A novel mutation in MIR96 in an Italian family with nonsyndromic inherited hearing loss

The conserved miR-183 microRNA (miRNA) family is essential for differentiation and function of the vertebrate inner ear. Recently, point mutations in the MIR96 gene were reported in 2 Spanish families with autosomal dominant nonsyndromic sensorineural hearing loss (AD-NSHL) and in a mouse model of NSHL. In this study, we screened 884 NSHL patients and 839 normal-hearing Italian controls for mutations in the miR-183 family and identified one putative novel mutation within MIR96 in a family with AD-NSHL. The detected variant replaces a highly conserved nucleotide in the seed region of the mature miR-96*, which is processed from the complementary strand of the miR-96 precursor and is predicted to reduce the stability of the pre-miRNA secondary structure. Although miR-96* has been detected in several vertebrate species, very little is known about its expression and function. To evaluate the effect of the detected mutation on miR-96/mir-96* processing, we transiently transfected HeLa cells with a vector expressing either the wild-type or the mutant miR-96 precursor, and measured the expression levels of mature miR-96, miR-96*, and pre-miR-96 by real-time PCR. Interestingly, expression levels of both miR-96 and miR-96* were significantly reduced in the mutant (85% reduction, p=0.0006 for miR-96 and 77% reduction, p=0.019, for miR-96*, respectively), whereas the precursor levels were unaffected by the mutation. Surprisingly, similar experiments performed on a vector expressing one of the previously reported miR-96 mutations, miR-96(+13G>A), showed a significant reduction of mature miR-96 but not mir-96* levels, suggesting that different mutations might act with at least partially different pathogenic mechanisms. In particular, as the novel mutation causes a reduction of both mature miRNAs but does not alter the expression of the miRNA precursor, it might impair precursor processing by creating an enlarged bulge close to the Dicer cleavage site. This was confirmed by transiently expressing a double-mutant vector carrying a second \u201ccompensatory\u201d mutation that reconstitutes the secondary structure of the miR-96 precursor. Indeed, both miR-96 and miR-96* levels were significantly increased in the double mutant compared to the single mutant, restoring mature miRNA expression at levels similar to the wild type. It will be important to determine if a quantitative defect in miR-96 expression is sufficient to cause NSHL, or if an alteration of the normal miR-96* expression/function is also contributing to the disease. This is currently being evaluated by in-vitro studies to identify miR-96* targets with relevant function/expression in the auditory system, and by in-vivo analysis of miR-96* expression in animal models