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Korean Society of Life Science (한국생명과학회)
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Identification of Homozygous Mutations in Two Consanguineous Families with Hearing Loss

청력 장애를 나타내는 두 근친 가계로부터 동형접합성 돌연변이의 분리

  • Lim, Si On (Department of Biological Sciences, Kongju National University) ;
  • Park, Hye Ri (Department of Biological Sciences, Kongju National University) ;
  • Jung, Na Young (Department of Biological Sciences, Kongju National University) ;
  • Park, Cho Eun (Department of Biological Sciences, Kongju National University) ;
  • Kanwal, Sumaira (Department of Biosciences, COMSATS University Islamabad) ;
  • Chung, Ki Wha (Department of Biological Sciences, Kongju National University)
  • 임시온 (공주대학교 생명과학과) ;
  • 박혜리 (공주대학교 생명과학과) ;
  • 정나영 (공주대학교 생명과학과) ;
  • 박초은 (공주대학교 생명과학과) ;
  • 수매라 칸월 (COMSATS 대학교(이슬라마바드) 바이오과학과) ;
  • 정기화 (공주대학교 생명과학과)
  • Received : 2021.03.19
  • Accepted : 2021.05.10
  • Published : 2021.05.30
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Abstract

Hearing loss is a group of clinically and genetically heterogeneous disorders characterized by congenital- to adult-onset deafness with frequent additional symptoms such as myopathy, nephropathy, and optic disorders. It is commonly divided into two types: syndromic, with no other symptoms, and nonsyndromic, with other symptoms. Autosomal recessive hearing loss is relatively frequent in Pakistan, which may be due in part to frequent consanguineous marriages. This study was performed by whole exome sequencing to determine the genetic causes in two Pakistani consanguineous families with autosomal recessive hearing loss. We identified a pathogenic homozygous variant (p.Leu326Gln in MYO7A) in a family with prelingual-onset hearing loss and two variants of uncertain significance (p.Val3094Ile in GPR98 and p.Asp56Gly in PLA2G6) in a family with early-onset hearing loss concurrent with muscular atrophy. The missense mutations in MYO7A and PLA2G6 were located in the highly conserved sites, and in silico analyses predicted pathogenicity, while the GPR98 mutation was located in the less conserved site, and most in silico analysis programs predicted its nonpathogenic effect. Homozygosity mapping showed that both alleles of the homozygous mutations identified in each family originated from a single founder; spread from this single source might be due to consanguineous marriages. This study will help provide exact molecular diagnosis and treatment for autosomal recessive hearing loss patients in Pakistan.

청력 장애(hearing loss)는 임상적 및 유전적으로 상당히 이질적인 질병들의 그룹으로, 일반적으로 증후군 유형(syndromic type)과 비증후군 유형(non-syndromic type)으로 구분된다. 상염색체 열성의 청력 장애 환자가 다른 나라들에 비해 파키스탄에서는 상대적으로 흔하게 관찰되는데, 그 원인으로는 빈번한 근친 결혼의 문화가 일부 관여할 것으로 여겨진다. 본 연구는 상염색체 열성의 청력 장애 환자가 있는 두 파키스탄의 근친 가계를 대상으로 전장 엑솜 서열분석(whole exome sequencing)을 실시하여 유전적 원인을 규명하기 위해 수행되었다. 환자의 유전체 분석 결과, 우리는 언어 습득전 발병(prelingual onset)의 청력 장애 가족으로부터 MYO7A 유전자에서 병원성으로 판단되는 동형접합성 돌연변이인 p.Leu326Gln을 분리하였으며, 조기 발병의 청력 장애와 동시에 근위축(muscular atrophy)을 나타내는 환자 가족에서는 병원성이 확실하지 않는 두 변이(variants of uncertain significance)를 GPR98 유전자(p.Val3094Ile)와 PLA2G6 유전자 (p.Asp56Gly)에서 각각 분리하였다. MYO7A 및 PLA2G6 유전자의 missense 돌연변이는 고도로 보존된 단백질 부위에 위치했으며, 인실리코 분석(in silico analysis)에서도 병원성을 예측하였다. 그러나, GPR98 유전자의 돌연변이는 보존성이 다소 낮은 부위에 위치하였으며, 대부분의 인실리코 분석도 비병원성으로 예측했다. 동형접합성 매핑(homozygosity mapping)을 실시하였을 때, 각 가계에서 분리된 동형접합성 돌연변이의 두 대립유전자가 모두 단일 기원에서 유래한 것으로 예측되었는데, 이것은 근친 결혼에 기인한 것으로 판단된다. 본 연구는 파키스탄의 상염색체 열성 청력 장애 환자들의 정확한 분자진단 및 치료에 도움을 줄 수 있을 것으로 기대된다.

Keywords

Introduction

Around 466 million people in the world (6.1% of the world’s population) suffer from disabling hearing impairment according to the estimation of the World Health Organization (WHO; https://www.who.int/pbd/deafness/ estimates/en/). Hearing loss is the most prevalent sensory defect, and it affects ~1/500 newborns [24]. Clinical severity of hearing loss is categorized from mild to profound hearing loss based on an audiometry test, and the ages of onset are very wide ranging from congenital to adult. Hearing loss is etiologically developed by heterogeneous pathology, such as genetic and environmental factors. Hearing loss can also be caused by infections, injuries and exposure to excessive noise. Approximate rate of 50% to 60% of people with hearing loss is seemed to be caused by genetic defects [9,24].

Hereditary hearing loss is a group of genetically and clinically heterogeneous sensorineural disorders [5]. It is commonly divided into two categories, syndromic hearing loss and non-syndromic hearing loss [35]. Non-syndromic hearing loss is a partial or total loss of hearing that is not associated with other symptoms. In contrast, people with syndromic hearing loss exhibit clinical impairments in at least one other organ. Among the disorders related to the syndromic hearing loss, Alport syndrome, Usher syndrome, Waardenburg syndrome, Pendred syndrome, and CHARGE syndrome are relatively prevalent. In addition to these syndromes, more than 700 genetic syndromes have been reported to have hearing loss [36].

People affected by non-syndromic hearing loss consist of approximately 80% autosomal recessive (DFNB), 15% autosomal dominant (DFNA), and less frequent mitochondrial or X-linked (DFNX) cases [43]. To date, more than 123 non syndromic hearing loss genes have been reported on the Hereditary hearing Loss website (https://hereditaryhearing loss.org/). Recent genome-wide association studies suggested that many genetic factors may have secondary causes or may be genetic modifiers [12,31]. Hearing loss genes encode variable proteins with a wide range of functions, which make molecular diagnosis and treatment difficult. Profound non-syndromic hearing loss affects about 1 in 2, 000 children prior to language acquisition (prelingual onset). About 80% of the cases have shown DFNB.

Hearing loss is a major public health concern, especially in developing or low-income countries, where treatments for hearing loss are more difficult and consanguinity increases the risk of recessive hearing loss. Two-thirds of people with hearing loss reside in developing countries [37]. DFNB is particularly frequent in Pakistan, which may be due in part to frequent consanguineous marriages [4, 8, 11, 25, 27, 29, 38].

Prelingual or early onset hearing loss could badly affect language acquisition, cognition, and emotional expression; therefore, early molecular diagnosis is very important. This study determined the genetic causes in two Pakistani consanguineous families with early onset hearing loss by whole exome sequencing (WES). As a result, we identified a p.Leu326Gln homozygous mutation in MYO7A as the underlying cause of hearing loss with prelingual onset. We also filtered out two homozygous mutations in GPR98 (p.Val3094 Ile) and PLA2G6 (p.Asp56Gly) as the candidates for pathogenic mutation in an early onset hearing loss family.

Materials and Methods

Subjects

The study included two consanguineous DFNB families containing three affected and six unaffected individuals (Fig. 1A, Fig. 1B). Whole blood samples were collected from the participants who were recruited from the Care Hospital Sahiwal, Pakistan. Written informed consent was provided by all the participants. For the minors involved in the study, the consent was provided by their parents. This study was approved by the Institutional Review Board for Kongju National University (KNU_IRB_2018-62).

SMGHBM_2021_v31n5_453_f0001.png 이미지

Fig. 1. Pakistani consanguineous families with hearing loss. (A, B) Pedigrees of DF11 family (A) and DF4 family (B). Square and circle mean male and female, re- spectively, and black and white symbols represent affected and unaffected in- dividuals, respectively. (C) Audiogram of air conduction for the patient in the DF11 family. PTA test was performed on the both ears (left: crosses, right: cir- cles).

Clinical examinations

Clinical information was obtained through clinical examinations including audiological testing and electroretinography. Clinical information was also partly obtained by history taking for the proband’s ancestors and relatives. Pure tone audiometry (PTA) was performed using air conduction at frequencies of 250 Hz, 500 Hz, 1 kHz, 2 kHz, 4 kHz, and 8 kHz. The severity of the hearing loss was classified into mild (26 to 40 dB), moderate to moderately severe (41 to 70 dB), severe (71 to 90 dB), and profound (above 91 dB) based on PTA.

DNA purification and paternity test

Genomic DNA was purified from whole blood by using the HiGene Genomic DNA Prep Kit (Biofact, Daejeon, Korea). Paternity was confirmed for two examined families by PCR amplification of STR markers using the PowerPlex Fusion System (Promega, Wisconsin-Madison, USA), and resolution and genotyping of the PCR products were done by the SeqStudio Genetic Analyzer and GeneMapper software, ver. 6 (Life Technologies-Thermo Fisher Scientific, Foster City, CA, USA).

Exome sequencing and variant mapping

WES and mitochondrial DNA (mtDNA) sequencing was performed for patients in the two examined families [16]. Exome was captured using the SureSelect Human All Exon kit, v4 (Agilent Technologies, Santa Clara, CA, USA), and sequencing was performed using the HiSeq 2500 Genome Analyzer (Illumina, San Diego, CA, USA). The hg19 genome assembly was used as the reference sequence for mapping (http://genome.ucsc.edu). Small nucleotide variants were called using the GATK (https://software.broadinstitute. org/gatk/) and SAMtools (http://samtools.sourceforge. net/). Minor allele frequencies (MAFs) were obtained from public human genome databases: 1000 Genomes Project (1000G; http://www.1000genomes.org/), Exome Sequencing Project (ESP; http://evs.gs.washington.edu/EVS/), and Genome Aggregation Database, v2.1.1 (gnomAD; https:// gnomad.broadinstitute.org/).

Whole mtDNA sequence was mapped to the human mtDNA genome using MToolBox, v1.0 (https://sourceforge. net/projects/mtoolbox/). Variants were assembled using the revised Cambridge reference sequence (rCRS) [2], which were then realigned to the hg19.

Pathogenicity of the variants was divided into five grades (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign) according to the guideline of the American College of Medical Genetics and Genomics (ACMG) [28]. Pathogenic candidate variants were confirmed by Sanger sequencing.

Conservation analysis and in silico prediction

Conservation analysis of the mutation sites and their neighboring sequences were performed by the MEGA-X, ver. 10.0.5 (http://www.megasoftware.net/). To predict the mutation effect, in silico analyses were performed using the PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), MUpro (http://www.ics.uci.edu/~baldig/mutation), PROVEAN (http:// provean.jcvi.org/), and Fathmm (http://fathmm.biocompute. org.uk/). Genomic evolutionary rate profiling scores (GERPs) were determined by the GERP++ program (http://mendel. stanford.edu/SidowLab/downloads/gerp/).

Determination of homozygous blocks

For the homozygous variants, homozygosity mapping was performed to determine whether two same alleles originated from a single founder. Homozygous blocks (HBs) were determined by haplotyping of SNPs distributed around the corresponding mutations from the WES data of the affected individuals by the method of Park et al. [26].

Results

Clinical manifestations

Clinical phenotypes were investigated for two Pakistani consanguineous families with early-onset bilateral hearing loss: DF11 family with prelingual onset and DF4 family with post-lingual onset. In the DF11 family with non-syndromic hearing loss, an 11-year-old boy was born from the consanguineous parents (Fig. 1A). He was delivered normally and showed no developmental or other disabilities except for hearing impairments. His onset was estimated to be prelingual (before 1 years old), and hearing loss degree was determined to be mild at the low frequency but moderately severe at the high frequency by the PTA test (Fig. 1C). His parents, elder sister (15 years old) and brother (13 years old) showed no symptom of hearing loss. No family history of deafness was taken in this family.

In the DF4 family having syndromic hearing loss current with muscular atrophy, two affected siblings consisting of a 7-year-old girl and 5-year-old boy were normally born with a full term pregnancy from the healthy unaffected parents (Fig. 1B). Their onset ages were determined to be at 2 and 2.5 years old, respectively. The degree of the hearing loss was estimated to be at the moderate to severe level in both affected individuals although the PTA test was not done. As an additional symptom, they showed considerably severe muscular atrophy. The proband (younger brother) could not walk without an aid at his examined age.

Identification of homozygous variants

From the analysis of the proband’s WES and subsequent Sanger sequencing of candidate variants, we filtered out three unreported or rare homozygous variants of pathogenic or uncertain significance in the two Pakistani deafness families (Table 1). However, no variant was considered to be pathogenic from the whole mtDNA sequencing data in both families.

Table 1. Pathogenic or rare variants in the DFNB genes from the Pakistani deafness patient

SMGHBM_2021_v31n5_453_t0001.png 이미지

`1,000G: 1,000 Genomes Project, B: benign, DFNB: autosomal recessive deafness, DVD: Deafness Variation Database, ESP: Exome Sequencing Project, GERP: genomic evolutionary rate profiling score, gnomAD: Genome Aggregation Database, INAD: infantile neuroaxonal dystrophy, NBIA: neurodegeneration with brain iron accumulation, P: pathogenic, UR: unreported, USH: Usher syndrome, VUS: variant of uncertain significance.

aReference DNA and protein sequences: GPR98: NM_032119.4 and NP_115495.3, PLA2G6: NM_001004426.3 and NP_001004426.1, MYO7A: NM_000260.4 and NP_000251.3.

bScores of PolyPhen-2 (PP2) 1, MUpro (MUp) <0, PROVEAN (PRO) <-2.5, and Faathmm (Fath) ≤-1.5 indicate pathogenic prediction (*denotes a "pathogenic" prediction).

In the DF11 family with hearing loss and no other symptom, a c.977T>A (p.Leu326Gln) homozygous mutation in MYO7A was found in the affected proband. The MYO7A p.Leu326Gln mutation was heterozygous in the unaffected parents and the elder sister and brother, indicating co segregation of the mutations with the affected member by the autosomal recessive inheritance mode (Fig. 1A). The MYO7A mutation was not reported in the public databases of 1000G, ESP, and gnomAD; however, it was registered in the dbNP (rs797044491) and GenBank (NC_000011.10). This mutation has been reported to be the underlying cause of Usher syndrome type 1B (USH1B; MIM 276900) several times [17,27]. Pathogenic prediction was suggested by all the in silico analysis programs (PolyPhen-2: 1.00, MUpro: -0.86, PROVEAN: -5.79, and Fathmm: -3.70).

In the DF4 family with hearing loss recurrent with muscular atrophy, the two affected individuals showed two homozygous variants, c.9280G>A (p.Val3094Ile) in GPR98 (MIM 602851) and c.167A>G (p.Asp56Gly) in PLA2G6 (MIM 603604). The unaffected parents were heterozygous for both mutations, which suggested cosegregation of the mutations with the affected members by the autosomal recessive inheritance mode (Fig. 1B). The p.Val3094Ile in GPR98 was reported in the 1000G, ESP, and gnomAD with the frequencies of 0.0465, 0.0650, and 0.0679, respectively. It was also registered in the dbNP (rs13157270) and GenBank (NC_000005. 10). In silico analysis of the GPR98 mutation predicted pathogenicity by the MUpro program (-0.59) but predicted reversely by the programs of PolyPhen-2 (0.01), PROVEAN (-0.09), and Fathmm (1.66). The p.Asp56Gly in PLA2G6 was not reported in the 1000G, ESP, and gnomAD and the dbSNP. It was predicted to pathogenic by three in silico analysis programs (PolyPhen-2: 1.00, MUpro: -0.98, and PROVEAN: -3.75).

The three missense mutations in the MYO7A, GPR98, and PLA2G6 were examined in all the participants by Sanger sequencing (Fig. 2). Amino acid sequences of the mutation sites and surrounding regions are highly conserved among vertebrate species in the MYO7A and PLA2G6 proteins but less conserved in the GPR98 protein (Fig. 3A). The p.Leu326Gln in MYO7A, p.Val3094Ile in GPR98, and p.Asp56Gly in PLA2G6 were located in the myosin motor, CalX-β domain and N-terminal domain, respectively (Fig. 3B).

SMGHBM_2021_v31n5_453_f0002.png 이미지

Fig. 2. Sequencing chromatograms of three missense mutations in the MYO7A, GPR98, and PLA2G6. (A) c.977T>A in MYO7A, (B) c.9280G>A in GPR98, and (C) c.167A>G in PLA2G6.

SMGHBM_2021_v31n5_453_f0003.png 이미지

Fig. 3. Conservation analysis and location of the identified mutations. (A) Conservation of the mutation sites among vertebrate species. The amino acids at the mutation sites and surrounding regions are highly conserved between vertebrate species. (B) Domain structures and location of the missense mutations. The p.Leu326Gln in MYO7A, p.Val3094Ile in GPR98, and p.Asp56Gly in PLA2G6 were located on the myosin motor domain, CalX-β domain, and N-terminal domain, respectively (IQ: isoleucine-glutamine motif, MyTH4: myosin tail homology 4, FERM: 4.1, ezrin, radixin and moesin domain, SH3: Src homology 3, SP: signal peptide, LamG/TspN/PTX: laminin G/amino–terminal thrombospondin–like/pentraxin, EAR: epi- lepsy-associated repeat, GPS: G protein-coupled receptor proteolysis site, TM: transmembrane domain, P: proline-rich motif, N: nucleotide binding domain, GTSTG: GXSXG lipase catalytic site, and CaM: calmodulin binding region).

HBs for the chromosomal regions around the corresponding mutation sites were determined by the haplotype analysis of SNPs obtained from WES data in the probands. In the neighboring region of the c.977T>A mutation in MYO7A on chromosome 11q13.5, a HB with an approximate size of 35 Mbp was observed from PLEKHB1 to ELMOD1. The lengths of the left and right blocks were estimated to be 4 Mbp and 31 Mbp, respectively (Fig. 4A). The neighboring region of the c.9280G>A mutation in GPR98 on chromosome 5q14.3 showed a relatively short HB with an approximate size of 3.2 Mbp from POLR3G to FAM172A. The left and right blocks were estimated to be 0.2 Mbp and 3 Mbp, respectively (Fig. 4B). In the neighboring region of the c.167A>G mutation in PLA2G6 on chromosome 22q13.1, a HB with an approximate size of 14 Mbp was observed from NOL12 to ACR. Based on the mutation site, the lengths of the left and right blocks were estimated to be 0.5 Mbp and 13 Mbp, respectively (Fig. 4C). The presence of the HBs strongly suggests that both alleles of the homozygous mutation identified in each family originated from a single founder.

SMGHBM_2021_v31n5_453_f0004.png 이미지

Fig. 4. Estimation of HBs for the neighboring chromosomal regions of MYO7A, GPR98, and PLA2G6 mutations detected from the consanguineous Pakistani families with DFNB. HBs were determined by haplotype analysis of SNPs distributed around the corresponding mutations from the WES data. (A) HB from the neighboring region of the c.977T>A mutation in MYO7A in DF11 family. (B) HB from the neighboring region of the c.9280G>A mutation in GPR98 in DF4 family. (C) HB from the neighboring region of the c.167A>G mutation in PLA2G6 in DF4 family.

Rare variants in hearing loss related genes

In addition to the three variants mentioned above, several rare variants were observed in genes related to deafness from the filtering of the WES data for affected probands in both families. Their exact nucleotides and amino acids changes, disorder type concerning the genes, allele frequencies in the public databases, GERP scores and in silico prediction values are provided in Table 2.

Table 2. Rare variants in hearing loss genes

SMGHBM_2021_v31n5_453_t0002.png 이미지

1,000G: 1,000 Genomes Project, ATS: Alport syndrome, B: benign, Cis: cis-arrangement of variants in a chromosome, DFNA: autosomal dominant deafness, DFNB: autosomal recessive deafness, ESP: Exome Sequencing Project, GERP: genomic evolutionary rate profiling score, gnomAD: Genome Aggregation Database, NS: nonsegregation, UR: unreported, VUS: variant of uncertain significance.

aReference DNA and protein sequences: COLAA3: NM_000091.5 and NP_000082.2, GPR98: NM_032119.4 and NP_115495.3, CDH23: NM_052836.4 and NP_443068.1, TECTA: NM_005422.4 and NP_005413.2, PTPRQ: NM_001145026.2 and NP_001138498.1, PDZD7: NM_024895.5 and NP_079171.1, GJB2: NM_004004.6 and NP_003995.2.

In the affected proband of the DF11 family, five rare variants with allele frequencies of < 0.1 were found in the deaf- ness-related genes: COL4A3 c.4484A>G (p.Gln1495Arg), GPR98 c.500T>C (p.Met167Thr), CDH23 c.800G>A (p.Arg267 His), TECTA c.3236A>G (p.Asp1079Gly), and PTPRQ c.1285C> G (p.Gln429Glu). All the variants were heterozygous in the proband and were nonsegregated with the affected members.

In the proband of the DF4, rare heterozygous variants were observed in two genes: [c.2314_2331delACGGCTGCG GCTACGGCT (p.772_777delSRSRSR) + c.3092G>A (p.Arg 1031His)] in PDZD7 and [c.56G>T (p.Ser19Ile) + c.457G>A (p.Val153Ile)] in GJB2. Two variants in PDZD7 were inherited from the unaffected mother, and two variants in GJB2 were inherited from the unaffected father. Thus, two variants in both genes were determined for which they were located as a cis-arrangement in the same chromosome and were not segregated with the affected individuals. The p.772_777delSRSRSR in PDZD7 was previously reported to be likely benign [33]. We considered these all rare variants from the two probands and nonpathogenic because of mainly the nonsegregation with the affected individuals and the inconsistency of the autosomal recessive inheritance model.

Discussion

This study tried to determine the genetic causes of two Pakistani consanguineous families with hearing loss. By WES, we identified the MYO7A p.Leu326Gln homozygous mutation in the DF11 family, and the GPR98 p.Val3094Ile and the PLA2G6 p.Asp56Gly in the DF4 family.

MYO7A encoding a protein classified as an unconventional myosin is expressed in the pigment epithelium and photoreceptor cells of the retina, embryonic cochlear and vestibular neuroepithelia. Weil et al. suggested that deafness and vestibular dysfunction in Usher syndrome are a result of a defect in the morphogenesis of the inner ear sensoryl stereocilia [41]. Transgenic mice showed restricted expression of mouse MYO7A and human MYO7A in the hair cells of the inner ear, cochlea, and vestibule [3]. The MYO7A protein has three domains: the N-terminal head or motor which possesses the ATP- and actin-binding sites, the neck or regulatory domain containing the IQ motif, and the tail domain [10]. Mutations in MYO7A are usually relevant with autosomal recessive non-syndromic deafness type 2 (DFNB2; MIM 600060) and USH1B (MIM 276900) [19, 40, 41]. In addition, they sometimes cause autosomal dominant deafness type 11 (DFNA11; MIM 601317) [19,34]. USH1B is the most common autosomal recessive disorder subtype characterized by sensorineural deafness, vestibular abnormalities, and retinitis pigmentosa (RP).

PLA2G6 encodes a ubiquitously expressed calcium-in-dependent phospholipase A2. PLA2G6 protein with a lipase motif and ankyrin repeats catalyzes the release of fatty acids by the hydrolysis of the sn-2 acyl-ester bonds in phospholipids [15,23]. Phospholipases A2 (PLA2s) catalyze hydrolysis of the sn-2 acyl-ester bonds in phospholipids, leading to the release of arachidonic acid and other fatty acids. PLA2G6 mutations are associated with the autosomal recessive allelic disorders of neurodegeneration with brain iron accumulation type 2B (NBIA2B; MIM 610217) and infantile neuroaxonal dystrophy type 1 (INAD1, also called NBIA2A; MIM 256600) which shows an earlier onset, axonal swelling, and spheroid bodies in the central nervous system [7, 14, 23]. The iPLA2β-KO mice revealed age-dependent neurological impairment with the formation of spheroids containing tubulovesicular membranes similar to human INAD [20].

GPR98, also called adhesion G protein-coupled receptor V1 (ADGRV1), encodes a large, Ca2+-dependent G protein coupled receptor (GPCR) expressed in the central nervous system [22]. The extracellular domain of the GPR98 particularly contains three repeated units of calcium-exchanger β motifs, which distinguishes it from other GPCRs [6]. GPR98 is a component of the ankle link complex required for the normal development of sensorineural cell hair bundles [21]. In mouse, Vlgr1 was expressed transiently around the developing hair bundles, and ankle links were not formed in the cochleae in the mice carrying Vlgr1 mutation [19]. Homozygous and digenic mutations in GPR98 have been reported to cause Usher syndrome type 2C (USH2C; MIM 605472), which are characterized by moderate to severe congenital or prelingual sensorineural hearing loss and later development of RP [1, 30, 42].

We believe that the p.Leu326Gln mutation in MYO7A is the underlying cause of the prelingual bilateral sensorineural hearing loss in the DF11 family, because it has been reported several times as the genetic cause and was fitted to the autosomal recessive inheritance mode [17,27]. Moreover, it was predicted to be pathogenic by all the in silico analysis pro- grams, and the mutation site is located in the well conserved myosin motor domain. Le Quesne Stabej et al. and Riazuddin et al. reported the same MYO7A mutation in Pakistani and British patients with USH1B, respectively [17,27]. In addi- tion, Sodi et al. reported a heterozygous mutation of p.Leu326Pro at the same amino acid residue of p.Leu326Gln in an Italian Usher syndrome patient who also has a heterozygous mutant of p.Arg1873Trp in MYO7A, indicating di genic causes [32]. The clinical symptoms of this patient are similar to previously reported cases with the MYO7A mutations in terms of severity and onset. However, our case did not exhibit retinal abnormality at his examined age of 11 years old. Because Usher syndrome type 1A (USH1A; MIM 276900) patients due to the MYO7A mutations have RP and hearing loss, the affected boy in this study may develop RP later. As a case report, a patient diagnosed with DFNB at the age of 10 in a Tunisian family was rediagnosed as USH1B after confirming the RP symptom at 25 years old [44].

In the DF4 family, we found p.Asp56Gly in PLA2G6 and p.Val3094Ile in GPR98 as cosegregating novel or rare muta- tions. The p.Asp56Gly mutation in PLA2G6 has not been reported in the public databases of dbSNP, 1000G, ESP, and gnomAD. The mutation site was located in the N-terminal domain, and three of four in silico analysis programs predicted its pathogenicity. Moreover, several cases have been reported to have concurrently hearing loss and muscular dystrophy among affected individuals with PLA2G6 mutations [13, 18, 39].

However, previous cases with the PLA2G6 mutations occasionally showed other symptoms of hereditary spastic par- aplegia, motor and mental deterioration, marked hypotonia, and visual disturbance in addition to hearing loss and muscular dystrophy. Thus, the p.Asp56Gly PLA2G6 mutation was determined as a “variant of uncertain significance (VUS)” although it might function as an underlying cause of the clinical phenotype. For the p.Val3094Ile mutation in GPR98, it has been reported at low but not very rare frequencies in the public databases, such as dbSNP (rs13157270), 1000G (0.0465), ESP (0.0650), and gnomAD (0.0679). The mutation site was located in a less conserved site, and most in silico analysis programs predicted its nonpathogenic effect. Therefore, we determined it as “VUS.” When we analyzed variants in the genes related with both phenotypes of hearing loss and muscular dystrophy, such as ABHD5, GGPS1, PRPS1, MPZ, SH3TC2, NEFL, ABHD12, COCH, MYH14, TRPV4, SSBP1, and GJB1, no pathogenic mutations were detected.

In conclusion, we identified three variants of pathogenic and uncertain significance as the putative underlying causes of the hearing loss in two Pakistani consanguineous families. Homozygosity mapping showed that both alleles of the homozygous mutations identified in each family originated from a single founder, which might be due to consanguineous marriages. This study will help provide exact molecular diagnosis and treatment for hearing loss patients in Pakistan.

Acknowledgments

We would like to thank the patients and their family members for their consent of participation and sample donation in this study. This work was supported by the grants of the National Research Foundation (2019R1A2C1087547, and 2021R1A4A2001389), Republic of Korea.

The Conflict of Interest Statement

The authors declare that they have no conflicts of interest with the contents of this article.

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