Nonmuscle myosin heavy-chain gene MYH14 is expressed in cochlea and mutated in patients affected by autosomal dominant hearing impairment (DFNA4)

Abstract

Myosins have been implicated in various motile processes, including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Different members of the myosin superfamily are responsible for syndromic and nonsyndromic hearing impairment in both humans and mice. MYH14 encodes one of the heavy chains of the class II nonmuscle myosins, and it is localized within the autosomal dominant hearing impairment (DFNA4) critical region. After demonstrating that MYH14 is highly expressed in mouse cochlea, we performed a mutational screening in a large series of 300 hearing-impaired patients from Italy, Spain, and Belgium and in a German kindred linked to DFNA4. This study allowed us to identify a nonsense and two missense mutations in large pedigrees, linked to DFNA4, as well as a de novo allele in a sporadic case. Absence of these mutations in healthy individuals was tested in 200 control individuals. These findings clearly demonstrate the role of MYH14 in causing autosomal dominant hearing loss and further confirm the crucial role of the myosin superfamily in auditive functions.

Figure 1

RT-PCR of the Myh14 gene in mouse cochlea. Specific oligonucleotides C1F (5′-AGCATTGGAAGAGGAGTCCC-3′) and C2R (5′-GCTCCAGTCGTTCTACAGC-3′), spanning coding exons 26–30, were used to detect the expression of Myh14 in mouse cochlea RNA, as well as in two mouse cell lines, B16 (melanoma) and ES cells. Cochlea RNA was kindly provided by K. Avraham (Tel Aviv). In brief, it has been prepared from inner ears dissected out of the temporal bones of mice at embryonic day (E) 14, E16, E18, and P0. Negative control was performed without cDNA aliquot in the reaction. Marker: 1-kb DNA ladder (BioLabs).

Figure 2

Immunohistochemistry of NMHC IIC in mouse neonatal cochlea. Gelatin tissues at P0 were sectioned (10 μm) and fixed with 4% paraformaldehyde. A, Negative control obtained using only biotin-conjugated immunoglobulin G secondary antibodies. B, Section immunolabeled with anti-NMHC IIC antibodies (Buxton et al. ; Golomb et al. 2004), revealing a high expression in the cells surrounding the scala media. The insert is enlarged in panel C. sv = scala vestibuli; sm = scala media; st = scala tympany; rm = Reissner’s membrane; oc = organ of Corti; stv = stria vascularis. No expression in the vestibular epithelia has been detected (data not shown). Scale bars = 20 μm.

Figure 3

MYH14 nonsense and missense mutations. A, Sequence data of the nonsense S7X mutation. B, Pedigree of the Belgian family with the R726S mutation and results showing its presence after AlwNI restriction-enzyme digestion. Haplotypes were constructed with markers around the MYH14 gene. Shared haplotypes are indicated with rectangles. MWM = molecular weight marker; N = normal. C, Pedigree of the Italian family with the L976F mutation and results showing sequence data in the proband (III:2) and in a healthy member (II:3).

Figure 4

ClustalW (European Bioinformatics Institute) alignment of the amino acid sequences of smooth muscle and nonmuscle myosins of class II; sequence alignments for MYH14 missense mutations. The residues mutated in NMHC IIC and their conservation across species are underlined. Hs = Homo sapiens; Rn = Rattus norvegicus; Mm = Mus musculus.