat genomic 9F- cagaacaagaaactctaagacagaga 250 9aR- attctctgactcccaaactcc genomic 10F- ttcaatgagggggctattttt 250 10R- actgcctgtggcaattctg genomic 11F- acaggaaattggcctgttct 850 12R- ACCCTTGACATGGTGCATTT PSMD7 cDNA 1F- ctgacgaaccggaagaagag 699 (7 EXs) 1R- atggacctggtttgtgatcc NM_002811 genomic 6F- tgagctgacttaaagcaaaaga 248 6R- ccttacactgtgtgacctctcg genomic 7F- acttgcctcacccaacaaag 628 7R- AACCCTCTAGCAGCACACTGA LOC348174 genomic 2F- CAGCCTGACTCCTGGAGATT 985 (13 EXs) 3R- accctggtcaagtggaagg NM_182619 genomic 4F- gcaggggagttgaatctttg 397 4R- aactgtagcctcttctgagatgg genomic 5F- ggtaagggagccctctgttc 298 5R- ggactctactttcgggtgga genomic 6F- tcccagggaatctccctatc 669 7R- tatgcagtgggcagactcag genomic 8F- gggaggggtattcttgctgt 827 9R- acccctgctttatgctgatg genomic 10F- cacacgtggcatcttaggc 700 11R- aaggatcagaccctcaaagg genomic 11aF- agctgctcaccccctttc 286 11aR- gcaggcccaagtgagaag genomic 12F- gatgcagggtcctgatgg 819 13R- GGTCTTAACCTGGCCCTTGT GLG1 genomic 1F- cgtctccaccccctcattta 615 (27 EXs) 1R- agcacagaggaagcaaggaa

NM_012201 cDNA 2F- acagcaacagcagcctcag 832 2R- gcgggttgtaagtgcttctc cDNA 3F- gggagcgtttttgtgaaaat 692

3R- tgcagctctaagagacggtgt cDNA 4F- ccagacagcctgcaaacata 797 4R- caccacgtccaccttctttt cDNA 5F- agctggtgcagatgaaggat 696 5R- cagcttcaggcaagagatga genomic 20F- tgggtgacacggtgagact 300 20R- aggtcactctacagccactgaa genomic 21F- gctccctttaggaaggtgga 677 22R- ccagtggaaacttctctgagc genomic 23aF- agagcttggctctgtttcaa 297 23aR- ccaaggtaggcagactacagc genomic 23bF- tttctgtgctcagtggggta 242 23bR(=23aR) ccaaggtaggcagactacagc genomic 24F- caacctgtcccaaagtggtc 397 24R- tccccagggaatgttgtcta genomic 25F- tgcaccctcttcctcatctt 249 25R- ttggtgaagtgttccatattttt genomic 26F- ccctccctctccaaaagaaa 378 26R- GAGAAGAGCGAGGTGAGTGG ZNRF1 genomic 1F- TTGACTCCCTCCCCCTTTAT 623 (5 EXs) 1R- tcgtgcactacattcccaaa NM_032268 genomic 2F- tgtcattcgagagtgtccacat 249 2R- gaggctcccacaaggactg genomic 3F- gcagctaagcagtcccctagt 286 3R- aaggagacagaatggctgga genomic 4F- aggaatctggccctctgaa 241 4R- actgcaggcccccaaagt ZFP1 genomic 1F- atagcggaggcattgacaag 582 (4 EXs) 1R- GGCCTTATGGGAGAAGGTTT NM_153688 genomic 2F- CAGCCATAAAGCAGCCATTT 823 2R- gAATTCCCATGTTCCCTCAA FLJ34389 cDNA 1F- gggaaagaaggtggaagagc 590 (11 EXs) 1R- ttcatgttgatttctaatcgtctca NM_152649 cDNA 2F- caggaagatcagcaggatgc 489 2R- cctatccaacagctccctca cDNA 4F- gacttccatgagtttgggaac 396 4R- ctctcccagcttcttgtcca (~3R) genomic 5F- agaggtgggctgcagacat 297 5R- gtctggcctgttctctcacc genomic 6F- gcctcatcagcccagttatg 240 6R- gagacactcgtgcccctct genomic 7F- tccctgccctttactcctct 296 7R- gtggtccttggaggagtttg genomic 9F- agccatcagcatccagagag 294 9R- cttggagctggtgaaaagga TERF2IP genomic 1F- CGGTGACAGCTCAGTCAGTT 849

(3 EXs) 1R- gctaccgaagatgccaagat NM_018975 genomic 2F- ccagagctcacacagcaaat 300 2R- gagccccaggtagatgaaaa genomic 3F- tggtgattaggagctggtttt 250 3R- AGGCCTGTGTAACTGTTGATAGA FA2H genomic 1F- ggatGTTAGAGGCGCTCAGG 400 (7 EXs) 1R- gggagtggaaggctgacg NM_024306 genomic 2F- ctctgccctcttccctcac 232 2R- cagctttggctccacacac genomic 3F- gggatgctgtaacgaggaaa 286 3R- atatctgattggcaccgtca genomic 4F- actctgtcaagctcctgtgc 229 4R- cagggaaagcactgaggtct genomic 5F- gctaccaggctgctatgcac 372 5R- ccccgtgagtcacatcaaac genomic 6F- agggagtggccagggttt 395 6R- tcgatggtagttggcaaataaa genomic 7F- ctaccctggcacccttcc 249 7R- CCAACCTTCTTAATGGGGTCT LDHD genomic 1F- atgggctgacagcagcagta 241 (11 EXs) 1R- ccaggaaacttgggatcaga NM_153486 genomic 2F- cacctggcaaggatgttgt 668 3R- acaggtgtgccaaggtgtg genomic 4F- acgtggccaggtcagagac 591 5R- ccaggaggtgcttttcacag genomic 6F- gtggagcagaaagcactgg 850 8R- gttgaggaaagggtttgtgg genomic 8F- gtatgctggggtggagtgg 300 8aR- tcagcctgctgctctgtg genomic 9F- agggctgaggcctagagaag 792 11R- CCGTGGCATGAAGAAAAGTT LDHD genomic 1F- aggcacacagggctataacg 214 (11 EXs) 1R- acagctgaactgggagctg NM_153486 genomic 2F- gccagtctaggtcccagaga 700 4R- ccgttaaggagcagtccatc genomic 5F- caccagggaaggagtctgtc 378 5R- ttcaataacgaacgggcagt genomic 6F- ccaacatcgtggtgatggt 348 6R- caggagggtgtggggttagt genomic 7F- ccctagactccaccaggtca 324 7R- agacacaggcagtgcagtca BCAR1 genomic 1F- gccagggcagaaggagat 409 (7 EXs) 1R- ggcttatttgcatggagagc NM_014567 genomic 2F- gtatcaaagctgggcacctc 842 2R- caaacacacagccactcctg

genomic 3F- cctgcccacctcctaaaag 685 4R- ctggagccagtctcttcctg genomic 5aF- acgtgtgtgtgtgtgtgtgg 672 5aR- CTGTGGCTCAGAGGGGCTAC genomic 5bF- GTTGCCCACCTTCTGGAC 661 5bR- caaatctccccctaagcaca genomic 6F- ggtggccactcagctcac 248 6R- aagagctgggggctcagg genomic 7F- caggtctcggcctgtagttc 681 7R- GTCCTGTCCCTAAGCCTGTG GABARAPL genomic g1F- CCGTCGTTGTTGTTGTGCT=1F 559 2 2R- cctatcacgggtcccagac (4 EXs) NM_007285 genomic 3F- agccatgtgaggccagag 300 3R- gagttccgtaagcagccaac genomic 4F- caggccatcctttttctcct 223 4R- AAAATGGCTGGCTATTTACAAGA ADAT1 genomic 3F- tgagaggctgtagcatcgtg 687 (11 EXs) 4R- ggagagtaaaacatggacactgg NM_012091 genomic 5F- gtggtaccttctggcaaagc 241 5R- gactcccaccccagtcct genomic 6F- agaggctttccccttaccac 300 6R- ggctacttcagccattagtgc genomic 7F- aaaacacactgctgggctct 835 7R- cctcagactgcctcttggaa genomic 8F- aggcatgcacctaaactgaa 846 9R- gttcacagccaccatctcct genomic 10F- ctggcaccttcaaacctaaa 250 10R- ccagaatcaaaacagttgtctacc genomic 11F- ctgtccagttgcttgctcag 347 11R- CAACAGAGATGCAAAGCTCA KARS genomic 1F- cgcaccttgacctggaaat 249 (14 EXs) 1R- atgtgcgtacccacgagag NM_005548 genomic g2F- catgtttttgataagttgctaggg 300 g2R- aagcctcatcagaagctttcc genomic 3F- ggtctgaccaggttcaacttc 297 3R- ccagaccttcccttgtgcta genomic 4F- cactgataccataaaatgacttctgg 273 4R- caaccatgtcccactccttc genomic 5F- ttgagtgagggttccagagg 575 6R- aaaaggaaggcaagggagag genomic 7F- catggtctgtccccaagg 250 7R- cccatctagccagtggtattc genomic 8F- ggcaacaagagcaaaactcc 821 10R- gaagagggaaccattcagca genomic 11F- catctggatgggagctgaga 247 11R- tgacaataaattgttaacaccactaa

genomic 12F- aaaaacaaaatttagctcattaggg 248 12R- aggctggtatttcctggtga genomic 13F- tgtcaggagcatctctggaa 400 13R- agtggcatggaactcctctg genomic 14F- aatctctggattttggtggtaa 300 14R- TGGAAAGCAGCACACAAGAA CNTNAP4 genomic 1F- gatctgatgggttgctcagg 162 (23 EXs) 1R- cgtttccatgctgctgataa NM_033401 genomic 2F- tgatgttgttgttttacttgttgg 280 2R- tgactgtatgcaaagataatgaaaaa genomic 3F- aagttccatagaggaatacagtcg 374 3R- aagagattggtaaacaagatgcag genomic 4F- aaaatgcagatcacacattgct 399 4R- tgtacaggcatgcacaaaaa genomic 5F- agtacgtatactgccttttctcaata 400 5R- gcactctgactgctaaaagataagc genomic 6F- tgaaagatggtggtccacag 397 6R- gggaaatgcttcacctgttc genomic 7F- tgaaaaattgatctgtgtgtgatg 395 7R- tgcatattgccaatcaaagaa genomic 8F- cgcggataatgtgagattga 432 8R- gcataaaatttggccacaga genomic 9F- tgagtgatgtgtatattgctgtttc 300 9R- aagacacgggggcactaat genomic 10F- tccctcctttaatacagaaaataatca 393 10R- gaatggggaagaggaaagaa genomic 11F- tgtctttctcatgaccaagtataaat 299 11R- tgctgagtttgcacagaaca genomic 12F- aaatttcaagatttgcggtatttc 300 12R- cgtcaacattctgcttatttca genomic 13F- tgactcagataaaaggtgattttga 393 13R- gacaccaccactttgcagac genomic 14F- tccaatcaatctttctccttttc 384 14R- TTGCACTAACTTGTTCAGTATCTGG genomic 15F- cattttaaatagcaaaagtgctaaaaa 398 15R- ggcgcaatgaaaagaatcag genomic 16F- gcacttcgccataggaactc 397 16R- tccatggtgatttgttattattgc genomic 17F- ggcctcagttttgtttggtc 396 17R- tgcatagaaaagaactgccaga genomic 18aF- tttgattctattttggggttgg 445 18aR- tgagaagccagaacacgaga genomic 19F- ttttaagtaagccgctctcaaag 282 19R- tgttcattatcaaatttctgcttga genomic 20F- tgcttcctgtcttctcaacaaa 248 20R- tgacgaagtgggtgtgtgtaa genomic 21F- tgcattagctcagaatcctcaa 394 21R- aaggtagcctgaaagccaca genomic 22aF- tcattgccattgacattgct 335

22R- gcctggcacttggagtatgt genomic 23bF- gacttattaagcaatgaagtcagca 377 23bR- AAGCCCACTGCCTGTACATT CHST5 genomic 1F- atgggtagggtagccgaatc 593 (3 EXS) 1R- CGTGCACAGTGTCTTGCATA NM_024533 genomic 2F- CTGTCCGCCTTTTTCAACTG 581 2R- ACCCGTGGGTGATGTTGT genomic 3F- AGATCCGCGCACTCTACG 395 3R- CCCTCTCCACCATGCAGT FLJ26184 genomic 2F- tgaaaagcaatgggagaatca 272 (4 EXs) 2R- gtgtccttgtctggctggtt NM_207385 genomic 3F- tgtgctcatggtattttcatatgtt 300 3R- acaggggtgagccaacac genomic 4F- gaccaggcattgtccaactt 499 4R- CCGATAGGAATGTGGAGGAA PMFBP1 genomic 2F- gcatagatccagctgggaaa 237 (21 EXs) 2R- tggaattttgaatcataaaacatga NM_031293 genomic 3F- caagtctctgtcaacctgttaagtg 299 3R- aagggagcagaggcagaagt genomic 4F- tggatgtgaatgactgaacaaa 426 4R- ctgctttctagtgggcttgg genomic 5F- ttacaaccgcatcctttcct 395 5R- gggtaaaggtttgggtttcc genomic 6F- ccatgttcatggaattctgaagta 299 6R- tccccatgtcttgattgagg genomic 7F- agggaatgcctgatgaattg 374 7R- ccaaccaagctcacacagat genomic 8F- gtggatgctgggcattttag 543 9R- gcaataatggtgggtgaagg genomic 10F- ggcttgagacaacctctcatt 400 10R- ggagagagaaggtggctgct genomic 11F- gggcacaaaatgaaaggaaa 394 11R- atgggaaatccttgctctcc genomic 12F- attgggtcagctccacaatc 292 12aR- agccactgctcctagtctgtg genomic 13F- tgtctgtatttttcccccaaa 696 14R- gggtcacagggttcaaattc genomic 15F- aggcacagagaatgcctcta 295 15R- ctcccgctaccttgacgtt genomic 16F- accctcctccattcaggact 782 17R- ttgggtggtcataggatgtg genomic 18F- cctgtgctcccacctctg 842 19R- ccctgctgacccttcttgt genomic 20F- tagcagggaaccaaccagag 400 20R- gccacaaaaggcctattttctt genomic 21F- catgttaccccagaggcaag 235 21R- GCTGATCCAGGTCAAGAGAGA

HPR genomic 1F- ggagaagggggagaagtgag 201 (5 EXs) 1R- ccatttgccctgtttctttg NM_020995 genomic 2F- accagctttccgctcctt 599 3R- accacccatcatggaaatgt genomic 4F- ttcttcttctttttaattcttctcctt 249 4R- ggaaggctgtgcctctagga genomic 5aF- ttcacccctttctcagatgg 466 5aR- TAACCCACACGCCCTACTTC genomic 5bF- TTGGGCTCATCAAACTCAAA 597 5bR- ACCCATCAGCTTCAAACCAC 5cF- CAGAAGGTGCTTGTTAATGAGAGA 495 5cR- GCTTTCAGGCAAGGGCTTC DHODH genomic 1F- ctaaggggcggagacaagag 248 (9 EXs) 1R- aggagaccccggaaacac NM_001361 genomic 2F- ccctctgtgtacctgggtgt 400 2R- cttggggttctggttttcaa genomic 3F- ccatgctttgagaaataccg 399 3R- gggttcctgaacattccaaa genomic 4F- tttttctcttgtttgaaaaagtcc 293 4R- ggtcctcagtggtttttgct genomic 5F- ctgtccacaggtggtttggt 383 5R- aggacagcttctgagggtga genomic 6F- cgctattgtggaaacccact 299 6R- agaacgccattctgggcta genomic 7F- ccttcgacctccagagaagc 599 8R- tctaaatgaacctctgcctaacttc genomic 9F- aacgtgggcttcctgtaaga 213 9R- GTCCCTCCTCTCATGATCCA CYB5-M genomic 1F- ggctctcaaggaaagtagtcg 300 (5 EXs) 1R- cccacatacactccacacagc NM_030579 genomic 2F- ggaaagtaacttgttggtttgctt 250 2R- tgcagctgttaagtagttttctctg genomic 3F- ccagaatatagtctcatgttccaaa 250 3R- tttttgcttggggaaaaagt genomic 4F- ttacagggttgacatagcttga 243 4R- gaattagttgtcaaactgcactca genomic 5F- aaaagcagttaaatttcccaaa 284 5R- CCCCCAAGCAAGTCTCTAGTT KIAA0174 genomic 2F- cccctaagaggtgttagttcctg 248 (10 EXs) 2R- ctggcctgaaagacccatta NM_014761 genomic 3F- cctgtaggccagatgcaagt 373 3R- acaggcctttcagcaactgt genomic 3aF- cacccagttcttcctgttgg 386 3aR- gttaggcagcagatccttgg genomic 4aF- tcacgaaaatggctagaagc 392 4aR- ggctgaaagggagaaacaaa

genomic 5F- ccaggaagaagcttaaagcag 850 6R- tttttgttttccaaagaaggtca genomic 6F- ttcgcatagtgaggtgagga (with 6R) 248 genomic 7F- tccatctcagactccgcttt 387 7R- gctaatggctcctgacaacc genomic 8F- ccttcccatacccaaacctt 229 8R- attttgctcgctcttcatgg genomic 9F- gctctgttgcatttggtgag 250 9R- ggaaacagtctctaagggaggaa genomic 10F- aaggttttctcctgtgttttga 358 10R- AAGGAGAAATTGCTCAGTCTCA EXOSC6 = genomic 1F- aacggaagaaccagtggatg 605 MTR3 1R- CCACCAGGTCGTACATCTCC (1 EX) NM_058219 genomic 2F- GCCTGCTCTGCGACTTCC 700 2R- CAGACCAAGTCCCACCATCT LOC92154 genomic 1F- cctccttccctccctcct 542 (15 EXs) 1R- cgctcagacaaggacacaca NM_138383 genomic 2F- cccaagtctggatggagcat 400 3R- tcccctactcttcctttcca genomic 4F- gaagagctggtggagatgga 381 4R- GATGAGGCTCTCCAGCAGTG genomic 5F- GCAGTTCACCAAgtgagtgg 486 6aR- aacaggttggagggtcctgt genomic 7F- tggtccagaacctccctatg 240 7R- tggagaggttccttccaatg genomic 8F- agccccagagtcctcactg 396 8R- gaagtgacccttcttggaggt genomic 9F- gaggaggggtgagagaggag 300 9R- cccctttactgttcccaacc genomic 10aF- cttctcctgggatgtggttc 278 10aR- caattttgctgggaacatcc genomic 11F- caccctcgtttgtcaacctc 396 11R- cagctatagtagtcccgcatgt genomic 12F- ccaaaactgttctctcctctcc 247 12R- caggccctttgtgcagtaat genomic 13F- tgtgattgcgaaccagagac 682 14R- cctcctggatggctaagtcc genomic 15F- acagctgggacttagccatc 497 15R- GAAGACGCACTCCTCACTGC genomic 15bF- CCATCGTCCCTGTGAAGAC 565 15bR- ctgggcctttgctctgagt FLJ21918 genomic 1F- CCCCGATACACGGTGTCC 651 (15 EXs) 2R- tcaaacaagagcccagtcct NM_024939 genomic 3F- cctccttcctcctaccctca 493 4R- ccacagcatgcacatactca genomic 5F- tgtctatggaggggagttgg 247

5R- gggaggagcaataggcattt genomic 6aF-cagaacttggtggggacac 392 7R- GAGTGCTACACCACCCctgt genomic 8F- GGCAGTCATCAGACCAGGAC 497 9aR- catgctcctccagacactca genomic 10F- atgggtgagtgtctggagga 817 11R- TCATCTGAATGAAGGCATCG genomic 12F- ggggtgtgtgttaggggtaa 673 13R- aaaggggatcgtgacctttc genomic 14F- agggaaacaggtctcggtct 469 15R- GCTGGTCTTCTGGACTCAGG CDH3 genomic 1F- CAAGAGCTGAGCGGAACAC 495 (16 EXs) 2R- ggtccacaccaaaatggtca NM_001793 genomic 3F- tcagaggactcttgtcagtcttg 247 3R- gctcctggccagcaattt genomic 4F- ccagccacccttttaactctt 300 4R- atgcaggcctttggatgg genomic 5F- gcccctcttcacagaggact 577 6R- acacagccaaggaaatcagg genomic 7F- gtagacagggctggagttgg 353 7R- acgtgggtcctcactgttct genomic 8F- gatcctcctctccatccaca 293 8R- aggcaaactttggtccttca genomic 9F- gttggatggaggcttctcag 360 9R- ggactagcccacggtagaca genomic 10F- agggcagcactgttgctagt 450 10R- gaacaaacgttggccatgat genomic 11F- ggtatgaggaggccctgaat 300 11R- ggaaacatgctgtgctgtgt genomic 12F- agagctgggcggtaaacag 389 12R- gtgtgcagaatcctggtgtg genomic 13F- tgtggaagccgtattctcaa 394 13R- tggtggcctcttagttccag genomic 14F- ctgcagttagaggggctctg 829 15R- atgcttgttctcctgtgtgg genomic 15F- gggtgtggggtgagatgtaa 300 (with 15R) genomic 16F- gagagaggggctcacagaga 399 16R- CTGACAAGCTCCGAAGTCCT PRMT7 cDNA 1F- CGCCACTACACTCGGATTTC 699 (19 EXs) 1R- CGTGGATGGGAAATAGCTTG NM_019023 cDNA 2F- tatgcacagctggtggagtc 650 2R- gtcctcagagcctggacgta cDNA 3F- atgagagagtccgccagatg 697 3R- ggctgctggaagtcaaaggt cDNA 4F- gtgcacatcatggacgacat 488 4R- agccctcaggccactttatt FLJ11126= cDNA 1F- gcagcgcatattttcacaag 649

DDX19L 1R- ccaatttattgcctcgaacg (12 EXs) NM_018332 cDNA 2F- aacatatgagctggcgcttc 663 2R- tggtggtcaccaaaaccttc cDNA 3F- ctgagtggggagatgatggt 451 3R- gttgtccttggggtgatgtc PKD1L3 genomic 1F- ataagacaagcccgccaata 490 (30 EXs) 1R- tggtttttctgtctacaaaagcag NM_181536

genomic 2F- ctgagtgggactgtctcaaaaa 297 2R- ccaatattctcacatgcactcc genomic 3F- cgtgcatgtaatgatggtttt 269 3R- cagtcttgtgcattcttttagca genomic 4F- tttgcattatttttagccatgaa 289 4R- tcttgcacgtaagtactttattttcc genomic 5F- tttcagagtatttggggaaatg 494 5R- ggctgtttccatggacttgt genomic 6F- caaggtcaacttggatgaaaa 299 6R- gcctcagattctctttttctgttt genomic 7F- aggcagaggttgcagtgagt 397 7R- gggagaggctgtgatacctg genomic 8F- ccttccaatcttctccaaaaca 500 9R- cacccaaatgcctacatgg genomic 10bF- tgatatatctttctagtggggtcactt 392 10bR- gcccagccctttaaaacac genomic 11aF- ggattttgaaagacatcaatgg 381 11aR- gaggctgcagtcaacaatga genomic 12F- tgactctagtttcatcatggtctca 399 12R- gctgccccaaatgagataac genomic 13F- tgtctgtcccctccttagca 379 13R- gctgcttttgacgaaggtgt genomic 14F- gttcatcctcctgctcctca 240 14R- ctcatgtggcagaagacacaa genomic 15F- gggataaacccaaagaattgtg 342 15R- tttggctcaaagctttactgtg genomic 16F- tctgagattacgtcttactacagacaa 300 16R- aaaaacaattcaatcgtgaacaa genomic 17F- gaggcttgggagttgctatg 395 17R- agaccagcctgggtaacaga genomic 18F- gagaggaatgactttgcgatt 351 18R- tctgtgcaacagagccagac genomic 19F- ttggtacaaatcctgggttaatg 296 19R- aagggagtgggaggcagat genomic 20F- gggaaaatatagcctcatcatcc 700 21R- gacacccagggacctcagtt genomic 22F- tgcccacctcttcctttaca 245 22R- agcaaggtggccagatctaa genomic 23F- ggaggtgctcacagtagatgc 285

23R- tcccttctggaaaaggtgct genomic 24F- ggaaaaatgaaaaagacatagagg 598 24R- cttattgaacagccttctttgc genomic 25F- ttggggttattcaaagacaaag 298 25R- ccaccacacctgggtaattt genomic 26F- gcgttcatcacacttgagca 298 26R- aaacaggaaactttagacatgagg genomic 27F- ttgacctcacaggactcagg 393 27R- atttctatgggaagcgatgc genomic 28F- ccacaaggaatttctgttcca 296 28R- agggggctgttttcataagg genomic 29bF- tgataagatcccaaacactagca 300 29bR- ccgtcatcttaggggcagt 30aF- tttactccactattagcagtaatttca 278 30R- ttttaaagctatgccatgcaa HYDIN genomic 2F- agcatttggatttcggaaga 359 (20 EXs) 2R- tgccaagtagcaactgtcattc NM_017558 genomic 3F- cctgaaagctgcaaacacaa 299 3R- cttattgttctggaattttggtg genomic 4F- tcttccccagtcatttttgtc 266 4R- tcaaagatccctcaagatctca genomic 5F- ttgatggatttcagtctgtgttt 294 5R- cagcactaccgcctctgtaa genomic 6F- gcaaaatgcctctcatcgac 373 6R- ccccaatgttatggtagcaa genomic 7F- ttcctccccgtctcacacta 281 7R- gcatcctcgtagcttggttc genomic 8F- tgcctaacttcagcttgaaaaa 400 8R- tggttgagttggctaaaaatg genomic 9F- gctgaggacatttccaggag 363 9R- cccaagtgagcgctcttatc genomic 10F- tgactgcagtaatcatttcactataca 343 10R- cacactcaggcaccacactt genomic 11F- gggaacatgatttggggtaa 297 11R- aacggcgaataaatgtgattg genomic 12F- gcaactttccctttgttgct 388 12R- gctaaaaaggcaataggttaaaacttc genomic 13F- tttttgtacttatccttgcccta 244 13R- acgggggttctttgtaaacc genomic 14F- aggggagattgagggtcaag 370 14R- gggcccatgactattttgaa genomic 15F- ctcccctcccccgtgtag 285 15R- caggaaggcaggacaaactc genomic 16F- gcgttttctgtatgtaaacaagc 250 16R- aagggaactcaatggaagaataa genomic 17F- ggggtgctcatccctattta 391 17R- gtgggcctcaaatgagaaag genomic 18F- gcccttgctttttattgaagg 300 18R- ttgcaaaaatagggccattg

genomic 19F- tttgaagtgtagagggctttga 399 19R- ttttctaaagtagaggtgatgctcag genomic 20F- cccaagggtgtagagctgaa 500 20R- AGCAACGGATGCTACTATGG genomic 20aR- TGTACACAATCCTTGAGAAACCA 469 (with 20F) FLJ10305 genomic g1F- GCTAAGGAGTCGCGAGGTT 231 (19 EXs) g1R- aagtaaccctggctgggaag NM_018052 genomic g2F- tcatctcccttcctcattgc 371 g2R- ggtgcggacagagtggtaat genomic g3F- aggcttgggaagggtcttt 386 g3R- ccaaggttccagaaggtcaa genomic g4F- tgaccctcgcctctgtgt 250 g4R- gaactgcacaccagggagta genomic 5F- caagtgattcagcgaggtca 219 5R- caaactggaggctggagaaa genomic 6F- aaacagggttctggagctga 236 6R- ctctgtgggcatgaagcac genomic 7F- gaaatggggctggtttgag 249 7R- cttctgcagaggtggcagt genomic 8F- catcccacgtgtatggtcaa 287 8R- gtgcagggtccctcaactt genomic 9F- tgggaatcacatttcagcaa 299 9R- ggcaactcctatgacatctgg genomic 10F- gtgcttgtcacaggggtctt 244 10R- gcgagtagaatttgccttgg genomic 11F- actccatgggagagggctat 686 12R- ctggccccaaagtgagtatt genomic 13F- caggtcagcacagctctcag 287 13R- gccatggtccagcctaagac genomic 14F- accctgtggacacataatcag 250 14R- tggcgtgaaagtgaaaagc genomic 15F- tcccaggctgagacacaga 291 15R- ccctggcatgcaggagac genomic 16F- gacttagacctgtggcccttc 297 16R- cgcctgttgggtggaaag genomic 17F- acaggaagccaggtgcag 244 17R- gacttcacccaccccactc genomic 18F- gtcggaggcaggcagtgt 300 18R- ttcctcaccaggctcctct genomic 19F- cactgccctccagcagtaac 295 19R- GCGTGACGACCCTTAGTGTT AARS cDNA 1F- GGACTCTGAGGGAGGAGCTG 574 (21 EXs) 1R- ATGTTGCCTGGGAGGATTTT NM_001605 cDNA 2F- tacttactttggcggggatg 798 2R- ccaggctctgaattttcctg cDNA 3F- aggacccagacatggtgaag 786 3R- tgagcctttctggtcagctt

cDNA 4F- gacgaagacccatcatgagc 671 4R- ttgagagtctcccgcaattc cDNA 5F- atcgctgaccttggagagg 600 5R- tcaaatgttcttgtagcagatga MGC34761 Genomic 1aF- GTGCACGGAGTGCCTGAC 223 (10 EXs) 1aR- cctagcacccactcctcaga NM_173619 Genomic 2F- ggcagggacacatgcttatt 287 2R- accctggtcaagtggaagg Genomic 3F- gcaggggagttgaatctttg 384 3R- tctgagatggaccagtggaa Genomic 4F- tgatagcgaaagctgtgtcc 297 4R- accaagggtgcagagcaa Genomic 5aF- tcccagggaatctccctatc 336 5R- gcccagtaggatgctaccac Genomic 6F- gggtggtagcatcctactgg 297 6R- tcaggaagtgtagcagcccta Genomic 7aF- tcttgctgtggttggcatt 299 7R- ttggaaagagaggcacacag Genomic 8F- caggtggccagaggaagg 298 8aR- AGGAACCACCTCTCACTCCA HP Genomic 1F- ccagggccaaagtttgtaga 236 (7 EXs) 1R- catttgcccgtttctttgtt NM_005143 Genomic 2bF- ctgtgaagcagggagactagc 2R- 187 agacccgagagggtcagagt Genomic 3F- cggttcactgggaacaattt 227 3aR- cacccatcatggaaatgtca Genomic 4F- gccttctcactctgctctgg 221 4R actggaaggctgtgcctcta Genomic 5F- cgctcatctgacttttcacg 248 5R- caccacccatcatggaaat Genomic 6F- tgtccccttttcctcttcct 247 6R- ggctgtgcctctaggacgtt Genomic 7aF- atgctttcacccctttctca 600 7aR- TCTTCTTTTCGGGGACTGTG Genomic 7bF- TGAAGTATGTCATGCTGCCTGT 392 7bR- CCCTCTTCCAGGCTGAAATC Genomic 7cF- CTGACCAAGACCAATGCATAA 361 7cR- TTCCAGGCTGAAATCTTGCT SMPD3 Genomic 3aF- tatctgacccactcccctga 574 (9 EXs) 3aR- CTTGTCTTCCAGCCGTGAAT NM_018667 Genomic 3bF- TTCTCGGCTTTCTCTTCTGG 619 3bR- CTGATTATGGTTGGGCGTCT Genomic 3cF- AAGCTGACGACCCTGTGC 672 3cR- actccaacctcccactccag Genomic 4F- ctactcggaccccagaatcc 584 5R- ggagcaggaattctttgagc Genomic 6F- ccgtgtgggacagcaaag 491

7R- ccagtccctgtctccacct Genomic 8F- cgcaggggagagatcttaaa 775 9R- CACTCGATGGAGGGGACAT FLJ13291 Genomic 1F- gcgggtcctaaagcagaact 750 (5 EXs) 2R- atgaaactgtgctgcgactg NM_032178 Genomic 3F- gtagggagccttccttcacc 400 3R- aatgttgttcgggagtggag Genomic 4F- caacttcccccagagcataa 247 4R- tatgcctgcacctcagtctc Genomic 5F- tgcattacaatcgggaacaa 293 5R- CTGGACTCAGAGCCCTCAAG FLJ20399 cDNA 1F- cagctgcgagatatcaaacaa 599 (17 EXs) 1R- tcttctagcgatggcaggat NM_017803 cDNA 2F- ctgccctgctgtcagacc 599 2R- cagctcctgtgtggtctcct cDNA 3F- ctgcttgtgccagatgctac 691 3R- atccacagccaccttagcac PSMB10 Genomic 1F- aggaagggtaaaggcgaaag 588 (8 EXs) 2R- AGAATGACCCCGTCctgag NM_002801. 2 Genomic 3F- ggaggggagccgagagtat 486 4R- cttctttctgtcccgccttc Genomic 5F- cacctaggagctgggcata 494 6R- ctgcccttactccactcacc Genomic 7F- ggcaagaccagattgggtaa 578 8R- caccccattccactcaagaa CTRL Genomic 1F- cctcaggcctggagcttta 249 (7 EXs) 1R- ttcttttgcccactctgtcc NM_001907 Genomic 2F- catgtaccctgttggggaag 695 4R- tgtcctgaattcctcgttcc Genomic 5F- cagacacggaggaaaagtgg 576 6R- ttggggaggtataccacagg Genomic 7F- CTCCATGATCTGTGCAGGTG 394 7R- AGCCTCTTCTTTCTCCTGAGC NFATC3 Genomic 1F- ccgacagtggaggcttagg 400 (11 EXs) 1R- atcctgcgagcgagaagc NM_004555 Genomic 2aF- tgacatacattttatatcccacttttt 591 2aR- TGTGAAAGCGATTCACAAGAA Genomic 2bF- TGCCAGCAGCATCTCTTCTA 799 2bR- gccaccaatgacccataaac Genomic 3F- aaatgttggtgctggcaaag 384 3R- agcccccaaaacaacagag Genomic 4aF- tggcatattgtaaaatgcatgg 442 4aR- tgaattgaattttccttctgga Genomic 5F- ttttgacctaattttgggtagttttt 390

5R- tgcctcagtttgcacttcag Genomic 6F- tcccaaaatttgcttctcttt 300 6R- tttgccccttagtttgaagtc Genomic 7F- tgatactatccatgcattagattttg 218 7R- catccataccataaagcagca Genomic 8F- tgctggagccttgtagttaaga 356 8R- gggcatctgttggtgttttc Genomic 9aF- gagtttgcccctcagcttac 599 9aR- TCCTGTTGAGCCTGAATTTG Genomic 9bF- TTCAGCAGGATGCAACTCTTT 674 9bR- ttgggagaataaagtccctgaa Genomic 10F- aaacaggctgggcactgtag 247 10R- ccactgcatgcaaataccag PSKH1 Genomic 2aF- gcctggctgtgctgactt 598 (3 EXs) 2aR- GTCTCGAACACCTCCACCAG NM_006742 Genomic 2bF- CCGTATGCCATCAAGATGATT 671 2bR- tctgactgagagctgcagga Genomic 3F- gctgagtagtggcctcatcc 480 3R- TATCTCCAGAGAGGGCATCG UNQ2446 Genomic 1F- cagctcccatccaatcacc 294 (3 EXs) 1R- tcctggagcccttacaactc NM_198443 Genomic 2F- ccagagtgctcccaggtaaa 684 3R- CAGAGCAGGGCTGGAGGTA RCD-8 Genomic 1F- gaagtggaggcggttggt 382 (29 EXs) 1R- cgggaggatccagagctagt NM_014329 Genomic 2F- gaaactctgaaaggggagagg 300 2R- ccatctcatgccttcagcta Genomic 3F- accttccccagtctggctat 628 4R- gggacatccagcagaacatt Genomic 5F- gcttctctgctactgcctga 841 7R- agaaagctgcacaggtgagg Genomic 8F- cattgccatcctcacttgg 600 10R- aggaagctacattcccaacc Genomic 11F- cagggttgggaatgtagctt 576 12R- CATGGGTACCATctgaaagga Genomic 13F- cagggtcagagctatgtgtcc 684 15R- ataagacctgtggcctgtgg Genomic 16F- CCTCCTTGCAGCAGgtactc 490 17aR- tccccacacttcacacacac Genomic 18F- tgccctgtctctcattactgc 600 18R- gccatggatgaatctatctgaa Genomic 19F- caggaggggcttcagataga 598 21R- acacaccagaccagggaatg Genomic 22F- cattccctggtctggtgtgt 588 23R- gtggagccatgagaagaagc Genomic 24F- ggggtacctgtcaagcttct 500 25R- aagaggaacagtaggaaagagga

Genomic 26F- tgggaggggttatcctcttt 598 27R- gagacacgtgacccttagcag Genomic 28F- gccacacagttcagacaagc 839 29R- GCCCAGCTAAACCTTCTGAC NUTF2 Genomic 2F- TGGCTGGTCACCTACTCTCA 245 (5 EXs) 2R- CCCAACATCCAAATTGATCC NM_005796 Genomic 3F- CAGAGTCTTTCCAGGGCCTTA 398 4R- AGCAGGGGTACAAGACTCTCC Genomic 5F- CCACTGAATTCCCTTCCTGT 249 5R- CATCTGGAGGAGTGGGAATAG THAP11 Genomic 1aF- GGCAGGAAGCGTATTCTGG 680 (1 EX) 1aR- TGCTGTCTACAGTGGCCTGA NM_020457 Genomic 1bF- GCAGCCGAACCTGGTATCT 600 1bR- CCCAGGAGTATAGAGGAGTTCAA FLJ13111 Genomic 1F- GGAGCATCGCGAGAGTTACA 294 (12 EXs) 1R- TTTCCTCTTCGAACAGAGTCG NM_025082 Genomic 2F- AGACCAAGACCCCTGATGAA 682 4R- CCAAGAAACTCCCGGACTAA Genomic 5F- CCCACCTTGGATTGTGCTAT 469 6R- GTCTCTCCTCCACCCACTCC Genomic 7F- GGCAGATGGAGGGATCTGT 299 7R- AACCCTGACTCAGCATCACC Genomic 8F- GGCCTGGAATACGCTGAGA 385 8R- CCCAACATGGACTCTGCTCT TSNAXIP1 Genomic 6F- GTAGGACGGGGTCTCACAGG 499 (13 EXs) 7R- GGTTCCCCAAAAGCTTGTCT NM_018430 Genomic 8F- AAGCTTTTGGGGAACCAACT 548 9R- AGGGACAAAGACCCCTCTCA Genomic 10F- GAGGGGTCTTTGTCCCTAGC 797 12R- CTGGGCTGAGCAGTGGAT Genomic 13F- GAGGGCACCTGGGAGTCT 397 13R- GGAGAATGGCATGGATTCAG MON1B Genomic 2aF- caagggggcctgactctataa 297 2aR- ccccgtccaacaatgaca Genomic 3F- gggcataggagacacttgga 3R- gaccatccacacacatggac Genomic 4aF- cagaagagccccactgtctc 533 4aR- ACCCAGTCGAGCAGCAAC Genomic 4bF- AGCCCAGGAGCGAAATGT 498 4bR- tgctgaacttcctggtttga Genomic 5F- tctagactgagggaggaaatgg 282 5R- agggaaaggaggcttgaagt Genomic 6F- actggggtaggcaggattg 393 6R- CCCATTGCTTAGGGAGACAA

VII.2.2. SNPs in the Candidate Genes for Otosclerosis in the OTSC4 Region on Chromosome 16q22.1-23.1

(SNP data taken from NCBI http://www.ncbi.nlm.nih.gov/SNP/; Revised: June 9, 2004) (Numbers of SNP rs and location are indicated only for SNPs found in database)

Gene SNP Amino Heterozy SNP rs Location Contig mRNA Nucleotide acid -gosity On contig

CDH1 1895c>a H632H Het NT_010498 NM_004360 2075t->c A692A Hom rs1801552 22471640 COG8 101a>g T14T Het rs11542583 22987613 NT_010498 NM_032382 WWP2 1632t->c L544L Hom NT_010498 NM_007014 2595c->t E846E Het AP1G1 2063c->a P688H Het rs904763 25387389 NT_010498 NM_001128 NQO1 98c->g T16T Hom NT_010498 NM_000903 122g->a E24E Het rs689453 23366572 566t->a I172I Het FLJ10079 1285t->c Y109H Het rs2549532 23775462 NT_010498 NM_017990 1573a->g I205V Het rs10852462 23778533 2041a->g T361A Het rs2432306 23784382 VPS4A 989a->g K287K Het rs1127231 22969162 NT_010498 NM_013245 ZNF23 1245g->c G144A Het NT_010498 NP_666016 COG4 637t->c L212L Het rs3762171 24160433 NT_010498 NM_015386 856g->a A285T Hom 1092c->t N363N Hom 1765g->a G588S Hom 2133g->a S710S Het rs11054 24129554 FLJ20511 469g->a V61V Hom NT_010498 NM_017853 FLJ11171 515c->t L60F Hom rs3096380 24933845 NT_010498 NM_018348 622g->a A95A Hom rs3826247 24933738 825a->t Y163F Hom 1584a->t N416S Hom rs3803704 24932776 2644a>g Q769A Het FLJ10520 696g->a T47T Hom rs4888262 28284657 NT_024797 NM_018124 1107t->a P184P Hom rs7188880 28279009 ZNF19 1098c->g H218Q Het rs8050871 25123995 NT_010498 NM_006961 FLJ12331 358g->a A68T Hom NT_010498 NM_024986 452c->a A99D Het rs32854 22682539 LOC348174 497g->a Q80Q Hom rs684036 23602459 NT_010498 NM_182619 529c->t T91I Het rs2549089 23602491 596a->g L113L Hom rs2549095 23602558 620c->t V121V Het 635g->a L126L Het 774g->a A173T Het rs2650549 23607104 843a->g V196I Het GLG1 1400a->g G458G Hom NT_024797 NM_012201 1424 g->a K466K Hom 1600c->t S525L Hom 1835a->g E603E Hom 2081c->t I685I Hom 3383c->t Y1119Y Het FA2H 949c->t P293P Hom rs2301865 28364604 NT_024797 NM_024306 CTRB1 682g->a V206I Het NT_024797 NM_001906

BCAR1 1595g->a, R491H Het NT_024797 NM_014567 1653t->c A510A Hom rs3169330 28883466 1917c->t H598H Het rs11545087 28883202 SLC12A4 2950a->c R961R Hom NT_010498 NM_005072 1948c->t L627L, Het 815g->a G193D Hom NT_010498 NM_012426 954a->t P239P Het 1142g->t W302L Hom 3224t->c I996I Het TAT 405g->a S103S Het NT_010498 NM_000353 LOC91862 815g->a K254K Hom NT_010498 NM_052858 HPR 420 g->t G130G Het NT_010498 NM_020995 ATBF1 793a->g E40E Hom NT_010498 NM_006885 887g->t A72S Het rs7193297 26608030 1013t->c A113A Hom 1096 a->g A141A Het 1937 g->c A422P Hom 2408a->g T579A Hom 2498t->c S608S Hom 2944a->g L757L Hom 2973t->g I767S Hom 3658c->g L995L Het 3712g->a Q1013Q Het 5119a->t A1482A Hom rs740178 26446334 9496a->g G2941G Hom rs699444 26441957 DHX38 1444c->t A436A Het rs1050363 25749213 NT_010498 NM_014003 283g->c L49L Het rs1050361 25744402 554a->g R140R Het rs1050362 25745014 1834g->a T566T Het rs2240243 25751760 2452c->a A772A Het rs2074626 25753383 DHODH 20a->c K7Q Het rs3213422 25656881 NT_010498 NM_001361 KIAA0174 236c->t H58H Het rs1049794 25564649 NT_010498 NM_014761 SMPD3 1812g->a P465P Het rs1868158 22013123 NT_010498 NM_018667

VII.2.3. Genotypic, Phenotypic and Familial Data of Connexin 26 and/or Connexin 30 Probands

Unique Ethnicity M Date of Date of GJB2 GJB2 Used 250 500 1.000 2.000 4.000 8000 FA in case Audio- Severity of /F birth audio- genotype genotype diagno Hz Hz Hz Hz Hz Hz or FA: metric HL/CI metry -stical other technique techni- patient nucleotide amino que S affected (PTA, ID level acid level relatives BERA, COR, VOR,

other + description)

35delG/ sibs, Profound, D6c Arab M 00.11.79 35delG fs/fs RE FA cousins CI 35delG/ Severe- D9c Arab F 00.00.95 10.9.97 35delG fs/fs RE 75dB 90dB 100dB 110dB S NB-FF profound 35delG/ Moderate D25c Ukraina M 00.1.98 18.9.98 35delG fs/fs RE 45dB 50dB 70dB 75dB 85dB FA aunt NB-FF -severe parents, sibs, uncles &aunts 35delG/ 105d (both Profound, D33 Morocco M 13.12.98 26.2.99 35delG fs/fs RE 90dB B Ø Ø Ø FA sides) PTA CI

35delG/ ∆30 (GJB6- fs/Cx30 RE, 105d Profound, D37c Ashkenazi M 00.2.93 7.11.93 D13S1830) del ASPCR 85dB B 115dB 120dB 120dB FA brother NB-FF CI

Ashkenazi 35delG/ Profound, D38c /Yemenite M 25.10.97 8.2.99 35delG fs/fs RE 95dB 95dB 100dB 100dB S PTA CI 167delT/ Profound, D40c Ashkenazi M 00.00.92 20.3.95 167delT fs/fs RE 75dB 90dB 115dB 120dB 115dB S PTA CI sister, parents, 35delG 105d many Profound, D45c Turkey M 00.5.93 17.1.97 /35delG fs/fs RE,Seq 90dB B 120dB Ø Ø FA relatives PTA CI 35delG/ parents, D48 Ashkenazi F adult 19.2.89 35delG fs/fs RE 95dB 95dB 100dB 95dB Ø Ø FA uncle PTA Profound 35delG/ uncle, Moderate D49 Ashkenazi M 00.00.58 19.2.89 167delT fs/fs RE 50dB 60dB 70dB 80dB 70dB 75dB FA cousins PTA -severe Iraq/ 35delG/ D51c Ashkenazi M 00.00.94 27.1.99 167delT fs/fs RE 80dB 95dB 105dB 115dB 110dB S PTA Profound T269C/ 18.11.9 IVS1+1 L90P/ RE, Mild- D54c Iraq M 31.8.95 8 (G->A) splice site Seq 25dB 30dB 45dB 70dB 65dB S PTA moderate 167delT/ D55 Ashkenazi F 00.3.50 17.6.99 167delT fs/fs RE 75dB 75dB 80dB 80dB 85dB 85dB S PTA Severe 167delT/ D56 Ashkenazi F adult 167delT fs/fs RE

35delG/ ∆30 (GJB6- fs/Cx30 RE, Profound, D59c Ashkenazi F 00.4.97 9.1.98 D13S1830) del ASPCR 70dB 80dB 90dB 105dB 105dB S NB-FF CI 167delT/ 100d D60c Ashkenazi M 6.12.94 27.1.99 167delT fs/fs RE 85dB B 105dB 95dB 100dB S PTA Profound 35delG/ sister, II Moderate D61c Ashkenazi M 00.3.93 17.2.99 167delT fs/fs RE 50dB 55dB 80dB 85dB 75dB FA cousin PTA -severe

167delT/ ∆30 (GJB6- fs/Cx30 RE, Severe- D62e Ashkenazi F 00.00.77 14.1.82 D13S1830) del ASPCR 85dB 90dB 90dB 100dB Ø FA sister PTA profound

∆30 (GJB6- D13S1830) /∆30 (GJB6- del/Cx30 D84 Ashkenazi F 31.8.71 D13S1830) del ASPCR Profound 35delG/ D86 Ashkenazi F 00.00.92 3.6.98 167delT fs/fs RE 60dB 55dB 60dB 60dB S PTA Moderate 167delT/ D90 Ashkenazi F 00.00.78 167delT fs/fs RE S Severe 35delG/ D91 Ashkenazi M 00.00.75 167delT fs/fs RE S Severe 51del12 insA/51 Severe- D96b Buchara M 00.00.71 17.7.96 del12insA fs/fs Seq 80dB 90dB 105dB 115dB Ø FA brother PTA profound

Egypt/ 35delG/ D101b Ashkenazi M 00.10.93 7.4.98 35delG fs/fs RE 80dB 95dB 115dB Ø Ø S PTA Profound 35delG/ RE, D103c Iraq/Egypt M 00.00.86 L90P fs/L90P Seq S Mild 28.12.9 35delG/ 100d D104 Ashkenazi M 00.00.20 8 167delT fs/fs RE 80dB B 100dB Ø Ø S PTA Profound Morocco/ 35delG/ RE, uncle, D122c Iraq F 00.12.95 28.6.00 L90P fs/L90P Seq 45dB 45db 60dB 55dB 50dB 60dB FA Iicousins PTA Moderate

ABR-Rt & Ashkenazi 167delT/∆ Lt - no /Ashkenaz 00.11.20 5.3.200 30 (GJB6- fs/Cx30 RE, response D125c i, syria M 00 1 D13S1830) del ASPCR S to 90dBHL Severe Ashkenazi , ABR-Rt & Morocco/ Lt - no Algeir,mo 00.3.200 35delG/ response D126 rocco M 1 35delG fs/fs RE FA cousins to130dBHL Profound

Morocco, Syria/ Syria, 35delG/ II D127 Morocco M child 35delG fs/fs RE FA cousins 233delC/ I205 Arab F 00.00.97 280InsA fs/fs Seq S Profound 167delT/ I212 Ashkenazi F Adult 167delG fs/fs RE FA sister Profound 35delG/ 110d I236 Ashkenazi F 9.3.90 9.2.95 167delT fs/fs RE 95dB B Ø Ø Ø FA brother PTA Profound 35delG/ S240 Ashkenazi M 18.3.91 167delT fs/fs RE S

35delG S244 Morocco F 00.00.75 35delG fs/fs RE 51del12 10.12.9 insA/51 Moderate T250 Buchara F 00.00.91 2 del12insA fs/fs Seq 50dB 70dB 85dB 85dB 90dB FA brother PTA -severe Yemenite/ Swedish (not 35delG/ T267 Jewish) F 2.10.91 35delG fs/fs RE FA cousin Profound 35delG/ T270 Yemenite M 8.10.64 30.3.90 35delG fs/fs RE 95dB 95dB 95d 95dB 100d 85dB FA cousin PTA Profound 167delT/ Moderate T274 Ashkenazi M 24.2.70 26.8.97 167delT fs/fs RE 55dB 65dB 85dB 95dB 90dB 85dB FA brother PTA -severe 35delG/ I285 Arab F 00.00.77 35delG fs/fs RE FA sister Profound 167delT/ Moderate T287 Ashkenazi M 00.00.81 18.1.99 167delT fs/fs RE 65dB 80dB 85dB 90dB 90dB 90dB S PTA to severe TEOAE-no response; BERA-no response 24.11.9 167delT/ great to I301 Ashkenazi M 23.9.99 9 167delT fs/fs RE FA uncle 130dBHL Profound V37I/ RE, Mild- Z365 Ashkenazi F 00.00.81 28.3.93 167delT V37I/fs Seq 30dB 35dB 55dB 60dB 50dB 70dB FA brother PTA moderate Moderate Iraq/ 35delG/ RE, (50dB- Z418 Tunisia F 00.00.91 L90P fs/L90P Seq FA sister 55dB)

167delT/ ∆30 (GJB6- fs/Cx30 RE, Profound, Z434 Ashkenazi M 00.00.97 D13S1830) del ASPCR S CI sibs, 35delG/ other Z442 Ashkenazi M 00.00.87 3.1.99 167delT fs/fs RE 85dB 95dB 110dB 110dB Ø Ø FA relatives PTA Profound

167delT/ ∆30 (GJB6- fs/Cx30 RE, T450 Ashkenazi M 1.9.86 6.1.97 D13S1830) del ASPCR 85dB 90dB 110dB Ø Ø Ø S PTA Profound

35delG/ ∆30 (GJB6- fs/Cx30 RE, Z453 Ashkenazi M 00.00.79 9.11.98 D13S1830) del ASPCR 85dB 95dB 110dB Ø Ø Ø FA brother PTA Profound 20.11.2 35delG/ Severe- Z463 Syria M 9.11.62 000 167delT fs/fs RE 70dB 90db 105dB Ø Ø Ø FA cousins PTA profound 167delT/ 100d Z464 Ashkenazi F 19.12.64 20.9.93 167delT fs/fs RE 75dB B Ø Ø Ø Ø S PTA Profound 35delG/ RE, Z477 Ashkenazi F 00.00.84 R32C fs/R32C Seq S Profound Ashkenazi NB-FF; /Ashkenaz BERA-Rt- i, 25.12.2 35delG/ 85dB, Lt- W483 Yemenite M 00.00.99 000 167delT fs/fs RE 80dB 95dB 110dB 115dB Ø Ø S NR 85dB Severe 51del12 Progressi insA/51 2nd ve, W600 Buchara F 00.00.94 del12insA fs/fs Seq FA cousins moderate

167delT/ 110d Profound, Z605 Ashkenazi F 00.00.93 7.10.99 167delT fs/fs RE 90dB B 115db 115dB Ø Ø S PTA CI parents, brother, Ashkenazi 35delG/ other Z614 /Syria F 00.00.83 23.8.99 167delT fs/fs RE 95dB 95dB 110dB 115dB Ø Ø FA relatives PTA Profound 51del12 5.6.00 insA/ Severe- W631 Buchara F 00.00.99 Ø W24X fs/W24X Seq 85dB 90dB 105dB Ø Ø S PTA profound 167delT/ Mild- Z683 Ashkenazi M 00.00.78 18.2.97 167delT fs/fs RE 30dB 40db 45dB 50db 60dB 60db S PTA moderate 35delG/ Profound, Z716 Ashkenazi M 00.00.94 167delT fs/fs RE S CI Severe- 5.12.20 167delT/ profound, Z717 Ashkenazi F 00.00.75 01 167delT fs/fs RE 60dB 85dB 110dB 120dB Ø Ø FA sister PTA CI 12.2.20 167delT/ T754 Ashkenazi M 00.00.97 02 167delT fs/fs RE 65dB 80dB 75dB 80dB 85dB FA sister PTA Severe 29.8.200 35delG/ Profound, T824 Ashkenazi F 0 167delT fs/fs RE S CI

167delT/ ∆30 (GJB6- fs/Cx30 RE, T851 Ashkenazi F 00.00.83 1.12.96 D13S1830) del ASPCR 100dB 95dB 110dB 115dB Ø Ø S PTA Profound 167delT/ T900 Ashkenazi M 00.00.74 167delT fs/fs RE FA sibs Profound 18.5.200 167delT/ T930 Ashkenazi M 1 167delT fs/fs RE S Profound 12.9.200 167delT/ Profound, T931 Ashkenazi M 2 167delT fs/fs RE FA sibs CI

VII.3. Manuscripts

0031-3998/04/5506-0995 PEDIATRIC RESEARCH Vol. 55, No. 6, 2004 Copyright © 2004 International Pediatric Research Foundation, Inc. Printed in U.S.A.

The R245X Mutation of PCDH15 in Ashkenazi Jewish Children Diagnosed with Nonsyndromic Hearing Loss Foreshadows Retinitis Pigmentosa

ZIPPORA BROWNSTEIN, TAMAR BEN-YOSEF, ORIT DAGAN, MOSHE FRYDMAN, DVORAH ABELIOVICH, MICHAL SAGI, FABIAN A. ABRAHAM, RIKI TAITELBAUM-SWEAD, MORDECHAI SHOHAT, MINKA HILDESHEIMER, THOMAS B. FRIEDMAN, AND KAREN B. AVRAHAM Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel [Z.B., O.D., M.F., K.B.A.]; Section on Human Genetics, Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Maryland 20850, USA [T.B., T.B.F.]; Danek Gertner Institute of Genetics, Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel [M.F.]; Department of Human Genetics, Hadassah Hebrew University Hospital and Medical School, Jerusalem 91120, Israel [D.A., M.S.]; Goldschleger Eye Institute, Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel [F.A.A.]; Department of Communication Disorders, Sackler School of Medicine, Tel Aviv University and Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel [R.T.-S., M.H.]; Department of Medical Genetics, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100, Israel. [M.S.]

ABSTRACT

Usher syndrome is a frequent cause of the combination of Ashkenazi population in Israel was estimated at 1%. Ashkenazi deafness and blindness due to retinitis pigmentosa (RP). Five Jewish children with profound prelingual hearing loss should be genes are known to underlie different forms of Usher syndrome evaluated for the R245X PCDH15 mutation and undergo oph- type I (USH1). In the Ashkenazi Jewish population, the R245X thalmologic evaluation to determine whether they will develop mutation of the PCDH15 gene may be the most common cause RP. Rehabilitation can then begin before loss of vision. Early use of USH1 (Ben-Yosef T, Ness SL, Madeo AC, Bar-Lev A, of cochlear implants in such cases may rescue these individuals Wolfman JH, Ahmed ZM, Desnick RK, Willner JP, Avraham from a dual neurosensory deficit. (Pediatr Res 55: 995–1000, KB, Ostrer H, Oddoux C, Griffith AJ, Friedman TB N Engl 2004) J Med 348: 1664Ð1670, 2003). To estimate what percentage of Ashkenazi Jewish children born with profound hearing loss will Abbreviations develop RP due to R245X, we examined the prevalence of the ERG, electroretinograms R245X PCDH15 mutation and its carrier rate among Ashkenazi SNHL, sensorineural hearing loss Jews in Israel. Among probands diagnosed with nonsyndromic RP, retinitis pigmentosa hearing loss not due to mutations of connexin 26 (GJB2) and/or USH1, Usher syndrome type 1 connexin 30 (GJB6), and below the age of 10, 2 of 20 (10%) USH2, Usher syndrome type 2 were homozygous for the R245X mutation. Among older non- USH3, Usher syndrome type 3 syndromic deaf individuals, no homozygotes were detected, NSHL, nonsyndromic hearing loss although one individual was heterozygous for R245X. The car- PCDH15, protocadherin 15 rier rate of the R245X mutation among the normal hearing ASPCR, allele-specific PCR

Usher syndrome accounts for more than 50% of the deaf- between the ages of 30 and 49 (2). This syndrome is defined by blind population (1), and approaches a prevalence of 1/10,000 SNHL, vestibular dysfunction, and progressive RP, eventually leading to blindness (3). Three clinical subtypes are described: Received August 12, 2003; accepted January 22, 2004. USH1 is the most severe, characterized by congenital profound Correspondence: Karen B. Avraham, Ph.D., Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, deafness, onset of RP in the first decade of life and constant Israel; e-mail: [email protected]. vestibular dysfunction. USH2 is distinguished from USH1 by a Supported by the Israel Ministry of Science, Culture and Sport (K.B.A.), the European Community (QLG2-1999-00988) (K.B.A.), and the National Institute on Deafness and mild to severe hearing loss with a downsloping audiogram Other Communication Disorders intramural research project ZO1 DC000039-06 (T.B.F.). configuration and intact caloric reflexes. USH3 is distinct from DOI: 10.1203/01.PDR.0000125258.58267.56 the others based upon the progressive nature and delayed onset

995 996 BROWNSTEIN ET AL. of the hearing loss and the occasional presence of vestibular of congenital NSHL, including children and adults, after ex- dysfunction (4, 5). cluding mutations in GJB2 and GJB6. Funduscopy and ERG Genes for seven USH1 loci have been mapped (4, 6) and five examinations were performed in two members of one family, have been cloned (7Ð11). Myosin VIIA (MYO7A) underlies under the age of 10, that were homozygous for the R245X USH1B, as well as NSHL DFNA11 and what was originally mutation, and in an additional 13-y-old proband diagnosed described as DFNB2, but is now considered to be atypical with USH1 based on congenital hearing loss and RP (not USH1 (10, 12Ð15). Mutations of cadherin 23 (CDH23) (8, 16) included among the 59 probands diagnosed with NSHL). are responsible for both USH1D and NSHL DFNB12. USH1C The project was approved by Helsinki (Institutional Review and NSHL DFNB18 are caused by allelic mutations of the Board) Committees at Tel Aviv University, Haim Sheba Med- USH1C gene, encoding harmonin (7). USH1G is caused by ical Center, Rabin Medical Center, Hadassah Medical Center, mutations of the USH1G gene, encoding SANS (11). Defects and the Wolfson Medical Center, Israel and by the NINDS/ in protocadherin 15, encoded by PCDH15, cause USH1F and NIDCD institutional review board at the National Institutes of NSHL DFNB23 (17Ð19). Health, Bethesda, MD, U.S.A. Blood samples were drawn after PCDH15 is located on chromosome 10q11.2-q21. Protocad- obtaining informed consent from each individual and, in case herins are thought to be involved in neural development, neural of individuals under 18 y of age, from their parents. circuit formation, and formation of the synapse (20). Mice Anonymous DNA samples from 505 hearing Ashkenazi carrying two mutations of Pcdh15 show disorganization in the Jews, obtained from Sheba Medical Center and from the Rabin orientation of the stereocilia, which are finger-like projections Medical Center, were used as controls. from the apical surface of hair cells of the inner ear and are necessary for mechanosensory transduction of sound, and lin- Experimental Methods ear and angular acceleration of the head (21). In the Ashkenazi Jewish population, the R245X mutation of Mutation exclusion of GJB2 and GJB6. The open reading PCDH15 accounts for more than half (58.3%) of USH1 cases frame of GJB2 was examined for mutations as described (22). The Israeli population is genetically heterogeneous and previously (25). Briefly, primers GJB2Ð1F, 5'-TCTTTTCCA- divides into three main ethnic groups, the Ashkenazi Jews GAGCAAACCGC-3', and GJB2Ð2R, 5'-GGGCAATGCGT- originating from Europe, the Sephardic Jews from the Medi- TAAACTGGC-3' amplified a 722 bp fragment that was se- terranean Sea area, and the Oriental Jewish community, who quenced and analyzed for the presence of mutations. For GJB6, came from the Middle East. In the Ashkenazi Jewish popula- a deletion [⌬(GJB6-D13S1830)] identified in Ashkenazi Jew- tion, two mutant alleles of connexin 26 (GJB2), 35delG and ish and Spanish hearing-impaired individuals (28, 29) was 167delT, account for the majority, but not all cases of nonsyn- analyzed in our DNA samples. Primers GJB6Ð1R, 5'- dromic recessive deafness (23Ð26). In addition, a deletion in TTTAGGGCATGATTGGGGTGATTT-3', designed 244 bp the connexin 30 (GJB6) gene, in trans configuration with one upstream of the proximal breakpoint of the deletion and GJB6- of the GJB2 mutations, has been shown to be associated with BKR-1, 5'-CACCATGCGTAGCCTTAACCATTTT-3', local- deafness in this population (27, 28). In the present study, we ized 216 bp downstream of the distal breakpoint of the dele- report the estimated prevalence of the R245X mutation among tion, amplified a 460 bp fragment encompassing the deletion Ashkenazi Jewish deaf probands in Israel, and carrier rates breakpoint if it was present. To positively detect a wild-type found in the population for this mutation. We also determined product, we designed a third primer localized 681 bp down- that the putative M1853L mutation (22) is a nonpathogenic stream of the GJB6Ð1R primer that was included in the same change in PCDH15. Since the R245X mutation of PCDH15, reaction, GJB6-RVS2, 5'-TCATCGGGGGTGTCAA- which is known to be involved in USH1F, is relatively com- CAAACA-3'. Wild-type DNA yielded a 681 bp band. mon in this population (22), some children incompletely diag- Mutation detection assays for R245X. To detect the R245X nosed with NSHL may have two R245X alleles. Molecular mutation by allele-specific PCR (ASPCR), two PCR reactions diagnosis of R245X would thus provide important presymp- were performed for each DNA sample, as described by Ben- tomatic detection of USH1, enabling optimal rehabilitation of Yosef et al. (22). We also used a restriction enzyme digestion communication in anticipation of loss of vision. assay because the R245X mutation creates an HphI restriction enzyme site. A 553 bp segment of PCDH15 exon 8 was METHODS PCR-amplified by the primers 361F (5'-ATA ACC ATG TTG GAC TGTTGTTTC-3') and 914R (5'-ATGTTTGCCAGGCT- Subjects GGTATCAAAC-3'). PCR cycling conditions were 95¡C for 5 Hearing-impaired probands were ascertained through genet- min, followed by a touchdown program of 60¡Cto56¡C and ics clinics at the Haim Sheba Medical Center, Rabin Medical 30 cycles of 95¡C for 45 s, 56¡C for 30 s, and 72¡C for 30 s, Center, Hadassah Medical Center, and the Wolfson Medical followed by two steps of 72¡C and 4¡C for 10 min each. The Center, Israel. Family histories were obtained, as well as a PCR product was digested with HphI (37¡C for 10 h) and complete clinical history of each affected individual to ensure separated by electrophoresis on a 2% agarose gel. Expected that no obvious environmental factors were involved in the product sizes are 553 bp for the wild-type allele and 372 bp and hearing loss. Audiograms were obtained from each proband. 181 bp for the mutant allele. Screening for the R245X mutation of PCDH15 was performed For sequence analysis, a 367 bp segment of exon 8 was in 59 Ashkenazi probands that were referred with a diagnosis amplified by PCR using primers F (5'-TGCCTAATTTC- PCDH15 MUTATION FORESHADOWS USH1F 997 TATAAACTACCTGTTG-3') and R (5'-CCCTGA AAATA- The family members of the two homozygous R245X young ATTTCGGACA-3'). The PCR products were purified by QIA- probands were also screened for R245X. Family T292 (Fig. quick PCR Purification Kit (QIAGEN, Valencia, CA, U.S.A.). 1A) includes parents, two deaf children, 4 and7yofage, both Sequencing was performed using the same primers as those with cochlear implants, and two unaffected children. Both used for PCR, the ABI Prism BigDye Terminator Cycle Se- parents were heterozygous for the R245X mutation, the two quencing Ready Reaction Kit (Applied Biosystems, Foster deaf children were homozygous for the mutation, one unaf- City, CA, U.S.A.) and an ABI 377 DNA sequencer. Mutations fected child was heterozygous and in the other unaffected child were confirmed by sequencing in both directions or by using at R245X was not detected. The older deaf child manifested a least two assays described above. delay in motor development and walked independently at 2 y, Detection of M1853L. A restriction enzyme digestion assay which is consistent with the USH1 phenotype (3). Hearing loss was performed for detection of the 5556A3C transversion was detected at 10 mo, and was found to be profound, with an that leads to M1853L. A 553 bp segment of PCDH15 exon 33 unaided corner audiogram (thresholds of 100 dBHL at 250 Hz was PCR-amplified by the primers F (5'-CTACCTCCATTTC- and 110 dBHL at 500 Hz). Aided thresholds were 60 dBHL at CAACTCCTCT-3') and R (5'-ATTTCATTGAATTTGGGG- 250 Hz and 90 dBHL at 500 Hz, with no response at higher TAAAAT-3'). PCR conditions were as described above, with frequencies. The child received a cochlear implant at the age of touchdown annealing temperatures of 56¡Cto51¡C and 45-s 3 y and 4 mo. He has used the implant for 4 y, with very good denaturation, annealing, and elongation times. The PCR prod- results in open set tests (60% in one-syllable-word identifica- uct was digested by TseI (65¡C for 10 h) and separated by tion and 80% in both two-syllable-word identification and in electrophoresis on a 2% agarose gel. A TseI site is inserted due words in sentences). He uses oral communication. to the mutation. Expected product sizes are 553 bp for the Ophthalmologic examination at age 7 revealed a corrected wild-type allele, and 427 bp and 126 bp for the mutant allele. visual acuity of 6/9 in each eye. The ocular media was trans- parent by slit lamp biomicroscopy and the intraocular pressure RESULTS was 12 mm mercury in both eyes. Color vision examined by R245X mutation detection. The PCDH15 gene contains 33 Ishihara pseudochromatic plates showed normal results, exons and is predicted to encode a protein of 1955 amino acids whereas the tangent screen visual field test witha5mmtest (18). The R245X mutation is caused byaCtoTtransition at object indicated severe peripheral visual field construction to position 733 in exon 8 of the PCDH15 gene, and leads to the about 20 degrees diameter for each eye. The retinal examina- substitution of an arginine codon by a stop codon (GenBank tion through the slit lamp in conjunction with a Volk aspheric accession number AY029237). Because this mutation was ϩ78 diopter lens revealed narrow retinal vessels with very fine reported only among Ashkenazi Jews, and was not detected in pigmentary stippling in the retinal periphery up to the vascular other Jewish ethnic groups (22), we ascertained 59 Ashkenazi arcade. ERG was performed with a Nicolet CA1000 instrument Jewish individuals diagnosed with congenital NSHL who did with Henkes type contact lens applied on the patient’s anes- not have mutations in GJB2 or in GJB6. Twenty of the thetized cornea after maximal pupillary dilatation. Standard probands were under the age of 10, and 39 were over 10 y old. full-field flash eye stimulation under light and dark adapting We chose this age for stratification because this is typically the conditions revealed severely reduced photopic responses and age of onset of loss of vision for individuals with USH1 (4). In nearly absent scotopic responses, consistent with Usher the first group of probands under 10, 18 of 20 were found to syndrome. have two wild-type PCDH15 alleles, whereas 2 of 20 (10%) In the 4-y-old child in Family T292, hearing loss was were homozygous for R245X. In the older group, no homozy- detected at 4 mo and was profound. He received a cochlear gotes for R245X were detected; 38 of 39 were wild type and 1 implant when he was 1 y and 3 mo old. He has used the implant of 39 (2.6%) was heterozygous (carrier) for R245X (Table 1). for 2 y and is able to identify two-syllable words only in closed The deaf individual heterozygous for the R245X mutation did set tests. Results of a funduscopic examination performed at not carry any other PCDH15 mutations in the reported 33 the age of 4 were normal in this child. An ERG was not exons of PCDH15, either suggesting that we have not yet available. found the partnering mutant allele of PCDH15 or this person is In Family T282 (Fig. 1A), the deaf children were 4 and 5 y a coincidental carrier of R245X. At least two different assays old and both underwent cochlear implantation. Both deaf were performed to confirm the presence of R245X (data not children were homozygous for R245X. The two deaf children shown). had delays in motor development and were reported by the

Table 1. R245X mutation frequencies among Ashkenazi Jewish probands in Israel diagnosed with deafness and carrier rate of this mutation in a control group Probands Number tested Wild type Heterozygous Homozygous Congenital deafness, under 10 y old 20 18 — 2† (10%) Congenital deafness, 10 y old and above 39 38 1* (2.6%) — Hearing individuals 505 500 5 (0.99%) — * The DNA from this deaf individual was sequenced for the reported coding exons of PCDH15 and a second mutation was not detected. † Probands from families T282 and T292 (see Fig. 1). 998 BROWNSTEIN ET AL. nated from Iraq (Oriental; Family Z305) and the second from Morocco (Sephardic; Family Z409). The parents in Family Z305 were heterozygous, and one deaf brother and one unaf- fected brother were homozygous for M1853L (Fig. 1B). In Family Z409, the father was heterozygous for M1853L but the mother was wild type. Two deaf and one hearing sibling were wild type (Fig. 1B). Therefore, based on our data, M1853L appears to be a benign polymorphism (i.e. not a pathogenic mutation of PCDH15).

DISCUSSION Our results confirmed the prevalence of the R245X muta- tion, associated with USH1 in the Ashkenazi Jewish popula- tion, as reported by Ben-Yosef et al. (22). The R245X mutation generates a protein translation stop codon in exon 8 of PCDH15, the gene encoding protocadherin 15, and presumably results in a truncated form of protocadherin 15. Alternatively, Figure 1. Pedigrees of families analyzed for mutations in PCDH15. Probands are indicated by arrows. (A) Families in which probands are homozygote for there may be no protein formed due to nonsense-mediated the R245X mutation of PCDH15. R refers to R245X mutation and ϩ refers to decay (30). Most significantly, we report that this mutation was the wild-type PCDH15 allele. Numbers refer to ages of each child. (B) found in children under 10 y of age who were inadvertently Families in which probands are heterozygous for the M1853L polymorphism diagnosed with NSHL, where hearing loss is the only symp- ϩ of PCDH15. M refers to the rare M1853L polymorphism and refers to tom, before the age that RP develops. Furthermore, we tested wild-type PCDH15 allele. a cohort of 59 individuals that included 39 over the age of 10 to exclude involvement of the R245X mutation in NSHL. We parents to have balance difficulties. In the older child, hearing did not discover a homozygote R245X mutation in any indi- loss was detected at 1 y and was profound. He received a viduals diagnosed with NSHL over the age of 10, further cochlear implant when he was 1 y and 10 mo old and has used demonstrating that this mutant allele of PCDH15 is associated the implant for 3 y and 5 mo. He has open set recognition for with RP in late childhood as well as hearing loss (7, 17, 22). familiar words. Among the 20 probands under the age of 10 y having no In the younger child, hearing loss was detected at 3 mo and mutations in GJB2 and GJB6, we identified two (10%) that was profound. She received a cochlear implant when she was were homozygous for the R245X mutation. The two probands 1 y and 6 mo old and has used her implant for 2 1/2 y and has homozygous for the R245X mutation manifested delays in very good open set results. Ophthalmologic examinations have motor development, which is characteristic of vestibular dys- not been performed in members of Family T282. function. One 7-y-old proband who underwent ophthalmologic One 13-y-old proband already diagnosed with USH1 based consultation showed an abnormal fundus and ERG, including on ophthalmologic examinations, including an ERG, was diffuse retinal dystrophy and visual field constriction, whereas found to be homozygous for the R245X mutation. This finding his 4-y-old brother’s results (homozygous for the R245X mu- further confirms that the R245X mutation is associated with tation) were within normal limits according to the funduscopy. USH1. Because onset of RP in USH1 is prepubertal, visual anomalies R245X carrier frequency. Anonymous DNA samples (n ϭ in the younger child may develop later. 505) of hearing Ashkenazi Jews were screened for the R245X In addition to the prevalence of the R245X mutation among mutation by the ASPCR assay and if the mutation was de- children diagnosed with NSHL, we found a carrier rate of 1% tected, it was confirmed by sequencing exon 8 of PCDH15. among the Ashkenazi Jewish population examined in Israel, Five out of 505 (0.99%; 95% confidence interval, 0Ð2%) which is comparable to 0.79Ð2.47% among Ashkenazi Jews, representative Ashkenazi Jews were heterozygous for R245X reported by Ben-Yosef et al. (22). This has led us to propose (Table 1). Because R245X was not detected among 293 non- that all Ashkenazi probands with NSHL under the age of 10 in Ashkenazi Jews screened elsewhere, and the mutation is as- whom GJB2 and/or GJB6 mutations have been excluded as the sumed to result from a founder effect in the Ashkenazi group reason for the hearing loss should be screened for the R245X (22), no other ethnic Jewish groups were screened. mutation to enable presymptomatic diagnosis of USH1. Be- Screening for M1853L. A putative PCDH15 mutation, cause a GJB2 mutant allele, 167delT, is carried by approxi- M1853L, was previously found in compound heterozygosity in mately 4% of the Ashkenazi Jewish population and another one Ashkenazi Jewish USH1 patient (22). Because carriers for allele, 35delG, is carried by 0.21Ð1.1% of this population M1853L were found for both Ashkenazi and non-Ashkenazi (23Ð26), individuals homozygous for the R245X mutation of Jews (with carrier rates higher in the latter group) (22), we PCDH15 might also be coincidental carriers of one of these screened non-Ashkenazi deaf probands (n ϭ 90) for the GJB2 mutations. The presence of only one GJB2 mutant allele M1853L putative mutation of PCDH15. Eighty-eight were should not preclude screening for R245X in a deaf Ashkenazi wild type and two were heterozygous for M1853L; one origi- Jewish child. PCDH15 MUTATION FORESHADOWS USH1F 999

Figure 2. Diagnostic algorithm for sporadic or recessively inherited SNHL with R245X testing for Ashkenazi Jewish children. If there is a family history of hearing loss, genetic evaluation should be performed; for sporadic cases, it is more cost-efficient to start with genetic testing and then extend the analysis only if no mutations are found. HL, hearing loss; SHL, syndromic hearing loss; CT, computed tomography; ESR, erythrocyte sedimentation rate; CBC, complete blood count.

Early diagnosis is especially crucial when deaf children will Acknowledgments. The authors thank the family members develop blindness as well. In such cases, oral language may be for their generous cooperation in this study and Mira Berlin, the optimal mode of communication inasmuch as sign lan- Bella Davidov, Miri Yanuv, Batsheva Bonne-Tamir, and An- guage and lip reading are both visual. There is a need to drew Griffith for their contributions. provide these children with the best hearing amplification available, with a preference for cochlear implantation if pos- REFERENCES sible, accompanied by intensive training and habilitation. A 1. Boughman JA, Vernon M, Shaver KA 1983 Usher syndrome: definition and estimate recent study of Usher syndrome suggests that auditory-oral of prevalence from two high-risk populations. J Chronic Dis 36:595Ð603 communication is more successful if cochlear implants are 2. Hope CI, Bundey S, Proops D, Fielder AR 1997 Usher syndrome in the city of Birmingham—prevalence and clinical classification. Br J Ophthalmol 81:46Ð53 implemented before the onset of retinal degeneration, in con- 3. Smith RJ, Berlin CI, Hejtmancik JF, Keats BJ, Kimberling WJ, Lewis RA, Moller junction with speech therapy (31). The earlier the diagnosis is CG, Pelias MZ, Tranebjaerg L 1994 Clinical diagnosis of the Usher syndromes. Usher Syndrome Consortium. Am J Med Genet 50:32Ð38 made, the better chance these children have for being able to 4. Petit C 2001 Usher syndrome: from genetics to pathogenesis. Annu Rev Genomics communicate optimally in society, even after they have lost a Hum Genet 2:271Ð297 5. Ness SL, Ben-Yosef T, Bar-Lev A, Madeo AC, Brewer CC, Avraham KB, Kornreich portion or all of their vision as well. In addition, early diagnosis R, Desnick RJ, Willner JP, Friedman TB, Griffith AJ 2003 Genetic homogeneity and of Usher syndrome might allow for an enhanced social and phenotypic variability among Ashkenazi Jews with Usher syndrome type III. J Med Genet 40:767Ð772 emotional adjustment for the family, recurrence risk counsel- 6. Mustapha M, Chouery E, Torchard-Pagnez D, Nouaille S, Khrais A, Sayegh FN, ing of at-risk couples, and the option for prenatal diagnosis for Megarbane A, Loiselet J, Lathrop M, Petit C, Weil D 2002 A novel locus for Usher syndrome type I, USH1G, maps to chromosome 17q24Ð25. Hum Genet 110:348Ð350 at-risk couples. 7. Ahmed ZM, Smith TN, Riazuddin S, Makishima T, Ghosh M, Bokhari S, Menon PS, We suggest a diagnostic algorithm for children presenting Deshmukh D, Griffith AJ, Friedman TB, Wilcox ER 2002 Nonsyndromic recessive deafness DFNB18 and Usher syndrome type IC are allelic mutations of USHIC. Hum with sporadic or recessively inherited SNHL, taking the Amer- Genet 110:527Ð531 ican College of Medical Genetics statement into consideration 8. Bork JM, Peters LM, Riazuddin S, Bernstein SL, Ahmed ZM, Ness SL, Polomeno R, Ramesh A, Schloss M, Srisailpathy CR, Wayne S, Bellman S, Desmukh D, Ahmed (32) (Fig. 2). The routine implementation of comprehensive Z, Khan SN, Kaloustian VM, Li XC, Lalwani A, Bitner-Glindzicz M, Nance WE, Liu tests, such as brain computed tomography, is cost ineffective XZ, Wistow G, Smith RJ, Griffith AJ, Wilcox ER, Friedman TB, Morell RJ 2001 Usher syndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 are and may be circumvented in many cases by less costly molec- caused by allelic mutations of the novel cadherin-like gene CDH23. Am J Hum Genet ular diagnostic tests (33). In addition, based on our data, we 68:26Ð37 9. Boeda B, El-Amraoui A, Bahloul A, Goodyear R, Daviet L, Blanchard S, Perfettini recommend testing for R245X in children under the age of 10 I, Fath KR, Shorte S, Reiners J, Houdusse A, Legrain P, Wolfrum U, Richardson G, of Ashkenazi Jewish descent with little or no family history of Petit C 2002 Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle. EMBO J 21:6689Ð6699 NSHL who are heterozygous or negative for a mutation of 10. Weil D, Kussel P, Blanchard S, Levy G, Levi-Acobas F, Drira M, Ayadi H, Petit C either GJB2 or GJB6. This molecular test may identify im- 1997 The autosomal recessive isolated deafness, DFNB2, and the Usher 1B syndrome are allelic defects of the myosin-VIIA gene. Nat Genet 16:191Ð193 pending RP before detection by ERG, and permit timely 11. Weil D, El-AmraouiA, Masmoudi S, Mustapha M, Kikkawa Y, Laine S, Delmaghani rehabilitation and genetic counseling for the parents. S, Adato A, Nadifi S, Zina ZB, Hamel C, Gal A, Ayadi H, Yonekawa H, Petit C 2003 1000 BROWNSTEIN ET AL.

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Prevalence and Evolutionary Origins of the del(GJB6-D13S1830) Mutation in the DFNB1 Locus in Hearing-Impaired Subjects: a Multicenter Study Ignacio del Castillo,1 Miguel A. Moreno-Pelayo,1 Francisco J. del Castillo,1,2 Zippora Brownstein,4 Sandrine Marlin,3 Quint Adina,5 David J. Cockburn,6 Arti Pandya,8 Kirby R. Siemering,9 G. Parker Chamberlin,11 Ester Ballana,12 Wim Wuyts,13 Andre´a Trevas Maciel-Guerra,14 Araceli A´ lvarez,1 Manuela Villamar,1 Mordechai Shohat,4,15 Dvorah Abeliovich,5 Hans-Henrik M. Dahl,9,10 Xavier Estivill,12 Paolo Gasparini,16 Tim Hutchin,7 Walter E. Nance,8 Edi L. Sartorato,14 Richard J. H. Smith,11 Guy Van Camp,13 Karen B. Avraham,4 Christine Petit,2 and Felipe Moreno1 1Unidad de Gene´tica Molecular, Hospital Ramo´n y Cajal, Madrid; 2Unite´deGe´ne´tique des De´ficits Sensoriels INSERM U587, Institut Pasteur, and 3Unite´deGe´ne´tique Me´dicale, Hoˆpital Trousseau, Paris; 4Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv; 5Department of Human Genetics, Hadassah Hebrew University Hospital, Jerusalem; 6DNA Laboratory and 7Molecular Medicine Unit, St. James’s University Hospital, Leeds, United Kingdom; 8Department of Human Genetics, Medical College of Virginia of Virginia Commonwealth University, Richmond; 9The Murdoch Childrens Research Institute, Royal Children’s Hospital, and 10Department of Paediatrics, University of Melbourne, Melbourne; 11Interdepartmental Human Genetics Program and the Department of Otolaryngology, University of Iowa, Iowa City; 12Genes and Disease Program, Center for Genomic Regulation, Pompeu Fabra University, Barcelona; 13Department of Medical Genetics, University of Antwerp, Antwerp; 14Centro de Biologia Molecular e Engenharia Gene´tica, Universidade Estadual de Campinas, Saˆo Paulo, Brazil; 15Department of Medical Genetics, Rabin Medical Center, Beilinson Campus, Petah Tikva, Israel; and 16Medical Genetics, Second University of Naples and Telethon Institute of Genetics and Medicine, Naples

Mutations in GJB2, the gene encoding connexin-26 at the DFNB1 locus on 13q12, are found in as many as 50% of subjects with autosomal recessive, nonsyndromic prelingual hearing impairment. However, genetic diagnosis is com- plicated by the fact that 10%–50% of affected subjects with GJB2 mutations carry only one mutant allele. Recently, a deletion truncating the GJB6 gene (encoding connexin-30), near GJB2 on 13q12, was shown to be the accompanying mutation in ∼50% of these deaf GJB2 heterozygotes in a cohort of Spanish patients, thus becoming second only to 35delG at GJB2 as the most frequent mutation causing prelingual hearing impairment in Spain. Here, we present data from a multicenter study in nine countries that shows that the deletion is present in most of the screened populations, with higher frequencies in France, Spain, and Israel, where the percentages of unexplained GJB2 het- erozygotes fell to 16.0%–20.9% after screening for the del(GJB6-D13S1830) mutation. Our results also suggest that additional mutations remain to be identified, either in DFNB1 or in other unlinked genes involved in epistatic inter- actions with GJB2. Analysis of haplotypes associated with the deletion revealed a founder effect in Ashkenazi Jews and also suggested a common founder for countries in Western Europe. These results have important implications for the diagnosis and counseling of families with DFNB1 deafness.

Hearing impairment is the most common sensory dis- sign, are highly heterogeneous, with 180 loci already order. In developed countries, 160% of the cases are reported and 30 genes identified so far (Hereditary Hear- due to genetic causes (Petit et al. 2001). Nonsyndromic ing Loss Homepage). Hearing impairment that manifests forms, in which the hearing deficit is the only clinical before speech acquisition (i.e., with prelingual onset) is mainly inherited in an autosomal recessive pattern, with 31 different loci and 16 currently identified genes (He- Received July 23, 2003; accepted for publication September 25, 2003; electronically published October 21, 2003. reditary Hearing Loss Homepage). Address for correspondence and reprints: Dr. Felipe Moreno, Unidad The DFNB1 locus for nonsyndromic, autosomal re- de Gene´tica Molecular, Hospital Ramo´ n y Cajal, Carretera de Col- cessive, prelingual hearing impairment (MIM 220290) menar, Km 9, 28034 Madrid, Spain. E-mail: fmoreno.hrc@salud was mapped to the 13q12 region (Guilford et al. 1994). .madrid.org ᭧ 2003 by The American Society of Human Genetics. All rights reserved. This locus contains the GJB2 gene (MIM 121011), en- 0002-9297/2003/7306-0022$15.00 coding connexin-26 (Cx26), a transmembrane protein 1452 Reports 1453 subunit of intercellular gap junctions (Kelsell et al. 1997). subjects who were homozygous for the deletion (del Cas- Six monomers of connexin bind together to form a hex- tillo et al. 2002; Pallares-Ruiz et al. 2002). In one study, amer (connexon), which, in turn, docks with another con- the deletion breakpoint junction was isolated and se- nexon on the surface of an adjacent cell to form an in- quenced, revealing the loss of a DNA segment of ∼342 tercellular gap-junction channel (Goodenough et al. 1996; kb, with one breakpoint inside the GJB6 coding region Kumar and Gilula 1996). In the cochlea, there are two (del Castillo et al. 2002). This deletion, named “del(GJB6- networks of gap junctions, the epithelial cell system and D13S1830),” was the accompanying mutation in ∼50% the connective tissue cell system, which are thought to be of the deaf GJB2 heterozygotes (del Castillo et al. 2002). involved in the recycling of potassium back into the coch- It remained to be determined whether the deletions de- lear endolymph, where it plays an essential role in sound tected by other groups (Lerer et al. 2001; Pallares-Ruiz transduction (Kikuchi et al. 2000). In fact, targeted in- et al. 2002) were also del(GJB6-D13S1830). activation of the GJB2 gene in the inner-ear epithelial These findings may indicate a digenic pattern of in- network led to hearing impairment in mice, demonstrat- heritance of hearing impairment from mutations involv- ing the key role of this network in cochlear function and ing GJB2 and GJB6. This hypothesis is supported by cell survival (Cohen-Salmon et al. 2002). Several different several facts: (i) both Cx26 and Cx30 are expressed in connexins have been shown to participate in these gap the same inner-ear structures (Lautermann et al. 1998, junction systems. To date, mutations in the genes encoding 1999), (ii) connexons composed of Cx26 can bind con- three of these connexins (GJB2 for Cx26, GJB6 for Cx30 nexons composed of Cx30 to form heterotypic gap-junc- [MIM 604418], and GJB3 for Cx31 [MIM 603324]) are tion channels (Dahl et al. 1996), (iii) a mutation in GJB6 known to result in hearing impairment (Kelsell et al. 1997; was reported in a case of autosomal dominant hearing Xia et al. 1998; Grifa et al. 1999). Among them, GJB2 impairment (Grifa et al. 1999), and (iv) Cx30-deficient stands out, because mutations in this gene account for as mice exhibit a severe constitutive hearing impairment many as 50% of all cases of prelingual hearing impair- and lack an endocochlear potential (Teubner et al. ment in many populations (Rabionet et al. 2000). More 2003). However, the fact that point mutations in GJB6 than 80 different mutations in GJB2 have been described have not yet been found in cases of autosomal recessive in subjects with hearing impairment (Connexin-Deafness hearing impairment argues against this hypothesis. An Homepage). Molecular testing for GJB2 mutations has alternative explanation is that the deletion may eliminate rapidly become the standard of care for the diagnosis and an upstream regulatory element for GJB2 that is essen- counseling of patients with nonsyndromic hearing im- tial for the normal expression of this gene in the inner pairment of unknown cause. ear. So far, such an element has not been found. Mutation screening of GJB2 in subjects with autosomal Given the association previously found between GJB2 recessive hearing impairment has, however, revealed an monoallelic mutations and the del(GJB6-D13S1830) unexpected problem—namely, a high number of patients mutation in several studies (Lerer et al. 2001; del Castillo carrying only one mutant allele. Exhaustive screening of et al. 2002; Pallares-Ruiz et al. 2002), we investigated the coding region (fully contained in exon 2), exon 1, and the contribution of this deletion to hearing impairment splice sites did not reveal any mutation in the second allele. in nine countries. A genetic analysis of five microsatellite These cases accounted for 10%–50% of all deaf subjects markers flanking the deletion breakpoints was also con- with at least one GJB2 mutation. These findings could ducted, to determine the haplotypes associated with the be attributed to intrinsic limitations in the techniques used del(GJB6-D13S1830) mutation and to explore its evo- for mutation screening, to the high frequency of carriers lutionary origins. The implications of our results for mo- for some GJB2 mutations, or to the existence of mutations lecular diagnosis of DFNB1 mutations are presented. in other noncoding parts of the gene that have not yet The study was performed on probands with nonsyn- been identified. However, it was also suspected that other dromic prelingual hearing impairment from nine coun- mutations might exist, in the DFNB1 locus but not in the tries (table 1). In Spain, Israel, and the United States, two GJB2 gene, that could provide an explanation for the high independent studies were performed. After getting written proportion of heterozygous affected subjects. Recently, informed consent, blood samples were obtained, and this hypothesis received experimental support from the DNA was extracted by standard procedures. Testing for finding of a novel class of mutations in the DFNB1 locus, the del(GJB6-D13S1830) mutation was performed using which were deletions not affecting GJB2 but truncating the previously reported primers GJB6-1R (forward) and the neighboring GJB6 gene, which encodes Cx30. These BKR-1 (reverse) (del Castillo et al. 2002), as well as a deletions were found accompanying in trans the only modification of the method to positively detect a wild- GJB2 mutant allele in heterozygous affected subjects type product by adding another reverse primer that is (double heterozygosity) (Lerer et al. 2001; del Castillo et located in the deleted segment of GJB6 (R2, 5-TCATCG- al. 2002; Pallares-Ruiz et al. 2002) and were also found GGGGTGTCAACAAACA-3). When these three primers to be the cause of deafness in three unrelated affected were used together, two different PCR products were ob- 1454 Am. J. Hum. Genet. 73:1452–1458, 2003

Table 1 Results from the Screenings for the del(GJB6-D13S1830) Mutation No. of (del[GJB6-D13S1830]ϩGJB2) Double Heterozygotes/ No. of del(GJB6-D13S1830) Country/Laboratory No. of GJB2 Heterozygotes Homozygotes/Total Screened Spain/Madrida 29/68 (42.6%)b 1/425 (.2%) ϩ 2 heterozygotesc Spain/Barcelona 5/35 (14.3%) 1/236 (.4%) Italy 0/31 (.0%) 0/238 (.0%) ϩ 1 heterozygotec France 23/60 (38.3%) 0/208 (.0%) ϩ 1 heterozygotec Belgium 2/19 (10.5%) 0/151 (.0%) United Kingdom 6/19 (31.6%) Not performed Israel/Tel Aviv 7/20 (35.0%) 1/191 (.5%) Israel/Jerusalem 5/7 (71.4%) Not performed United States/Virginia 14/88 (15.9%) 1/486 (.2%) ϩ 4 heterozygotesc United States/Iowa 7/95 (7.4%) Not performed Brazil 2/9 (22.2%) Not performed Australia 2/29 (6.9%) Not performed a This work expands the results of a previous study (del Castillo et al. 2002). b By performing haplotype analysis for genetic markers from 13q12, linkage to DFNB1 was excluded in 11 cases. After correction, the figures are 29/57 (50.9%). c Number of del(GJB6-D13S1830) heterozygotes with no mutation in GJB2. tained—GJB6-1RrR2 (681 bp) and GJB6-1RrBKR-1 factors cannot be excluded (for instance, differences in (460 bp)—allowing for discrimination between wild-type the proportion of probands who were the offspring of subjects (681-bp product), homozygotes for the deletion intermarriages among deaf people). (460-bp product), and heterozygotes (both products) in Before the screenings reported here, subjects with only a single test. In all subjects carrying the deletion, the PCR one mutant GJB2 allele accounted for 11.2%–51.5% of product that contains the breakpoint junction of the de- those with (one or two) mutations in GJB2 in our co- letion was sequenced, to confirm that the breakpoint junc- horts of cases. The lowest percentage was observed in tion was identical in all cases. Thus, we confirmed that Italy (11.2%), a result that is consistent with the absence all the deletions reported by Lerer et al. (2001) were of the deletion in that screening. In screenings performed del(GJB6-D13S1830). In an independent study, it was on large cohorts (150 subjects) in other countries, data also shown that the deletions reported by Pallares-Ruiz were, in general, rather homogeneous (22.1%–35.1%). et al. (2002) were del(GJB6-D13S1830) (A. F. Roux, per- Differences between the two screenings in Spain are sonal communication). probably due to regional bias in the Barcelona cohort Two different screenings were performed, the first on (40% of cases are from the same Spanish region, Can- deaf subjects carrying only one GJB2 mutant allele, tabria). It is noteworthy that screenings not including which had been found during routine testing for GJB2 exon 1 and splice sites show percentages that are similar mutations (tables 1 and 2); and the second on subjects to those that do include them, a result that suggests a with nonsyndromic prelingual hearing impairment, car- low frequency for mutations in these noncoding parts rying no mutation in GJB2 (table 1). The frequency of of the gene. the del(GJB6-D13S1830) allele among DFNB1 alleles is As expected, the observed frequencies for the del(GJB6- shown in table 3. D13S1830) allele correlate with the percentage of cases The del(GJB6-D13S1830) allele is most frequent in with only one mutant GJB2 allele that were elucidated Spain, France, the United Kingdom, Israel, and Brazil, by the finding of the deletion (table 1). The highest figures accounting for 5.9%–9.7% of all the DFNB1 alleles correspond to France, Spain, Israel, and the United King- (table 3). Its frequency is lower in Belgium and Australia dom (31.6%–71.4%). Differences between the two Israeli (1.3%–1.4%), and it has not been found among Italian studies, both performed on Ashkenazi Jews, are likely to GJB2 unelucidated heterozygotes. In the United States, be due to the small size of the cohorts that were analyzed. two different screenings yielded different results: a mod- On the other end, Australia, the United States, and Bel- erate frequency (4.5%, Virginia cohort) or a low fre- gium show much lower percentages of elucidated cases quency similar to those of Belgium and Australia (1.6%, (6.9%–15.9%). A slightly higher percentage was found Iowa cohort). A likely explanation for the higher fre- in an independent study performed in the United States quency observed in the Virginia cohort is that it contains (Stevenson et al. 2003), with the deletion accounting for a higher proportion of individuals of Spanish descent 20% of GJB2 deaf heterozygotes. After screening for the than does the Iowa cohort, but the contribution of other deletion, cases that remain not elucidated fell to 16.0%– Reports 1455

Table 2 Impact of Screening for del(GJB6-D13S1830) on the Elucidation of Cases with Only One Mutant GJB2 Allele

NO. OF MONOALLELIC SUBJECTS/ NO. OF MONOALLELIC ϩ NO. OF BIALLELIC SUBJECTSb Before Screening After Screening COUNTRY/LABORATORY GJB2 TESTINGa for the Deletion for the Deletion Spain/Madrid Ex2-CR, Ex1, SpS 68/244 (27.9%) 39/244 (16.0%)c Spain/Barcelona Ex2-CR, Ex1, SpS 35/68 (51.5%) 30/68 (44.1%) Italy Ex2-CR 31/278 (11.2%) 31/278 (11.2%) France Ex2-CR, Ex1, SpS 60/177 (33.9%) 37/177 (20.9%) Belgium Ex2-CR 19/86 (22.1%) 17/86 (19.8%) United Kingdom Ex2-CR 19/64 (29.7%) 13/64 (20.3%) Israel/Tel Aviv Ex2-CR, Ex1, SpS 20/75 (26.7%) 13/75 (17.3%) Israel/Jerusalem Ex2-CR, Ex1, SpS 7/44 (15.9%) 2/44 (4.5%) United States/Virginia Ex2-CR, Ex1, SpS 88/251 (35.1%) 74/251 (29.5%) United States/Iowa Ex2-CR, Ex1, SpS 95/305 (31.1%) 88/305 (28.9%) Brazil Ex2-CR, Ex1, SpS 9/21 (42.9%) 7/21 (33.3%) Australia Ex2-CR, Ex1, SpS 29/102 (28.4%) 27/102 (26.5%) a Ex2-CR p exon 2 coding region; Ex1 p entire exon 1; SpS p splice sites. b “Monoallelic” refers to subjects carrying only one mutant GJB2 allele; “biallelic” refers to subjects carrying two mutant GJB2 alleles. c After excluding cases not linked to DFNB1 according to haplotype analysis, results were 28/244 (11.5%).

20.9% in France, Spain, and Israel (results from larger We investigated the evolutionary origins of the dele- cohorts) and are now closer to data from Italy (11.2%) tion by studying haplotypes associated with this muta- (table 2). Moreover, in the screening performed by the tion. Five microsatellite markers closely flanking the de- Madrid team in Spain, genotyping and haplotype analysis letion breakpoints were selected for this study. Their for genetic markers close to GJB2 allowed us to exclude relative order and physical distances were as follows: linkage to DFNB1 in 11 of 39 unelucidated cases, with D13S1835–117 kb–(TG)n–42 kb–D13S141–68 kb– the percentage of unelucidated cases then decreasing to (GAAA)n–4 kb–deletion proximal breakpoint–309 kb– 11.5% (table 2). In contrast, these figures remain high in deletion distal breakpoint–233 kb–D13S1831. Note all the other countries (19.8%–33.3% in Belgium, the that the deletion size, according to the latest sequencing United Kingdom, Australia, the United States, and Brazil). data, is 309 kb (National Center for Biotechnology In- It is remarkable that Spanish subjects carrying a single formation database, Homo sapiens genome view, build mutation in GJB2 but without linkage to DFNB1 (co- incidental carriers) account for at least 4.5% (11 of 244; Table 3 data from the Madrid laboratory) of cases with GJB2 mutations. It must be taken into account that linkage to Frequency of the del(GJB6-D13S1830) Allele among the DFNB1 Alleles DFNB1 cannot be tested in simplex (sporadic) cases, im- peding the detection of additional coincidental carriers, No. of del(GJB6-D13S1830) Country/Laboratory Alleles/Total No. of DFNB1 allelesa and so this figure undoubtedly is higher. However, even if we assume a higher percentage of coincidental carriers, Spain/Madrid 31/408 (7.6%) it clearly emerges that other DFNB1 mutations remain Spain/Barcelona 7/72 (9.7%) Italy 0/494 (0%) to be identified in most countries. France 23/280 (8.2%) The frequency of the del(GJB6-D13S1830) mutation Belgium 2/138 (1.4%) in all populations is not high enough to result in a large United Kingdom 6/102 (5.9%) number of homozygous subjects. They represent !0.5% Israel/Tel Aviv 9/126 (7.1%) of all cases of prelingual hearing impairment without Israel/Jerusalem 5/84 (6.0%) USA/Virginia 16/356 (4.5%) mutations in GJB2 (table 1; none were born from con- USA/Iowa 7/434 (1.6%) sanguineous parents). It is noteworthy that four screen- Brazil 2/28 (7.1%) ings detected del(GJB6-D13S1830) heterozygotes with- Australia 2/150 (1.3%) out accompanying DFNB1 mutation (table 1), which a Only cases in which both DFNB1 alleles were identified are to be added to the cases lacking elucidation. contribute to this total. 1456 Am. J. Hum. Genet. 73:1452–1458, 2003

Table 4 Haplotypes Associated with the del(GJB6-D13S1830) Mutation

GENOTYPE FOR CEPH ALLELE IN HAPLOTYPE HETEROZYGOSITYb INDIVIDUAL MARKERa (%) A1 A2 A3 A4 B1 B2 B3 B4 B5 B6 C1 D1 E1 E2 F1 134702 D13S1835 78 134 134 134 136 132 132 134 136 136 138 136 134 138 165 138 134/136

(TG)n 65 204 204 204 204 208 208 208 208 208 208 210 204 208 208 208 206/208 D13S141 55 124 124 124 124 124 124 124 124 124 124 124 126 126 126 124 126/126

(GAAA)n 79 209 209 209 209 209 209 209 209 209 209 209 209 209 209 205 209/216 D13S1831 84 96 103 105 105 87 105 105 87 105 105 105 105 105 105 101 99/101

a Relative order and physical distances are as follows: D13S1835–117 kb–(TG)n–42 kb–D13S141–68 kb–(GAAA)n–4 kb–deletion proximal breakpoint–309 kb–deletion distal breakpoint–233 kb–D13S1831. b Calculated from 100 Spanish control chromosomes.

33, contig NT_009799). Conditions for the PCR am- their data with those reported in this work, we provide plification of these markers have been reported elsewhere allele sizes for individual 134702, available from CEPH (Hudson et al. 1992; Kibar et al. 1999; Lerer et al. 2001). (Dib et al. 1996) (table 4). Cases suitable for haplotype analysis were genotyped for In 51 of 52 chromosomes carrying the deletion, we these markers, but we report here only those cases in observed association with allele 209 from marker which the haplotype associated with the deletion could (GAAA)n, which is at a distance of only 4 kb from the be determined unambiguously. These included 52 non- deletion proximal breakpoint (frequency of this allele: related chromosomes: 28 from Spain (including 1 of 0.415 in Spain and 0.364 in Ashkenazi Jews). When con-

Russian origin), 9 from Israel, 5 from France, 3 from sidering a core haplotype constituted by markers (TG)n, the United Kingdom, 3 from the United States, 2 from D13S141, and (GAAA)n, we could define six different Brazil, and 2 from Australia (tables 4 and 5). Allele sizes haplogroups, A–F (table 4). Finally, an expanded hap- were determined by DNA sequencing of a control sam- lotype with all the five markers revealed 15 variants as- ple, which was shared by all the laboratories in this sociated with the deletion (table 4). When examining the multicenter study and was used as a standard in geno- geographic distribution of haplotypes (table 5), it is re- typing assays. To allow other laboratories to compare markable that all nine Israeli chromosomes belong to

Table 5 Distribution of Haplotypes Associated with the Deletion in Different Populations Haplotype N Distribution Group A: A1 1 Spain (Russian origin) A2 1 Australia A3 21 11 Spain (10 Madrid, 1 Barcelona) 8 Israel (6 Tel Aviv, 2 Jerusalem), 2 France A4 1 Israel (Jerusalem) Total 24 Group B: B1 7 Spain (Madrid) B2 2 Spain (Madrid) B3 1 Spain (Madrid) B4 1 Spain (Madrid) B5 3 2 Spain (Madrid), 1 United Kingdom B6 9 2 Spain (Madrid), 2 France, 1 United Kingdom, 1 Australia 1 United States (Virginia), 2 Brazil (Portuguese origin) Total 23 Group C (C1) 1 France Group D (D1) 1 United States (Virginia) Group E: E1 1 United Kingdom E2 1 Spain (Madrid) Total 2 Group F (F1) 1 United States (Virginia) Total 52 Reports 1457 group A (eight A3 and one A4; these two haplotypes differ ciones de Padres y Amigos de los Sordos, for their enthusiastic only in D13S1835). Spanish haplotypes are mainly con- support of this research. F.J.d.C. and M.V. were recipients of centrated in groups A and B (11 A3, 15 B, and 1 E2), fellowships from the Comunidad de Madrid. A.A. was a recip- and the two Brazilian chromosomes are B6. Most chro- ient of a fellowship from Fondo de Investigaciones Sanitarias. mosomes from other countries (France, the United King- This work was supported by European Community grant QLG2-CT-1999-00988, Comisio´ n Asesora Interministerial de dom, and Australia) also belong to groups A and B. It is Ciencia y Tecnologı´a of Spanish Ministerio de Ciencia y Tec- noteworthy that two of three chromosomes from the nologı´a grant SAF99-0025 (to F.M.), Spanish Fondo de Inves- United States belong to groups D and F. tigaciones Sanitarias grants FIS 00/0244 and FIS PI020807 (to Our results show a clear founder effect for the I.d.C.), a grant from the Flemish Fund for Scientific Research del(GJB6-D13S1830) mutation in Ashkenazi Jews in Is- (to G.V.C.), a grant from the Israel Ministry of Science, Culture rael. Although the size of the sample should be increased and Sport (to K.B.A.), a grant from The Garnett Passe and to reach firmer conclusions in most of the analyzed pop- Rodney Williams Memorial Foundation (to H.H.M.D.), Na- ulations, our data also suggest a common founder for tional Institutes of Health grant RO1-DC02842 (to R.J.H.S.), the deletion in some countries in western Europe, since and a Consiglio Nazionale della Ricerche–Genomica Funzionale it is very scarce in Italy and has a low frequency in grant (to P.G.). Belgium, whereas it is quite frequent in Spain and France, in Brazilian subjects of Portuguese origin, and— Electronic-Database Information although to a lesser extent—in the United Kingdom (in all of these cases, the deletion is mainly associated with The accession number and URLs for data presented herein haplogroups A and B). The fact that the Ashkenazi af- are as follows: fected subjects share a common haplotype along 464 kb contrasts with the diversity of haplotypes found in pop- Connexin-Deafness Homepage: http://www.crg.es/deafness/ ulations from western Europe, suggesting an older origin Hereditary Hearing Loss Homepage, http://www.uia.ac.be/ dnalab/hhh/ for the deletion in these countries. The existence of hap- National Center for Biotechnology Information, http://www logroup F also suggests that the deletion could have .ncbi.nlm.nih.gov/ (for contig NT_009799) other independent origins. Online Mendelian Inheritance in Man (OMIM), http://www Inherited hearing impairment shows almost unparal- .ncbi.nlm.nih.gov/Omim/ (for DFNB1, GJB2, GJB6, and leled genetic heterogeneity, not only in terms of numbers GJB3) of genes and mutations, but also in prevalence of specific mutations among different populations. Therefore, data from genetic epidemiological studies are essential for the References design of molecular diagnostic protocols well suited for Cohen-Salmon M, Ott T, Michel V, Hardelin J-P, Perfettini I, each population. Our study provides some conclusions Eybalin M, Wu T, Marcus DC, Wangemann P, Willecke K, that should serve to improve the molecular diagnosis of Petit P (2002) Targeted ablation of connexin 26 in the inner DFNB1 cases. Given the simplicity of the test for the ear epithelial gap junction network causes hearing impair- detection of the del(GJB6-D13S1830) mutation, it should ment and cell death. Curr Biol 12:1106–1111 be performed for all subjects with prelingual hearing im- Dahl E, Manthey D, Chen Y, Schwarz HJ, Chang YS, Lalley pairment, at least in populations having higher frequen- PA, Nicholson BJ, Willecke K (1996) Molecular cloning and functional expression of mouse connexin-30, a gap junction cies. Our work also highlights the importance of per- gene highly expressed in adult brain and skin. J Biol Chem forming haplotype analysis, when possible, in subjects 271:17903–17910 with only one mutant GJB2 allele, to identify carriers del Castillo I, Villamar M, Moreno-Pelayo MA, del Castillo whose hearing impairment might be due to mutations in FJ, A´ lvarez A, Tellerı´a D, Mene´ndez I, Moreno F (2002) A a different gene or to hypothetical epistatic interactions deletion involving the connexin 30 gene in nonsyndromic between GJB2 mutations and other unlinked gene(s). This hearing impairment. N Engl J Med 346:243–249 step would also help to concentrate research on the elu- Dib C, Faure´ S, Fizames C, Samson D, Drouot N, Vignal A, cidation of cases consistent with linkage to DFNB1 by Millasseau P, Marc S, Hazan J, Seboun E, Lathrop M, Gya- investigating other parts of GJB2 (promoter, 3 UTR) and pay G, Morissette J, Weissenbach J (1996) A comprehensive by searching for hypothetical point mutations in GJB6 genetic map of the human genome based on 5,264 micro- or other DNA rearrangements in the DFNB1 locus. satellites. Nature 380:152–154 Goodenough DA, Goliger JA, Paul DL (1996) Connexins, con- nexons, and intercellular communication. Annu Rev Bio- Acknowledgments chem 65:475–502 Grifa A, Wagner C, D’Ambrosio L, Melchionda S, Bernardi We thank the patients and their relatives, for their kind co- F, Lo´ pez-Bigas N, Rabionet R, Arbones M, Della Monica operation in this study, and Federacio´ n Espan˜ ola de Asocia- M, Estivill X, Zelante L, Lang F, Gasparini P (1999) Mu- 1458 Am. J. Hum. Genet. 73:1452–1458, 2003

tations in GJB6 cause nonsyndromic autosomal dominant O, Frank HG, Jahnke K, Winterhager E (1998) Expression deafness at DFNA3 locus. Nat Genet 23:16–18 of the gap-junction connexins 26 and 30 in the rat cochlea. Guilford P, Ben Arab S, Blanchard S, Levilliers J, Weissenbach Cell Tissue Res 294:415–420 J, Belkahia A, Petit C (1994) A non-syndromic form of neu- Lerer I, Sagi M, Ben-Neriah Z, Wang T, Levi H, Abeliovich rosensory, recessive deafness maps to the pericentromeric D (2001) A deletion mutation in GJB6 cooperating with a region of chromosome 13q. Nat Genet 6:24–28 GJB2 mutation in trans in non-syndromic deafness: a novel Hudson TJ, Engelstein M, Lee MK, Ho EC, Rubenfield MJ, founder mutation in Ashkenazi jews. Hum Mutat 18:460 Adams CP, Housman DE, Dracopoli NC (1992) Isolation Pallares-Ruiz N, Blanchet P, Mondain M, Claustres M, Roux and chromosomal assignment of 100 highly informative hu- AF (2002) A large deletion including most of GJB6 in re- man simple sequence repeat polymorphisms. Genomics 13: cessive non syndromic deafness: a digenic effect? Eur J Hum 622–629 Genet 10:72–76 Kelsell DP, Dunlop J, Stevens HP, Lench NJ, Liang JN, Parry Petit C, Levilliers J, Hardelin JP (2001) Molecular genetics of G, Mueller RF, Leigh IM (1997) Connexin 26 mutations in hearing loss. Annu Rev Genet 35:589–646 hereditary non-syndromic sensorineural deafness. Nature Rabionet R, Gasparini P, Estivill X (2000) Molecular genetics 387:80–83 of hearing impairment due to mutations in gap junction Kibar Z, Lafrenie`re RG, Chakravarti A, Wang J, Chevrette M, genes encoding beta connexins. Hum Mutat 16:190–202 Der Kaloustian VM, Rouleau GA (1999) A radiation hybrid Stevenson VA, Ito M, Milunsky JM (2003) Connexin-30 de- map of 48 loci including the Clouston hidrotic ectodermal letion analysis in connexin-26 heterozygotes. Genet Test 7: dysplasia locus in the pericentromeric region of chromosome 151–154 13q. Genomics 56:127–130 Teubner B, Michel V, Pesch J, Lautermann J, Cohen-Salmon Kikuchi T, Adams JC, Miyabe Y, So E, Kobayashi T (2000) M, So¨ hl G, Jahnke K, Winterhager E, Herberhold C, Har- Potassium ion recycling pathway via gap junction systems delin JP, Petit C, Willecke K (2003) Connexin30 (Gjb6)- in the mammalian cochlea and its interruption in hereditary deficiency causes severe hearing impairment and lack of en- nonsyndromic deafness. Med Electron Microsc 33:51–56 docochlear potential. Hum Mol Genet 12:13–21 Kumar NM, Gilula NB (1996) The gap junction communi- Xia JH, Liu CY, Tang BS, Pan Q, Huang L, Dai HP, Zhang cation channel. Cell 84:381–388 BR, Xie W, Hu DX, Zheng D, Shi XL, Wang DA, Xia K, Lautermann J, Frank HG, Jahnke K, Traub O, Winterhager E Yu KP, Liao XD, Feng Y, Yang YF, Xiao JY, Xie DH, Huang (1999) Developmental expression patterns of connexin26 JZ (1998) Mutations in the gene encoding gap junction pro- and -30 in the rat cochlea. Dev Genet 25:306–311 tein b-3 associated with autosomal dominant hearing im- Lautermann J, ten Cate WJ, Altenhoff P, Gru¨ mmer R, Traub pairment. Nat Genet 20:370–373 Downloaded from jmg.bmjjournals.com on 12 March 2006

A novel deletion involving the connexin-30 gene, GJB6del( -d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment

F J del Castillo, M Rodríguez-Ballesteros, A Álvarez, T Hutchin, E Leonardi, C A de Oliveira, H Azaiez, Z Brownstein, M R Avenarius, S Marlin, A Pandya, H Shahin, K R Siemering, D Weil, W Wuyts, L A Aguirre, Y Martín, M A Moreno-Pelayo, M Villamar, K B Avraham, H-H M Dahl, M Kanaan, W E Nance, C Petit, R J H Smith, G Van Camp, E L Sartorato, A Murgia, F Moreno and I del Castillo

J. Med. Genet. 2005;42;588-594 doi:10.1136/jmg.2004.028324

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LETTER TO JMG A novel deletion involving the connexin-30 gene, del(GJB6- d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment F J del Castillo, M Rodrı´guez-Ballesteros, A A´lvarez, T Hutchin, E Leonardi, C A de Oliveira, H Azaiez, Z Brownstein, M R Avenarius, S Marlin, A Pandya, H Shahin, K R Siemering, D Weil, W Wuyts, L A Aguirre, Y Martı´n, M A Moreno-Pelayo, M Villamar, K B Avraham, H-H M Dahl, M Kanaan, W E Nance, C Petit, R J H Smith, G Van Camp, E L Sartorato, A Murgia, F Moreno, I del Castillo ......

J Med Genet 2005;42:588–594. doi: 10.1136/jmg.2004.028324

earing impairment is a common and highly hetero- geneous sensory disorder. Genetic causes are thought Key points to be responsible for more than 60% of the cases in H 1 developed countries. In the majority of cases, non-syndromic N DFNB1 deafness, caused by mutations in the gene hearing impairment is inherited in an autosomal recessive encoding connexin-26 (GJB2), is the most frequent pattern.2 Thirty eight different loci and 20 genes for subtype of autosomal recessive non-syndromic hearing autosomal recessive non-syndromic hearing impairment impairment. Molecular testing for GJB2 mutations has (ARNSHI) have been identified to date.3 become a standard diagnostic approach for subjects In many populations, up to 50% of all cases of ARNSHI are with this disorder. However, 10–50% of affected caused by mutations in the DFNB1 locus (MIM 220290) on subjects with GJB2 mutations carry only one mutant 13q12.4 This locus contains the GJB2 gene (MIM 121011), allele. 5 encoding connexin-26 (Cx26), which belongs to a family of N A 309 kb deletion truncating the GJB6 gene (encoding transmembrane proteins with about 20 members in humans. connexin-30) was shown to be the accompanying Hexamers of connexins (connexons) are displayed in the mutation in up to 50% of deaf GJB2 heterozygotes in plasma membrane. Docking of connexons on the surfaces of different populations. We report the molecular char- two adjacent cells results in the formation of intercellular gap acterisation of the breakpoint junction of a novel 232 junction channels.6 Several different connexins, including kb deletion in the DFNB1 locus, del(GJB6-D13S1854), Cx26, have been shown to participate in the complex gap which was also found in trans with pathogenic GJB2 junction networks of the cochlea.78 It has been postulated that these networks play a key role in potassium homeostasis, mutations in affected subjects. The deletion arose by which is essential for the sound transduction mechanism.9 unequal homologous recombination, involving an AluY Given the high prevalence of DFNB1 deafness, molecular sequence inside GJB6 intron 2, a mechanism which testing for GJB2 mutations has become the standard of care might generate other deletions at DFNB1. for the diagnosis of patients with non-syndromic hearing N We developed a novel diagnostic test for the combined impairment of unknown cause.10 However, the finding of a detection of del(GJB6-D13S1830) and this new large number of affected subjects with only one GJB2 mutant del(GJB6-D13S1854) in a single PCR assay. The allele complicates the molecular diagnosis of DFNB1 deaf- del(GJB6-D13S1854) mutation accounts for 25.5% of ness. In different studies, these have accounted for 10–50% of the affected GJB2 heterozygotes which remained deaf subjects with GJB2 mutations.4 It was hypothesised that unresolved after screening for del(GJB6-D13S1830) there could be other mutations in the DFNB1 locus but in Spain, 22.2% in the UK, 6.3% in Brazil, and 1.9% in outside the GJB2 gene. This hypothesis gained support by the northern Italy. It was not found in affected GJB2 finding of a deletion in the DFNB1 locus outside GJB2 but heterozygotes from France, Belgium, Israel, the truncating the neighbouring GJB6 gene (MIM 604418), Palestinian Authority, USA, or Australia. which encodes connexin-30 (Cx30), another component of N Haplotype analysis revealed a common founder for the the gap junction networks of the cochlea. This deletion, mutation in Spain, Italy, and the UK. Our data further named del(GJB6-D13S1830), was found in affected subjects support the complexity of the genetic epidemiology of either in homozygosity or in double heterozygosity with a non-syndromic hearing impairment. GJB2 mutation.11–13 Isolation and sequencing of the deletion breakpoint junction revealed the loss of a DNA segment initially thought to be 342 kb in size but currently estimated to be 309 kb.12 14 Italy.14 Recent studies have found, however, that the deletion In a multicentre study, it was shown that the del(GJB6- is present in northern Italy at frequencies similar to those of D13S1830) mutation is most frequent in Spain, France, the other European countries (15 and Murgia A, Leonardi E, United Kingdom, Israel, and Brazil (5.9–9.7% of all DFNB1 alleles); it is less frequent in the USA, Belgium, and Australia Abbreviations: ARNSHI, autosomal recessive non-syndromic hearing (1.3–4.5% of all DFNB1 alleles), and is very rare in southern impairment

www.jmedgenet.com Downloaded from jmg.bmjjournals.com on 12 March 2006 Letter 589 unpublished data). The deletion was also found in other and Australia. After getting written informed consent, blood studies in the USA16–19 and Germany,20 but not in Austria,21 samples were obtained and DNA was extracted by standard Turkey,22 23 or China.24 Although the finding of the del(GJB6- procedures. D13S1830) mutation provided an explanation for the hearing Novel microsatellite markers were developed in the DFNB1 impairment in as many as 30–70% affected GJB2 hetero- region by searching for tandem repeats of the CA dinucleo- zygotes in some populations, it has become evident that other tide in sequence contig NT_024524.13 (National Center for DFNB1 mutations remain to be identified in most countries.14 Biotechnology Information database, Homo sapiens genome Here we report the molecular characterisation of a novel view, build 34) and by designing flanking primers: deletion, also truncating the GJB6 gene, but resulting in the loss of a DNA segment shorter than in del(GJB6-D13S1830). N marker D13S1853: forward primer 59-CAGACTGGCAC- AAACTTAACTG-39; reverse primer, 59-TGTACATCTCTT- METHODS CTTACATTCATGT-39 (annealing temperature, 56˚C); This study was done on probands with ARNSHI and their N marker D13S1854: forward primer, 59-CTCCATCCTGGG- relatives from Spain, Italy, France, Belgium, the United TGACAGAGTGAG-39; reverse primer, 59-AGGAAGAGCT- Kingdom, Israel, the Palestinian Authority, the USA, Brazil, GGGGTTGCTAAGAA-39 (annealing temperature, 58˚C).

A

D13S1316 D13S141 (GAAA)n D13S175 (GGAA)n (CA)n D13S1854 D13S1853 D13S1830

50 kb CEN TEL

GJA3 GJB2 GJB6 CRYL1

S N B S N S S S SN

0 5 10 kb

GJB6 gene 3’UTR CDSExon 2 Exon 1

Exon 3 Probe 2R

C I-1 I-2

II-1 Control

del bp (~14 kb) 9416 wt (~7.5 kb) 6557

Figure 1 Map of the DFNB1 region on 13q12, and Southern blot analysis of family E079. (A) Map of a 600 kb DNA segment including the DFNB1 locus. The positions of polymorphic genetic markers are indicated by vertical bars. Genes in the region are depicted as horizontal bars or arrows. GJA3 encodes connexin-46 (MIM 121015) and CRYL1 codes for l-crystallin. The two breakpoints of the del(GJB6-D13S1854) mutation are marked by vertical arrows, and the extent of the deletion is indicated by the dashed line. An empty arrowhead indicates the distal end of the previously reported del(GJB6-D13S1830) mutation. (B) Physical map of a 10 kb DNA segment containing the GJB6 gene. Restriction sites are indicated by vertical bars. N, NsiI; S, SspI. The structure of the GJB6 gene is shown below the map. Exons are depicted as boxes, introns as thin lines. 39-UTR, 39 untranslated region; CDS, GJB6 coding region. A vertical arrowhead marks the deletion breakpoint internal to GJB6. The position of probe 2R, used in the Southern blot analysis, is indicated below the gene. (C) Southern blot analysis of family E079 with probe 2R on NsiI digests of genomic DNA. Polymerase chain reaction amplification of probe 2R and Southern blotting experiments were carried out as reported.12 An approximately 7.5 kb band (wt) is revealed in all subjects of family E079 and in the control. In addition, a novel 14 kb band (del), created by the deletion, is revealed in affected subject II:1 (double heterozygote, 35delG in GJB2/del(GJB6-D13S1854)), and in his father, I:1 (del(GJB6-D13S1854) carrier). This band is absent in the control subject and in the proband’s mother, I:2 (35delG carrier).

www.jmedgenet.com Downloaded from jmg.bmjjournals.com on 12 March 2006 590 Letter

A CRYL1 gene, GJB6 gene, intron 4 intron 2

TEL CEN C A G G A GA AT G G C G T G A AC C CCCAG G G A G G C G G A G T T G G T G

B

Alu GJB6 GAGATGGAGTCTAGCTCTGTTGCCCAAGCTGGAGTACCGTGGCACGATCT 50

Alu CRYL1 GAGACGGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGCTATCT 50 Break site Alu GJB6 CGGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCGTTCTCCCGCCTCA 100

Alu CRYL1 CGGCTCACTGCAAGCTCCGCCACCCGGGTTCACGCCATTCTCCTGCCTCA 100

Alu GJB6 GCCTCTCTGAGCAGCTGGGACTACAGGCGCCTGCCACCAAGCCCGGCTAA 150

Alu CRYL1 GCCTCCCA-AGTAGCCGGGACTACAGGCGCCCGCCACTACGCCCGGCTAA 149

Alu GJB6 TTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGATGG 200

Alu CRYL1 TTTTTTGTATTTTTAGTAGAGATGGGGTTTCACTGTGTTAGCCAGAATGG 199

Alu GJB6 TCTCGATCTCCTGACCTCATGATCCGCCCCCCTTGGCCTCCCAAAGTGCT 250

Alu CRYL1 TCTCGATCTCCTGACATCGTGATCTGCCCGTCTCCGCCTCCCAAAGTGCT 249

Alu GJB6GGGATTACAAGCATGAGCCACCGCGCCCGGCC 282

Alu CRYL1GGGATTACAGACGTGAGCCACTGCGCCCCGCC 281

Figure 2 Breakpoint junction of the del(GJB6-D13S1854) deletion. Sequencing of the breakpoint junction was done directly on the polymerase chain reaction product obtained with primers DelBK1 and DelBK2. We used sequencing primer DelBK3, 59-TCTTCTGTATGTATGTCACTTTTCA-39. (A) Electropherogram of a DNA segment containing the breakpoint junction (boxed) and flanking sequences. The relative positions of the centromere (cen) and telomere (tel) of the long arm of are indicated to show the orientation of the sequenced DNA segment. (B) Alignment of the sequences flanking the proximal and distal deletion breakpoints. The alignment was performed using BLASTN software (National Center for Biotechnology Information), which reported an identity of 88% along 282 nucleotides.

RESULTS Conversely, haplotype analysis revealed no segregation We reported previously on 39 unrelated Spanish subjects inconsistencies, but there was heterozygosity for marker with ARNSHI, who were heterozygous for one GJB2 mutant D13S1830 in either the proband or the parents of all four allele and did not carry the del (GJB6-D13S1830) mutation.14 cases. Together, these data suggested the existence of (at All these cases carried alleles which are considered unam- least) a novel deletion at the DFNB1 locus, involving marker biguously pathogenic.10 After excluding 11 cases not linked to D13S175 but not D13S1830. DFNB1 on the basis of haplotype analysis of siblings, there To simplify the search for the deletion breakpoints, we remained 28 unelucidated heterozygotes.14 We genotyped the assumed that the deletion in these four cases would be the proband, parents, and siblings from these 28 cases for same. Haplotype analysis for other microsatellite markers microsatellite markers D13S17525 and D13S1830,26 both of from the DFNB1 locus11 (fig 1A) revealed inconsistencies in which are deleted in the del(GJB6-D13S1830) mutation the segregation of alleles, which allowed us to map the (fig 1A). In two multiplex cases (S591, S630) and two telomeric breakpoint distal to (CA)n, and the centromeric simplex cases (E079, E262), haplotype analysis revealed breakpoint between markers (GAAA)n and D13S175. These inconsistencies in the segregation of alleles of marker results suggested that the centromeric breakpoint could be D13S175, suggesting the presence of an unamplifiable allele. inside the GJB6 gene. We tested this hypothesis by Southern The same results were obtained when using an alternative blotting (fig 1, panels B and C). Probe 2R was assayed on SspI primer pair12 flanking the microsatellite at D13S175. digests of genomic DNA from the proband and parents of

www.jmedgenet.com Downloaded from jmg.bmjjournals.com on 12 March 2006 Letter 591 case E079 in order to investigate whether the GJB6 coding the sequence stretch containing GJB6 intron 1, exon 2, and region, fully contained in exon 3 (fig 1B), was intact. This intron 2 with the sequence spanning the interval between probe did not show any change in dosage of GJB6 exon 3 or in D13S1854 and D13S1853. This analysis revealed the existence the SspI restriction pattern when comparing deletion carriers of a 282 bp Alu sequence inside GJB6 intron 2 sharing 88% with control subjects (data not shown). To investigate identity with another Alu repeat located in direct orientation whether the deletion could involve other parts of the GJB6 inside the D13S1854–D13S1853 interval. We designed pri- gene, we assayed probe 2R on NsiI digests of genomic DNA mers flanking this candidate breakpoint junction, and a from deletion carriers and control subjects (fig 1, panels B polymerase chain reaction (PCR) product of about 560 bp and C). In addition to the expected 7.5 kb band, a novel 14 kb was obtained only from DNA samples of deletion carriers. band, created by the deletion, was observed in the deletion Sequencing of this PCR product revealed the deletion carriers (fig 1C). As this 14 kb band has the expected size breakpoint junction (fig 2A), which was the same in all four based upon the predicted restriction map, these findings led studied cases. us to conclude that the deletion truncated GJB6. This novel deletion was named del(GJB6-D13S1854). To locate the deletion distal breakpoint, we searched for Examination of the breakpoint junction supported the novel microsatellite markers in the interval between (CA)n hypothesis that it originated from homologous recombina- and D13S1830 (see Methods). In all four cases with the tion between two Alu sequences which belong to the Y deletion, genotyping and haplotype analysis revealed hetero- subfamily, as shown by RepeatMasker software27 (fig 2B). zygosity and consistent segregation for marker D13S1853, The proximal repeat is located in GJB6 intron 2, and the distal but inconsistencies in the segregation of alleles of marker repeat is in intron 4 of the gene encoding l-crystallin (CRYL1, D13S1854 (fig 1A). These data placed the distal deletion GenBank AF077049). The exact breakpoints could not be breakpoint between D13S1854 and D13S1853, an interval of determined as the breaks could have taken place at any point about 9.5 kb. Thus we undertook a BLASTN comparison of of two identical 14 bp stretches (fig 2B). The deletion spans

A

(GAAA)n D13S175 (GGAA)n (CA)n D13S1854 D13S1853 D13S1830

CEN TEL

GJB6 DelBK2 BKR-1

Exon 3 Exon 2 Exon 1

GJB6-1R DelBK1 Cx30Ex1A Cx30Ex1B

B ABCDE

del(GJB6-D13S1854) 564 bp del(GJB6-D13S1830) 460 bp

GJB6 exon 1 333 bp

Figure 3 Single test for the detection of del(GJB6-D13S1830) and del(GJB6-D13S1854). The rationale of the method is to amplify DNA segments containing the breakpoint junction of each deletion, as well as a segment containing GJB6 exon 1, which is used as a control to check the efficiency of the polymerase chain reaction (PCR) and to distinguish heterozygosity v homozygosity for any of the two deletions (GJB6 exon 1 is removed by both deletions). (A) Schematic drawing showing the location of the primers used in the multiplex PCR assay. GJB6-1R, 59- TTTAGGGCATGATTGGGGTGATTT-39, and BKR-1, 59-CACCATGCGTAGCCTTAACCATTTT-39 (for amplification of the del(GJB6-D13S1830) breakpoint junction); DelBK1, 59-TCATAGTGAAGAACTCGATGCTGTTT-39, and DelBK2, 59-CAGCGGCTACCCTAGTTGTGGT-39 (for amplification of the del(GJB6-D13S1854) breakpoint junction); Cx30Ex1A, 59-CGTCTTTGGGGGTGTTGCTT-39, and Cx30Ex1B, 59- CATGAAGAGGGCGTACAAGTTAGAA-39 (to amplify GJB6 exon 1). PCR was carried out using the following programme: one cycle of denaturation at 95˚C for five minutes; five touchdown cycles of denaturation at 94˚C for 40 seconds, and annealing for 40 seconds at 65˚C for the first cycle and a 1˚C reduction per cycle; 25 cycles of denaturation at 94˚C for 40 seconds, and annealing at 60˚C for 40 seconds; and a final extension step of 72˚C for seven minutes. The reaction took place in a final volume of 15 ml, at a final concentration of 1.5 mM MgCl2, using Fast Start Taq DNA polymerase (Roche). (B) Separation of the PCR products by electrophoresis in a 1.5% agarose gel. The position of the PCR products corresponding to the deletion breakpoint junctions and to GJB6 exon 1 are indicated by arrows on the left, and their sizes in base pairs (bp) are shown on the right. All the PCR products obtained in this multiplex reaction were sequenced to confirm their identities and validate the test. A, Wild type (wt); B, del(GJB6-D13S1854)/ wt heterozygote; C, del(GJB6-D13S1830)/del(GJB6-D13S1854) compound heterozygote; D, del(GJB6-D13S1830)/wt heterozygote; E, del(GJB6- D13S1830) homozygote.

www.jmedgenet.com Downloaded from jmg.bmjjournals.com on 12 March 2006 592 Letter

Table 1 Results from the screenings for the del(GJB6-D13S1854) mutation

No of DFNB1 heterozygotes carrying del(GJB6-D13S1854)/No of DFNB1 Accompanying DFNB1 mutant allele Country/laboratory heterozygotes (No of cases)

Spain 12/47 (25.5%) 35delG (10), V37I (1), del(GJB6- D13S1830) (1) Italy 1/53 (1.9%) 35delG (1) France 0/40 Belgium 0/20 United Kingdom 4/18 (22.2%) 35delG (4) Israel 0/11 USA/Virginia 0/92 USA/Iowa 0/88 Brazil 1/16 (6.3%) V37I (1) Australia 0/27

232 kb. It could create a chimeric gene by joining CRYL1 Palestinian Arab unrelated subjects with ARNSHI, who did intron 4 with GJB6 intron 2. The hypothetical chimeric mRNA not carry any GJB2 mutation. would contain the first three exons of CRYL1 and GJB6 exon DNA sequencing confirmed that the breakpoint junction 3. However, no product is likely as there is an in-frame stop was the same in all the positive cases found in Spain, Italy, codon in between the two open reading frames. the United Kingdom, and Brazil. We investigated the We developed a single test for the detection of both evolutionary origins of the deletion by studying haplotypes deletions, which is useful for routine molecular diagnosis associated with this mutation (table 2). All chromosomes (fig 3). The del (GJB6-D13S1854) mutation was found in carrying the deletion share a core haplotype composed of trans in 12 of 47 Spanish unrelated affected subjects which allele 209 from marker (GAAA)n (frequency of this allele in were unresolved DFNB1 heterozygotes (25.5 %) (table 1). Spain, 0.415), and allele 204 of marker D13S1853 (frequency Their hearing impairments ranged from mild to profound. of this allele in Spain, 0.411) (table 2). These markers are Interestingly, one subject was a compound heterozygote for very close to the deletion breakpoints, at distances of only 9 the two deletions (fig 3, lane C). We also screened 604 and 6 kb, respectively. An expanded haplotype with all the additional Spanish unrelated subjects with ARNSHI who four markers revealed four variants associated with the were not carriers of DFNB1 mutations. One of these was deletion, the most frequent being haplotype A, from which found to be heterozygous for del(GJB6-D13S1854). The the other three could have arisen through single recombina- deletion was not found in 100 control subjects with normal tion events (table 2). Our results show that all the studied hearing. After this screening, the unelucidated heterozygotes chromosomes carrying the del (GJB6-D13S1854) mutation in in our sample represent 12.2% of the total number of subjects Spain, the United Kingdom, and Italy share a common with at least one DFNB1 mutation (36/295). Excluding 11 founder. cases not linked to DFNB1, this figure drops to 8.5% (25/295). With a frequency of 2.2% (12/548), the del(GJB6-D13S1854) DISCUSSION mutation is among the five most common DFNB1 alleles in The hypothesis of digenic inheritance of DFNB1 hearing our Spanish sample. impairment has received theoretical support from several A multicentre study was conducted to investigate the observations. Both Cx26 and Cx30 are expressed in the same prevalence of the novel deletion in different countries inner ear structures.28 29 Moreover, connexons composed of (table 1). The del(GJB6-D13S1854) mutation was found to Cx26 can bind connexons composed of Cx30 to form account for 22.2% of affected GJB2 heterozygotes who were heterotypic gap junction channels.30 It was also reported that unresolved after screening for del(GJB6-D13S1830) in the a GJB6 mutation results in autosomal dominant hearing United Kingdom, for 6.3% in Brazil, and for 1.9% in northern impairment in humans,31 and that Cx30 deficient mice lack Italy. It was not found in screening carried out on samples the endocochlear potential and have a severe constitutive from France, Belgium, Israel, the USA, or Australia. The novel hearing impairment.32 However, the fact that point mutations deletion was not found in 159 Israeli Jewish and 40 in GJB6 have not yet been found in cases of ARNSHI in

Table 2 Haplotypes associated with the del(GJB6-D13S1854) mutation

Haplotype* Genotype for CEPH individual Marker Heterozygosity` (%) ABCD134702

(TG)n 65 208 204 206 208 206/208 (GAAA)n 79 209 209 209 209 209/216 D13S1853 66 204 204 204 204 202/202 D13S1830 71 153 153 153 156 153/156 Number and geographical distribution of 6 Spain 1 Spain 1 Spain 2 Spain haplotypes (n = 14) 2UK 1UK 1 Italy Total: 9 Total: 2 Total: 1 Total: 2

*We only report here those cases in which the haplotype associated with the deletion could be determined unambiguously. Allele sizes were determined by DNA sequencing of a control sample, which was used as a standard in genotyping assays. To allow other laboratories to compare their data with those reported in this work, we provide allele sizes for individual 134702, available from CEPH.25 Relative order and physical distances are as follows: (TG)n – 110 kb – (GAAA)n – 9 kb – deletion proximal breakpoint – 232 kb – deletion distal breakpoint – 6 kb – D13S1853 – 60 kb – D13S1830. `Calculated from 100 Spanish control chromosomes.

www.jmedgenet.com Downloaded from jmg.bmjjournals.com on 12 March 2006 Letter 593 humans argues against this hypothesis. In addition, Cx26+/2/ illustrate the complexity of the genetic epidemiology of non- Cx30+/2 double heterozygous mice have only a moderate syndromic hearing impairment. hearing impairment,33 in contrast with the phenotype observed in humans, where most double heterozygotes for ...... del(GJB6-D13S1830) and a GJB2 mutation have severe or Authors’ affiliations ´ profound hearing impairment.11–13 15 16 19 20 34 35 F J del Castillo*, M Rodrı´guez-Ballesteros*,AAlvarez, L A Aguirre, An alternative hypothesis postulates the existence of a cis Y Martı´n, M A Moreno-Pelayo, M Villamar, F Moreno, I del Castillo, Unidad de Gene´tica Molecular, Hospital Ramo´n y Cajal, Madrid, Spain acting regulatory element which would activate the expres- D Weil, C Petit, Unite´deGe´ne´tique des De´ficits Sensoriels INSERM sion of GJB2 in the inner ear. This regulatory element would U587, Institut Pasteur, Paris, France have been removed by the deletions, and its absence would T Hutchin, Clinical Chemistry, Birmingham Children’s Hospital, have dramatic effects on the expression of GJB2, to the point Birmingham, UK that an otherwise normal allele would behave as a null allele. E Leonardi, A Murgia, Department of Paediatrics, University of Padua, Both hypotheses can be combined—that is, the main Padua, Italy pathogenic effect of the deletions might be caused by the C A de Oliveira, E L Sartorato, Centro de Biologia Molecular e GJB2 expression deficit, but haploinsufficiency for Cx30 may Engenharia Gene´tica (CBMEG), Universidade Estadual de Campinas, Saˆo Paulo, Brazil contribute to worsening of the phenotype. H Azaiez, M R Avenarius, R J H Smith, Interdepartmental Human The 232 kb sequence stretch removed by del(GJB6- Genetics Program and the Department of Otolaryngology, University of D13S1854) is still too large to search for a regulatory element. Iowa, Iowa City, Iowa, USA Molecular characterisation of other DNA rearrangements in Z Brownstein, K B Avraham, Department of Human Genetics and the DFNB1 locus leading to hearing impairment may help to Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel define a smaller interval. Under the hypothesis of the Aviv, Israel regulatory element, it is predicted that another class of S Marlin, Unite´deGe´ne´tique Me´dicale, Hoˆpital Trousseau, Paris, France deletions, leading to hearing impairment but not truncating A Pandya, W E Nance, Department of Human Genetics, Medical College of Virginia of Virginia Commonwealth University, Richmond, GJB6, might also be present in the DFNB1 locus. After Virginia, USA screening for the deletions so far reported, affected GJB2 H Shahin, M Kanaan, Life Sciences Department, Bethlehem University, heterozygotes still represent 8–30% of all subjects with Bethlehem, Palestinian Authority mutations in GJB2 in different populations (14 and this K R Siemering, The Murdoch Children’s Research Institute, Royal study). These figures are far from what should be expected if Children’s Hospital, Melbourne, Australia these GJB2 heterozygotes were just coincidental carriers. W Wuyts, G Van Camp, Department of Medical Genetics, University of Although hypothetical epistatic interactions between GJB2 Antwerp, Antwerp, Belgium mutations and other unlinked gene(s) might contribute to H-H M Dahl, Department of Paediatrics, University of Melbourne, Melbourne, Australia this situation, additional mutations in DFNB1, not yet identified, are also likely to exist. The AluY sequence *These authors contributed equally to this work contained in GJB6 intron 2 has the potential of generating We thank the patients and their relatives for their kind cooperation in this deletions affecting this gene, by homologous recombination study, and FIAPAS for their enthusiastic support of this research. FJdC with other highly similar repeats along the DFNB1 locus. Alu/ and MV were recipients of fellowships from the Comunidad de Madrid. Alu recombination leading to deletion is a common disease MRB and AA were recipients of fellowships from Fondo de causing mechanism.36 Investigaciones Sanitarias. LA was a recipient of a fellowship from the Both del(GJB6-D13S1830) and del(GJB6-D13S1854) inac- Organizacio´n Nacional de Ciegos Espan˜oles. This work was supported by grants from the European Community (QLG2-CT-1999-00988), tivate the CRYL1 gene and remove the sequence interval CAICYT of Spanish Ministerio de Ciencia y Tecnologı´a (SAF2002- between GJB6 and CRYL1, where no additional genes have 03966, to FM), Spanish Research Network on the Genetic and been reported so far. The CRYL1 gene is widely expressed, and Molecular Bases of Hearing Disorders (FIS G03/203, to FM), its product, l-crystallin, shows similarity with 3-hydroxyacyl- Programa Ramo´n y Cajal (to IdC), Spanish Fondo de Investigaciones CoA dehydrogenase.37 The contribution of l-crystallin to Sanitarias (FIS PI020807, to IdC), the Israel Ministry of Science and DFNB1 hearing impairment, if any, remains enigmatic. To Technology (to KBA), and the National Institutes of Health (RO1- DC02842, to RJHS). date, subjects carrying either del(GJB6-D13S1830) or del(GJB6-D13S1854) do not present with any eye disorder Competing interests: none declared (12 and this study). Correspondence to: Dr Ignacio del Castillo, Unidad de Gene´tica Our multicentre study reveals significant differences in the Molecular, Hospital Ramo´n y Cajal, Carretera de Colmenar, Km 9, frequency of each of the deletions, and also different patterns 28034 Madrid, SPAIN; [email protected] of geographical distribution. The del(GJB6-D13S1830) muta- tion, found in many populations over the world, is much more frequent than del(GJB6-D13S1854), which is for the REFERENCES present restricted to a few countries. Both mutant alleles are 1 Petit C, Levilliers J, Hardelin JP. Molecular genetics of hearing loss. Annu Rev Genet 2001;35:589–646. frequent in Spain and the United Kingdom (the combined 2 Friedman TB, Griffith AJ. 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GJB2 Mutations and Degree of Hearing Loss: A Multicenter Study Rikkert L. Snoeckx, Patrick L. M. Huygen, Delphine Feldmann, Sandrine Marlin, Franc¸oise Denoyelle, Jaroslaw Waligora, Malgorzata Mueller-Malesinska, Agneszka Pollak, Rafal Ploski, Alessandra Murgia, Eva Orzan, Pierangela Castorina, Umberto Ambrosetti, Ewa Nowakowska-Szyrwinska, Jerzy Bal, Wojciech Wiszniewski, Andreas R. Janecke, Doris Nekahm-Heis, Pavel Seeman, Olga Bendova, Margaret A. Kenna, Anna Frangulov, Heidi L. Rehm, Mustafa Tekin, Armagan Incesulu, Hans-Henrik M. Dahl, Desire´e du Sart, Lucy Jenkins, Deirdre Lucas, Maria Bitner-Glindzicz, Karen B. Avraham, Zippora Brownstein, Ignacio del Castillo, Felipe Moreno, Nikolaus Blin, Markus Pfister, Istvan Sziklai, Timea Toth, Philip M. Kelley, Edward S. Cohn, Lionel Van Maldergem, Pascale Hilbert, Anne-Franc¸oise Roux, Michel Mondain, Lies H. Hoefsloot, Cor W. R. J. Cremers, Tuija Lo¨ppo¨nen, Heikki Lo¨ppo¨nen, Agnete Parving, Karen Gronskov, Iris Schrijver, Joseph Roberson, Francesca Gualandi, Alessandro Martini, Genevie`ve Lina-Granade, Nathalie Pallares-Ruiz, Ce´u Correia, Grac¸a Fialho, Kim Cryns, Nele Hilgert, Paul Van de Heyning, Carla J. Nishimura, Richard J. H. Smith, and Guy Van Camp*

Hearing impairment (HI) affects 1 in 650 newborns, which makes it the most common congenital sensory impair- ment. Despite extraordinary genetic heterogeneity, mutations in one gene, GJB2, which encodes the connexin 26 protein and is involved in inner ear homeostasis, are found in up to 50% of patients with autosomal recessive nonsyndromic hearing loss. Because of the high frequency of GJB2 mutations, mutation analysis of this gene is widely available as a diagnostic test. In this study, we assessed the association between genotype and degree of hearing loss in persons with HI and biallelic GJB2 mutations. We performed cross-sectional analyses of GJB2 genotype and audiometric data from 1,531 persons, from 16 different countries, with autosomal recessive, mild- to-profound nonsyndromic HI. The median age of all participants was 8 years; 90% of persons were within the age range of 0–26 years. Of the 83 different mutations identified, 47 were classified as nontruncating, and 36 as truncating. A total of 153 different genotypes were found, of which 56 were homozygous truncating (T/T), 30 were homozygous nontruncating (NT/NT), and 67 were compound heterozygous truncating/nontruncating (T/ NT). The degree of HI associated with biallelic truncating mutations was significantly more severe than the HI associated with biallelic nontruncating mutations (P ! .0001 ). The HI of 48 different genotypes was less severe than that of 35delG homozygotes. Several common mutations (M34T, V37I, and L90P) were associated with mild- to-moderate HI (median 25–40 dB). Two genotypes—35delG/R143W (median 105 dB) and 35delG/dela(GJB6- D13S1830) (median 108 dB)—had significantly more-severe HI than that of 35delG homozygotes.

Introduction and mitochondrial (!1%) inheritance also occur. In 30% of cases, additional physical findings lead to the diag- Hearing impairment (HI) affects 1 in 650 newborns (Mehl nosis of 1 of 1400 syndromes in which hearing loss can and Thomson 2002), which makes it the most frequent be a clinical component. In the remaining 70% of cases, congenital sensory impairment. In most of these cases, nonsyndromic HI is diagnosed (Morton 1991). the inheritance pattern is autosomal recessive (80%), al- Nonsyndromic HI is extraordinarily heterogeneous— though autosomal dominant (17%), X-linked (2%–3%), ∼100 localizations have been reported across the genome as sites of genes causally related to nonsyndromic HI, and 37 different genes encoding proteins with a wide Received June 22, 2005; accepted for publication September 8, 2005; electronically published October 19, 2005. variety of functions have been identified. Despite this Address for correspondence and reprints: Dr. Guy Van Camp, De- degree of heterogeneity, variants of one gene, GJB2 partment of Medical Genetics, University of Antwerp, Universiteit- (MIM 121011), account for up to 50% of cases of splein 1, B-2610 Antwerp, Belgium. E-mail: [email protected] autosomal recessive nonsyndromic HI in many world * The authors’ affiliations can be found in the Acknowledgments. ᭧ 2005 by The American Society of Human Genetics. All rights reserved. populations, which makes GJB2 the gene most fre- 0002-9297/2005/7706-0007$15.00 quently associated with this condition (Kenneson et al.

945 946 Am. J. Hum. Genet. 77:945–957, 2005

2002). A few specific mutations in GJB2 also have been phenotype correlations, but only a few associations have described in families with autosomal dominant HI, with been recognized, a limitation reflecting the large number and without skin manifestations, although their preva- of genotypes and a small number of affected patients lence is low (Denoyelle et al. 1998). in most series. GJB2 encodes the connexin 26 (CX26) protein, a In the largest study reported earlier, we investigated member of the connexin family of highly related gap- possible genotype-phenotype correlations in a data set junction proteins. Connexins oligomerize to form hexa- of 277 unrelated hearing impaired persons with biallelic meric hemichannels called “connexons,” which are pres- GJB2 mutations and showed that persons homozy- ent in the plasma membrane, where they can bind with gous for the 35delG mutation have significantly more- connexons from adjacent cells to form functional gap severe HI than do 35delG/non-35delG compound het- junctions (Bruzzone et al. 1996). These junctional chan- erozygotes. Persons with two non-35delG mutations nels are permeable to ions and small metabolites with have even less severe HI. Because of data set size, we molecular masses up to 1,200 Da (Harris and Bevans were able to develop only a few specific genotype-phe- 2001). They can be composed of one (homomeric) or notype correlations (Cryns et al. 2004). This limitation more (heteromeric) connexins, thereby modifying selec- prompted us to complete this large multicenter study to tive permeability of gap junctions (Stauffer 1995). describe more-detailed genotype-phenotype associations Expression of GJB2 has been documented in a variety for this frequent form of hereditary HI. of cells and tissues. In the cochlea, CX26-containing gap ϩ junctions are proposed to maintain K homeostasis by Material and Methods ferrying Kϩ away from the hair cells during auditory transduction (Kikuchi et al. 1995). Recently, it has been Patient Recruitment shown that the intercellular transduction of the second messenger inositol triphosphate (IP3) is also essential for Persons with congenital HI were sequentially accrued the perception of sound (Beltramello et al. 2005). In the from otolaryngology departments and genetics units. All epidermis, CX26-containing gap junctions play a role patient information was obtained between 1980 and in coordinated keratinocyte growth and differentiation 2003. Individuals with syndromic, unilateral, acquired, (Choudhry et al. 1997), which explains the skin abnor- or dominant types of HI were excluded from this study. malities associated with autosomal dominant HI in per- The clinical evaluation included a complete history, phys- sons who segregate a few mutations in GJB2 that affect ical examination, and audiometry. Information on eth- the first extracellular domain of CX26 and exert a dom- nicity was obtained by a combination of self-reporting inant negative effect by impairing interconnexon dock- and physician assessment. Most Arabs were collected by ing (Maestrini et al. 1999; Heathcote et al. 2000; Al- the Pediatric Molecular Genetics Unit, Ankara Univer- varez et al. 2003). sity School of Medicine (Ankara, Turkey), and the De- Many HI-causing mutations of GJB2 have been re- partment of Human Genetics and Molecular Medicine, ported (Connexin-Deafness Home Page), some of which Tel Aviv University (Tel Aviv). The latter research group are very common and others of which are extremely also collected the majority of Ashkenazi Jews. The Af- rare. The mutation spectrum diverges substantially rican and Asian participants, as well as some Arabs, were among populations, as reflected by specific ethnic biases immigrants. Informed consent to allow genetic testing for common mutations like 35delG among whites (car- was obtained from all participants or from the parents rier rate of 2%–4%) (Zelante et al. 1997; Estivill et al. of minors. 1998; Green et al. 1999), 235delC in the Japanese (car- We collected audiometric data from 1,718 patients rier rate of 1%–2%) (Abe et al. 2000; Kudo et al. 2000), with biallelic GJB2 mutations, including del(GJB6- 167delT in the Ashkenazi Jewish population (carrier D13S1830). Of those, 187 patients were excluded be- rate of 7.5%) (Morell et al. 1998), and V37I in Taiwan cause of additional clinical features or because audio- (carrier rate of 11.6%) (Hwa et al. 2003). Despite this metric data were incomplete. Of the 187, 79 were per- variability, the combined frequency of all GJB2 muta- sons whose data were collected using auditory-brain- tions is sufficiently high in most populations to make stem response (ABR) audiometry (five laboratories), mutation analysis of this gene a clinically useful, and which does not provide complete frequency-specific therefore widely available, genetic test. thresholds. To avoid bias, data from sibships with mul- The HI in persons with biallelic GJB2 mutations ranges tiple affected siblings were reduced to one randomly cho- from mild to profound and is most commonly nonpro- sen hearing impaired individual per family. In the final gressive (Denoyelle et al. 1999; Murgia et al. 1999). Gen- analysis, detailed audiometric data from 1,531 persons erally, phenotypic variability has been attributed to un- were included in this study. All samples were anony- known modifier genes or environmental factors. Pre- mized, to safeguard patient identity and to preclude the vious reports have attempted to address GJB2 genotype- ability to link a given genotype and audiogram to a Snoeckx et al.: GJB2 Genotype-Phenotype Correlation 947

Table 1 Table 1 (continued) Frequencies of Mutations in Study Participants Mutation No. (%) of Alleles Mutation No. (%) of Alleles Nontruncating: M1V 3 (.10) T8M 1 (.03) 284insdupCACGT 1 (.03) G12V 2 (.07) 290insA 2 (.07) K15T 2 (.07) 299-300delAT 4 (.13) I20T 2 (.07) 310del14 52 (1.70) V27I 10 (.33) 313insGG 1 (.03) R32C 2 (.07) 328delG 1 (.03) R32H 4 (.13) 333-334delAA 5 (.16) M34I 1 (.03) 355del9 1 (.03) M34T 123 (4.01) 383ins7 2 (.07) I35S 1 (.03) 511-512insAACG 3 (.10) V37I 75 (2.45) 558dup46 1 (.03) A40E 4 (.13) 631delGT 2 (.07) G45E 1 (.03) W24X 47 (1.53) V52L 1 (.03) W44X 1 (.03) V63M 2 (.07) E47X 43 (1.40) W77R 15 (.49) Q57X 9 (.29) Q80P 2 (.07) C64X 1 (.03) I82M 4 (.13) Y65X 2 (.07) V84L 5 (.16) W77X 6 (.20) L90P 57 (1.86) Q124X 2 (.07) L90V 1 (.03) Y155X 1 (.03) M93I 1 (.03) W172X 2 (.07) V95M 16 (.52) C211X 2 (.07) H100L 1 (.03) del(GJB6)-D13S1830 51 (1.67) H100P 1 (.03) IVS1ϩ1GrA 23 (.75) H100Y 3 (.10) NOTE.—Alleles in bold italics had frequencies 11%. S113R 1 (.03) E114G 4 (.13) delE120 23 (.75) specific individual. Institutional approval for this study R127H 2 (.07) was obtained from the University of Antwerp. L132V 1 (.03) S138N 1 (.03) S139N 3 (.10) Audiometric Evaluation I140S 1 (.03) R143Q 1 (.03) All patients underwent otoscopic examination and au- R143W 13 (.42) diometric testing. In most cases, hearing levels were de- E147K 9 (.29) M151R 1 (.03) termined by pure-tone audiometry, which was completed V153I 5 (.16) using a diagnostic audiometer in a soundproof room, in D159N 1 (.03) accordance with International Standards Organization C174R 1 (.03) (ISO 8253-1-3) standards. Five centers also reported be- R184P 21 (.74) havioral testing results for 117 patients (7.6%), and one R184W 4 (.13) S199F 1 (.03) center reported the use of steady-state evoked potentials N206S 11 (.36) (SSEPs) for 23 patients (1.5%). SSEPs are electrophysio- T208P 2 (.07) logical measures of hearing acuity used extensively in Truncating: Australia, Asia, and Canada. Because the auditory re- 28delC 1 (.03) sponse is phase locked to changes in a continuous tonal 31del14 1 (.03) 31del38 3 (.10) stimulus, a higher average sound pressure level can be 35insG 4 (.13) delivered than is possible with click stimuli; as a result, 35delG 2,218 (72.44) SSEPs can provide an estimate of hearing sensitivity in 51del12insA 5 (.16) children who demonstrate no response to ABR testing. 167delT 91 (2.97) Pure-tone averages (PTAs) in those cases were recorded 176del16 1 (.03) 235delC 19 (.62) as the SSEP best response (always 100–120 dB). 269delT 1 (.03) The binaural mean PTA for air conduction at 0.5, 1,

269insT 7 (.23) and 2 kHz (PTA0.5,1,2kHz) was used to compare subgroups (continued) of patients. Average thresholds in the range of 21–40 948 Am. J. Hum. Genet. 77:945–957, 2005

Figure 1 Relative frequencies of the degree of HI in the three classes of genotypes. The actual number of participants is given in parentheses. The three classes were biallelic truncating (T/T), compound heterozygous truncating/nontruncating (T/NT), and biallelic nontruncating (NT/ NT). There were significant differences among the three classes, with x2 testing (P ! .0001 ). An additional distinction between 35delG and non- 35delG mutations in the T/T and T/NT classes was made, but statistical analysis (x2 test) revealed no significance among these subgroups (data not shown). The number of persons is shown under each subgroup. dB were defined as “mild HI,” in the range of 41–70 heterozygosity with W24X in two unrelated persons, dB as “moderate HI,” in the range of 71–95 dB as “se- these variants were found only once in compound het- vere HI,” and 195 dB as “profound HI” (Smith et al. erozygosity with other GJB2 mutations (table 1). Vari- 2005). ants of debatable pathogenicity, like M34T and V37I, were also included. GJB2 Mutation Analysis Identified allele variants were classified as truncating or nontruncating mutations. The group of truncating Various methods—including DNA sequencing, SSCP, mutations contained nonsense mutations and deletions, denaturing gradient gel electrophoresis, and denaturing insertions, and duplications that introduced a shift in high-performance liquid chromatography—were used for reading frame. The splice-site mutation (IVS1ϩ1GrA) mutation analysis of GJB2. In some cases, the 35delG and one large deletion, del(GJB6-D13S1830), were also mutation was detected by an allele-specific PCR or by classified as truncating, on the basis of published data restriction-enzyme digestion of the PCR product (Scott (del Castillo et al. 2002; Shahin et al. 2002). The group et al. 1998; Storm et al. 1999). The exact nature of all of nontruncating mutations contained amino acid sub- GJB2 variants was confirmed by DNA sequencing. PCR stitutions and one inframe deletion. Although a trans- was used to detect del(GJB6-D13S1830), as described lated protein can be made in the presence of these mu- by del Castillo et al. (2002). We considered all allele tations, for some amino acid substitutions, it is possible variants of GJB2 listed as nonsyndromic HI mutations that functional activity is lost. on the Connexin-Deafness Home Page to be potentially pathologic. We also included novel allele variants not Statistical Analysis yet listed on that Web site, including M34I, I35S, L132V,

S138N, I140S, M151R, and T208P. For all these mu- PTA0.5,1,2kHz was used to compare subgroups of pa- tations, at least 50 controls with normal hearing were tients. Major genotype-based pairwise comparisons were tested, to determine whether we should exclude the mu- done between PTA0.5,1,2kHz thresholds with use of persons tations because they are common polymorphisms. With homozygous for the 35delG mutation as the reference the exception of T208P, which was found in compound group. Fisher’s exact test was performed with2 # 2 con- Snoeckx et al.: GJB2 Genotype-Phenotype Correlation 949 tingency tables of appropriately dichotomized data at Of the remaining 74 mutations with a frequency 11%, the median (50th percentile [P50]), 25th percentile (P25), 27 had frequencies 10.1%; the remainder were very rare, or 5th percentile (P5) of the frequency distribution for with frequencies !0.1%. It is possible that the two mu- ϩ r PTA0.5,1,2kHz of the reference group. Given the multiple tations del(GJB6-D13S1830) and IVS1 1G A were comparisons made, we focused on the most clearly sig- underrepresented in this study. This might be because nificant test results. many patients had been ascertained before del(GJB6- The group of 35delG homozygotes was used as a ref- D13S1830) was characterized (Lerer et al. 2001; del erence, or “standard,” group, because that group is well Castillo et al. 2002); also, IVS1ϩ1GrA lies outside the defined and is present in most of the previously reported GJB2 coding region and so was not included in mutation studies of GJB2 mutations as the cause of HI. Threshold screens by all laboratories. data (PTA0.5,1,2kHz) for all groups with a specific biallelic The most prevalent mutations in population subgroups combination of mutations were screened for the presence were 35delG (65% of mutant alleles) in Arabs, 167delT of progression by performing analysis of the linear regres- (84%) in the Jewish Ashkenazi population, and V37I sion of threshold on age (at audiometry, in years). If (37%) in Asians. Persons of African origin did not carry there was no significant correlation coefficient (P ! .025 ) a “common” mutation. Of the eight unrelated Roma combined with a positive slope, it was concluded that Gypsies, six were homozygous for W24X, one was a com- there was no significant progression. pound heterozygote for W24X/35delG, and one carried the M34T/R127H combination. These findings are in ac- Results cordance with the study reported by Minarik et al. (2003) and Seeman et al. (2004), which showed that W24X is Study Sample a prevalent mutation in the Roma population. The study sample consisted of 638 (41.7%) males and Comparison of HI between Different Genotype Classes 607 (39.6%) females; sex information was not available for 286 patients (18.7%). The median age of all par- By classifying variants as truncating or nontruncating, ticipants was 8 years; 90% of persons were within the we defined three genotype classes: biallelic truncating (T/ age range of 0–26 years (total age range, 0–70 years). T) (1,183 persons [77.3%]; 56 genotypes [37%]), bial- The majority of participants were white (90%), with lelic nontruncating (NT/NT) (95 persons [6.2%]; 30 ge- other subgroups fractionally represented—that is, Arabs notypes [20%]), and compound heterozygous truncat- (4.8%), Ashkenazi Jews (2.4%), Asians (1.8%), Africans ing/nontruncating (T/NT) (253 persons [16.5%]; 67 ge- (0.5%), and Roma Gypsies from the Czech Republic notypes [44%]). The HI across classes is nonrandomly (0.5%). Because the different ethnic subgroups were too distributed (x2 testing,P ! .0001 ) (fig. 1), with the HI small for detection of a statistical difference between in the biallelic T/T class more severe than in the T/NT groups, we used the sample set as a whole for statistical class, which is more severe than in the NT/NT class. evaluation. Linear regression analysis of thresholds on Within the T/T and T/NT classes, we distinguished be- age in the entire study sample and in subsamples defined tween 35delG and non-35delG mutations. This distinc- by genotypes did not show significant progression in any tion had little impact on the distribution of HI, which samples; therefore, age was not used as a variable for remained similar irrespective of the nature of the muta- the analyses. This finding is in concordance with many tion (35delG vs. non-35delG) (x2 testing: T/T class P p other studies (Denoyelle et al. 1999; Orzan et al. 1999; .39; T/NT classP p .42 ). Loffler et al. 2001), although progression of hearing loss In the T/T class, 59%–64% of persons had profound cannot be definitively excluded, given the cross-sectional HI, 25%–28% had severe HI, 10%–12% had moderate nature of the regression analysis. A slight degree of asym- HI, and 0%–3% had mild HI. In the T/NT class, 24%– metry was found in this study sample; however, the dif- 30% of persons had profound HI, and 10%–17% had ! ference in PTA0.5,1,2kHz between the two ears was 15 dB severe HI, with a shift toward severe-to-profound de- in 90% of the persons. grees of HI in persons with two non-35delG mutations, although this difference was not significant (x2 testing, GJB2 Mutation Spectrum P p .42). More than half (53%) of persons in the NT/ NT class had only a mild degree of HI, and only one A total of 83 different mutations were identified, which person in five in this class had severe-to-profound HI. were subclassified as 47 nontruncating and 36 truncat- ing mutations (table 1). These mutations were associated Comparison of HI between Specific GJB2 Genotypes with 153 different genotypes, of which 56 were homo- zygous truncating (T/T), 30 were homozygous nontrun- To investigate hearing thresholds by genotype, we con- cating (NT/NT), and 67 were compound heterozygous structed scatter diagrams showing the binaural mean truncating/nontruncating (T/NT). The nine most com- PTA0.5,1,2kHz for each person within each genotype class mon mutations, including 35delG, had a frequency 11%. (fig. 2). The reference group (35delG/35delG) is included

Snoeckx et al.: GJB2 Genotype-Phenotype Correlation 951 in each panel to facilitate visual comparisons, and bial- Table 2 lelic GJB2 genotypes are listed in descending order of Genotypes of Study Subjects Whose Degree of HI Was Significantly PTA0.5,1,2kHz. In figure 2, we have not included genotypes Different from That of the Reference Group (35delG/35delG) that were present in only one or two persons. Instead, Mutation PTA Pa these data are listed in table 2 and table 3. Table 2 contains 0.5,1,2kHz the genotypes of study subjects whose degree of HI was Truncating/truncating (T/T): 35delG/631delGT 22 .0022 significantly different from that of the reference group, 35delG/W77X 32 .0034 and table 3, those that were not significantly different. 35delG/W172X 45 .0191

The associated P value, for comparison of the PTA0.5,1, 310del14/W24X 55 .0416 with the reference group. is included in table 2 only. 310del14/Q57X 49 .0247 2kHz ϩ r These results must be interpreted with some caution be- W24X/IVS1 1G A 53 .0337 Truncating/nontruncating (T/NT): cause of small sample sizes. 35delG/I20T 52 .0337 Within the T/T genotype class shown in figure 2A, 35delG/H100Y 47 .0213 only two genotypes differed significantly from the ref- 35delG/M101T 32 .0034 erence group, persons segregating 35delG/del(GJB6- 35delG/S139N 29 .0022 D13S1830) had significantly more HI (median 35delG/A40E 50 .0045 p ! 35delG/G160S 16 .0011 PTA0.5,1,2kHz 108 dB;P .0001 ), whereas persons seg- 31del38/L90P 53 .0337 regating 35delG/IVS1ϩ1GrA had significantly less HI 167delT/V37I 50 .0281 p ! (medianPTA0.5,1,2kHz 64 dB;P .0001 ). Among per- 167delT/L90P 45 .0191 sons with T/NT genotypes (fig. 2B), one genotype, 235delC/V37I 29 .0022 35delG/R143W, showed significantly more HI than did 310del14/V37I 30 .0034 310del14/L90P 25 .0045 the reference group, and eight genotypes had signifi- Y65X/L90V 30 .0034 cantly less HI. The three genotypes with the least HI IVS1ϩ1GrA/L90P 48 .0247 p ! ϩ r were 35delG/V37I (medianPTA0.5,1,2kHz 40 dB; P IVS1 1G A/R184P 40 .0112 p Nontruncating/nontruncating (NT/NT): .0001), 35delG/M34T (median PTA0.5,1,2kHz 34 dB; P ! .0001), and del(GJB6-D13S1830)/M34T (median M34T/V95M 5 .0011 p ! M34T/R143W 41 .0112 PTA0.5,1,2kHz 25 dB;P .0001 ). In the T/NT geno- M34T/V153I 28 .0022 type class, the threshold distribution in persons with V37I/R143W 23 .0022 35delG/L90P suggests a bimodal distribution (fig. 2B). V37I/G160S 20 .0011 Seven 35delG/L90P compound heterozygotes had V37I/N206S 24 .0022 PTA 1 95dB, and 34 had PTA ! 65 dB, V63M/D159N 38 .0079 0.5,1,2kHz 0.5,1,2kHz L90P/S139N 22 .0022 with the PTA0.5,1,2kHz of only one person falling between L90P/V153I 38 .0079 the two values (65–95 dB). Among the NT/NT genotype V95M/L90P 72 .0165 class, participants with three genotypes showed HI sig- V153I/T8M 1 .0011 nificantly different from that of the reference group: T123N/T123N 56 .0028 p ! M34T/M34T (medianPTA0.5,1,2kHz 30 dB;P .0001 ), NOTE.—All genotypes were present in only one participant except p ! V37I/V37I (medianPTA0.5,1,2kHz 27 dB;P .0001 ), for the genotypes 35delG/I20T, 310del14/L90P, V95M/L90P, and p ! T123N/T123N (shown in bold italics), which were present in two and M34T/V37I (medianPTA0.5,1,2kHz 23 dB; P .001). participants. a P values were calculated by comparing the median PTA of Figure 3 shows the P50, P10, and P90 of hearing 0.5,1,2kHz each genotype with the median PTA0.5,1,2kHz of the reference group, by thresholds in audiogram format for genotypes with HI use of Fisher’s exact test. significantly different from that of the reference group. We did not plot P10 and P90 values whenn ! 10 . Ge- notypes represented by a small number of persons or a 35delG homozygote reference group (i.e., mildly down- large variation in threshold (n ! 5SD and1 25 dB) also sloping), although the rather flat configuration seen with were excluded. It is noteworthy that the audiogram slope the 35delG/IVS1ϩ1GrA genotype is an exception. of nearly all genotypes is fairly similar to that of the The distribution of the degree of HI for the most prev-

Figure 2 Scatter diagrams of the PTA0.5,1,2kHz of groups with specific genotypes. The genotypes were divided into three classes, truncating/ truncating (A), truncating/nontruncating (B), and nontruncating/nontruncating (C). The genotypes are shown (left to right) in descending order of group median PTA0.5,1,2kHz. The dividing lines used to dichotomize the threshold data in any group, as well as the reference group (“ref”), are shown as horizontal lines at 100 dB (∼P50 of the reference group), 85 dB (∼P25), and 55 dB (∼P5). The P value indicated above the panel frame relates to the Fisher’s exact test applying this dichotomy to both the reference group and the test group. HLphearing loss; “del(GJB6)” represents the del(GJB6-D13S1830 mutation. 952 Am. J. Hum. Genet. 77:945–957, 2005

Table 3 the predicted gating properties of IVS1ϩ1GrA, V37I, Infrequent Study GJB2 Genotypes, with HI Not and L90P are discrepant with the degree of HI we ob- Significantly Different from That of the Reference served. Expression studies have demonstrated complete Group (35delG/35delG) loss of channel activity for V37I and L90P (Bruzzone The table is available in its entirety in the online et al. 2003; Skerrett et al. 2004), although we found edition of The American Journal of Human Genetics. these mutations to be associated with mild-to-moderate HI, and functional studies of 35delG and IVS1ϩ1GrA у do not yield detectable CX26 protein and mRNA, re- alent genotypes (n 10 ) is given in table 4. Of 18 spectively (D’Andrea et al. 2002; Shahin et al. 2002), genotypes, 10 (shown in bold italics) had an HI that which is inconsistent with our observation that 35delG/ was significantly different from that of the homozygous IVS1ϩ1GrA compound heterozygotes had significantly 35delG reference group. less-severe HI (P ! .0001 ) compared with 35delG homozygotes. For the M34T allele variant, data are Discussion even more contradictory. Some functional studies have demonstrated a complete loss of channel activity for In this study of persons segregating GJB2-related deaf- this mutation, whereas other studies have shown that ness, we found truncating mutations of GJB2 to be as- this variant affects neither the permeability of dyes nor sociated with a greater degree of HI than were nontrun- the formation of stable connexons (Oshima et al. 2003; cating mutations. Several of the common genotypes in Skerrett et al. 2004). this study group were associated with mild-to-moderate The difficulty in data interpretation is illustrated by HI, which suggests that complete GJB2 mutation screen- functional studies of V84L, which have shown no ap- ing, including IVS1ϩ1GrA and del(GJB6-D13S1830), preciable effect on channel properties, although the mu- should be offered to all children with nonsyndromic HI, tation has been proven to be clearly associated with HI regardless of severity. in humans (D’Andrea et al. 2002; Bruzzone et al. 2003). The pathogenicity of missense mutations depends on A recent study by Beltramello and colleagues (2005), many factors, including the position of the mutation in which reviews the limitations of our knowledge of gap- the protein and the nature of the substitution. For ex- junction function, reports that the V84L mutant causes ample, a change in an amino acid that is positioned in HI due to an impaired permeability to IP3. The spread- 2ϩ a functional domain or that is conserved in related genes ingofanIP3-mediated Ca signal is essential for the or species is likely to be pathogenic. However, given the propagation of Ca2ϩ waves in cochlear supporting cells. complex structure and function of gap junctions, it is This discovery implicating IP3 as an essential component extremely difficult to predict pathogenicity of some mis- for the perception of sound is very promising for future sense mutations. This limitation reflects our incomplete studies of GJB2 mutations, especially when functional understanding of the molecular basis for gap-junction studies and clinical data are discordant (i.e., for M34T function, and it is for this reason that data from animal and V37I). models and recombinant expression systems, although GJB6 is unique because of its chromosomal localiza- valuable for the investigation of mutations, should be tion within 50 kb of GJB2. A frequent mutation in- extrapolated to humans with caution. cluded in this study, del(GJB6-D13S1830), leaves the Gap-junction channels are permeable not only to ions GJB2 coding region intact but deletes a large region but also to small metabolites with relative molecular close to GJB2 and truncates GJB6. This deletion is fre- masses up to ∼1,200 Da (Harris and Bevans 2001), with quently found in compound heterozygosity with a GJB2 differences in ionic selectivity and gating mechanisms mutation, and the associated HI is assumed to be caused among gap junctions that reflect the existence of 120 either by the deletion of a putative GJB2 regulatory different connexin isoforms in humans. Of the handful element or by digenic inheritance (del Castillo et al. of GJB2 mutations that have been tested in recombi- 2002). Pure digenic inheritance, however, seems un- nant expression systems, most show a loss of function likely, since compound heterozygosity with a GJB2 mu- due to altered sorting (G12V, S19T, 35delG, and L90P), tation has not been found for other GJB6 mutations. inability to induce formation of homotypic gap-junc- We found del(GJB6-D13S1830) to be one of two muta- tion channels (V37I, W77R, S113R, delE120, M163V, tions associated with HI that is significantly worse than R184P, and 235delC), or interference with translation that of the homozygous 35delG reference group (the (R184P). Most GJB2 mutations, however, have not been other is 35delG/R143W). The 33 35delG/del(GJB6- studied, and their impact on gap-junction function re- D13S1830) compound heterozygotes we ascertained mains speculative. showed the highest median PTA0.5,1,2kHz (108 dB) in this Of the reported functional studies, some results are study. The regulatory-element hypothesis could explain in apparent contradiction with our data. In particular, this finding, since deletion of this element would both Snoeckx et al.: GJB2 Genotype-Phenotype Correlation 953

Figure 3 Audiogram format for genotypes with an HI that was significantly different from that of the reference group of 35delG homozygotes. Only genotypes represented by a minimum of five persons and with an SD !25 dB are included. Median (P50) threshold (solid line) and P10 and P90 thresholds (dashed lines) are shown only forn 1 10 . The reference group (“Ref”) is included and is shaded in gray. “del(GJB6)” represents the del(GJB6-D13S1830 mutation. abolish GJB2 expression and inactivate one GJB6 allele. mal dominant mutation (Kelsell et al. 1997), consistent If GJB6 partially substitutes for GJB2 inner ear func- with the study by White and colleagues (1998), in which tion, as has been suggested (Ahmad et al. 2003), this it was reported to have a dominant negative effect over substitution would then be less efficient, thus leading to wild-type CX26 in Xenopus oocytes. These dominant more-severe HI. effects were later attributed to an artifact in the ex- The 35delG/L90P genotype was associated with a bi- pression levels of mutant and wild-type RNA that was modal distribution of the binaural mean PTA0.5,1,2kHz not controlled in the exogenous system (Wilcox et al. (fig. 2B). Although, as a whole, those with this genotype 2000). Other reports list the M34T allele as an auto- had significantly less HI than did the reference group, somal recessive mutation in the presence of other GJB2 a small group of 35delG/L90P compound heterozygotes mutations or in the homozygous condition (Wilcox et had severe-to-profound HI. The generally mild charac- al. 2000; Houseman et al. 2001; Kenneson et al. 2002; ter of the L90P mutation was corroborated by six ad- Wu et al. 2002), whereas other studies have stated that ditional compound heterozygous L90P combinations this variant is not pathogenic (Griffith et al. 2000; Feld- that all had significantly less HI than did the reference mann et al. 2004). If M34T is indeed a polymorphism, group—that is, combinations involving S139N, V153I, persons with the 35delG/M34T genotype are carriers of 31del38, 167delT, IVS1ϩ1GrA, and 310del14. Only only one GJB2 mutation (35delG), and their HI must two genotypes involving L90P (i.e., L90P/R143Q and be caused by other unidentified mutations in GJB2 or L90P/delE120) failed to show a significant difference by other genes. Because of the large phenotypic vari- from the 35delG/35delG genotype. ability among genetic causes of HI, we would expect a The M34T variant was described first as an autoso- highly variable degree of HI in these persons, with a 954 Am. J. Hum. Genet. 77:945–957, 2005

Table 4 Degree of HI for the Most-Prevalent Genotypes (n у 10 )

NO.(%)OF SUBJECTS, BY HI SEVERITY NO. OF MUTATION AND GENOTYPE SUBJECTS Mild Moderate Severe Profound Truncating/Truncating (T/T): 35delG/del(GJB6-D13S1830) 33 0 (0) 1 (3) 5 (15) 27 (82) 35delG/35delG 889 9 (1) 89 (10) 222 (25) 569 (64) W24X/W24X 12 0 (0) 0 (0) 3 (25) 89 (75) 35delG/W24X 13 0 (0) 0 (0) 5 (38) 8 (62) 35delG/E47X 29 0 (0) 1 (3) 10 (34) 18 (63) 35delG/310del14 42 0 (0) 3 (7) 11 (26) 28 (67) 35delG/167delT 45 2 (4) 4 (9) 13 (29) 26 (58) 167delT/167delT 17 0 (0) 3 (18) 5 (29) 9 (53) 35delG/IVS1ϩ1GrA 16 1 (6) 9 (56) 3 (19) 3 (19) Truncating/nontruncating (T/NT): 35delG/R143W 10 0 (0) 0 (0) 0 (0) 10 (100) 35delG/W77R 11 0 (0) 3 (27) 2 (18) 6 (55) 35delG/R184P 15 0 (0) 5 (34) 5 (33) 5 (33) 35delG/delE120 11 1 (9) 4 (36) 3 (28) 3 (27) 35delG/L90P 42 20 (48) 14 (33) 1 (2) 7 (17) 35delG/V37I 20 11 (55) 9 (45) 0 (0) 0 (0) 35delG/M34T 38 26 (68) 11 (29) 1 (3) 0 (0) Nontruncating/nontruncating (NT/NT): M34T/M34T 16 13 (81) 1 (6) 2 (13) 0 (0) V37I/V37I 18 10 (55) 7 (39) 1 (6) 0 (0)

NOTE.—Genotypes with HI that is significantly different from that of the reference group (35delG/ 35delG) are shown in bold italics. range from mild to profound. However, all persons with Acknowledgments a 35delG/M34T genotype had mild-to-moderate HI, This study was supported by grants from the University of with a median PTA0.5,1,2kHz of 34 dB. Persons homozy- Antwerp, the Vlaams Fonds voor Wetenschappelijk Onderzoek gous for M34T had an even lower median PTA0.5,1,2kHz value (30 dB). M34T is reported to have a high fre- and the Interuniversity Attraction Poles program P5/19 of the Belgian Federal Science Policy (to G.V.C.), and the National quency in the general white population, comparable to Institutes of Health (National Institute on Deafness and Other that of 35delG (Green et al. 1999; Roux et al. 2004). Communication Disorders [NIDOCD] R01-DC02842) (to The lower frequency of M34T, compared with 35delG, R.J.H.S.). Additional grants are from the Internal Grant in the patient sample of this study may reflect reduced Agency of the Ministry of Health of the Czech Republic (IGA penetrance or possible ascertainment bias toward more- MH CR NM 7417-3), the Israel ministry of science and tech- severe HI, since persons with mild HI are less likely to nology (to K.B.A.), the Turkish Academy of Sciences in the see an otorhinolaryngologist for audiologic or genetic framework of the young scientist award program (MT/TUBA- testing. Although some studies report that V37I is not GEBIP/2001-2-19), the Polish State Committee for Scientific pathogenic (Kelley et al. 1998; Kudo et al. 2000; Hwa Research (3P05A15024), the Programa Ramo´ n y Cajal of Min- et al. 2003; Wattanasirichaigoon et al. 2004), we docu- isterio de Ciencia y Tecnologı´a (to I.d.C.), the Spanish Fondo mented an association with mild HI in 9 of 10 genotypic de Investigaciones Sanitarias (FIS G03/203 and FIS PI020807), combinations in our study sample. This result is con- the G. Passe & R. Williams Foundation (to H.H.M.D), and sistent with other studies of this allele (Abe et al. 2000; NIDOCD grant DC005248 (to M.A.K.). This study was ini- Wilcox et al. 2000; Kenna et al. 2001; Lin et al. 2001; tiated through the European Union GENDEAF study group. Marlin et al. 2001). Author affiliations.—Department of Medical Genetics, Uni- versity of Antwerp (R.L.S., N.H., and G.V.C.), and Depart- In spite of the genotype-phenotype correlations we ment of Otorhinolaryngology, University Hospital of Ant- observed, significant phenotypic variability within geno- werp (P.V.d.H.), Antwerp; Departments of Otorhinolaryngol- types remains. This variability may reflect the effect of ogy (P.L.M.H. and C.W.R.J.C.) and Human Genetics (L.H.H.), modifier genes and/or environmental factors that lead Radboud University Nijmegen Medical Centre, Nijmegen, The to incomplete penetrance and variable expression (Na- Netherlands; Services de Biochimie et de Biologie Mole´culaire deau 2001). If modifier genes are involved, their char- (D.F.) and d’Oto-Rhino-Laryngologie et de Chirurgie Cervico- acterization will be essential for refining genotype-phe- Faciale (F.D.) and Unite´deGe´ne´tique Me´dicale (S.M.), Hoˆ pital notype correlations and improving the accuracy of phe- d’Enfants Armand-Trousseau, AP-HP, Paris; Clinic of Diabe- notype prediction. tology, Neonatology and Birth Defects (J.W.) and Department Snoeckx et al.: GJB2 Genotype-Phenotype Correlation 955 of Forensic Medicine (R.P.), Medical University of Warsaw, Ph.D. Program in Genetics, University of Iowa, Iowa City Institute of Physiology and Pathology of Hearing (J.W. and (C.J.N. and R.J.H.S.). M.M.-M.), and Departments of Audiology and Laryngology (E.N.-S.) and Medical Genetics (J.B. and W.W.), Institute of Web Resources Mother and Child, Warsaw; International Center of Hearing and Speech, Kajetany, Poland (A. Pollak); Department of Pe- The URLs for data presented herein are as follows: diatrics, Rare Disease Center, University of Padua (A. Murgia), and Child Audiology, Otosurgery Unit, University Hospital Connexin-Deafness Home Page, http://davinci.crg.es/deafness/ Padua (E.O.), Padua, Italy; Medical Genetics Unit, Department Online Mendelian Inheritance in Man (OMIM), http://www of Medicine, Istituti Clinici di Perfezionamento (P.C.), and .ncbi.nlm.nih.gov/Omim/ (for GJB2) Department of Otorhinolaryngology, Service of Audiology, University of Milan, Policlinico Hospital, Instituto di Ricerca References e Cura a Carattere Scientifico (U.A.), Milan; Departments of Medical Genetics, Molecular and Clinical Pharmacology Abe S, Usami S, Shinkawa H, Kelley PM, Kimberling WJ (2000) (A.R.J.) and Hearing, Speech and Voice Disorders (D.N.-H.), Prevalent connexin 26 gene (GJB2) mutations in Japanese. 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חרשות הינה הליקוי החושי הנפוץ ביותר באדם. כאחד מתוך 1000 תינוקות נולדים לוקים בשמיעתם.

ליקוי שמיעה מתקדם (פרוגרסיבי) שכיח בקרב 10% מאוכלוסית גילאי ה- 65, ומגיע לכ50%- מהאוכלוסיה

המגיעה לגיל 80. חרשות תורשתית מהווה כ60%- מכלל החרשות וכוללת 30% חרשות סינדרומית ו70%-

חרשות לא סינדרומית. החרשות הרצסיבית היא החרשות השכיחה ביותר ומהווה כ80%- מכלל החרשות הלא-

סינדרומית. למעלה ממאה גנים מעורבים בחרשות, עד עתה מופו 96 ומתוכם שובטו 43 גנים.

במחקר שערכתי בשנות השמונים הגעתי לאומדן סטטיסטי של מספר הלוקוסים לגנים לחרשות

רצסיבית לא סינדרומית באוכלוסיה הישראלית הכללית ובעדות השונות בישראל שמוצאן מאירופה (אשכנזים),

אסיה (מזרחיים) ואפריקה (ספרדים). האומדן נמצא שווה ל8-9- באוכלוסיה הכללית. אנליזה סטטיסטית של

נישואים תוך עדתיים ובין עדתיים הובילה לאומדן של 7 ו22- לוקוסים בהתאמה, מה שמוביל למסקנה שבעדות

שונות קיימים לוקוסים שונים (Brownstein et al. 1991). בשנים האחרונות, עם התפתחות הגנטיקה, מתאפשר

לחקור מולקולרית את מספר הגנים ותפקודם ואת החלבונים המקודדים על ידם. מטרת עבודת הדוקטוראט שלי

היתה לתרגם את האומדן למספר הלוקוסים לחרשות באוכלוסיה הישראלית, שקבלתי בשיטות סטטיסטיות, לזיהוי

מולקולרי ואיפיון של הגנים.

עד עתה ידועות מוטציות ב- 4 גנים האחראיות לליקוי שמיעה לא סינדרומי באוכלוסית ישראל. הגנים

המעורבים הם קונקסין GJB2) 26), קונקסין GJB6) 30), מיוזין MYO3A) IIIA) ו- POU4F3. שני הגנים

האחרונים זוהו כל אחד במשפחה אחת גדולה בלבד. לעומת זאת, מוטציות בקונקסין 26 אחראיות לכ39%-

מהחרשות המולדת הלא סינדרומית. שתי המוטציות השכיחות ביותר הן 35delG ו- 167delT שאחראיות ל-

72% מהמוטציות בקונקסין, עם שכיחות נשאים של 2.9-7.5% בקרב האוכלוסיה האשכנזית.

מוטצית החסר בקונקסין del(GJB6-D13S1830) ,30, מופיעה בעיקר עם מוטציה הטרוזיגוטית

בקונקסין 26 אצל חרשים. בישראל זיהיתי את המוטציה אצל 7 חרשים שהם הטרוזיגוטים כפולים למוטצית החסר

בקונקסין 30 ולמוטציה נוספת בקונקסין 26 ואילו רק חרש אחד נמצא הומוזיגוט למוטציה זו בקונקסין 30.

תוצאות מחקר זה התפרסמו כחלק ממחקר רב מרכזי (Del Castillo et al. 2003). לפי מחקר זה מוטצית החסר (del(GJB6-D13S1830 שכיחה ביותר בספרד, צרפת, בריטניה, ישראל וברזיל, כאשר השכיחויות נעות בין

5.9% ל- 9.7% מתוך האללים של DFNB1, והיא נדירה בדרום איטליה.

מחקר רב תחומי נוסף בו השתתפנו בדק שכיחות של מוטצית חסר שניה בקונקסין del(GJB6- 30

(D13S1854 במדינות שונות (del Castillo et al. 2005). בדקתי 159 חרשים יהודים ישראלים שנשללו אצלם

מוטציות בקונקסין 26. המוטציה החדשה בקונקסין 30 לא זוהתה אצל אף לא אחד מקבוצת מחקר זו.

מתוך 222 חרשים ישראלים שנבדקו במעבדתנו למוטציות בקונקסין 26 וקונקסין 30, אני בדקתי 79

ועיבדתי את כל הנתונים של כל קבוצת המחקר. חמישים ושישה חרשים נמצאו הומוזיגוטים או הטרוזיגוטים

מורכבים (compound heterozygotes) למוטציות בקונקסין 26, ו- 21 היו הטרוזיגוטים. אצל שבעה מתוך

ההטרוזיגוטים למוטציות בקונקסין 26 זוהתה גם מוטצית החסר (del(GJB6-D13S1830 בקונקסין 30. חרש

אחד היה הומוזיגוט למוטציה זו.

שישים וארבעת החרשים עם מוטציות בקונקסין היוו חלק ממחקר רב מרכזי עולמי שמנה אוכלוסיה

של 1531 חרשים כתוצאה ממוטציות בקונקסין (Snoeckx et al. 2005). מטרת המחקר היתה לבדוק קורלציה

בין גנוטיפ לפנוטיפ, דהיינו קשר בין חומרת ליקוי השמיעה למוטציות ספציפיות. מסקנות המחקר היו שליקוי

שמיעה חמור יותר מופיע כאשר שני האללים המוטנטים מקודדים חלבון קטום (truncating mutations) אילו

שני אללים מוטנטים שאינם פוגעים באורך החלבון (non-truncating mutations) מעורבים בליקויי שמיעה

פחות חמורים. מאחר והמוטציות השכיחות בישראל נמנות על הסוג הראשון, הפוגע באורך החלבון הנוצר, רוב

החרשות הקשורה בקונקסין היא חמורה עד עמוקה.

רוב הילדים בעלי החרשות העמוקה באוכלוסית המחקר עברו השתלה קוכלארית, מה שהוביל אותנו

למחקר נוסף הבודק את הקורלציה בין חרשות הקשורה בקונקסין ובין תפיסת דיבור לאחר ההשתלה. חמישים

ילדים עם שתל קוכלארי עברו אנליזה גנטית במעבדתנו ומתוכם 30 נמצאו מתאימים להשתתף במחקר. ל- 17

ילדים מתוך ה30- היתה חרשות רצסיבית כתוצאה ממוטציות בקונקסיו 26 ו/או קונקסין 30 ואצל 13 הילדים

הנותרים לא נמצאו מוטציות בגנים אלה. כל ילד בקבוצת הקונקסין הותאם לילד בקבוצה השניה, בה לא זוהו

מוטציות בגנים אלה, לפי גיל השתלה, משך השימוש בשתל וצורת התקשורת של הילד. בשתי הקבוצות לא היו

לילדים בעיות רפואיות או קוגניטיביות נוספות על החרשות. תפיסת הדיבור נבדקה במכון לשמיעה ודיבור בבית החולים תל השומר. נעשתה אנליזה רטרוספקטיבית של תפיסת הדיבור 6, 12, 24, 36 ו48- חודשים לאחר

ההשתלה. חומר המבחן נבחר לפי גיל הילד והיכולות השפתיות והקוגניטיביות שלו. שתי הקבוצות הראו שיפור

משמעותי בתוצאות תפיסת דיבור לאחר השתלה ולא נצפו הבדלים משמעותיים בין הקבוצות. התוצאות שקבלנו

במחקר זה דומות לאלה שהתקבלו בחלק מהמחקרים האחרים שנעשו בנושא זה, אם כי היו מחקרים שהראו

תוצאות טובות יותר בקבוצת הקונקסין. ההבדלים בין המחקרים יכולים לנבוע מגודל אוכלוסיה קטן מדי או

קריטריונים שונים לבחירת קבוצת הביקורת. המסקנה הנובעת מהממצאים שלנו היא שכאשר בחירת הקבוצות

נעשית בצורה קפדנית, וההבדל היחיד בין קבוצת המחקר וקבוצת הביקורת הוא האטיולוגיה, תתקבלנה תוצאות

דומות של תפיסת דיבור לאחר שתל קוכלארי. בקבוצת הביקורת שלנו, למרות שסיבת החרשות אינה ידועה, סביר

להניח שברוב המקרים החרשות היא גנטית, במיוחד כאשר מדובר בחרשות משפחתית. אם מוטציות בגנים הלא

ידועים פוגעות באוזן הפנימית הרי שניתן לצפות לאותה השפעה כמו של מוטציות בקונקסין. גם כאשר מדובר

בגורמי חרשות סביבתיים המערבים פגיעה סנסורית ולא מרכזית, נצפה לתוצאות תפיסת דיבור דומות לאלה של

קבוצת הקונקסין, מה שיכול להסביר את תוצאות המחקר.

מוטציות הגורמות לחרשות סינדרומית נמצאו בישראל בגנים המעורבים בתסמונת אשר - USH1,

USH2 וUSH3-. מוטציות חדשות בגן USH2A זוהו אצל יהודים ממוצא אירני וממוצא מרוקאי. המוטציה

N48K בגן USH3A זוהתה בקרב יהודים אשכנזים. אני אפיינתי את המוטציה R245X בגן PCDH15 המעורב

ב- USH1F אצל יהודים אשכנזים בישראל. זיהיתי את המוטציה בקרב ילדים יהודים אשכנזים מתחת לגיל 10,

לפני התחלת רטיניטיס פיגמנטוזה, שהיו מאובחנים בטעות כבעלי חרשות לא סינדרומית. 10% (2/20) נמצאו

הומוזיגוטים למוטציה R245X. באוכלוסיה האשכנזית השומעת בישראל מצאתי שכיחות נשאים של 1%

(Brownstein et al. 2004). המסקנה היא שנחוצה סריקה מוקדמת למוטציה זו בקרב ילדים יהודים אשכנזים

הנחשבים כבעלי חרשות לא סינדרומית. אנו מציעים לבדוק מוטציה זו אצל כל הילדים האשכנזים מתחת לגיל 10,

הנחשבים כבעלי חרשות לא- סינדרומית ושנשללו אצלם מוטציות בקונקסין. בדיקה מולקולרית זו עשויה לגלות

את תסמונת אשר ולצפות התפתחות של רטיניטיס פיגמנטוזה לפני שניתן לגלותה בבדיקת ERG, ותאפשר יעוץ

גנטי, הסתגלות מוקדמת ושיקום מוצלח יותר. בנוסף, חקרתי את הבסיס המולקולרי לחרשות במשפחה ישראלית עם אוטוסקלרוזיס (OTSC4)

וליקוי שמיעה פרוגרסיבי (DFNA51). אוטוסקלרוזיס שונה מליקויי השמיעה שנידונו לעיל בכך שהוא מאופיין

בליקוי שמיעה מתקדם (פרוגרסיבי), המתחיל כליקוי שמיעה הולכתי בגיל מבוגר. אוטוסקלרוזיס הוא ליקוי גרמי

בעצם הטמפורלית הגורם לליקוי שמיעה מתקדם ששכיחותו 0.2%-1% באוכלוסיה הלבנה. גיל הופעת ליקוי

השמיעה הוא 20-40 שנה. ההורשה היא אוטוזומלית דומיננטית עם חדירות חלקית. עד עתה מופו ארבעה לוקוסים

לאוטוסקלרוזיס על כרומוזומים OTSC2) 7q34-36 ,(OTSC3) 6p21.3-22.3 ,(OTSC5) 3q22-24) ו-

OTSC1) 15q25-q26). אני מיפיתי ואפיינתי קלינית את הלוקוס OTSC4 במשפחת O הישראלית. המשפחה

עברה אנליזה אודיולוגית של כל לקויי השמיעה וסריקה גנומית בעזרת סמנים גנטים למפוי הלוקוס

לאוטוסקלרוזיס. 24 בני משפחת O, לקויי שמיעה ובעלי שמיעה תקינה, השתתפו במחקר. ניתוחים שעברו 3 בני

משפחה הלוקים בשמיעתם אישרו את האבחנה של אוטוסקלרוזיס אצל בני משפחה אלה. ליקוי שמיעה של בני

המשפחה האחרים אובחן כאוטוסקלרוזיס על פי קריטריונים מקובלים כמו בדיקות שמיעה והיסטוריה רפואית

ומשפחתית. שונות גדולה מאוד באיפיוני החרשות נצפתה בין בני המשפחה הלוקים בשמיעתם. השלבים השונים

של התפתחות האוטוסקלרוזיס באו לידי ביטוי אצל בני משפחה שונים בסוג ליקוי השמיעה, חומרתו וצורת

האודיוגרמה שהתקבלה. הייחוד של מחקר זה הוא בשונות הגדולה שנצפתה בין פרטים שונים באותה משפחה ואצל

בני משפחה מסוימים אף בין שתי האוזניים של אותו פרט, ואת כל השונות הזו ניתן להסביר בשינויים המתרחשים

במוקדים האוטוסקלרוטים.

מיפיתי את הלוקוס החדש OTSC4 לכרומוזום 16q21-23.2. האנליזה הסטטיסטית שהובילה ל-

LOD score של 3.973 ב- θ= 0, אשרה את התאחיזה. אזור המיפוי שאורכו Mb 9-10, כולל 93 גנים. מכיוון

שהאטיולוגיה של אוטוסקלרוזיס אינה ברורה, גנים רבים באזור המיפוי יכולים להיות מועמדים טובים כמעורבים

באוטוסקלרוזיס, ביניהם, גנים שיש להם תפקידים במערכת החיסונית ואחרים המעורבים בבניית עצם. ריצפתי את

כל 93 הגנים באזור המיפוי של OTSC4 ולא מצאתי מוטציה באף לא אחד מהם. הכישלון במציאת מוטציה

בריצוף כל הגנים באזור יכול להיות מיוחס לקומבינציה בין כמה גורמים: 1) מוגבלות של הטכניקות לסריקת

מוטציות, 2) נוכחות מוטציות באזורים רגולטורים באינטרונים או בכל מקום אחר על הכרומוזום. ניסויים העוסקים

בתפקוד של חלבונים ובודקים הצטברות של עודף חלבון לעומת חסר של חלבון עשויים לעזור לזהות את גן

המטרה. מאחר ורוב הגנים באזור התאחיזה יכולים להיות מועמדים טובים למעורבות באוטוסקלרוזיס, לא יהיה מציאותי לבדוק את כולם במונחים של ביטוי חלבונים ותפקודם. התפתחות המחקר בתחומים כמו אזורים

רגולטורים, אינטראקציה גנית וטכניקות חדשות עשויה להוביל לשיבוט הגן OTSC4. זיהוי הגן יעזור לגלות את

האטיולוגיה של אוטוסקלרוזיס, את מבנה החלבון ותפקודו, ויקדם את אפשרויות האבחון, טיפול ומניעה של

אוטוסקלרוזיס.

Brownstein, Z., T. Ben-Yosef, O. Dagan, M. Frydman, D. Abeliovich, M. Sagi, F. A. .1 Abraham, R. Taitelbaum-Swead, M. Shohat, M. Hildesheimer, T. B. Friedman and K. B. Avraham (2004). The R245X mutation of PCDH15 in Ashkenazi Jewish children diagnosed with nonsyndromic hearing loss foreshadows retinitis pigmentosa. Pediatr .Res, 55, 995-1000 Brownstein, Z., Y. Friedlander, E. Peritz and T. Cohen (1991). Estimated number of loci .2 for autosomal recessive severe nerve deafness within the Israeli Jewish population, .with implications for genetic counseling. Am J Med Genet, 41, 306-312 del Castillo, F. J., M. Rodriguez-Ballesteros, A. Alvarez, T. Hutchin, E. Leonardi, C. A. de .3 Oliveira, H. Azaiez, Z. Brownstein, M. R. Avenarius, S. Marlin, A. Pandya, H. Shahin, K. R. Siemering, D. Weil, W. Wuyts, L. A. Aguirre, Y. Martin, M. A. Moreno-Pelayo, M. Villamar, K. B. Avraham, H. H. Dahl, M. Kanaan, W. E. Nance, C. Petit, R. J. Smith, G. Van Camp, E. L. Sartorato, A. Murgia, F. Moreno and I. del A novel deletion involving the connexin-30 gene, del(GJB6- .(Castillo (2005 d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects .with DFNB1 non-syndromic hearing impairment. J Med Genet, 42, 588-594 F. J. Del Castillo, Z. Brownstein, S. Marlin, Q. ,Del Castillo, I., M. A. Moreno-Pelayo .4 Adina, D. J. Cockburn, A. Pandya, K. R. Siemering, G. P. Chamberlin, E. Ballana, W. Wuyts, A. T. Maciel-Guerra, A. Alvarez, M. Villamar, M. Shohat, D. Abeliovich, H. Hutchin, W. E. Nance, E. L. Sartorato, R. J. .H. Dahl, X. Estivill, P. Gasparini, T Smith, G. Van Camp, K. B. Avraham, C. Petit and F. Moreno (2003). Prevalence and evolutionary origins of the del(GJB6-D13S1830) mutation in the DFNB1 locus in .J Hum Genet, 73, 1452-1458 hearing-impaired subjects: a multicenter study. Am Snoeckx, R. L., P. L. M. Huygen, D. Feldmann, S. Marlin, F. Denoyelle, J. Waligora, M. .5 Mueller-Malesinska, A. Pollak, R. Ploski, A. Murgia, E. Orzan, P. Castorina, U. Ambrosetti, E. Nowakowska-Szyrwinska, J. Bal, W. Wiszniewski, A. R. Janecke, D. Nekahm-Heis, P. Seeman, O. Bendova, M. A. Kenna, A. Frangulov, H. L. Rehm, M. Tekin, A. Incesulu, H.-H. M. Dahl, D. du Sart, L. Jenkins, D. Lucas, M. Bitner- Blin, M. .Glindzicz, K. B. K B Avraham, Z. Brownstein, I. del Castillo, F. Moreno, N Pfister, I. Sziklai, T. Toth, P. M. Kelley, E. S. Cohn, L. Van Maldergem, P. Hilbert, A.-F. Roux, M. Mondain, L. H. Hoefsloot, C. W. R. J. Cremers, T. Lopponen, H. Lopponen, A. Parving, K. Gronskov, I. Schrijver, J. Roberson, F. Gualandi, A. Martini, G. Lina-Granade, N. Pallares-Ruiz, C. Correia, G. Fialho, K. Cryns, N. Hilgert, P. Van de Heyning, C. J. Nishimura, R. J. H. Smith and G. Van Camp (2005). GJB2 mutations and degree of hearing loss: a multicenter study. Am J Hum Genet., In .Press

תוכן הענינים

I. מבוא 1

I.1. אנטומיה ופיזיולוגיה של האוזן 2 I.1.1. האוזן החיצונית 3 I.1.2. האוזן התיכונה 3 I.1.3. האוזן הפנימית 5 I.2 . תפקוד האוזן 10 I.2.1. המכניקה של השמיעה 10 I.2.2. מערכת שיווי המשקל 13 I.3. ליקויי שמיעה 15 I.3.1. אודיומטריה בסיסית ומושגים אודיולוגים 16 I.3.1.1. בדיקת pure-tone 16 I.3.1.2. סוגים של ליקויי שמיעה 17 I.3.1.3. סוגי אודיוגרמות 18 I.3.2. בדיקות שמיעה נוספות 19 I.3.2.1. בדיקות הולכה ((Impedance 19 I.3.2.2. בדיקות שמיעת דיבור 19 I.3.2.3. בדיקת ABR 21 I.4. גנים לחרשות לא-סינדרומית באוכלוסית ישראל 21 I.4.1. קונקסין 26 וקונקסין 30 23 I.4.2. קורלציה בין חרשות הקשורה לקונקסין ובין תוצאות השתל הקוכלארי 26 I.5. תסמונת אשר 27 I.5.1. גנים המעורבים בתסמונת אשר באוכלוסיה הישראלית 28 28 I.5.2.. PCDH15 I.6. אוטוסקלרוזיס 29

II. מטרות המחקר 33

III. חומרים ושיטות 34

III.1. איסוף אוכלוסית המחקר 34 III.1.1. מכתבים, טפסים ושאלונים 34 III.1.2. הערכה קלינית 34 III.1.3. בדיקות שמיעה 34 III.1.4. איסוף דמים 35 III.2. אנליזה מולקולרית 36 III.2.1. שיטות 36 III.2.1.1. פרוטוקולים לבופרים, תמיסות וג'לים 36 III.2.1.2. ראגנטים 38 III.2.1.3. מכשור 39 III.2.1.4. ערכות 40 III.2.2. שיטות כלליות 40 III.2.2.1. הפקת דנ"א 40 III.2.2.1.1. הפקת דנ"א על ידי ערכה 40 III.2.2.1.2. הפקת דנ"א בטכניקה של השקעת מלח ((salting-out 40 III.2.2.2. הכנת שורות תאים 41 III.2.2.3. הפקת רנ"א מרקמות ומשורות תאים 41 42 III.2.2.4. RT-PCR III.2.2.5. פרוטוקול בסיסי של PCR רב שלבי ((42touchdown III.2.2.5.1l. מרכיבי ראקצית ה- PCR 42 III.2.2.5.2. תכנית ה- PCR 42 III.2.2.6. אלקטרופורזה של תוצרי PCR בג'ל אגרוז 43 III.2.2.7. ריצוף דנ"א 43 III.2.2.7.1. ניקוי תוצרי PCR 43 III.2.2.7.2. הפקת דנ"א מג'ל אגרוז 43 III.2.2.7.3. ריצוף תוצרי ה- PCR המנוקים 44 44 III.2.2.8. SDS-PAGE III.2.2.8.1. סימון רדיואקטיבי של דנ"א 44 III.2.2.8.2. צביעת כסף של דנ"א 44 III.2.3. אנליזה של מוטציות בגן GJB2 (קונקסין 26) 46 III.2.3.1. זיהוי מוטציות בגן GJB2 על ידי עיכול באינזימי חיתוך 46 III.2.3.1.1t. איתור המוטציה delG35 על ידי עיכול באנזים החיתוך Bsl1 46 III.2.3.1.1. 1. פרוטוקול אמפליפיקציה 46 III.2.3.1.1. 2. תכנית ה- PCR 46 III.2.2.3.1. 3. עיכול אנזימתי 47 III.2.3.1.2. איתור המוטציה delT167 על ידי עיכול באנזים החיתוך Mwo1 47 III.2.3.1.2.1. פרוטוקול אמפליפיקציה 47 III.2.3.1.2.2. תכנית ה- PCR 47 III.2.3.1.2.3. עיכול אנזימתי 48 III.2.3.1.3. זיהוי המוטציה IVS1+1(G->A) באקסון 1 של הגן GJB2 על ידי עיכול אנזימתי 48 III.2.3.1.3.1. עיכול אנזימתי 49 III.2.3.2. זיהוי מוטציות בגן GJB2 על ידי ריצוף 49 III.2.3.2.1. פרוטוקול אמפליפיקציה...... 49 III.2.3.2.2. תכנית ה- PCR 50 III.2.3.2.3. ניקוי וריצוף תוצרי PCR 50 III.2.3.2.4. זיהוי מוטציות בגן GJB2 בעזרת ביואינפורמטיקה 50 III.2.4. זיהוי המוטציה ((GJB6-D13S1830בקונקסין 30 51 III.2.5. זיהוי המוטציה R245X בגן PCDH15 52 III.2.5.1. פרוטוקול (Allele-specific PCR (ASPCR 52 III.2.5.2. זיהוי המוטציה R245X על ידי עיכול אנזימתי 53 III.2.5.3. זיהוי המוטציה R245X על ידי ריצוף אקסון 8 בגן PCDH15 54 III.2.6. אוטוסקלרוזיס 54 III.2.6.1. משפחת O 54 III.2.6.2. שלילת תאחיזה 55 III.2.6.3. אנליזת תאחיזה 56 III.2.6.3.1. סריקה גנומית 56 III.2.6.3.2. מיפוי הלוקוס OTSC4 56 III.2.6.3.3. אנליזה הפלוטיפית 57 III.2.6.4. גנים מועמדים ((candidate 58 58 III.2.6.4.1. Mutation analysis

IV. תוצאות 60

IV.1. משפחת O 60 IV.1.1. בחירת המשפחה 60 IV.1.2. הערכה אודיולוגית 61 IV.1.2.1. גיל הופעת החרשות 61 IV.1.2.2. סימטריה של ליקוי השמיעה 61 IV.1.2.3. סוג ליקוי השמיעה וחומרתו 61 IV.1.2.4. צורת האודיוגרמה 66 IV.1.2.5. התקדמות ליקוי השמיעה ותוצאות הניתוחים 66 IV.1.2.6. בדיקות Immittance 69 IV.1.3. שלילת תאחיזה 70 IV.1.4. אנליזת תאחיזה 70 IV.1.5. גנים מועמדים 75 IV.2. קונקסין 26 וקונקסין 30 76 IV.2.1. שכיחות המוטציה del(GJB6-D13S1830) באוכלוסיה הישראלית 76 IV.2.2. מחקר רב מרכזי על שכיחות המוטציה del(GJB6-D13S1830) בלוקוס DFNB1 בקרב אוכלוסיות של לקויי שמיעה 77 IV.2.3. מחקר רב מרכזי על שכיחות המוטציה (del(GJB6- D13S1854 בלוקוס DFNB1 בקרב אוכלוסיות של לקויי שמיעה 77 IV.2.4. מוטציות בגנים GJB2 ו- GJB6 באוכלוסיה הישראלית 77 IV.2.5. קורלציה בין גנוטיפ-פנוטיפ במוטציות קונקסין 78 IV.2.6. קורלציה בין חרשות הקשורה לקונקסין ותוצאות שתל קוכלארי 79 IV.3. איפיון מוטציות בגן PCDH15 באוכלוסית החרשים בישראל 80 IV.3.1. זיהוי המוטציה R245X 80 IV.3.2. שכיחות נשאים למוטציה R245X 82 IV.4. איסוף משפחות גדולות נוספות עם ליקוי שמיעה 83 IV. 4.1. משפחת Z 83

V. דיון 85

V.1. משפחת O 85 V.1.1. איפיון פנוטיפי של אוטוסקלרוזיס תורשתי בלוקוס OTSC4 85 V.I.2. מיפוי אוטוסקלרוזיס תורשתי ללוקוס OTSC4 88 V.2. קונקסין 26 וקונקסין 30 94 V.2.1. מוטציות בגנים GJB2 ו- GJB6 באוכלוסיה הישראלית 94 V.2.2. שכיחות המוטציה (del(GJB6-D13S1830 94 V.2.3. קורלציה בין גנוטיפ- פנוטיפ במוטציות קונקסין 96 V.2.4. קורלציה בין חרשות הקשורה לקונקסין ותוצאות שתל קוכלארי 98 V.3. מוטציות בגן PCDH15 באוכלוסית החרשים בישראל 99 V.4. משפחת Z 103

VI. מחקרים עתידיים...... 104

VII. רשימת ספרות...... 107

.VIII נספחים...... 124..

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