**Background:** Sensorineural age-related hearing loss (ARHL) affects a large proportion of the elderly population, with an estimated heritability of 35–70%. Despite large-scale genome-wide association studies, the genetic risk factors remain largely unknown, and ARHL is considered a complex, polygenic disorder. Two hypotheses to explain the missing heritability are the additive burden of many small-effect variants or a limited number of rare large-effect variants. Recent whole exome sequencing studies have identified rare variants in Mendelian hearing loss genes as risk factors for adult-onset hearing loss. Usher syndrome is a rare autosomal recessive disorder characterized by congenital sensorineural hearing impairment, progressive retinitis pigmentosa, and sometimes vestibular dysfunction. It is caused by mutations in at least 13 genes, including USH1G and ADGRV1. The protein products of these genes belong to the same functional network involved in stereocilium biogenesis, hair bundle organization, and mechanoelectrical transduction in cochlear hair cells.
**Methods:** A 73-year-old female with progressive bilateral high-frequency hearing loss that began around age 60 and culminated in profound deafness in her seventies presented to the Audiology Unit of the Policlinico Ca’ Granda in Milan. She had minimal benefit from hearing aids, no noise exposure, no vestibular symptoms, and normal ocular and vestibular evaluations. Her sister, three years older, was also affected by severe progressive sensorineural hearing loss. After genetic counseling and informed consent, genomic DNA was extracted from saliva samples. Diagnostic clinical exome next-generation sequencing (TruSight One Expanded 6794 genes panel, Illumina NextSeq500) was performed on the proband. After filtering common variants with population frequency >1%, an unbiased prioritization using HPO phenotypic descriptors and a targeted analysis of 78 genes involved in non-syndromic hereditary deafness was conducted. Variants were classified according to ACMG-AMP criteria. The identified variants were confirmed by direct Sanger sequencing in both siblings. Structural predictions for the missense mutation in ADGRV1 were performed using ColabFold (AlphaFold2-MMseqs2) and RoseTTAFold algorithms, and effects on protein stability were predicted with DynaMut2, Polyphen, SIFT, and CADD.
**Key Results:** NGS sequencing revealed two heterozygous variants in the proband: a rare truncating variant in USH1G (NM_173477.5: c.1162G>T, p.E388X) with a population frequency of 0.0008% (8 × 10^-6), and a previously undescribed missense variant in ADGRV1 (NM_032119.4: c.13418T>A, p.I4473N). No other variants associated with hereditary deafness were detected among the 78 analyzed genes. The novel ADGRV1 variant was absent from a population of 2400 subjects without hearing loss. Sanger sequencing confirmed both variants in heterozygosis in both siblings. The USH1G truncating variant is predicted to lack the C-terminal SAM domain and PDZ-binding motif, and was classified as pathogenic (class 5) according to ACMG criteria. The ADGRV1 missense variant falls within the 30th Calx-β domain. Structural predictions indicated that the I4473N substitution disrupts a hydrophobic cluster normally formed by I4473 and I4451, creates a steric clash with G4447, and may impair Ca2+ binding. DynaMut2 predicted a decrease in domain stability by 0.22 kcal/mol. The variant was classified as likely pathogenic (class 4) based on absence from population databases (PM2), co-segregation with deafness (PP1), location in a well-established functional domain (PM1), moderate conservation, large Grantham distance, and structural predictions (PP3).
**Clinical Implications:** This study provides a novel example of digenic inheritance of age-related hearing loss involving heterozygous variants in two Usher syndrome genes. The USH1G and ADGRV1 protein products belong to the same functional network involved in stereocilium organization and function in cochlear hair cells. The authors propose that the two defective alleles act additively through combined or complex haploinsufficiency, resulting in the profound deafness observed in both siblings. This case expands the spectrum and complexity of phenotypic consequences of Usher gene mutations beyond simple Mendelian inheritance of classical Usher syndrome. It highlights that different mutations of the same gene may either cause syndromic deafness or constitute a risk factor for adult-onset hearing loss. The findings underscore the importance of investigating rare variants in Mendelian hearing loss genes as potential contributors to ARHL, and suggest that digenic inheritance should be considered in familial cases of late-onset hearing loss. The study was limited to two siblings, as other family members were not available for genetic testing. The parents were deceased and reportedly had no hearing problems, suggesting each parent likely carried one variant allele. This report adds to the growing body of evidence that ARHL has a complex genetic architecture with shared etiology between Mendelian and complex inheritance.