A Splice-Site Variant in ACY1 Associated with Congenital Hearing Loss: Clinical, Biochemical, and Zebrafish...

Variants in the aminoacylase 1 (ACY1) gene are associated with diverse clinical phenotypes, but their roles in hearing loss remain unclear. This study aimed to investigate the potential role of ACY1 in hearing loss. Exome sequencing was performed on 396 patients with sensorineural hearing loss. We identified a homozygous splicing variant, c.1063-1G>A, in ACY1 as a plausible candidate variant in a 5-year-old girl with congenital hearing loss and normal developmental milestones over a 4-year follow-up. Functional assays, including urinary organic acid analysis and aminoacylase 1 enzyme activity testing in Epstein-Barr virus (EBV)-transformed lymphoblasts, confirmed aminoacylase 1 deficiency in the patient. In vitro splicing assays showed that the c.1063-1G>A variant activated a cryptic splice site, causing aberrant splicing, a frameshift, and a premature stop codon. A zebrafish model with morpholino-mediated acy1 knockdown revealed a significant reduction in hair cells and impaired auditory function, which were effectively rescued by wild-type but not mutant human ACY1 mRNA. Furthermore, transcriptomic profiling of zebrafish auditory hair cells, combining fluorescence-activated cell sorting (FACS) with RNA sequencing, revealed downregulation of critical inner ear development genes, including gfi1ab and atoh1a/b, which was validated by RT-qPCR. These results provide clinical and experimental evidence supporting the functional impact of the ACY1 c.1063-1G>A variant and its potential involvement in congenital hearing loss. Our findings support ACY1 as a candidate gene for hereditary hearing impairment, although additional unrelated patients and independent validation are needed to further establish the gene-disease relationship.