HUMAN MUTATION Mutation in Brief #804 (2005) Online MUTATION IN BRIEF Identification of a Novel Frameshift Mutation in the DFNB31/WHRN Gene in a Tunisian Consanguineous Family with Hereditary Non-Syndromic Recessive Hearing Loss Abdelaziz Tlili1, Ilhem Charfedine2, Imed Lahmar3, Zaineb Benzina4, Ben Amor Mohamed2, Dominique Weil5, Nabil Idriss3, Mohamed Drira2, Saber Masmoudi1, and Hammadi Ayadi1* 1Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Sfax, Tunisia; 2Service d'O.R.L., C.H.U. H. Bourguiba de Sfax, Sfax, Tunisia; 3Service d'O.R.L., C.H.U. de Mahdia, Mahdia, Tunisia; 4Service d’Ophtalmologie, C.H.U. H. Bourguiba de Sfax, Sfax, Tunisia; 5Unité de Génétique des Déficits Sensoriels, Institut Pasteur de Paris, Paris, France *Correspondence to: Pr. Hammadi Ayadi, Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine, 3029 Sfax, Tunisia; Tel: 216 74 241 888 (350) ; Fax: 216 74 461 403; E-mail: [email protected] Communicated by Henrik Dahl Approximately 80% of hereditary hearing loss is non-syndromic. Non-syndromic deafness is the most genetically heterogeneous trait. The most common and severe form of hereditary hearing impairment is autosomal recessive non-syndromic hearing loss (ARNSHL), accounting for approximately 80% of cases of genetic deafness. To date, 22 genes implicated in ARNSHL have been identified. Recently a gene, DFNB31/WHRN, which encodes a putative PDZ scaffold protein called whirlin, was found to be responsible for the ARNSHL DFNB31. We found evidence for linkage to the DFNB31 locus in a consanguineous Tunisian family segregating congenital profound ARNSHL. Mutation screening of DFNB31/WHRN revealed four nonpathogenic sequence variants and a novel frameshift mutation [c.2423delG] + [c.2423delG] that changed the reading frame and induced a novel stop codon at amino acid 818 ([p.Gly808AspfsX11] + [p.Gly808AspfsX11]). To determine the contribution of the DFNB31 locus in the childhood deafness, we performed linkage analysis in 62 unrelated informative families affected with ARNSHL. No linkage was found to this locus. From this study, we concluded that DFNB31/WHRN is most likely to be a rare cause of ARNSHL in the Tunisian population. © 2005 Wiley-Liss, Inc. KEY WORDS: non-syndromic hearing loss; DFNB31; WHRN; mutation INTRODUCTION Hearing loss is the most common sensory disorder in humans. The incidence of congenital hearing loss is estimated at 1 in 1,000 births, of which approximately 60% of cases are attributed to genetic factors (Morton, 1991; Gorlin et al., 1995). Autosomal recessive non-syndromic childhood/prelingual hearing loss (ARNSHL) accounts for approximately 80% of genetic hearing loss cases (Morton, 1991). Fifty-one loci causing ARNSHL have been mapped (DFNB) and 22 genes have been identified (http://www.uia.ac.be./dnalab/hhh/ and http://www.gene.ucl.ac.uk/nomenclature/). One locus for ARNSHL, DFNB31 (MIM# 607084), has previously been reported on chromosome 9q32-34 in a large family from Jordan (Mustapha et al., 2002). The region syntenic Received 26 July 2004; accepted revised manuscript 31 January 2005. © 2005 WILEY-LISS, INC. DOI: 10.1002/humu.9333 2 Tlili et al. to the DFNB31 interval, located on the murine chromosome 4, contains the locus of the recessive mutant whirler (wi) (Fleming et al., 1994 ; Rogers et al., 1999). A mutation in the Whrn gene (MIM# 607928) was recently found in the whirler mouse mutant. Whrn encodes a novel PDZ domain-containing protein, whirlin, involved in the elongation and maintenance of mechano-sensory stereocilia of both inner and outer hair cells. This protein contains three PDZ domains and a prolin-rich domain. The DFNB31/WHRN is predicted to encode a 907 amino acid protein. In the DFNB31 family, a nonsense mutation ([p.Arg778X] + [p.Arg778X]) in exon 10 of the human ortholog of Whrn gene was shown to be the causal mutation, which is likely to result to a truncated protein lacking the third PDZ domain (Mburu et al., 2003). Here, we describe evidence for linkage to the DFNB31 locus in a Tunisian consanguineous family with congenital profound ARNSHL and identify a novel frameshift mutation, [c.2423delG] + [c.2423delG] which results in a premature stop codon at position 818 ([p.Gly808AspfsX11] + [p.Gly808AspfsX11]). In addition, through an extensive genetic linkage study performed on ARNSHL-affected Tunisian families, we show that DFNB31/WHRN does not represent a major genetic contributor in causing ARNSHL in the Tunisian population. MATERIALS AND METHODS Subjects We analysed 63 Tunisian families in which non-syndromic hearing impairment is transmitted as an autosomal recessive trait. Individuals were evaluated for hearing loss by clinical examination and audiological tests including pure tone audiometry and brainstem evoked response audiometry. Air-conduction pure-tone average (ACPTA) thresholds in the conversational frequencies (0.5, 1 and 2 kHz) were calculated for each ear, and were used to define the severity of the hearing loss according to the better hearing ear: mild (25 dB_ACPTA_39 dB), moderate (40 dB_ACPTA_69 dB), severe (70 dB_ ACPTA_89 dB) and profound (ACPTA_90 dB). All of the affected individuals underwent examination for defects in other clinical features that could indicate that deafness was syndromal. In particular, no vestibular defects were detected. Appropriate informed consent was obtained from all subjects. Genotyping and mutation analysis Genomic DNA was extracted using a standard phenol-chloroform technique. PCR reactions for microsatellite markers were carried out in 25 µl with 60 ng of genomic DNA, 0.8 µM of each primer, 50 µM of each dNTP, 1.5 mM MgCl2, 5 mM KCl, 10 mM Tris-HCl, pH 8.8 and 1 U of Taq DNA polymerase. In general, PCR conditions were: 96°C for 5 min, followed by 35 cycles of: 94°C for 1 min, 55°C for 40 s and 72°C for 1 min, with a final 7 min extension step at 72°C. PCR products were analysed on 6% denaturing polyacrylamide gels, transferred onto N+-Hybond membrane (Amersham) and hybridized with a polyAC probe labeled with α32P dCTP. Mutation detection was performed by PCR amplification of each of the 12 coding exons of DFNB31/WHRN (GenBank NM_015404.1) including intron-exon junctions. Once purified, the PCR products were directly sequenced with one of the primers used for the amplification using an ABI 3100-Avant automated DNA sequencer (Applied Biosystems, USA). The oligonucleotide primers used for PCR amplification of DFNB31/WHRN exons and intron/exon junctions have been previously described (Mburu et al., 2003). Mutation and polymorphisms have been described according to the HGVS website (http://www.hgvs.org/mutnomen/). For nucleotide numbers +1 is the A of the ATG translation initiation codon. RESULTS AND DISCUSSION Seven individuals (4 affected and 3 unaffected) from a consanguineous Tunisian family affected with ARNSHL, were investigated (Fig. 1). The audiometric tests showed a loss of hearing greater than 90 dB for all deaf individuals. All affected individuals are otherwise healthy without dysmorphic or other abnormal findings indicative of syndromic deafness. In order to map the gene responsible for deafness, we performed linkage analyses with microsatellite markers closely bordering 21 identified DFNB genes (http://www.uia.ac.be./dnalab/hhh/). For each locus we genotyped 3 markers selected based on published linkage studies. Using homozygosity mapping, a linkage was detected with the markers D9S1776 and D9S154 corresponding to the DFNB31 locus (Mburu et al., 2003). The four affected siblings were homozygous for these two markers. Linkage was confirmed by two point linkage analysis with Zmax = 2.42 at θ = 0.00 for both markers (Fig. 1). A Novel Mutation in the DFNB31/WHRN Gene 3 Figure 1. Haplotype analysis of DFNB31 family with nonsyndromic hearing impairment. Blackened circles and squares indicate affected members. Haplotypes for three polymorphic markers from the 9q32 chromosomal region and obligate recombination events are indicated. The haplotype assumed to carry the disease allele is indicated by the black bar. A mutation in the DFNB31/WHRN gene was recently identified causing deafness in a DFNB31 family (Mburu et al., 2003). Therefore, we examined the DNA of affected individuals for deleterious mutations in that gene. Mutation screening of all 12 coding exons and their intron-exon junctions of the DFNB31/WHRN gene in affected members of the Tunisian family revealed a 1-bp deletion, at position 2423 bp of the cDNA: [c.2423delG] + [c.2423delG] (GenBank NM_015404.1) (Fig. 2A). The deletion was also found in a heterozygous state in both parents (BT31 and BT33) and the unaffected sister (BT30) (Fig. 2C). 4 Tlili et al. Figure 2. 2422delG (or 2423 delG) mutation in the DFNB31/WHRN gene; A: Affected individual, B: Hearing individual, C: Heterozygous individual. The complementary strand is shown. This mutation produces a frameshift resulting in a premature stop codon. It is likely to lead to an unstable mRNA, which might be degraded by RNA surveillance mechanism (Culbertson, 1999), and the predicted truncated whirlin protein would be consequently non-translated. However, the possibility that this frameshift deletion leads to a truncated protein cannot be ruled out. This mutation affects both the long isoform of the protein (GenBank NP_056219.1) as well as its short isoform (GenBank BAB14275.1) which would encode for truncated proteins 818 amino-acid ([p.Gly808AspfsX11] + [p.Gly808AspfsX11]) and 467 amino-acid ([p.Gly457AspfsX11] + [p.Gly457AspfsX11]) long respectively with 11 COOH-terminal amino-acid residues not in-frame. In addition to this disease-causing mutation, we identified 4 exonic non-pathogenic variants: a T to C transition at position 1353 ([c.1353T>C] + [c.1353T>C]) which was silent ([p.Gly451Gly] + [p.Gly451Gly]), and a transversion of a C to an A at position 2417 ([c.2417C>A] + [c.2417C>A]) and two T to C transitions at nucleotides 1838 ([c.1838T>C] + [c.1838T>C] and 2348 [c.2348T>C] + [c.2348T>C]) which substituted a proline to a glutamine ([p.Pro806Gln] + [p.Pro806Gln]), a methionine to a threonine ([p.Met613Thr] + [p.Met613Thr]) and a valine to an alanine ([p.Val783Ala] + [p.Val783Ala]), respectively.