A novel mutation within the MIR96 gene causes nonsyndromic inherited hearing loss in an Italian family by altering pre-miRNA processing

MiR-96 is part of the conserved miR-183 microRNA (miRNA) family, which plays essential functions in the vertebrate inner ear. Point mutations within the seed region of this miRNA were recently reported to cause autosomal dominant nonsyndromic sensorineural hearing loss (AD-NSHL) in 2 Spanish families. We screened 882 NSHL patients and 836 Italian controls and identified 1 putative novel mutation within the MIR96 gene in a family with AD-NSHL. Although located outside the mature miR-96 sequence, the detected variant replaces a highly conserved nucleotide within the companion miR-96* (processed by the opposite strand of the miRNA precursor), and is predicted to reduce the stability of the pre-miRNA hairpin. Therefore, we evaluated the effect of the detected mutation on miR-96/miR-96* biogenesis by transient expression in HeLa cells of a vector encoding either the wild-type or the mutant miR-96 precursor, followed by real-time RT-PCR. We found that both miR-96 and miR-96* levels were significantly reduced in the mutant, whereas the precursor levels were unaffected. Moreover, miR-96 and miR-96* expression levels were restored by a compensatory mutation that reconstitutes the secondary structure of the pre-miR-96 hairpin, demonstrating that this mutation impairs precursor processing, probably interfering with Dicer cleavage. Finally, we evaluated the effect of the novel mutation on the regulation of 7 selected miR-96 and/or miR-96* putative targets by luciferase reporter assays. The expression of the mutant pre-miR-96 led to a significantly reduced silencing of 3 miR-96/miR-96* predicted targets. In particular, for one of these targets, MYRIP, we demonstrated by site-specific mutagenesis that miRNA-mediated regulation is strictly dependent on the integrity of the miR-96 target site. Moreover, miR-96-mediated downregulation of MYRIP 3’UTR could be restored by expressing the pre-miR-96 double mutant, which has a wild-type secondary structure, indicating that the altered precursor processing can significantly impact on the normal regulation of miR-96 targets. In conclusion, we provide further evidence of the involvement of miR-96 mutations in human deafness and demonstrate that a quantitative defect in this miRNA may be sufficient to cause NSHL.