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Deafness is the most frequent sensory disorder. With over 90 genes and 110 loci causally implicated in non-syndromic hearing loss, it is phenotypically and genetically heterogeneous. Here, we investigate the genetic etiology of deafness in four families of Iranian origin segregating autosomal recessive non-syndromic hearing loss (ARNSHL). We used a combination of linkage analysis, homozygosity mapping, and a targeted genomic enrichment platform to simultaneously screen 90 known deafness-causing genes for pathogenic variants. Variant segregation was confirmed by Sanger sequencing. Linkage analysis and homozygosity mapping showed segregation with the DFNB57 locus on chromosome 10 in two families. Targeted genomic enrichment with massively parallel sequencing identified causal variants in PDZD7: a homozygous missense variant (p.Gly103Arg) in one family and compound heterozygosity for missense (p.Met285Arg) and nonsense (p.Tyr500Ter) variants in the second family. Screening of two additional families identified two more variants: (p.Gly228Arg) and (p.Gln526Ter). Variant segregation with the hearing loss phenotype was confirmed in all families by Sanger sequencing. The missense variants are predicted to be deleterious, and the two nonsense mutations produce null alleles. This report is the first to show that mutations in PDZD7 cause ARNSHL, a finding that offers addition insight into the USH2 interactome. We also describe a novel likely disease-causing mutation in CIB2 and illustrate the complexity associated with gene identification in diseases that exhibit large genetic and phenotypic heterogeneity.

Hearing loss is the most common sensory disorder worldwide and affects 1 of every 500 newborns. In developed countries, at least 50% of cases are genetic, most often resulting in nonsyndromic deafness (70%), which is usually autosomal recessive (∼80%). Although the cause of hearing loss is heterogeneous, mutations in GJB2 gene at DFNB1 locus are the major cause of autosomal recessive nonsyndromic hearing loss (ARNSHL) in many populations. Our previous study showed that mutations of GJB2 gene do not contribute to the major genetic load of deafness in the Iranian population (∼16%). Therefore, to define the importance of other genes in contributing to an ARNSHL phenotype in the Iranian population, we used homozygosity mapping to identify regions of autozygosity-by-descent in 144 families which two or more progeny had ARNSHL but were negative for GJB2 gene mutations. Using flanking or intragenic short-tandem repeat markers for 33 loci we identified 33 different homozygous variations in 10 genes, of which 9 are novel. In aggregate, these data explain ∼40% of genetic background of ARNHSL in the Iranian population.

MYO15A is located at the DFNB3 locus on chromosome 17p11.2, and encodes myosin-XV, an unconventional myosin critical for the formation of stereocilia in hair cells of cochlea. Recessive mutations in this gene lead to profound autosomal recessive nonsyndromic hearing loss (ARNSHL) in humans and the shaker2 (sh2) phenotype in mice. Here, we performed a study on 140 Iranian families in order to determine mutations causing ARNSHL. The families, who were negative for mutations in GJB2, were subjected to linkage analysis. Eight of these families showed linkage to the DFNB3 locus, suggesting a MYO15A mutation frequency of 5.71% in our cohort of Iranian population. Subsequent sequencing of the MYO15A gene led to identification of 7 previously unreported mutations, including 4 missense mutations, 1 nonsense mutation, and 2 deletions in different regions of the myosin-XV protein.

Mutations in GJB2, encoding connexin 26 (Cx26), cause both autosomal dominant and autosomal recessive nonsyndromic hearing loss at the DFNA3 and DFNB1 loci, respectively. Most of the over 100 described GJB2 mutations cause autosomal recessive nonsyndromic hearing loss. Only a minority has been associated with autosomal dominant hearing loss. In this study, we present two families with autosomal dominant nonsyndromic hearing loss caused by a novel mutation in GJB2 (p.Asp46Asn). Both families were ascertained from the same village in northern Iran consistent with a founder effect. This finding implicates the D46N missense mutation in Cx26 as a common cause of deafness in this part of Iran mandating mutation screening of GJB2 for D46N in all persons with hearing loss who originate from this geographic region.

To the Editor: Hearing impairment is the most common genetic sensory defect in humans worldwide. It is estimated that profound hearing loss occurs in 4 out of every 10,000 children [Morton 1991]. Seventy percent of hereditary SNHL is nonsyndromic (DFN) and in at least 80% of these cases the inheritance is autosomal recessive (DFNB) [Smith et al., 2005]. Genetic studies have shown that mutations in 67 loci and 25 genes are associated with DFNB [Van Camp and Smith, 2008]. Further understanding of the molecular mechanisms associated with SNHL will assist in the development of therapeutics for hereditary hearing loss in the future. Mutations in the RDX gene that encodes the radixin protein have recently been shown to cause DFNB24 hearing loss in consanguineous Pakistani and Indian families [Khan et al., 2007]. In this study, we report a novel splice site mutation in the RDX gene in a consanguineous Iranian family with autosomal recessive non-syndromic hearing loss (ARNSHL). This mutation is only the fourth to be identified at the rare DFNB24 ARNSHL locus and the second that is predicted to lead to nonsense mediated decay or a truncated version of the radixin protein. A five-generation Iranian family with apparent congenital ARNSHL was studied (Fig.1). Family members in the fourth and fifth generations had severe-to-profound hearing loss suggesting autozygosity by descent (Fig.2). A complete physical examination and audiologic testing was completed on consenting family members and blood samples were obtained for DNA extraction. Liver function testing on affected individual V:2 showed total bilirubin (0.4 mg/dl), direct bilirubin (0.1 mg/dl), alanine transaminase (41 U/ml), aspartate transaminase (35 U/ml) and alkaline phosphatase (229 U/L), all normal. Human Research Institutional Review boards at the Welfare Science and Rehabilitation University and Iran University of Medical Sciences and the University of Iowa approved all procedures. Fig. 1 Pedigree of the Iranian family with ARNSHL. Open symbols = unaffected; filled symbols = ARNSHL. The genotypes and haplotypes for affected individuals and carriers are shown. Fig. 2 Audiograms from affected family members. All affected individuals displayed severe-to-profound bilateral ARNSHL. Genomic DNA from individuals III:1, IV:3, IV:4, IV:5, IV:6, IV:7, IV:8, V:1, V:2, V:3, and V:6 (Fig.1) was genotyped for 50,000 SNPs using Affymetrix (Santa Clara, CA, USA) 50K XBA GeneChips at the Translational Genomics Research Institute (Pheonix, Arizona). All genotypes were determined using the BRLMM genotyping algorithm [Di et al., 2005; Rabbee and Speed 2006]. Genotyping data were examined with PEDSTATS [Wigginton and Abecasis 2005] for Mendelian inheritance errors and MERLIN [Abecasis et al., 2002] for errors based on inferred double recombination events between tightly linked markers. Because the size of the pedigree was above the threshold for efficient implementation of the Lander-Green multipoint linkage mapping algorithm, a smaller subset of the pedigree was used which only considered the descendents of individuals III:1, III:2, III:3, III:4 (Fig.1). An autosomal, genome-wide parametric linkage analysis was carried out as the pedigree showed affected males and females. All linkage analyses were performed with MERLIN. The Lander-Green multipoint linkage mapping algorithm assumes linkage equilibrium. The markers were too close together to be in linkage equilibrium, so a smaller marker set was created using an in-house program (linkdatagen.pl;http://bioinf.wehi.edu.au/ software/). The binsize was set to 0.5 cM (i.e. the highest heterozygosity marker was chosen in each 0.5 cM interval) and 6140 autosomal markers were selected. Linkage analyses were carried out using the following parameters for the disease allele a, and normal allele A - Pr(a)=0.0001, Pr(disease|aa)=0.999, Pr(disease|AA or Aa)=0.001 - reflecting a highly penetrant, autosomal recessive disease model. The genetic map used was generated by Affymetrix using interpolation based on the empirical deCode, Marshfield and SNP Linkage (SLM1) genetic maps [Broman et al., 1998; Kong et al., 2002]. The physical map used was derived from the UCSC database using the NCBI Build 36.1 (March 2006; http://genome.ucsc.edu/cgi-bin/hgGateway). In the genome-wide analysis 38 Mendelian (.07% error rate) and 168 double recombinant errors were detected. A peak LOD score of 4.13 was achieved for a region on chromosome 11 (Fig.3) and there were no other regions where the LOD score exceeded 3. The critical region spanned approximately 3.6 cM between markers rs1789819 (109,185,632 bp) and rs10502160 (112,122,938 bp) (Fig.1). All four affected individuals included in the mapping were homozygous for a region between 108.814 cM and 112.368 cM on chromosome 11 (11q22.3-23.1). Fig. 3 Graphic showing the only significant LOD score achieved in the genome-wide analysis on chromosome 11. The dashed line indicates threshold (LOD score of 3). The dotted line at LOD score of 3.6 represents the Lander-Kruglyak threshold. The peak LOD score ... The critical region contained the known ARNSHL locus DFNB24. The RDX gene was amplified and sequenced using previously reported gene-specific primers [Khan et al., 2007]. A novel homozygous splice site mutation (c.698+1G>A) in intron 7 was found to segregate in affected family members (Fig.4). The +1G nucleotide of the 5’ donor splice site is invariant among all eukaryotes [Shapiro and Senapathy, 1987]. The c.698+1G>A mutation is predicted to result in read-through into intron 7 and a stop codon immediately following the amino acids encoded by exon 7. The last residue encoded by exon 7 would remain a lysine (K) due to the redundancy of the genetic code (AAG > AAA) and the next codon created would be a stop (TAA). This is predicted to result in a severely truncated protein lacking part of the FERM3 domain and the entire α- and c-terminal (CTD) domains (Fig.5), although the mutant mRNA may be subject to nonsense mediated decay and in this case no mutant protein would be produced. The c.698+1G>A mutation was not observed in 53 (106 chromosomes) Iranian and 133 (266 chromosomes) CEPH control individuals. Fig. 4 Sequence chromatograms showing the novel c.698+1G>A mutation in the RDX gene. Fig. 5 Domain structure of the RDX gene showing mutations known to cause DFNB24 hearing loss in humans. The novel splice site mutation described in this study (c.698+1G>A) is boxed. Radixin and ezrin are present in the hair cell stereocilia of the mouse inner ear [Kitajiri et al., 2004; Pataky et al., 2004]. While it is known that radixin functions as a linker between sub-cellular actin and the plasma membrane [Niggli and Rossy 2008], the exact role of this protein in the stereocilia of hair cells is unclear. Current knowledge of the role of these proteins in the inner ear comes from study of a radixin knockout mouse model. RDX −/− mice exhibit syndromic hearing impairment and hyperbilirubinemia [Kikuchi et al., 2002; Kitajiri et al., 2004]. The murine liver phenotype does not appear to model the human phenotype. One affected individual in the family reported here and another in a previously reported DFNB24 family have been shown to have normal liver function [Khan et al., 2007]. In conclusion, we have identified the first splice site mutation in the RDX gene and the first mutation known to cause DFNB24 hearing loss in the Iranian population. Our data also suggest that, in contrast to the murine model, liver dysfunction is not a component of the human phenotype. DFNB24 appears to be a rare form of ARSNHL as only four families have been reported. Further characterization of the molecular events triggered by this new mutation may increase our understanding of the function of radixin protein in the inner ear.

Hereditary hearing loss (HHL) is a very common disorder. When inherited in an autosomal recessive manner, it typically presents as an isolated finding. Interestingly and unexpectedly, in spite of extreme heterogeneity, mutations in one gene, GJB2, are the most common cause of congenital severe-to-profound deafness in many different populations. In this study, we assessed the contributions made by GJB2 mutations and chromosome 13 g.1777179_2085947del (the deletion more commonly known as del (GJB6-D13S1830) that includes a portion of GJB6 and is hereafter called Δ(GJB6-D13S1830)) to the autosomal recessive non-syndromic deafness (ARNSD) genetic load in Iran. Probands from 664 different nuclear families were investigated. GJB2-related deafness was found in 111 families (16.7%). The carrier frequency of the 35delG mutation showed a geographic variation that is supported by studies in neighboring countries. Δ(GJB6-D13S1830) was not found. Our prevalence data for GJB2-related deafness reveal a geographic pattern that mirrors the south-to-north European gradient and supports a founder effect in southeastern Europe. © 2005 Wiley-Liss, Inc.