A 250-kb Microdeletion Identified in Chromosome 16 Is Associated With Non-Syndromic Sensorineural Hearing Loss in a South Indian Consanguineous Family
Article information
Abstract
Background and Objectives
Hereditary hearing loss is the most common genetic disorder in children. Nearly 120 genes associated with auditory impairment have been identified. Although the disease is clinically and genetically complex, the chances of identifying deafness-causing loci increase when studying consanguineous families.
Materials and Methods
Whole-exome sequencing was performed to identify genetic variants underlying sensorineural hearing loss in affected individuals from a family with third-degree consanguineous practices.
Results
A homozygous deletion of 250.285 kb was identified in the 16p12.2 region encompassing three genes, METTL9, IGSF6, and OTOA, and a partial deletion of the NPIPB4 gene co-segregated within the family.
Conclusions
This study highlighted the genetic heterogeneity of hearing loss in consanguineous families. Future research should focus on the OTOA mutational spectrum in South Indian populations with hearing loss.
Introduction
Genetic hearing loss is the most common sensory defect, particularly among children [1]. Approximately 2.7 cases out of every 1,000 newborns are affected by sensorineural hearing loss [2]. In India, the prevalence of hearing loss is 6.3%, according to the World Health Organization (WHO) [3]. There is a high prevalence of consanguinity practice in India, particularly in Southern regions due to ethnic customs and socioeconomic beliefs [4,5]. Due to the high inbreeding co-efficient, offspring of parental consanguinity tend to carry causal variants, making them an ideal population for understanding and studying genetic disorders [6,7]. Based on the etiology, hearing loss (HL) is classified into syndromic forms and non-syndromic forms, affecting the inner ear and causing sensorineural HL [8]. Clinical severity of HL varies from mild to profound, with ages of onset ranging from congenital to adult. Non-syndromic hearing loss (NSHL) can follow autosomal and sexlinked, mitochondrial patterns of inheritance. Nearly 70% of NSHL follows autosomal recessive patterns of inheritance [8,9]. To date, more than 120 genes are associated with NSHL (https://hereditaryhearingloss.org/). Among these, GJB2 is the most common gene associated with autosomal recessive NSHL in different ethnicities. Many studies have implicated those mutations in DFNB loci (autosomal recessive NSHL causing loci), particularly GJB6, CDH23, SLC26A4, and MYO15A, to be highly associated with sensorineural hearing loss [9]. Pathogenic mutations in NSHL genes could be single nucleotide variants, small insertions/deletions (indels), splice site variants, and copy number variations (CNVs). CNVs of STRC and OTOA genes are found to be the most prevalent in NSHL patients [10,11]. Due to the phenotypic complexity and genetic heterogeneity of NSHL, the molecular diagnosis remains difficult. Therefore, investigating the correlation of phenotype-genotype of NSHL is more appropriate in consanguineous populations [10,12]. Whole exome sequencing (WES) enables the identification of single nucleotide variants (SNV), small insertion and deletions (indels), and structural variants including CNV in the coding regions of the genome, making it an ideal approach for investigating the genetic architecture of NSHL [13]. Therefore, in this study, we performed WES for two siblings affected by NSHL and their unaffected parents.
Subjects and Methods
Patient history and clinical evaluation
The probands (II-1, II-2), an 18-year-old female and a 17-year-old male, who visited ENT outpatient department were siblings affected with sensorineural hearing loss. The probands were born of consanguineous marriage, wherein the parents were first cousins with normal hearing ability. A complete medical history was collected. Clinical examination including otoscopy followed by pure tone audiometry (PTA) was performed for air and bone conduction of the participants. Based on the PTA criteria, the severity of the HL was categorized into moderate (41 to 70 dB), severe (71 to 90 dB), and profound (above 91 dB).
WES and CNV analysis
Genomic DNA was extracted from whole blood using a DNA QIAmp mini kit (Qiagen, Hilden, Germany). Targeted gene enrichment of exonic regions was done by Twist Biosciences Exome Capture Kit (San Francisco, CA, USA). DNA libraries were prepared and sequenced by Illumina HiSeq4500 sequencer Illumina (San Diego, CA, USA) with an average 100×coverage. The reads obtained were aligned to the human reference genome (GRCh38) using a Burrows-Wheeler Aligner (BWA-MEM, v 0.7.10) (Supplementary Fig. 1 in the online-only Data Supplement). Integrative Genomics Viewer (IGV; https://igv.org/) was used to visualize the read alignment and confirm the variant calls. Sentieon DNAseq (San Jose, CA, USA) was used for duplicate removal, base recalibration, and indel re-alignment. Sentieon haplotype caller identified variants in the samples and were deeply annotated by the VariMAT pipeline. Gene annotation of the variants was performed using the VEP program against the Ensembl release 99 (www.ensembl.org) human gene model. In addition to variants and indels, CNVs were also detected based on the read depths of the sequenced data. ExomeDepth tool compared the read distribution of the test sample with the normal reference (EST00000646100.2) to determine the CNV regions. ExomeDepth tool has been shown to detect CNV with a higher sensitivity percentage [14]. Variants identified in both coding and non-coding regions were annotated based on a set of databases like ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), OMIM (https://www.omim.org/), HGMD (https://www.hgmd.cf.ac.uk/ac/index.php), LOVD (https://www.lovd.nl/), DECIPHER (https://www.deciphergenomics.org/), and SwissVar (https://bio.tools/swissvar). The pathogenicity of the variants was classified based on the guidelines for the interpretation of sequence variants from the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology [15]. Common variants were filtered based on allele frequency in 1000 Genome Phase 3, gnomAD (v3.1 & 2.1.1), dbSNP (GCF_000001405.38), 1000 Japanese Genome, TOPMed (Freeze_8), Genome Asia, and Indian population database (MedVarDb v2.1). The functional prediction of clinically relevant variants was evaluated by SIFT, PolyPhen2, and MutationTaster2.
Ethical approval
This study was approved by the Institutional Human Ethical Committee of Aarupadai Veedu Medical College & Hospital (IEC.No: AV/IEC/2020/010), and informed consent was obtained from participants included in the study. This study was performed in line with the principles of the Declaration of Helsinki.
Results
Clinical findings
In the probands, an auditory finding showed type A tympanogram, indicating no middle ear dysfunction by immittance testing. PTA confirmed the affected individuals with moderately severe sensorineural hearing loss (Supplementary Fig. 2 in the online-only Data Supplement). The affected siblings showed no dysmorphic features or clinical deformities. The parents had normal hearing ability (Supplementary Fig. 2 in the online-only Data Supplement).
Genetic analysis
The genetic analysis of the family with two siblings affected by autosomal recessive NSHL carrying OTOA deletion is shown in Table 1. The two affected individuals showed homozygous deletion of 250.285 kb in 16p12.2 region [chr16: g.21597256-21847541del], encompassing 3 genes including METTL9, IGSF6, OTOA, and partial deletion of NPIPB4 gene (Figs. 1 and 2). CNV co-segregated between father and mother in the heterozygous state (Fig. 3). As per Franklin (https://franklin.genoox.com/clinical-db/home) and ACMG guidelines, 250 kb microdeletion was classified as a pathogenic variant (PVS1) [15]. Additionally, two compound heterozygous variants c.3815-3816del, c.4910C>G in the TRIOBP gene were detected in the son and the mother. A novel heterozygous variant in the COL4A6 gene was identified in the affected daughter and also in the father. In silico pathogenicity analyses have predicted COL4A6 c.4277C >T, TRIOBP variants c.4910C>G, and c.3815-3816del to be damaging.

Genetic variants identified by whole exome sequencing in family members affected and unaffected by hearing loss

Homozygous OTOA CNV identified in proband II-1 (daughter). Integrative Genome Viewer (IGV) shows homozygous OTOA deletion in the proband II. Red and blue rectangles indicate pair-end reads generated by sequencer, which were compared and visualized by IGV. CNV, copy number variation.

Homozygous OTOA CNV identified in Proband II-2 (son). Integrative Genome Viewer (IGV) shows homozygous OTOA deletion in the proband II-2. Reference sample was used for comparison. Red and blue rectangles indicate pair-end reads generated by sequencer, which were compared and visualized by IGV. CNV, copy number variation.
Discussion
This study reports, for the first time, a 250 kb microdeletion in Chr16 resulting in the whole deletion of 3 genes METTL9, IGSF6, OTOA, and partial deletion of NPIPB4 gene in NSHL subjects of South Indian origin. Microdeletions occur recurrently in a critical region of Chr16p12.2 via non-allelic homologous recombination owing to the presence of three breakpoints (BP), leading to segmental duplications/deletions [11]. BP1 and BP2 flank the three genes: METTL9, IGSF6, and OTOA; while BP2 and BP3 span about 520 kb and comprise 7 genes: PDZD9, C16orf52, VWA3A, POLR3E, UQCRC2, EEF2K, and CDR2 [16]. The 16p12.2 region is designated as a “hot spot” for genomic deletion [17]. The frequency of OTOA-associated HL is high in Middle-Eastern countries such as Iran, Israel, and Qatar [12]. Previous studies reporting OTOA CNVcausing NSHL are shown in Table 2. An extensive study by Sugiyama, et al. [12] identified OTOA-associated HL from sporadic 2,262 Japanese autosomal recessive HL patients and prevalence was found to be 0.3% (7/2,262). Among seven probands, two cases were found to have a homozygous deletion in OTOA gene, four cases carry heterozygous OTOA deletions in trans to SNVs, and one case with homozygous SNV (c.647T>C). This study also identified the audiometric characteristics of the affected individuals to be of mid-frequency [12]. The frequency of 16p12.2 OTOA deletion in the heterozygous state was identified to be about 1% in the Palestinian population [18]. To date, two studies have reported an association between OTOA gene deletion and HL in nonconsanguineous families [19,20]. Segregation analysis revealed compound heterozygous OTOA deleterious variant p. Trp741* in an Italian family wherein two siblings were affected by NSHL [20]. The molecular diagnosis of 131 patients affected with autosomal recessive NSHL in the Western European population emphasized that screening of OTOA CNV in the comprehensive analysis of NSHL [21]. However, no point mutation or CNV of OTOA has been reported in the Indian HL subjects.
OTOA encodes OTOAncorin, a glycosyl phosphatidylinositol protein with a molecular weight of 120 kDa essential for the anchoring of tectorial membrane (TM) with the apical surface of hair cells in the cochlear duct [22]. OTOA links the tectorial membrane with the spiral limbus of the cochlear wall required for creating an endo cochlear potential by shearing [22,23]. Mutations in the OTOA gene affect the anchoring of TM with inner hair cells as well as the detachment of TM, from the cochlear wall [23]. Zwaenepoel, et al. [23] first reported the mutation of OTOA underlying prelingual onset of severe to profound hearing loss (DFNB22 loci).
In the present study, probands carrying OTOA CNV show similarities with a phenotypic spectrum of “distal 16p12.2 microdeletion.” Distal 16p12.2 microdeletions are extremely rare compared to recurrent 520 kb 16p12.2 deletions, which occur often [16]. The prevalence of distal 16p12.2 deletion as shown in gnomAD (DEL_CHR16_2A34FF15) is 0.00040%. The common characteristics of 520 kb 16p12.2 microdeletion include developmental problems such as autism, delayed speech, intellectual disability, hypotonia, short stature, microcephaly along with dysmorphic facial pattern, congenital cardiac defects, and epilepsy, as well as psychiatric illnesses such as schizophrenia [11,24]. However, phenotypic features of 16p12.2 microdeletion vary between individuals [25]. The clinical phenotypes of distal 16p12.2 microdeletion include autism, dyslexia, intellectual disability, delayed speech and development, along with hearing impairment due to OTOA gene deletion [16]. Phenotypes associated with the chromosomal rearrangements vary even among affected members of the same family, and sometimes, individuals with the deletion have no physical or developmental abnormalities [11]. In this study, probands showed no clinical deformities other than NSHL following the autosomal recessive pattern of inheritance. The probands in this study showed different audiograms. In an earlier study, Sugiyama, et al. [12] found OTOA-associated HL in 7 out of 2,262 Japanese sporadic autosomal recessive HL patients. This study summarized audiometric characteristics of these seven probands ranging from moderate to severe HL with audiometric configuration of mid-frequency. Among 7 patients, three adult probands had progressive HL. A proband in the study, seven years of age, showed an asymmetric pattern of SNHL. Similar to the study, proband II-2 also had asymmetrical hearing loss (moderately severe HL in the right ear and mild HL in the left ear) in our study. These changes are due to environmental or genetic factors including aging. The genotype-phenotype correlation of CNVs is complex and unclear due to the incomplete penetrance and variable expressivity leading to differences in the severity of genetic disorders necessitating crucial measures for diagnosis and treatment [26].
Additionally, COL4A6 novel heterozygous variant was identified in the probands affected with NSHL in this study. COL4A6 gene found in the Chr X encodes collagen type IV alpha-6 chain protein expressed in the basement membrane of the inner ear and plays a crucial part in the development of cochlea. Mutations in the gene associated with X-linked hearing loss (DFX4) [8,27]. A novel variant c.4277C>T identified in COL4A6 gene segregated with carrier mother and affected son in this study.
A novel compound heterozygous variation, two-base pair deletion in exon 7 (chr22: g.37726371-37726372del) and p. Thr1637Ser in exon 9 of TRIOBP gene located in Chr22 possess, was identified in this study. Multiple isoforms of TRIOBP have been discovered. It encodes a protein called TRIO and F actin-binding protein BP, primarily modulating the assembly of the actin cytoskeleton in the inner ear. Mice deficient in TRIOBP result in thin actin rootlets affecting the mechanism of hearing [1,2,28]. Mutations found in the study phenotypically correlated by segregation analysis are shown in Fig. 4. The current study highlights the genetic complexity of non-syndromic hearing loss in a South Indian family.

Pedigree of the family studied showing heterozygous parents and affected children for OTOA deletion. Pedigree chart of the affected family shows probands II-1 and II-2 born to consanguineous parents, affected with non-syndromic hearing loss due to complete OTOA gene deletion in homozygous state. The parents of the affected siblings and carriers of OTOA gene deletion exhibited with normal hearing.
In conclusion, CNVs also contribute to NSHL and their identification must be included in genetic screening for hearing loss. The results of the study provide insights into the genetic basis of hearing disorders highlighting the future investigation to elucidate the underlying mechanism and therapeutic strategies. Future studies elucidating the mutational spectrum of OTOA genes causing hearing loss are warranted.
Supplementary Materials
The online-only Data Supplement is available with this article at https://doi.org/10.7874/jao.2024.00038.
Supplementary Fig. 1.
Reference region of 16p12.2 used for CNV comparison in the study.
Supplementary Fig. 2.
Pure tone audiograms of the probands (II1& II2) and parents (I1-father & I2-mother). A: Proband II1. B: Proband II2. C: Father I1. D: Mother I2.
Notes
Conflicts of Interest
The authors have no financial conflicts of interest.
Author Contributions
Conceptualization: Jayakumar Swetha, Sambandam Ravikumar. Data curation: Arulmozhi Sakthignanavel, Aarthi Manoharan, Priyadharshini Arunagiri. Formal analysis: Jayakumar Swetha, Priyadharshini Arunagiri. Funding acquisition: Arulmozhi Sakthignanavel, Sambandam Ravikumar. Investigation: Arulmozhi Sakthignanavel, Aarthi Manoharan, Priyadharshini Arunagiri. Methodology: Arulmozhi Sakthignanavel, Chandramohan Govindasamy. Project administration: Jayakumar Swetha, Priyadharshini Arunagiri. Software: Chandramohan Govindasamy, Jayakumar Rangarajalu. Supervision: Sambandam Ravikumar, Chandramohan Govindasamy. Visualization: Jayakumar Swetha, Jayakumar Rangarajalu. Writing—original draft: Jayakumar Swetha, Aarthi Manoharan. Writing—review & editing: Aarthi Manoharan, Jayakumar Rangarajalu, Sambandam Ravikumar. Approval of final manuscript: all authors.
Funding Statement
The research leading to these results received funding from Indian Council of Medical Research under Adhoc Research Project No.5/4-5/06/ENT/2020-NCD-I entitled “Genetic Screening to identify the association between consanguineous marriage with deafness in Puducherry and Tamil Nadu.”
Acknowledgements
The author would like to thank the participants in the study. We thankfully acknowledge the School of Rehabilitation and Behavioural Sciences, AVMC Puducherry for audiometric analysis.