Refined Mapping of a Gene for Autosomal Dominant

Original Paper Eur J Hum Genet 1997;5:397-405 Received: June 10, 1997 Revision received: August 28. 1997 Accepted: September I, 1997 L. Van Laera, G. Van Campa Refined Mapping of a Gene for D. van Zuijien b, E.D. Greene M Verstrekena,d, l Schattemand Autosomal Dominant Progressive P. Van de Heyningd, W Balemansa P. Couckea, J.B. Greinwalde Sensorineural Hearing Loss (DFNA5) to R.J.B. Smith e, E. Huizingb a 2-cM Region, and Exclusion of a P. Willems a Candidate Gene That Is Expressed in a Department of Medical Genetics, University of Antwerp, Belgium; the Cochlea b Department of Otorhinolaryngology, University Hospital of Utrecht, The Netherlands; c National Human Genome Research Institute, National Institutes of Health, Bethesda, Md., USA; d Department of Otorhinolaryngology, Abstract University Hospital of Antwerp, Belgium; A gene for an autosomal dominant form of progressive sensorineural hearing e Department of Otorhinolaryngology, loss (DFNA5) was previously assigned by us to a 15-cM region on chromo University ofIowa Hospitals and Clinics, some 7p15. In this study, the DFNA5 candidate region was refined to less than Iowa City, Iowa, USA 2 cM, and completely cloned in a Y AC contig. The HOXAI gene located in 7p15 was considered to be a good candidate gene for DFNA5 as it harbours mutations leading to developmental defects of the inner ear in mice. However, KeyWords the refinement of the candidate region ofDFNA5 excludes the HOXAI gene Sensorineural progressive as a candidate for D FNA5. We cloned a novel candidate gene (CG 1, candidate hearing loss gene 1), which is expressed in human fetal cochlea, from the DFNA5 candi DFNA5 date region. The complete cDNA sequence of CGl, encoding a 423 amino Chromosome 7p15 acid protein of unknown function, was determined. Mutation analysis of the Refinement CG 1 gene in DFNA5 patients, however, could not reveal a disease-causing Candidate gene CG 1 mutation. Introduction drugs, and presbyacusis with predisposing genetic factors [3]. A first requirement to unravel these interactions Hearing loss is the most common sensory impairment between genetic and environmental factors is a better in developed countries. Approximately 1 child in every understanding how single gene mutations lead to progres 1,000 is born deaf or develops profound prelingual hear sive hearing loss [1]. Large pedigrees showing autosomal ing loss in early childhood, and it is estimated that in half dominant hearing loss, which is nearly always progressive of these cases the hearing impairment is caused by genetic and postlingual, offer an opportunity to identify these factors [1]. Progressive late-onset hearing loss is much genes by positional cloning, and therefore represent excel more frequent than prelingual deafness. By the age of 65 lent working models to unravel the complex process of years, 1 person in 6 has functionally significant hearing progressive late-onset hearing loss. The genes involved in impairment, increasing to 1 person in 2 by the age of 80 autosomal dominant hearing loss are given a DFNA pre years [2]. This progressive late-onset hearing loss probably fix followed by a consecutive number, in the order of dis is the result of an interaction of various environmental covery. Recessive deafness genes are given a DFNB prefix factors such as infection (otitis), acoustic trauma, ototoxic in an analogous manner. Up to now, 13 DFNA loci and © 1997 S. Karger AG, Basel Guy Van Camp KAR.GER. Department of Medical Genetics Fax+41 61 306 1234 This article is also accessible online at: University of Antwerp (VIA), Universiteitsplein I E-Mail [email protected] http://BioMedNet.com/karger B-26 10 Antwerp (Belgium) www.karger.com Tel. 323 8202585, Fax 323 8202566, E-Mail [email protected] Branch 1 Branch 6 Fig. 1. Pedigree of the DFNAS family. Affected individuals are represented by solid symbols. Individuals whose affection status is uncertain are represented with? in the respective symbols. Spouses were omitted from the figure. Family branches 1, 4 and S are excluded from the figure, as these branches contain no additional patients. 17 DFNB loci have been localised [4]. A list of all pub branches 2 and 3 of the DFNAS family. This enabled us to lished loci, including detailed genetic and clinical infor reduce the DFNAS candidate interval to a 2-cM region, mation, and their respective references can be found on which was completely cloned in a Y AC contig. Further line in the Hereditary Hearing loss Homepage (http:// more, we identified a novel gene that is expressed in the www-dnalab.uia.ac.be/dnalab/hhh; downloaded in Au cochlea from the DFNAS candidate region. Mutation gust 1997). Although 13 DFNA loci and 17 DFNB loci analysis could not reveal a disease-causing mutation in have already been found, only a single gene for autosomal this DFNAS candidate gene. dominant and 2 genes for autosomal recessive hereditary hearing loss have been identified. Recently, mutations in the connexin 26 gene were found in dominant as well as in Materials and Methods recessive families, identifying connexin 26 as the DFNA3 Clinical Studies as well as the DFNBI gene [S]. Mutations in the myosin The pedigree of the extended Dutch family linked to DFNAS, VilA gene were proven to be the cause of DFNB2 reces and the clinical aspects ofthe hearing loss have been reported before sive non-syndromic deafness [6, 7]. [8-14]. Briefly, the hearing loss in the Dutch family is non-syndromic In a previous study we have reported the localisation of and starts at the high frequencies at an age between S and IS years. the DFNAS gene in branch 6 of an extended Dutch family The hearing loss is progressive and with increasing age also the mid dle and lower frequencies become affected. The pattern of inheri to a IS-cM candidate region between D7S493 and tance is autosomal dominant. For the initial localisation of the D7S632 on chromosome 7plS [8]. This DFNAS family DFNAS gene to chromosome 7plS, 88 members of branch 6 were has been followed during the past decades, and the examined [8]. In this study, 32 members of branch 2, 71 members of DFNAS hearing loss has been characterised as a progres branch 3, and 6 additional members of branch 6 were examined sive hereditary perceptive deafness, starting in the high (fig. 1). Otologic examination, pure-tone audiometry, and clinical diagnosis were performed as described before [8]. Blood samples for frequencies at an age between S and IS years [9-14]. In DNA extraction were collected after informed consent. the present study, linkage analysis was performed in 398 Eur J Hum Genet 1997;5:397-405 Van Laer et al. Table 1. Primer sequences to amplify CGl cDNA Primer Sequence Nucleotide position CGl-RI 5'-CGAGAAGATGACGTCACAGTAGC-3' -119 ~ -97 CGI-R2 5'-CGAACGGTGCAGACTGAAGACG-3' -46 ~ -25 CGI-R3 5'-CTGGAGCTTCAACTAACAGGAAGG-3' 254 ~ 277 CGI-R4 5'-CCAGAGATATTCCAATGTCATCCAG-3' 153 ~ 177 CGl-R5 5'-CCAGAGGAATTGAGGCTTGAATACC-3' 463 ~ 487 CGI-R6 5'-GAAAAGAAACTTCTGGAAGGAATTG-3' 337 ~ 361 CGl-R7 5'-CTAAA TATATCAACT AAAGTAGCTTTGC-3' 583 ~ 610 CGl-R8 5'-GCTGCAGCCTCTACCAGTTCAG-3' 817 ~ 838 CGl-R9 5'-CCTTTGGAGGTGGCAGTTCTGTG-3' 1004 ~ 1026 CGl-RlO 5'-CCACTTCTCTGTCAGCCTCAAGC-3' 1109~ 1131 CGI-Rll 5'-CGAACATCCCGGTGCTAGG-3' 60 ~ 78 CGl-SI 5'-CGTGCATAATTTTTCAGTTCCATTC-3' 1510 ~ 1486 CGl-S2 5'-CATCTCTGTATGAATCACTCAAAGC-3' 1338 ~ 1314 CGl-S3 5'-GGTGTGAATAACACATTATCTGTTGC-3' 1166 ~ 1141 CGl-S4 5'-GGCTTCCCAAATCCAAAAGCTGG-3' 878 ~ 856 CGl-S5 5'-GCAGGTGCTGCTTGATTTACTCC-3' 650 ~ 628 CGl-S6 5'-GAGAATGTGAGCCCGGACTACC-3' 1057 ~ 1036 CGl-S7 5'-CTGTAAGTTATTGCTGGTTAAGAAG-3' 516 ~ 492 CGl-S8 5'-CCCTGATGATTCCCAAACCTCC-3' 393 ~ 372 CGl-S9 5'-GCAAATGGGTTCTCAGACAATCC-3' 308 ~ 286 CGl-SlO 5'-CCCCATGGTGTGGATTTGGAGA-3' 209 ~ 188 CGl-S11 5'-CCTGAAGGCTGCTGCTGC-3' 122 ~ 105 Microsatellite Analysis Institute (NIH), was included. Y ACs for the region were selected from Polymorphic markers of chromosome 7p were analysed radioac the WHITEHEAD/MIT database (YAC contig WC-633 and YAC con tively using standard procedures [15]. PCR consisted of27 cycles of tig WC7.3, downloaded in January 1997). YAC clones were obtained 1 min denaturation (94 ° C), 1 min annealing at temperatures ranging from the UK HGMP Resource Centre. Thirty-five PCR cycles were from 55 to 60°C depending on the marker, and 1 min extension performed for all selected polymorphic markers on all Y ACs. (72°C), followed by a final lO-min extension step. PCR products were separated on a 6% polyacrylamide gel, and subjected to autora RNA Isolation and Nested RT-PCR diography. Total RNA was isolated from EBV-transformed cell lines using Trizol (Life Technologies), according to the Manufacturer's Guide Linkage Analysis lines. cDNA synthesis was performed using the random hexamers Two-point lod scores were calculated using the LINKAGE soft provided in the SuperScript Preamplification System following the ware package version 5.1 [16]. The frequency of hearing loss due to Instruction Manual (Life Technologies). Resulting cDNAs were DFNA5 mutations was set at 11100,000. Penetrance was assumed to directly used as templates in the PCR reactions. A first PCR (30 be 98%, and the risk of phenocopies was set at 2%. Recombination cycles) was performed using CGl primer pair Sl-Rl (table 1). The frequencies were assumed equal for males and females. The number resulting PCR product was diluted 50-fold and used in a nested PCR of alleles was set at the observed number of alleles in the pedigree (N), consisting of 2 5 cycles. The nested PCR was performed using various and allele frequencies were set equal at liN. combinations of CG l-R and CG I-S primers (table 1). Haplotypes were constructed assuming a minimum number of recombinational events. Single-Stranded Conformation Polymorphism Nested RT-PCR was performed in the presence of a-32P-dCTP Construction ofa YAC Contig (ICN; 3,000 Cilmmol).

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