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

Abstract

The miR-96, miR-182 and miR-183 microRNA (miRNA) family is essential for differentiation and function of the vertebrate inner ear. Recently, point mutations within the seed region of miR-96 were reported in two Spanish families with autosomal dominant non-syndromic sensorineural hearing loss (NSHL) and in a mouse model of NSHL. We screened 882 NSHL patients and 836 normal-hearing Italian controls and identified one putative novel mutation within the miR-96 gene in a family with autosomal dominant NSHL. Although located outside the mature miR-96 sequence, the detected variant replaces a highly conserved nucleotide within the companion miR-96*, and is predicted to reduce the stability of the pre-miRNA hairpin. To evaluate the effect of the detected mutation on miR-96/mir-96* biogenesis, we investigated the maturation of miR-96 by transient expression in mammalian cells, followed by real-time reverse-transcription polymerase chain reaction (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 could be restored by a compensatory mutation that reconstitutes the secondary structure of the pre-miR-96 hairpin, demonstrating that the mutation hinders precursor processing, probably interfering with Dicer cleavage. Finally, even though the mature miR-96 sequence is not altered, we demonstrated that the identified mutation significantly impacts on miR-96 regulation of selected targets. In conclusion, we provide further evidence of the involvement of miR-96 mutations in human deafness and demonstrate that a quantitative defect of this miRNA may contribute to NSHL.

Figure 1.

The novel pre-miR-96(+57T>C) mutation identified in an Italian family with autosomal dominant non-syndromic hearing loss. (A) Multiple alignment of pre-miR-96 sequences (annotated in miRBase) from different vertebrate species obtained using the CLUSTALW software. The nt position +57 within the human pre-miR-96 is indicated by an arrow. Mature miR-96 and miR-96* sequences are boxed, and their seed regions are underlined. hsa, Homo sapiens; ppa, Pan paniscus; ptr, Pan troglodytes; ggo, Gorilla gorilla; mml, Macaca mulatta; mne, Macaca nemestrina; sla, Saguinus labiatus; mdo, Monodelphis domestica; bta, Bos taurus; mmu, Mus musculus; rno, Rattus norvegicus; oan, Ornithorhynchus anatinus; fru, Fugu rubipres; tni, Tetraodon nigroviridis; dre, Danio rerio. (B) Pedigree of the Italian NSHL family carrying the miR-96(+57T>C) mutation. Black symbols indicate affected subjects. The numbers within the symbols represent: age of onset of NSHL (black symbols) and present age (empty symbols). Presence or absence of the mutation is indicated below the genetically analyzed subjects. (C) Electropherogram depicting the pre-miR-96 sequence surrounding the mutated nt. (D) Audiograms showing the progression of the hearing impairment in the proband (III-2). The age at which each audiometric record was obtained is indicated. Each graph point represents the average hearing loss for the right and left ears.

Figure 2.

Different functional consequences of the miR-96(+57T>C) and the known miR-96(+13G>A) mutations on miR-96/miR-96* biogenesis. (A) Predicted secondary structures of wild-type, mutant (+57T>C and +13G>A) and double-mutant (+23A>G+57T>C and +13G>A+66C>T) miR-96 precursors obtained by using the mfold algorithm. The nt positions involved in the mutations are boxed. The double mutants were created to restore the pre-miR-96 secondary structure, in order to verify whether the defects in miRNA expression were dependent on correct folding of the hairpin precursor. Dicer cleavage sites are indicated by arrows. (B) The positions of the 5′ primers used for real-time RT–PCR to quantify the levels of mature miRNA and pre-miRNA species. Primer sequences are listed in Supplementary Material, Table S2. (C) The miR96(+57T>C) impairs the processing of pre-miR-96 to its mature forms, and is rescued by a compensatory mutation (+23A>G) restoring the correct hairpin folding. (D) The known miR96(+13G>A) impairs mature miR-96, but not miR-96* levels, independently from the correct folding of the miR-96 precursor. The effect of the analyzed mutants on pre-miR-96 processing was evaluated by quantitative real-time RT–PCR using the ▵▵CT method. Variations in the expression levels of mature miRNAs and pre-miR-96 in the miR-96(+57T>C) and miR-96(+23A>G+57T>C) (C), or the known miR-96(+13G>A) and miR-96(+13G>A+66C>T) (D) mutants were compared with the wild-type samples (set as 1). Bars stand for mean ± SEM (represented as percentage of variation) of six independent experiments, each performed in triplicate in different days on different cell batches and with different plasmid preparations. The results were analyzed by unpaired t-test (*P< 0.05; **P< 0.01; ***P< 0.001).

Figure 3.

The miR96(+57T>C) mutation impairs the regulation of MYRIP by reducing mature miR-96 levels. (A) Downregulation of MYRIP 3′UTR is only dependent on miR-96, and is impaired in the miR96(+57T>C) mutant. The MYRIP 3′UTR reporter vectors (wild-type, or Δ96 and Δ96* mutants) were cotransfected with plasmids expressing either the wild-type or the +57T>C mutant pre-miR-96. Mock samples were transfected with the empty psiCHECK2 and psiUX vectors. (B) Restoration of correct pre-miR-96 folding rescues the regulation of MYRIP 3′UTR. The wild-type MYRIP 3′UTR reporter vector was cotransfected with the wild-type, the +57T>C or the double-mutant (+23A>G+57T>C) pre-miR-96 expression constructs. Relative luciferase activity is expressed as mean ± SEM of six independent experiments, each performed in triplicate in different days on different cell batches and with different plasmid preparations. The luciferase activity of the empty psiCHECK2 plasmid (A) or of the corresponding psiCHECK2-3′UTR plasmid (B) is set as 1. The results were analyzed by unpaired t-test (*P< 0.05; **P< 0.01; ***P< 0.001).