Novel Homozygous Mutation in the MYO15A Gene in Autosomal Recessive Hearing Loss

AUTHORS

Farah Talebi 1 , Farideh Ghanbari Mardasi 2 , * , Javad Mohammadi Asl 3

AUTHORS INFORMATION

1 Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, IR Iran

2 Shoushtar Faculty of Medical Sciences, Shoushtar, IR Iran

3 Department of Medical Genetics, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran

ARTICLE INFORMATION

Zahedan Journal of Research in Medical Sciences: 18 (10); e4256
Published Online: September 28, 2016
Article Type: Case Report
Received: October 6, 2015
Revised: November 11, 2015
Accepted: September 24, 2016
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Abstract

Background: Hearing loss is one of the most common sensory disorders, which can be syndromic and non-syndromic. So far, 61 genes and more than 100 genetic loci have been identified in ARNSHL.

Materials and Methods: In this case report study, in order to verify the ARNSHL in a patient with several clinical symptoms and study the variations of the MYO15A gene for the first time in Southwest Iran, the MYO15A gene was entirely sequenced. Coding exons analysis of MYO15A gene was performed by exon direct sequencing.

Results: A novel homozygous missense mutation, c.9698T > G in exon 59 of the MYO15A gene was founded by Molecular genetic testing in the patient.

Conclusions: This novel mutation results in substituting a Leusin for an Arginin (p.L3233A). It seems that this change is predicted to affect the function of the myosin XVa protein negatively, maybe by disturbing its interaction with whirlin.

Keywords

Hearing Loss MYO15A Novel Mutation Sequencing

Copyright © 2016, Zahedan University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.
1. Introduction

Hearing loss is one of the most common sensory disorders that can significantly affected quality of life [1]. Syndromic and non-syndromic hearing loss display in autosomal dominant, autosomal recessive, Y-linked, X-linked or mitochondrial pattern of inheritance [2]. Autosomal recessive nonsyndromic hearing loss (ARNSHL) accounts for up to 80% of cases of NSHL [3]. To date, 61 genes and more than 100 genetic loci have been identified in ARNSHL (http://hereditaryhearingloss.org/).

The most frequently genes involved in ARNSHL are those encoding myosin XVA (MYO15A, MIM# 602666), gap junction protein beta 2 (GJB2, MIM# 121011), solute carrier family 26 (anion exchanger) member 4 (SLC26A4, MIM# 605646), transmembrane channel- like 1 (TMC1, MIM# 606706), otoferlin (OTOF, MIM# 603681) and cadherin-related 23 (CDH23, MIM#605516), each of which has been contained more than 20 various mutations that most of them have been detected in consanguineous families [4]. In present study, we report a case of novel mutation discovered from the direct mutation screening of all exons in the MYO15A gene in an Iranian patient with hearing loss disorder (HLD).

2. Case Presentation

The patient was a 24-years-old male, the only-child of Iranian consanguineous couple (Figure 1A). He was diagnosed with congenital hearing loss and had no dysmorphic features. There was no significant history of hearing loss in this family and no history of systemic disease in the patient.

A, Pedigree of the family; B, The result of genetic sequencing, showing the homozygous missense mutation CTG > CGG in exon 59 of the patient’s MYO15A gene
Figure 1. A, Pedigree of the family; B, The result of genetic sequencing, showing the homozygous missense mutation CTG > CGG in exon 59 of the patient’s MYO15A gene
2.1. Molecular Analysis

Genomic DNA was extracted from peripheral leukocytes of the patient by the standard salting out protocol, and the PCR was conducted under the following conditions: 200 μM deoxyribonucleotide triphosphates (dNTPs), 100 ng genomic DNA, 2.5 units supertaq polymerase, 1.5 mm MgCl, and 25 pmoL each primer (Table 1). Amplification Carried out in 25 μL volumes and 35 cycles: 94°C for 1 minute, 64°C for 35 seconds and 72°C for 45 seconds. Direct sequencing of the 66 exons performed by using the big dye terminator cycle sequencing ready reaction kit on an ABI Prism 3700 automated genetic analyzer. Finally, the sequencing reactions were carried out and the sequences were compared to the reported gene sequence using the BLASTN program.

Table 1. Primers used for amplification of the MYO15A gene
ExonForward Primers (5' - 3')Reverse Primer (5' - 3')Amplicon Size, bp
2Multiple - available upon request
3ATG ACC AAG CCA GGG GTCCTC TGG CTG GGA GGG TG223
4GAC CCA TGC CAG AAC CAGAGA AAT CTG TGC GTC CCA CC204
5ATC TGT CCG GAT GGA AAC AGTCT GAC TCA TGG CTC AGG TG311
6 - 7GGG AGG TGT GGG AGC TTA GTCG GGA GTA CAT GAG GTG TG499
8TCC TGG AGA GAG TGG TGG TCCTA GGA CAG GCC TTT GGA TG239
9 - 10GGG TGT CCC CAG CTA TGCTAT CTG TAC CTC CCA CCC CG435
11GTT CTC ATC TGC AGC CCA CTAAA CTC ACC CTC CCC AAA TC365
12CAA CTC AGG CCA CCA CAC TAAAA ACA GGA ACA AGT GAT ATG TGC381
13GAC TAC TGG CAT GAG CCA CATGA CCC AGG GAC AGA GAG AG335
14 - 15GCT TTC CGG AGG CAG AGGAG GGA GGC GAG ACC TTG385
16AGG GAA GGT AGG GGC AAACTG TCT CCA AGG AGG TCC AC231
17ATT CAA CAT GGG AGG GAG GTGA GGA CAT GAG GCT GAG AG269
18ATA GTG AGG TTG CCA CCA GGTCT CCA ACA GCT AGC AGC AC262
19TCC CTC CTA GGA TAG ACA GAG AGAAG GCA GGC TGG GTG TG212
20TTC CTC CTC ATT TCG GTC TCCAA GGT CAC ACA GCA TGG G441
21-22TTC CTC CTC ATT TCG GTC TCCAA GGT CAC ACA GCA TGG G441
23TAG CAG ACA CCT CGG GTA GGGAC TCA GTA GTT GTG GAC CCC241
24CTT AGT CCA GCC TCC TGG CTTC AGG CGT GAC CTC TCC297
25AGG GCC TCT CTA CCT TTT GGCTA AGT GCC CTT TCC CCT TC219
26 - 28GTG CCG GTC GTC ACC TCCCC AGG GCA AGG ACA ATG569
29CAC AGA GCA GTG GGT CCA GCTC ATG GCC CAG TTT CAG G231
30GGG GAC TGG AAG GAA CAA CCTT TAA GAC CCT GCC TTG GG368
31CAG CCC TCA GCC CCA AGACT GGG CCC TGC TGA CTC300
32GCA CAG CCA AAC TGG ACT CCCT TCT GCC TGG GAG TGG566
33TCT GTT CAT GTT TAG GGT CTG GCTC AGC CTG TCC CAG CAG396
34 - 35GGA GAA AGC CAC TGA ATA CCA GGAG AAG CTC TCA GGT CAC CC553
36 - 38AGT GTC AGG TGC CTG TTG CTCC TCT TTA CAG CTT GTG TCT CC620
39 - 40TCT GGA GTC CCA GAG AGC AGGGG CCA TGA TGG ACA CTC549
41 - 42ATG TGA TGG GAA AGG GAG ACCTG TGC CCA CAG ACT TCC TC460
43ACT CTA GCC TGG GGG ACA ACCCC AAG TCC TAG ACC CTC CT320
44CCC AGG AGG ACA GAA AAA GGGGG AGG GGG AGA TTC AAT AA356
45AGT ATA GTC CAG CCT GGG TCCCTG GCT GTG CCT CTG ACT G202
46TGG CCA TCT CAT CCA TTT CTCAC AGC TAG GAG CTG CAC AC397
47GAA CCA GCT GGA CAC ACA GAAAA TGG GTT TGC TTC AAT GG458
48GGG CAG GAC AGG ATC AGA AGAGG GAG ATC CCT GTT GCT G291
49 - 50CTA GGC CTC TGG GAG TGGCAC CAC GAG TGG GTG AAA C400
51CCC CTT AGT CAC AAG ACA AGA CTTA TCC CCA CTC GCC TCA C319
52CTA GGG GTT CGC TTG TCA GTAGT GGG GCC TCC GAG ACT295
53TGT GAG GCT CAT TTC AGT GCAGG GTG CTG AGA ATC AGA GG352
54 - 55TGT GTC CCC TTT CTG TTC TGTGA TAG ATG GGG AAA CTG AAC C534
56GTG CCC ACC CTG TTC TTA TGCCT CCT GGA GCA TGG ACA C222
57TCT CAG CTC AAT CCC AGG AGTCC ACC CAG TCC CCA AG271
58ATG GGG GAG TAA ATG CCT TCGGC TTG TGT CTC CCA TTC AT594
59CAG GAG ACA AGG GCT GTC CCTG GAG CCT GGG CTG TC214
60AGA AGG ACA GAG GTC AAG CCAAA TCT GGG TGG AGG GC236
61AAG CTG TGT CCC AGA ACA GGACA GGG CCT GAA TCA TGA AC418
62TGA GAG GGC AGG GTT GCCAT GCA TGT CCC CAG GTC271
63ACA GTG AGG ATT GCC TGA GCTAC CCA TCC TCC ATG ACC AC269
64AGC CCA GAG AAG CTA TGC AGAGG CTC AGA GGA GGG AAG AG374
65TGG TTG AGA CTA TCC TCG CCGAC CTG ACC TAT CTT GGA GCC271
66CAA GGT AAG AGC TGG GGA AGTTG ATC CTG AGA GGT TCA GTG240

The effect of Candidate variant in protein structure and phylogenetic conservation was predicted by using bioinformatic tools like PolyPhen-2 (PolymorphismPhenotyping v2).

3. Results

Sequencing analysis of the patient, after comparison with MYO15A reference sequence in 1000 Genomes database, demonstrated a novel mutation, a homozygous missense mutation, c.9698T > G in exon 59 of the MYO15A gene (Figure 1B). The c.9698T > G mutation is novel and has not been previously described in HLD.

The novel homozygous missense mutation was predicted to be possibly damaging by in silico prediction of the recognized variant, Polyphen 2 (probably damaging, score 1.00).

4. Discussion

We analyzed an affected person with HLD with PCR and direct sequencing of coding exons of the MYO15A gene. As a result we identified a genetic variant of MYO15A in ARNSHL patient. The mutation identified in our patient involved a novel homozygous mutation, c.9698T > G in exon 59, which results in substituting a Leusin to an Arginin (p.L3233A) in the ferm domain and tail region of the Myosin protein. So, this exchange amino acid results in alter a nonpolar amino acid to polar positively charged that can modify interaction of tail region of myosin with membranous compartments and change its movement to actin filaments. This change is predicted to negatively affect the usual function of the myosin XVa protein by in silico. Overall, it seems that this amino acid has an important role in the myosin-XV protein, and mutation at this site results in pathogenicity and deafness.

MYO15A has 66 exons and its coding protein, myosin XVa, has a critical role in formation of stereocilia in hair cells of the cochlea [5]. Myosin XVa in the organ of Corti is localized completely at the tips of stereocilia and is anactin-activated ATPase that uses the hydrolysis of ATP to move on actin filaments. The tip of a stereocilium is one of the proposed sites of mechano-electrical transduction and the site of stereocilia growth [6]. Myosin XVa is required for proper function and formation of the mechanotransduction machinery. All myosins are composed of one or two heavy chains and several light chain. The tails of the myosins presumed to bind to membranous compartments, which would be moved relative to actin filaments [7].

Twenty-nine mutations have been described in MYO15A in HGMD (http:// www.hgmd.org).

In summary, this is the first case with hearing loss in south-west of Iran confirmed by genetic analysis involving a novel MYO15A gene mutation. Further studies are required to understand the structural and functional changes of proteins involved in this disorder and their relations with phenotypic spectrum. Genotype-phenotype relations of MYO15A mutations and degree of hearing loss suggest that mutations in all 66 exons cause intense deafness.

Footnotes
References
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