|Year : 2020 | Volume
| Issue : 2 | Page : 84-88
Identification of a novel compound heterozygous mutation in OTOG in a chinese family with severe hearing impairment
Wen-Ya Yan1, Fan Xu2, Bing Li1
1 GMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, Guangzhou 511436, China
2 Department of Ophthalmology, People's Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, China
|Date of Submission||20-Nov-2019|
|Date of Decision||03-Jan-2020|
|Date of Acceptance||02-Apr-2020|
|Date of Web Publication||26-Jun-2020|
GMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, Taoyuan Road, Guangzhou 511436
Source of Support: None, Conflict of Interest: None
Objectives: Hearing loss is a worldwide disease. In 50% of the patients, hearing loss is caused by genetic problems associated with GJB2, MTRNR1, SLC26A4, and other genes. Considering the recent development and cost reduction of whole-exome sequencing, it is possible to filter out the normal genes and find which among the more novel genes contributed to the loss of hearing.
Methods: After prescreening all individuals for GJB2, MTRNR1 and SLC26A4 mutations, whole-exome sequencing was performed in the proband, and the pathogenic variant was confirmed via Sanger sequencing.
Results: The compound-heterozygous variant namely c.8076G>C:p.E2692D and c.6362T>C:p.V2121A in OTOG was identified as a candidate gene of a consanguineous Kazakh family.
Conclusion: This is the first reported case of severe deafness caused by an OTOG compound-heterozygous variant in the world and the first case of deafness caused by an OTOG variant in China. This discovery identified the important contribution of OTOG toward deafness and expanded the spectrum of variants responsible for human hearing loss.
Keywords: Genetics; Hearing Loss; OTOG; Whole-exome Sequencing
|How to cite this article:|
Yan WY, Xu F, Li B. Identification of a novel compound heterozygous mutation in OTOG in a chinese family with severe hearing impairment. Reprod Dev Med 2020;4:84-8
|How to cite this URL:|
Yan WY, Xu F, Li B. Identification of a novel compound heterozygous mutation in OTOG in a chinese family with severe hearing impairment. Reprod Dev Med [serial online] 2020 [cited 2020 Jul 8];4:84-8. Available from: http://www.repdevmed.org/text.asp?2020/4/2/84/288023
| Introduction|| |
The genetic etiology of hearing loss is often divided into two forms, namely, syndromic hearing loss and nonsyndromic hearing impairment. Syndromic hearing loss is associated not only with hearing problems but also with malformations of the external ear and other organ systems. No systemic abnormalities have been found in people suffering from nonsyndromic hearing impairment other than the defects in the inner or middle ear.
About 60% of prelingual deafness is caused by inherited genetic factors, which are typically divided into four types: autosomal (both recessive and dominant), sex linked, and mitochondrial inheritance. The proportions are 75%–85%, 15%–24%, and 1%–2%, respectively. Among autosomal recessive nonsyndromic forms, GJB2 accounts for the most common causative gene. A variant of STRC is the second most common.
There are many genes and proteins responsible for nonsyndromic hearing loss, including adhesion protein PTPRQ (DFNB84), transport protein MOY3A (DFNB30), proteins of synapses SLC17A8 and OTOF (DFNB9), electromotility SLC26A5 (DFNB61), cytoskeleton ESPN (DFNB36) and DIAPH 1 (DFNA1), ion homeostasis and gap junctions TJP2, KCNQ4 (DFNA2), and BSND (DFNB73), and others.
Thirteen genes are located in the DFNB18 locus (MIM 602092). Of these, variants in USH1C (MIM 605242) and OTOG (MIM 604487) are capable of causing DFNB18-related hearing loss. So far, the only known compound heterozygous OTOG mutations c.6347C>T (p. Pro2116Leu) and c.6559C>T (p. Arg2187X) were identified in one Spanish family which have a moderate hearing impairment and vestibular dysfunction.
As a newly discovered deafness gene, OTOG (DFNB18B) codes to otogelin protein. Otogelin is one of the three noncollagenous glycoproteins (α-tectorin, β-tectorin, and otogelin) of the acellular gelatinous structures. These structures cover the sensory epithelia of the inner ear (IE). They also cover the tectorial membrane (TM) of the cochlea as well as the otoconial membranes (OM) in the utricle and saccule and the cupulae that cover the cristae.
Kazakhs are a special ethnic group in China. Because of their special geographical environment, intermarriage is common, resulting in high rates of genetic diseases.
In this study, whole-exome sequencing was performed on a 10-year-old female proband with severe-to-profound nonsyndromic hearing impairment. A new autosomal recessive compound heterozygous variant consisting of c.8076G>C:p.E2692D and c.6362T>C:p.V2121A on OTOG has been identified as the genetic pathogenic cause of deafness in the Kazakh family. p.V2121A is a known variant that causes a missense mutation on the VWFD4 domain, whereas p.E2692D, another novel variant, leads to a nonsynonymous variant on chain otogelin. This is the first reported case of a variant caused by the new deafness gene OTOG in China.
| Methods|| |
A proband and her parents were recruited from the Kazakhs who live in Aletai City, Xinjiang Uygur Autonomous Region, China. The family's medical history and written informed consent were obtained from all participating individuals. The phenotype was identified via physical examination, otoscopy, and pure tone audiometry (sound frequencies between 250 and 8,000 Hz). The entire study was approved by the Ethics Committee of the Medical College, Fudan University.
Genomic DNA extraction
DNA samples from the affected patient and her family members were obtained according to the QIAamp DNA Blood Mini Kit (Qiagen) protocol. The product's concentration was then measured (using the Nanodrop 1000 spectrophotometer, Thermo Scientific) and stored in a −20°C freezer. The most prevalent deafness genes GJB2, SLC26A4, and MT-RNR1 were excluded by polymerase chain reaction (PCR) amplification and bidirectional sequencing of the affected member.
DNA library preparation was started via fragment genomic DNA by sonication using TargetSeq® Enrichment Kit (iGeneTech) according to the manufacturer's instructions. After the ends were repaired and the 3′-ends were adenylated, the single DNA fragments were ligated with adapter oligonucleotides.
Hybridization-based targeted enrichment
The sample library was initially pooled followed by the denatured double-stranded DNA library. The biotinylated probes were added and hybridized to the targeted regions. Streptavidin beads were used to enrich the DNA and then eluted for hybridization. Sequencing was implemented by loading captured genomic samples onto the NovaSeq 6000 platform.
Map reads to the UCSC reference genome (hg19, 2009); find SNPs and InDels by GATK, Samtools, and Varscan software; annotate the SNPs and InDels sites via ANNOVAR software; screen the results by crowd frequency (1000g_EAS, ExAC_EAS, gnomAD_exome_EAS) and prediction software (SIFT_pred, Polyphen2_HDIV_pred, Polyphen2_HVAR_pred, MutationTaster_pred, PROV EAN); mutation screen (screen against the 1,000 Genomes public variant databases, dbSNP, and the National Heart, Lung, and Blood Institute [NHLBI] Exome Sequencing Project Exome Variant Server [http://evs.gs.was hington.edu/EVS] for high-quality functional variants, including nonsense, missense, splice-site, and indel variants that cause or may cause disease). The pattern of recessive inheritance helps narrow the search for such candidate variants with only homozygous or compound heterozygous.
PCR primers were designed to amplify the genomic DNA of all family members with the National Center for Biotechnology Information Primer-BLAST: OTOG-2692-forward 5′-GCCAAGTACGAGTGTGGTGA-3′, OTOG-2692-rev erse 5′-GTAGCAACACAAGCACGCAA-3′, OTOG-2121-forward 5′-GAGTCATGGGATCAGCGGAG-3′, a nd OTOG-2121-rever se 5′-CTTTGCCTCTGGTACCCTGG-3′.
A 3730XL sequencer (Applied Biosystems) was used to conduct Sanger sequencing to check if any variation of these targeted NSHL genes follows the laws of Mendelian inheritance and cosegregated between phenotype and genotype. One hundred healthy Kazakhs and 1000 Chinese controls were tested as well.
Bioinformatic tools, including SIFT (http://sift.bii.a-star.edu.sg/), PPH 2 (http://genetics.bwh.harvard.edu/pph 2/), MutTas (https://www.mutationtaster.org/), PROVEAN (http://provean.jcvi.org), 1KG_eas (http://browser. 1000genomes.org/), Ex AC (http://exac.broadinstitute.org/), Gnome AD East Asian (http://gnomad.broadinstitute.org), and Genomes of populations in northern China (https://diseasedx.virgilbio.com), were used to identify function changes between wild type and mutant.
The protein sequencing of the two types was submitted to PyMOL software (http://www.pymol.org/) for molecular modeling. If necessary, more amino acids were removed from the query sequence compared to those from the template sequence to improve the reliability of the results.
| Results|| |
Clinical characteristics of the affected individuals
According to a detailed medical history collection, in [Figure 1]a, the parents have a normal level of hearing, but the II: 1 family member has deafness. The hearing level of the daughter's left ear exceeded 100 dB (profound level, 100% hearing impairment). The right ear exceeded 87 dB (severe lever, 80% hearing impairment). II: 1 has severe-to-profound hearing loss [Figure 1b].
|Figure 1: (a) Pedigree of the Chinese Kazakh minority family with nonsyndromic autosomal recessive hearing loss. Darkened symbols denote affected individuals. (b) Audiograms of affected member of the family. S: Severe.|
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Result of whole-exome sequencing
To discover the genetic background of this family, performing whole-exome sequencing was performed on the DNA sample of II: 1. The assessment of library quality is reliable: QC rate is 97.07%, mapped reads is 101.67M, accurate mapping rate is 99.48%, coverage rate is 99.89%, target mean depth is 123.92, and 20X coverage rate is 97.72%. After data analysis, the 1,000 Genomes public variant databases, dbSNP, and the NHLBI Exome Sequencing Project Exome Variant Server were used to screen the quality of potential candidate variations about nonsense, missense, splice-site, and indel variants. Consequently, no homozygous variants were found, but 25 high-quality compound heterozygous variants met the conditions of 1000g_EAS, ExAC_EAS, and gnomAD_exome_EAS frequency, all being less than 0.01 or not available. Among 25 high-quality compound heterozygous variants, there are two variants on gene OTOG, DMXL2, CUX2, DENND2C, DNAH14, FAT4, KNTC1, MPDZ, NLN, PCDHB10, PRR14L, and three variants on PKHD1L1.
Identification of compound heterozygous mutations in OTOG
Based on the principle of cosegregation of phenotypes and genotypes, we screened and confirmed that there is only one novel compound heterozygous state (c.6362T>C: p.V2121A and c.8076G>C: p.E2692D), which demonstrated a significant genetic basis in OTOG gene causative deafness [Figure 2]a and [Table 1]. Amino acid valine changed to alanine, leading to a nonsynonymous mutation on the VWFD4 domain. Aspartic acid replaced glutamic acid, which led to a nonsynonymous mutation on chain otogelin [Figure 2]b. Two mutation sites were conserved across species [Figure 2]c. To be even more convincing, detailed information included frequency and bioinformatic predictions about the two sites as shown in [Table 2]. One hundred healthy Kazakhs and 1,000 Chinese controls were tested for these two variants, and none was found to be in existence among them.
|Figure 2: (a) DNA sequence chromatograms showing two compound heterozygous mutation c.8076G>C:p.E2692D, c.6362T>C:p.V2121A in affected individuals II: 1, heterozygous mutation c.6362T>C:p.V2121A in normal individual I: 1 and heterozygous mutation c.8076G>C:p.E2692D in a normal individual I: 2. (b) Schematic representation of the structure of OTOG. The black arrow (above) points to the mutations identified in this study. (c) Protein alignment of OTOG in different species, Homo sapiens, Macaca mulatta, Bos taurus, and Mus musculus, which shows the conservation of the residue p.V2121A and p.E2692D.|
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|Table 2: Frequency and function prediction of the OTOG mutation identified in II:1|
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In this study, one of the identified known variants (p.V2121A) led to a missense mutation on the VWFD4 domain. After the removal of some spare amino acids, we submitted both wild-type and mutant sequencing of this part to the PyMOL software and chose the PDB: c6n29A crystal structure of monomeric von Willebrand factor d′d3 assembly as the template for the model structure. For comparison purposes, wild-type and mutant three-dimensional (3D) models overlapped with each other [Figure 3]a. The variation changed the β-sheet neighboring that would make the original structure unstable.
|Figure 3: (a) After overlap, the wild-type (shown in green) and mutation type (shown in blue) 3D models of p.V2121A. Two differences are shown in rectangles with a black border, and the red part denotes the location of the variation. (b) The wild-type (shown in green) and mutation type (shown in blue) 3D models of p.E2692D. The red part represents the location of the variation. 3D: Three dimensional.|
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Another mutation (p.E2692D) led to a nonsynonymous mutation on the otogelin chain. Following the same workflow, the wild-type and mutant 3D structure are shown in [Figure 3]b based on the template d1u5 ma, and the PyMOL software indicated no difference.
| Discussion|| |
The mammalian IE mainly consists of two broad compartments: the vestibular apparatus that comprises the saccule and the utricle and the three semicircular canals. The cochlea consists of sensory hair cells and supporting cells. When hearing a sound or accelerating, supporting cells will provide mechanical stimulation to the hair bundles of the sensory cells. Each neuroepithelium in the IE is covered with an acellular gelatinous membrane. For example, the TM covers the organ of Corti. Another example is that the OMs cover the maculae of the utricle and saccule. Finally, there is also a large amount of gelatinous component forming a dome-shaped cupula over the cristae of the semicircular canals. Otogelin protein encoded by OTOG is one of the noncollagenous components of the acellular gelatinous structures covering the six sensory epithelia of the IE. In the vestibule, otogelin helps anchor the OM and the cupula to the neuroepithelia. In the cochlea, otogelin is required to organize the fibrillar network of the TM, ensuring that the outer hair cells respond effectively to the movement of the basilar membrane, and provides feedback at the appropriate gain and timing required for amplification, which may play a role in determining the resistance of the membrane to sound stimulation.
The identified known variant (p.V2121A) caused a missense mutation on the VWFD4 domain. This variation could cause the original VWFD4 structure to become unstable due to a change in β-sheet neighboring. Another mutation (p.E2692D) led to a nonsynonymous mutation on chain otogelin. Both were highly conserved between different species. VWFD4 was supposed to be involved in protein–protein interaction as well as in normal multimerization and optimal secretion. A case of deafness caused by an OTOG mutation was first reported in 2012 and three pathogenic variations were discovered in OTOG later, one of which was a homozygous 1bp deletion (c.5508delC, p. Ala1838Profs*31) and the other was a compound heterozygous state (c.6347C>T, p. Pro2116Leu and c.6559C>T, p. Arg2187*).
p.V2121A occurred on the VWFD4 domain; however, much about the function of VWFD4 in otogelin is still unknown to us. The functions of the VWFD4 domain need to be deeply investigated. The specific role and related mechanism of otogelin in the TM need to be clarified in further studies too.
In conclusion, as this method of targeted enrichment and Sanger sequencing has been adopted, we detected and validated a compound heterozygous defect of OTOG in a Kazakh family. One is c.8076G>C that causes the p.E2692D transition and the other is c.6362T>C located on the VWFD4 domain that causes the p.V2121A transition. This is the first case of severe deafness caused by an OTOG mutation in the world and the first study into the OTOG variation spectrum in a deafness-suffering pedigree in China. This case report about OTOG expands the spectrum of mutations responsible for human hearing loss. The novel mutation can improve the genetic counseling offered to those affected by OTOG mutations. This will assist clinics to give better a genetic diagnosis and more effective therapeutic approaches in the future. Screening programs for newborns can add these new mutation sites to evaluate the genetic causes for NSHL and to provide the appropriate gene therapy. Meanwhile, it encourages researchers to explore the complex mechanism in OTOG.
We appreciate all the participants for their cooperation and especially Drs. Lei Wang and Qing Sang for their contributions to patient recruitment and sample collection.
Blood samples were from the State Key Laboratory of Genetic Engineering and the MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai, China.
Financial support and sponsorship
This study was supported by the National Natural Science Foundation of China (No. 81560166).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]