Hearing impairment (HI) is the most frequent sensory defect. Genetic causes are involved in two thirds of prelingual cases. Moreover, the autosomal recessive HI frequency is increased in countries where there is a high rate of consanguinity, such as in North African Mediterranean countries. This population shares several features, including history and social behavior, that promote the spread of founder mutations. HI is characterized by tremendous heterogeneity in both the genetic and clinical aspects. The identification of the causal mutation is important for early diagnosis, clinical follow-up, and genetic counseling. Addressing the extreme genetic heterogeneity of HI using classic molecular methods would be expensive and time-consuming. We designed a cost-effective North African Deafness chip for rapid and simultaneous analysis of 58 mutations using multiplex PCR coupled with dual-color arrayed primer extension. These mutations are found in North African HI patients and are distributed over 31 exons and five introns in 21 distinct genes. Assay specificity was initially optimized using 103 archived DNA samples of known genotypes. Blind validation of HI-unrelated patients revealed mutant alleles in 13 samples, and these mutations were confirmed by Sanger sequencing. The North African Deafness chip allows for simultaneous genotyping of eight different samples, at a minimal cost and in a single day, and is therefore amenable to large-scale molecular screening of HI in North Africa.

Background: Otosclerosis (OTSC) is among the most common causes of a late-onset hearing loss in adults and is characterized by an abnormal bone growth in the otic capsule. Alteration in the osteoprotegerin (OPG) expression has been suggested in the implication of OTSC pathogenesis. Methods: A case-control association study of rs2228568, rs7844539, rs3102734 and rs2073618 single nucleotide polymorphisms (SNPs) in the OPG gene was performed in a Tunisian-North African population composed of 183 unrelated OTSC patients and 177 healthy subjects. In addition, a multilocus association and a meta-analysis of existing studies were conducted. Results: Rs3102734 (p = 0.013) and rs2073618 (p = 0.007) were significantly associated with OTSC, which were predominantly detected in females after multiple corrections. Among the OPG studied SNPs, the haplotypes A-A-C-G (p = 0.0001) and A-A-CC (p = 0.0004) were significantly associated with OTSC in females. Multilocus association revealed that the SNPs: rs2073618 in OPG, rs1800472 in TGFβ1, rs39335, rs39350 and rs39374 in RELN, and rs494252 in chromosome 11 showed significant OTSC-associated alleles in Tunisian individuals. In addition, meta-analysis of the rs2073618 SNP in Tunisian, Indian and Italian populations revealed evidence of an association with OTSC (OR of 0.826, 95% CI [0.691-0.987], p = 0.035). Conclusions: Our findings suggest that rs3102734 and rs2073618 variants are associated with OTSC in North African ethnic Tunisian population. Meta-analysis of the rs2073618 in three different ethnic population groups indicated an association with OTSC.

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Otosclerosis (OTSC) is a complex bone disorder of the otic capsule, which causes conductive hearing impairment in human adults. The dysregulation of the signaling axis mediated by the receptor activator of nuclear factor-kappa-B (RANK), RANK ligand (RANKL), and osteoprotegerin has been widely attributed to the context of metabolic bone disorders. While genetic associations and epigenetic alterations in the TNFSF11 gene (RANKL) have been well-linked to metabolic bone diseases of the skeleton, particularly osteoporosis, they have never been addressed in OTSC. This study aimed to assess whether the genetic association of rs1021188 polymorphism in the upstream of TNFSF11 and the DNA methylation changes in its promoter CpG-region reveal the susceptibility of OTSC. Peripheral blood DNA samples were collected from unrelated Tunisian-North African subjects for genotyping (109 cases and 120 controls) and for DNA methylation analysis (40 cases and 40 controls). The gender-stratified analysis showed that the TNFSF11 rs1021188 C/T was associated with OTSC in men (p = 0.023), but not in women (p = 0.458). Individuals with CC genotype were more susceptible to OTSC, suggesting an increased risk to disease development. Using publicly available data, the rs1021188 was within a cluster grouping the subpopulations with African ethnicity. Moreover, 26 loci in the TNFSF11 gene were in linkage disequilibrium with rs1021188, revealing relative similarities between different populations. Significant differences in both DNA methylation and unmethylation status were detected with 4.53- and 4.83-fold decreases in the global DNA methylation levels in female and male OTSC groups, respectively. These changes could contribute to an increased risk of OTSC development. Bioinformatic analyses indicated that each of the rs1021188 variations and the DNA methylation changes in the promoter CpG-sites within TNFSF11 may play an important role in its transcription regulation. To our knowledge, this is the first study that investigates an independent effect of the rs1021188 polymorphism and DNA hypomethylation of TNFSF11 promoter in OTSC. Genetic and epigenetic changes in the regulatory regions of TNFSF11 could offer new molecular insights into the understanding of the complexity of OTSC.

Background Otosclerosis (OTSC) is among the most common causes of a late-onset hearing loss and alteration in the osteoprotegerin (OPG) expression was suggested in the implication of OTSC pathogenesis. A case-control association study of single nucleotide polymorphisms (SNP) in the OPG gene was performed in a Tunisian-North African population composed of 183 unrelated OTSC patients and 177 healthy subjects.Results Rs3102734 and rs2073618 were significantly associated with OTSC which were predominantly detected in females after multiple corrections. The haplotypes A-A-C-G (p = 0.0.001) and A-A-C-C (p = 0.0004) were significantly associated with OTSC suggesting a reduced risk in females. Multilocus association revealed that: rs2073618 in OPG, rs39335, rs39350 and rs39374 in RELN, rs494252 in chromosome 11 and rs1800472 in TGFβ1 showed significant OTSC-associated alleles in the Tunisian samples. In addition, meta-analysis for rs2073618 SNP in Tunisian, Indian and Italian populations revealed evidence of association of the rs2073618 polymorphism with OTSC (Odds ratios of 0.826 (95% CI [0.691–0.987], p = 0.0035)).Conclusions Our findings suggest that rs3102734 and rs2073618 variants are associated with OTSC in an additional North African ethnic Tunisian population. Meta-analysis for rs2073618 based on three ethnic population revealed evidence of association with OTSC.