Congenital hearing impairment (HI) is a genetically highly heterogeneous disorder in which prompt recognition and intervention are crucial to optimize outcomes. In this study, we used exome sequencing to investigate a large consanguineous Pakistani family with eight affected individuals showing bilateral severe-to-profound HI. This identified a homozygous splice region variant in STX4 (c.232 + 6T>C), which causes exon skipping and a frameshift, that segregated with HI (two-point logarithm of odds (LOD) score = 5.9). STX4, a member of the syntaxin family, is a component of the SNARE machinery involved in several vesicle transport and recycling pathways. In silico analysis showed that murine orthologue Stx4a is highly and widespread expressed in the developing and adult inner ear. Immunofluorescent imaging revealed localization of STX4A in the cell body, cell membrane and stereocilia of inner and outer hair cells. Furthermore, a morpholino-based knockdown of stx4 in zebrafish showed an abnormal startle response, morphological and developmental defects, and a disrupted mechanotransduction function in neuromast hair cells measured via FM1–43 uptake. Our findings indicate that STX4 dysfunction leads to HI in humans and zebrafish and supports the evolutionary conserved role of STX4 in inner ear development and hair cell functioning.

Intellectual disability (ID), which affects around 2% to 3% of the population, accounts for 0.63% of the overall prevalence of neurodevelopmental disorders (NDD). ID is characterized by limitations in a person’s intellectual and adaptive functioning, and is caused by pathogenic variants in more than 1000 genes. Here, we report a rare missense variant (c.350T>C; p.(Leu117Ser)) in HACE1 segregating with NDD syndrome with clinical features including ID, epilepsy, spasticity, global developmental delay, and psychomotor impairment in two siblings of a consanguineous Pakistani kindred. HACE1 encodes a HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1), which is involved in protein ubiquitination, localization, and cell division. HACE1 is also predicted to interact with several proteins that have been previously implicated in the ID phenotype in humans. The p.(Leu117Ser) variant replaces an evolutionarily conserved residue of HACE1 and is predicted to be deleterious by various in silico algorithms. Previously, eleven protein truncating variants of HACE1 have been reported in individuals with NDD. However, to our knowledge, p.(Leu117Ser) is the second missense variant in HACE1 found in an individual with NDD.

Otitis media is the most frequently diagnosed disease and a leading cause of hearing loss in young children. However, genetic contributors to susceptibility and pathogen–host–environment interactions in otitis media remain to be identified. Such knowledge would help identify at-risk individuals and effectively monitor, diagnose, and treat patients with otitis media. Through exome and Sanger sequencing, we identified a rare, deleterious splice variant SLPI c.394+1G>T co-segregating with otitis media in a large pedigree, with a genome-wide significant maximum LOD score of 4.59. Alternative splicing of SLPI was observed in saliva RNA of variant carriers. In bulk mRNA-seq data from an independent cohort of children with otitis media, SLPI was co-expressed with genes involved in infection, immune response, inflammation, and epithelial cell organization. After inoculation of non-typeable Haemophilus influenzae, Slpi was upregulated in polymorphonuclear leukocytes and epithelial cells of mouse middle ears. Furthermore, in the human middle ear, Haemophilus was significantly enriched in non-carriers, whereas Family-XI-Incertae-Sedis and Dialister were significantly enriched in variant carriers. Given the role of SLPI in immune modulation and host defense in mucosal epithelia, our findings support the SLPI variant as modulating susceptibility to otitis media.

COCH is the most abundantly expressed gene in the cochlea. Unsurprisingly, mutations in COCH underly deafness in mice and humans. Two forms of deafness are linked to mutations in COCH , the well-established autosomal dominant nonsyndromic hearing loss, with or without vestibular dysfunction (DFNA9) via a gain-of-function/dominant-negative mechanism, and more recently autosomal recessive nonsyndromic hearing loss (DFNB110) via nonsense variants. Using a combination of targeted gene panels, exome sequencing and functional studies, we identified four novel pathogenic variants (two nonsense variants, one missense and one inframe deletion) in COCH as the cause of autosomal recessive hearing loss in a multi-ethnic cohort. To investigate whether the non-truncating variants exert their effect via a loss-of-function mechanism, we used mini-gene splicing assays. Our data showed both the missense and inframe deletion variants altered RNA-splicing by creating an exon splicing silencer and abolishing an exon splicing enhancer, respectively. Both variants create frameshifts and are predicted to result in a null allele. This study confirms the involvement of loss-of-function mutations in COCH in autosomal recessive nonsyndromic hearing loss, expands the mutational landscape of DFNB110 to include coding variants that alter RNA-splicing, and highlights the need to investigate the effect of coding variants on RNA-splicing.

Hearing loss (HL) is a common sensory disorder, with syndromic forms accounting for ~30% of genetic cases. Due to phenotypic and genetic heterogeneity, accurate diagnosis remains challenging. Exome sequencing (ES) offers a powerful tool to uncover the underlying genetic causes. This study aimed to investigate the genetic basis of syndromic hearing loss (SHL) in North African Moroccan patients through ES. Seven individuals with suspected SHL were recruited from Hassan II University Hospital, Fez. After clinical and audiological assessments, ES was performed to identify causal genetic variants. Two of the participating individuals had Usher syndrome, and one each had Cornelia de Lange syndrome, Wolfram syndrome, Jervell and Lange-Nielsen syndrome, CHARGE syndrome, and Waardenburg syndrome. The causes of all these syndromes were determined, with pathogenic variants in MYO7A, USH1G, SMC1A, WFS1, KCNQ1, CHD7, and MITF. Across the combined cohort of reported Moroccan SHL cases, variants in CHD7 and MYO7A were among the most frequently observed, while USH1G and MITF variants were rare. This study enhances the understanding of SHL in North Africa, revealing a high level of locus and allelic heterogeneity. Examining disparate populations yields novel insights into the etiology of SHL, which can subsequently enhance genetic diagnosis and tailored management strategies.