Novel splice-site variants in TMPRSS3 impair hearing via exon skipping and abrogated protease activity

Hearing loss (HL) is genetically heterozygous, making its genetic diagnosis challenging. Identification of novel HL-associated genes and variants will enhance our understanding of the molecular mechanisms and improve genetic diagnosis. TMPRSS3, encoding a transmembrane serine protease, is implicated in autosomal recessive nonsyndromic hearing loss (ARNSHL), designated as DFNB8 (postlingual onset) or DFNB10 (prelingual onset). Although over 100 pathogenic TMPRSS3 variants have been reported, only seven splice-site variants have been documented to date.

This study investigates the molecular etiology of ARNSHL in three Chinese family and functionally characterizes five novel TMPRSS3 variants, including one non-canonical splice-site variant, two canonical splice-site variants, and two missense variants.

Whole-exome sequencing and gene panel sequencing identified candidate variants, followed by validation with Sanger sequencing. Functional analyses included minigene splicing assays to evaluate the variants' effect on mRNA splicing and a yeast-based functional assay to assess their impacts on protease activity.

Three families carrying compound heterozygous TMPRSS3 variants were identified. The proband with c.205 + 5G>C/c.923T>C (p.Met308Thr) presented with progressive, postlingual, high-frequency-predominant, nonsyndromic sensorineural hearing loss, consistent with DFNB8. Probands with c.572 + 1G>A/c.967G>A (p.Val323Met) and c.1348-2A>G/c.271C>T (p.Arg91Ter) exhibited prelingual, nonsyndromic sensorineural hearing loss. Functional studies revealed that all five novel variants (c.205 + 5G>C, c.923T>C, c.572 + 1G>A, c.967G>A, and c.1348-2A>G) led to reduced protease activity. Notably, the splice-site variants caused exon skipping and resulted in a more pronounced loss of activity compared to the missense variants.

This study expands the spectrum of TMPRSS3 variants and provides mechanistic insights into their pathogenicity. Intriguingly, splice-site variants led to a more severe impairment of enzymatic activity compared to missense variants, providing molecular evidence for the considerable genotype-phenotype heterogeneity observed in TMPRSS3-associated hearing loss.