The Promise of Whole-Exome Sequencing in Detecting Novel Variants of the MYO15A Gene in Two Iranian Pedigrees with Non-Syndromic Hearing Loss, A descriptive experimental study

Background: Clinical genetic diagnosis of non-syndromic hearing loss (NSHL) is quite challenging. With regard to its high heterogeneity as well as large size of some genes, it is also really difficult to detect causative mutations using traditional approaches. One of the recent technologies called whole-exome sequencing (WES) has been thus developed in this domain to remove the limitations of conventional methods. Methods: This study was a report on two unrelated pedigrees with multiple affected cases of hearing loss (HL). Accordingly, clinical evaluations and genetic analysis were performed in both families. Results: The implementation of WES to uncover autosomal recessive non-syndromic hearing loss (ARNSHL) and its related variants was reported in the present study. Two novel variants of MYO15A i.e. c.T6442A:p.W2148R and c.10504dupT:p.C3502Lfs*15 were correspondingly identified and then segregations were confirmed using Sanger sequencing. According to online prediction tools, both identified variants seemed to have damaging effects. Conclusion: This study further supported the effectiveness of WES for genetic diagnosis of ARNSHL as a first approach.


Introduction
Hearing impairment is considered as an etiologically heterogeneous sensory deficiency. In this regard, genetic hearing loss (HL) has been divided into syndromic and non-syndromic types. Considering the high rate of consanguineous marriages in the Middle East, autosomal recessive non-syndromic hearing loss (ARNSHL) is 3 reportedly more prevalent. [1] However; due to the wide variety of pathogenic genes associated with non-syndromic hearing loss (NSHL), including both nuclear and mitochondrial ones, the disease includes diverse patterns of inheritance comprised of autosomal dominant, autosomal recessive, mitochondrial, and X-linked recessive.
Disease heterogeneity has been admittedly recognized as the most important challenge in genetic diagnosis of NSHL. Diagnostic approaches which have been relied on conventional methods based on genetic testing of the most common genes, often fail to determine the exact genetic cause of the disease in many countries including Iran. [2] In heterogeneous populations like Iran, the distribution of mutations in the gap junction beta-2 protein, also known as connexin 26, (i.e. GJB2) gene as a major cause of ARNSHL can be extremely diverse depending on patients' ethnicities. The prevalence rate of GJB2-related hearing loss (HL) has been reported by 38.3% in northern Iran, but the percentage of such variations has been found rarely in southern regions. [2] Various frequencies of causative mutations, compound heterozygotes, as well as nuclear modifier genes can also render the molecular diagnosis of ARNSHL as a challenge. [2,3] Moreover, mutation screening in large genes such as MYO15A can be impeded following the use of traditional approaches based on Sanger sequencing. [4] Today, molecular genetic testing on the basis of multi-gene screening such as whole-exome sequencing (WES) are being used instead of traditional diagnostic procedures. [5] In this study; WES was employed as a first approach to find genetic causes of NSHL in two distinct pedigrees, manifesting an autosomal recessive type of NSHL.

Subjects and Clinical Evaluations
The study was approved by the Ethics Committee of Shahrekord University of Medical Sciences (IR.SKUMS.REC.1397.008), Iran. Two Iranian families from Hamedan Province with hearing impairments with no additional symptoms were thus studied. Moreover, informed written consent was taken from both families and the proband from each family was further subjected to clinical evaluations of the inner ear accompanied by pure-tone audiometry (PTA).

Molecular Analysis
Genomic deoxyribonucleic acid (DNA) was extracted from whole peripheral blood of Data analysis was correspondingly performed using an in-house developed pipeline, adopted from Genome Analysis Tool Kit v3.6 and ANNOVAR software. [6] Homozygous missense, start codon change, splice site, nonsense, stop loss, and indel variants with minor allele frequency <1% were further filtered in dbSNP

Clinical and molecular findings
The first family pedigree is displayed in Figure 1A. Accordingly, four members of the pedigree including two affected and two unaffected individuals who consented to be included in this study are indicated. In this family, the proband (V: 4) was a 21-yearold woman with congenital HL. No additional abnormal phenotypic features were also observed in the proband, including visual impairments or any limb and facial malformations. The parents were consanguineous and both showed normal hearing.
According to the audiogram, the proband is suffering from congenital profound deafness ( Figure 1B).
A three-generation pedigree, depicted in Figure 2A, was presented as the second family with ten members, six males and fourteen females, suffering from ARNSHL.
The proband was a 25-year-old male individual, born as the second child of nonsanguineous healthy parents, who had been diagnosed for congenital HL when he 6 was one year old. At the age of 5, he had gone through bilateral cochlear implant surgery based on physical examinations and audiometry testing. Audiogram analysis also confirmed HL to be profound in the proband (II: 3) ( Figure 2B).  (Table 1).

Considering all limitations in
Additionally, the results of Sanger sequencing confirmed the presence of p.W2148R variant in MYO15A gene in the proband and other affected members who were studied, but the unaffected sister (V.5) and her father were found heterozygous for the mutation ( Figure 4A).
The findings of Sanger sequencing also revealed co-segregation of c.10504dupT variant in the second family ( Figure 4B). The affected proband was thus homozygous, whereas his unaffected sister (II:5) and his father (I:1) were heterozygous for this locus and his unaffected brother (II:2) lacked the variant.
It should be noted that these two variants were absent in 50 ethnically-matched control cases. 7

Discussion
Based on WES data as well as segregation and genotype-phenotype correlational study, mutations in MYO15A were identified as the causes of NSHL in the first and second families examined.
MYO15A, a new branch of the myosin superfamily protein-coding gene, has thus a role in stereo cilia formation. [7] This gene is considered as the third leading cause of ARNSHL in many populations, [8] including Iran, with a reportedly prevalence rate ranged from 4.8-9.6%. [9][10][11] Mutations in this gene are accordingly associated with severe-to-profound HL. Screening of 66 coding exons through Sanger sequencing is expensive and more time-consuming. On the contrary, high-throughput techniques can save time and money [12].
MYO15A is a different form of myosins with long N-terminal extensions preceding the conserved motor domain [13]. The novel c.T6442A: p variant is located in the first domain of myosin tail homology4 (MTH4) and is also conserved between different species (data not shown). The substitution of a highly conserved (Table 1) hydrophobic amino acid non-polar tryptophan for arginine can thus lead to loss of the positive charge on the arginine side-chain.
The c.T6442A:p.W2148R variant in the arginine side-chain of MYO15A can accordingly alter protein structure and interfere with actin binding to the plasma membrane as well as microtubule binding. In this respect, interaction of MyTH4 domain and PDZ domain of whirlin proteins seems essential for formation of a complex of actin microfilaments at the stereo cilia tips. Moreover, interaction of MYO15A and EPS8 can have similar activities in acting binding of cochlear hair cell stereo cilia. [14,15] In the second studied family, WES could successfully detect a novel homozygous insertion variant i.e. c.10504dupT:p.C3502Lfs*15 in MYO15A gene, co-segregated with the disease within the pedigree. This variant between the second FERM and PDZ domains could also lead to a reading frame shift at position 10504 and a stop codon (p.C3502Lfs*15) with truncation and translation of mRNA resulting in lack of its conserved amino acids at C-terminal (data not shown) .
Today, the topic of oligogenic inheritance traits has made a big wave in diagnostic medicine; since, in many monogenic diseases, it represents that there is not just one gene affected phenotype which causes new challenges in diagnosing these diseases and it can be more complicated for diseases with heterogenic pathology in which many genes are involved [16].

Conclusion
It was concluded that accurate mutational analysis of MYO15A, as a third cause of HL in Iran is of utmost importance for genetic counseling and even prenatal diagnosis. [17] As stated before, traditional approaches are not affordable for clinical applications. The present study confirmed that WES is a reliable first approach for screening of MYO15A variants in patients with hearing impairments.

Consent for publication: Informed consent for publication was obtained.
Availability of data and materials: Dr. Samira Asgharzadeh had full access to the study and take full responsibility for the integrity and accuracy of data analysis.   Identification of c.T6442A:p.W2148R and c.10504dupT:p.C3502Lfs*15 variants in impaired-a