The current molecular genetic diagnostic rates for hereditary hearing loss (HL) vary considerably according to the population background. Pakistan and other countries with high rates of consanguineous marriages have served as a unique resource for studying rare and novel forms of recessive HL. A combined exome sequencing, bioinformatics analysis, and gene mapping approach for 21 consanguineous Pakistani families revealed 13 pathogenic or likely pathogenic variants in the genes GJB2, MYO7A, FGF3, CDC14A, SLITRK6, CDH23, and MYO15A, with an overall resolve rate of 61.9%. GJB2 and MYO7A were the most frequently involved genes in this cohort. All the identified variants were either homozygous or compound heterozygous, with two of them not previously described in the literature (15.4%). Overall, seven missense variants (53.8%), three nonsense variants (23.1%), two frameshift variants (15.4%), and one splice-site variant (7.7%) were observed. Syndromic HL was identified in five (23.8%) of the 21 families studied. This study reflects the extreme genetic heterogeneity observed in HL and expands the spectrum of variants in deafness-associated genes.

Background: MYO3A, encoding the myosin IIIA protein, is associated with autosomal recessive and autosomal dominant nonsyndromic hearing loss. To date, only two missense variants located in the motor‐head domain of MYO3A have been described in autosomal dominant families with progressive, mild‐to‐profound sensorineural hearing loss. These variants alter the ATPase activity of myosin IIIA.Methods: Exome sequencing of a proband from a three‐generation German family with prelingual, moderate‐to‐profound, high‐frequency hearing loss was performed. Segregation analysis confirmed a dominant inheritance pattern. Regression analysis of mean hearing level thresholds per individual and ear was performed at high‐, mid‐, and low‐frequencies.Results: A novel heterozygous missense variant c.716T>C, p.(Leu239Pro) in the kinase domain of MYO3A was identified that is predicted in silico as disease causing. High‐frequency, progressive hearing loss was identified.Conclusion: Correlation analysis of pure‐tone hearing thresholds revealed progressive hearing loss, especially in the high‐frequencies. In the present study, we report the first dominant likely pathogenic variant in MYO3A in a European family and further support MYO3A as an autosomal dominant hearing loss gene.

CDC14A encodes the Cell Division Cycle 14A protein and has been associated with autosomal recessive non-syndromic hearing loss (DFNB32), as well as hearing impairment and infertile male syndrome (HIIMS) since 2016. To date, only nine variants have been associated in patients whose initial symptoms included moderate-to-profound hearing impairment. Exome analysis of Iranian and Pakistani probands who both showed bilateral, sensorineural hearing loss revealed a novel splice site variant (c.1421+2T>C, p.?) that disrupts the splice donor site and a novel frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene CDC14A, respectively. To evaluate the pathogenicity of both loss-of-function variants, we analyzed the effects of both variants on the RNA-level. The splice variant was characterized using a minigene assay. Altered expression levels due to the c.1041dup variant were assessed using RT-qPCR. In summary, cDNA analysis confirmed that the c.1421+2T>C variant activates a cryptic splice site, resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20) and the c.1041dup variant results in a defective transcript that is likely degraded by nonsense-mediated mRNA decay. The present study functionally characterizes two variants and provides further confirmatory evidence that CDC14A is associated with a rare form of hereditary hearing loss.

Genetic heterogeneity and consanguineous marriages make recessive inherited hearing loss in Iran the second most common genetic disorder. Only two reported pathogenic variants (c.323G>C, p.Arg108Pro and c.419A>G, p.Tyr140Cys) in the S1PR2 gene have previously been linked to autosomal recessive hearing loss (DFNB68) in two Pakistani families. We describe a segregating novel homozygous c.323G>A, p.Arg108Gln pathogenic variant in S1PR2 that was identified in four affected individuals from a consanguineous five generation Iranian family. Whole exome sequencing and bioinformatics analysis of 116 hearing loss-associated genes was performed in an affected individual from a five generation Iranian family. Segregation analysis and 3D protein modeling of the p.Arg108 exchange was performed. The two Pakistani families previously identified with S1PR2 pathogenic variants presented profound hearing loss that is also observed in the affected Iranian individuals described in the current study. Interestingly, we confirmed mixed hearing loss in one affected individual. 3D protein modeling suggests that the p.Arg108 position plays a key role in ligand receptor interaction, which is disturbed by the p.Arg108Gln change. In summary, we report the third overall mutation in S1PR2 and the first report outside the Pakistani population. Furthermore, we describe a novel variant that causes an amino acid exchange (p.Arg108Gln) in the same amino acid residue as one of the previously reported Pakistani families (p.Arg108Pro). This finding emphasizes the importance of the p.Arg108 amino acid in normal hearing and confirms and consolidates the role of S1PR2 in autosomal recessive hearing loss.

Neural stem cells (NSCs) have previously been described up to the adult stage in the rat cochlear nucleus (CN). A decreasing neurogenic potential was observed with critical changes around hearing onset. A better understanding of molecular factors affecting NSCs and neurogenesis is of interest as they represent potential targets to treat the cause of neurologically based hearing disorders. The role of genes affecting NSC development and neurogenesis in CN over time on hearing capacity has remained unclear. This study investigated the mRNA abundance of genes influencing NSCs and neurogenesis in rats’ CN over time. The CN of rats on postnatal days 6, 12, and 24 were examined. Real-time quantitative polymerase chain reaction arrays were used to compare mRNA levels of 84 genes relevant to NSCs and neurogenesis. Age- and hearing-specific patterns of changes in mRNA abundance of neurogenically relevant genes were detected in the rat CN. Additionally, crucial neurogenic factors with significant and relevant influence on neurogenesis were identified. The results of this work should contribute to a better understanding of the molecular mechanisms underlying the neurogenesis of the auditory pathway.

EPS8L2, encoding EPS8 signaling adaptor L2 protein, is critical for stereocilia maintenance in cochlear hair cells. Four previously published families have replicated autosomal recessive non-syndromic hearing loss (DFNB106), yet the mutational and clinical spectrum remains poorly described. This study expands the mutational and clinical spectrum of EPS8L2 -associated hearing impairment by identifying five additional individuals in four families, more than doubling the number of reported cases to date, while going deeper into clinical aspects. These include two frameshift variants c.818_827dup, p.(Ala279Glyfs*36) and c.1430dup, p.(Val478Serfs*25), the first nonsense variant c.1878C > A, p.(Tyr626Ter), and the first missense variant with a spliceogenic effect c.767C > G, p.(Thr256Arg). Functional analysis confirmed that the c.767C > G variant causes exon 9 skipping, leading to a frameshift r.701_768del, p.(Gly234Alafs*55). A shared 4.17 Mb homozygous region among two unrelated Iranian families with this variant suggested a founder effect. These individuals uniquely showed U-shaped audiograms as a new association of more severe middle frequency hearing loss. Clinically, affected individuals presented moderate to moderately severe hearing loss that was mostly progressive, with an onset starting between the prelingual stage, extending postlingually. A younger sibling of the first European proband diagnosed with DFNB106 passed newborn hearing screening but was identified with compound heterozygous variants, underscoring the importance of early genetic testing. The progressive nature of EPS8L2 -related hearing loss is unusual for recessive non-syndromic forms and highlights the need for early monitoring and intervention. This work emphasizes the need for early molecular diagnosis and long-term clinical follow-up, while describing the natural history of EPS8L2 that will be important for future therapeutic development.