Mutations of GJB2 which encodes connexin 26, contribute to 6–7% of profound deafness in Pakistan. We investigated the involvement of GJB2 mutations in a cohort of 84 pedigrees and 86 sporadic individuals with moderate or severe hearing loss. Individuals in eight consanguineous families and four sporadic cases (9.52% and 4.65%, respectively) were homozygous or compound heterozygous for p.W24X or p. W77X mutations in GJB2. These two variants are also among the most common mutations known to cause profound deafness in South Asia. The association of identical mutations with both profound and less severe phenotype of hearing loss suggests that alleles of other genes modify the phenotype due to these GJB2 nonsense mutations. Our study demonstrates that GJB2 mutations are an important contributor to aetiology of moderate to severe hearing loss in Pakistan.

center for Mendelian Genomics & Sadaf naz 1* Hearing loss affects 380 million people worldwide due to environmental or genetic causes. Determining the cause of deafness in individuals without previous family history of hearing loss is challenging and has been relatively unexplored in pakistan. We investigated the spectrum of genetic variants in hearing loss in a cohort of singleton affected individuals born to consanguineous parents. Twentyone individuals with moderate to severe hearing loss were recruited. We performed whole-exome sequencing on DNA samples from the participants, which identified seventeen variants in ten known deafness genes and one novel candidate gene. All identified variants were homozygous except for two. Eleven of the variants were novel, including one multi-exonic homozygous deletion in OTOA. A missense variant in ESRRB was implicated for recessively inherited moderate to severe hearing loss. Two individuals were heterozygous for variants in MYO7A and CHD7, respectively, consistent with de novo variants or dominant inheritance with incomplete penetrance as the reason for their hearing loss. our results indicate that similar to familial cases of deafness, variants in a large number of genes are responsible for moderate to severe hearing loss in sporadic individuals born to consanguineous couples. Approximately 50% of hearing loss cases has a genetic etiology. Of these, 70% are nonsyndromic and the remaining 30% are syndromic 1,2. Nonsyndromic hearing loss is most frequently inherited as an autosomal recessive trait 3 and the genetics of profound deafness in families has been well characterized 4. However, few studies have explored the prevalence of sporadic, moderate to severe hearing loss in the broader population. De novo variants or variants with incomplete penetrance, recessively inherited X-linked variants and mitochondrial variants may occur in individuals with no family history of hearing loss 5,6. The etiology of severe to profound deafness in sporadic cases has been explored in some populations and variants in GJB2 (OMIM 121011) and SCL26A4 (OMIM 605646) have been found to be the major contributors 6-9. Worldwide, only a few comprehensive studies have been carried out on single affected individuals exhibiting moderate to severe hearing loss. These studies have shown that variants in GJB2, OTOG (OMIM 604487), STRC (OMIM 606440), TECTA (OMIM 602574) and SERPINB6 (OMIM 173321) are more frequent in individuals with moderate or moderate to severe hearing loss in Chinese, Korean, Japanese and American populations 5,10-12. So far, no extensive study has been conducted to elucidate the genetic etiology of moderate to severe, sporadic hearing loss in Pakistan. We present the first report on the contribution of different deafness genes in the etiology of hearing loss of sporadic individuals born to consanguineous parents. Methods Ascertainment and audiological assessment. This study was approved by Institutional Review Board

Hereditary deafness is clinically and genetically heterogeneous. We investigated deafness segregating as a recessive trait in two families. Audiological examinations revealed an asymmetric mild to profound hearing loss with childhood or adolescent onset. Exome sequencing of probands identified a homozygous c.475G>A;p.(Glu159Lys) variant of CLDN9 (NM_020982.4) in one family and a homozygous c.370_372dupATC;p.(Ile124dup) CLDN9 variant in an affected individual of a second family. Claudin 9 (CLDN9) is an integral membrane protein and constituent of epithelial bicellular tight junctions (TJs) that form semipermeable, paracellular barriers between inner ear perilymphatic and endolymphatic compartments. Computational structural modeling predicts that substitution of a lysine for glutamic acid p.(Glu159Lys) alters one of two cis‐interactions between CLDN9 protomers. The p.(Ile124dup) variant is predicted to locally misfold CLDN9 and mCherry tagged p.(Ile124dup) CLDN9 is not targeted to the HeLa cell membrane. In situ hybridization shows that mouse Cldn9 expression increases from embryonic to postnatal development and persists in adult inner ears coinciding with prominent CLDN9 immunoreactivity in TJs of epithelia outlining the scala media. Together with the Cldn9 deaf mouse and a homozygous frameshift of CLDN9 previously associated with deafness, the two bi‐allelic variants of CLDN9 described here point to CLDN9 as a bona fide human deafness gene.