Recurrent and Novel Pathogenic Variants in Genes Involved with Hearing Loss in the Pakistani Population

Abstract

Background: Molecular diagnostic rates for hereditary hearing loss vary by genetic ancestry, highlighting the importance of population-specific studies. In Pakistan, where consanguineous marriages are prevalent, genetic research has identified many autosomal recessive genes, advancing understanding of rare and novel hearing loss mechanisms. This study aimed to identify pathogenic genetic variants in 31 families from Azad Kashmir, Pakistan, presenting non-syndromic hearing loss.

Methods: We conducted exome sequencing and bioinformatics analysis, and targeted gene sequencing on 31 Pakistani families with hearing loss.

Results: We identified ten pathogenic, three likely pathogenic variants, and one variant of uncertain significance, comprising six nonsense, four missense, three frameshift, and one deep intronic variant, across ten hearing loss-associated genes (MYO15A, GJB2, SLC26A4, TMC1, HGF, TMIE, SLC19A2, KCNE1, ILDR, PCDH15 and MYO6) in 25 families. The overall diagnostic rate, including families with pathogenic and likely pathogenic variants, was 77.4%. GJB2 was the most frequently affected gene, identified in seven families. Thirteen out of 14 identified variants were homozygous. Notably, we identified two novel variants: MYO15A (NM_016239.4, DFNB3) c.870C>G, p.(Tyr290*) and MYO6 (NM_016239.4, DFNB37) c.3465del, p.(Pro1156Leufs*9). Additionally, we identified c.10475dupA, p.(Leu3493Alafs*25) in MYO15A (NM_016239.4, DFNB3) and c.617T>A, p.(Leu206*) in SLC26A4 (NM_000441.2, DFNB4), previously documented in ClinVar but unpublished. We also propose SLC19A2 as a candidate gene presenting as non-syndromic hearing loss, despite its association with thiamine-responsive megaloblastic anemia syndrome.

Conclusion: Our work expands the genotypic and phenotypic spectrum of hearing loss by emphasizing the importance of investigating under-represented groups to identify unique genetic variants and clinical characteristics. Such efforts deepen understanding of genetic diversity in under-represented populations to improve diagnosis and treatment strategies.

Fig. 1

Pedigrees of 25 families, illustrating the segregation of variants identified. Genotypes are indicated as +/+ for wild-type. +/− for heterozygous, and −/− for homozygous individuals in the respective families. Affected individuals are represented by filled black squares (males) and circles (females), while deceased individuals are indicated with a diagonal line. The variants in bold have never been published in the literature

Fig. 2

Audiometric air conduction thresholds from the 14 probands of each Pakistani family revealed a consistent phenotype of prelingual, severe-to-profound deafness

Fig. 3

Sanger sequencing electropherograms showed 14 variants identified in affected individuals. Each variant is compared to a control in the family except PKHL-03, PKHL-10, PKHL-16, PKHL-17, and PKHL-23, where only affected individuals supported in segregation. The variant sites are boxed in red. The variants in bold have never been reported in the literature

Fig. 4

Overview of the genes and their variants identified in this cohort. (a) The number and type of variants identified, with colour codes corresponding to the genes in the panel. (b) The contribution of each gene to the overall cohort

Fig. 5

Structural modelling of three single amino acid substitutions in the genes TMC1 (a), SLC26A4 (b), and TMIE (c), mapped onto their respective three-dimensional protein structures from the Alphafold protein structure database (https://alphafold.ebi.ac.uk/). The substituted residues are marked and labelled in orange, while the rest are in navy blue. Dashed lines indicate hydrogen bonds, and “O” in parentheses represents the main chain carbonyl group. TM2 refers to the second transmembrane helix of TMIE. The wild-type structures are shown on the left, and mutant structures on the right in a, b and c