Spectrum of Structural Variations in Non-Syndromic Hearing Loss in an Indian Population

Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss. We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology. We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1-22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs. We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.

Hearing loss affects about 70 million people worldwide, which can be inherited as either syndromic or non-syndromic forms. Approximately 50% of all childhood deafness is caused due to gene defects. The Non-syndromic hearing loss (NSHL) is extremely heterogenous trait inherited as autosomal recessive, autosomal dominant and X-linked and also due to mitochondrial mutations. In the present review genes causing non-syndromic hearing loss (NSHL) are classified based on their molecular function. GJB2 is most prominent deafness causing gene among the different populations including India, followed by other genes. The recent technological advances in target-enrichment methods and next generation sequencing has overcome the barriers possessed by the earlier methods and laid the path for comprehensive analysis of all the known genes causing non-syndromic hearing loss. The review mainly emphases on the genes causing non-syndromic hearing loss in Indian population and their molecular function. Identifying the genes responsible for hearing loss enables otolaryngologists, geneticist to apply molecular diagnosis by offering genetic testing which helps in prevention of the condition of non-syndromic hearing loss, proper diagnosis, management, pre-natal diagnosis and to offer genetic counselling.

Prevalence of mitochondrial DNA mutations in sporadic patients with nonsyndromic sensorineural hearing loss

The onset of Non Syndromic Hearing Loss (NSHL) typically occurs without any other symptoms and can vary from person to person, even within the same family. Hearing loss can be unilateral or bilateral and can range from mild to profound degrees of hearing loss. Epidermodysplasia Verruciformis (EDV) is a dermatologic condition in which patients show reduced immunologic ability to defend and eradicate certain types of Human Papillomavirus (HPV), leading to persistent infection and an increased lifetime risk of developing cutaneous dysplasia and malignancy. Both conditions have a genetic background. However, their concurrent occurrence is very rare. Therefore, a case study is presented of a four-year-old female child who visited the dermatology outpatient department with a chief complaint of hearing loss and white-coloured lesions covering her arms, neck, back, and chest for the last two years. The hearing loss was gradual, not apparent at birth, and progressed slowly to complete hearing loss. A final diagnosis of EDV with Non Syndromic Sensorineural Hearing Loss (NSSNHL) was made based on clinical and histological examination.

On the basis of strong research, universal newborn screening should be performed before age 1 month with repeat or follow-up testing for those who do not pass performed before age 3 months and intervention started before age 6 months. On the basis of strong research and consensus statement, tympanostomy tubes should be considered for individuals with bilateral persistent middle ear effusion for 3 months or greater and a documented conductive hearing loss. On the basis of consensus statement, all children with suspected hearing loss should have an age appropriate hearing test. On the basis of strong research, the most common form of congenital hearing loss is genetic. Most of this is nonsyndromic hearing loss.

Objective: To study connexin 26 (Cx26) gene mutations among autosomal recessive non-syndromal hearing loss in Kuwaiti patients and evaluate their effect on phenotypes. Subjects and Methods: This cross sectional study included 100 patients aged between 6 months and 18 years, who were referred to the Sheikh Salem Al-Ali Centre for audiology and speech evaluation of autosomal recessive non-syndromic sensorineural hearing loss confirmed by clinico-genetic evaluation and a battery of diagnostic tests. Gene profiling and sequencing were performed to detect the presence and nature of Cx26 mutation. Results: Of the 100 patients, mutation of Cx26 gene was detected in 15 patients (15%) of which 9 (60%) cases were heterozygous and 6 cases (40%) were homozygous. Eighty per cent of the 15 Cx26 positive cases resulted from the 35delG mutation. Among the heterozygous cases, 6 (66.6%) were positive for 35delG. All 6 homozygous patients were positive for the 35delG mutation. A significant correlation was found between genetic findings (p = 0.013) and family history (p = 0.029), as well as the onset (p = 0.015), course (p = 0.033), degree and configuration of hearing loss (p = 0.001). Conclusion: Among the selected Kuwaiti population sample, the Cx26 gene mutation was responsible for 15% of autosomal recessive non-syndromic sensorineural hearing loss. We recommend that screening for Cx26 gene mutation be considered in the screening strategy of patients with non-syndromic childhood hearing loss for counselling and management purposes.

Mutations in the connexin 26 (Cx26) or gap junction beta 2 gene are the leading cause of hereditary nonsyndromic sensorineural hearing loss in Caucasians. The Cx26 coding region of 68 children with nonsyndromic sensorineural hearing loss was sequenced to determine the frequency and type of Cx26 mutations in this population. Screening was also performed for a common connexin 30 (Cx30) or gap junction beta 6 mutation (del [GJB6-D13S1830]). Children also underwent audiological testing to determine whether any correlation exists between Cx26 mutations and severity of hearing loss. In all, 68 children with nonsyndromic sensorineural hearing loss were screened for Cx26 and Cx30 mutations by polymerase chain reaction and direct sequencing. Genomic DNA was amplified by polymerase chain reaction using primers that flank the entire Cx26 coding region. Screening for the 342-kb Cx30 deletion was performed using primers that amplified the breakpoint junction of the deletion. The amplicons were then sequenced in both directions and analyzed for mutations. Audiometric testing, including pure-tone audiometry and auditory evoked brainstem response, was also performed to determine the degree of hearing loss. Twenty-seven of 68 children tested had mutations in Cx26 with 35delG being the most prevalent. Ten additional Cx26 mutations were detected including a novel compound heterozygote. Two children were heterozygous for the Cx30 del (GJB6-D13S1830) mutation. Cx26 and Cx30 mutations were present in 41.2% of children tested in the study population. Audiometric data supported previous studies demonstrating a greater degree of hearing loss in subjects who are homozygous for the 35delG mutation.

BackgroundGenetic testing is essential to the diagnosis of nonsyndromic bilateral sensorineural hearing loss (BSNHL), where pathogenic variants in GJB2 are the most common cause. Current testing strategies often fail to provide a comprehensive diagnosis and typically require the use of multiple testing methodologies. This study evaluated the diagnostic utility of genome sequencing (GS) in a cohort with heterozygosity for GJB2 pathogenic variants and BSNHL.MethodsA retrospective cohort of 23 individuals with BSNHL and a heterozygous pathogenic variant in GJB2 underwent targeted GJB2 resequencing and variant reinterpretation. Those without biallelic GJB2 variants upon single gene reanalysis proceeded to exome sequencing (ES) using a large virtual panel of hearing loss–associated genes. Subjects with no definitive diagnosis from ES subsequently underwent GS. Variants were interpreted using hearing loss–specific ACMG guidelines and published literature.ResultsThree individuals were diagnosed with biallelic pathogenic variants upon GJB2 single gene reanalysis. ES identified a definitive or likely diagnosis in five different hearing loss–related genes in 5/20 (25%) individuals, while two additional cases remained inconclusive due to novel or ambiguous variants in two other hearing loss–associated genes. GS of the remaining 15 cases yielded diagnoses in three individuals, including the identification of deletions in LOXHD1 and STRC, and a recently characterized 125 kb deletion overlapping CRYL1, which refines a critical upstream regulatory region associated with GJB2-related hearing loss. Overall, 11/23 (48%) individuals received a diagnosis with our stepwise testing approach, with GS providing sequencing coverage of all findings.ConclusionGS improves diagnostic yield in patients with BSNHL, capturing both SNVs and CNVs missed by ES and targeted testing, and supports its adoption as a comprehensive first-tier diagnostic test for nonsyndromic hearing loss.

An update of common autosomal recessive non-syndromic hearing loss genes in Iranian population

BackgroundHearing loss is a multifactorial condition with the etiology of hereditary and/or environmental factors, which leads to various degrees of hearing impairment. One of the most common mutations associated with aminoglycoside-induced non-syndromic hearing impairment in the mitochondrial 12S ribosomal RNA gene is the A1555G mutation, which has been reported in several ethnic groups. Hereditary hearing impairment is very common in the Arab community. As far as we know, the frequency of the A1555G mutation in the Egyptian population has not been extensively documented. This study aimed to investigate the presence of the mitochondrial A1555G mutation in Egyptian children diagnosed with non-syndromic sensorineural hearing loss, to provide insight into its possible contribution to hearing impairment in this population.Material and methodsA cross-sectional observational study was conducted on 30 Egyptian children (aged 9 months to 12 years) with confirmed non-syndromic sensorineural hearing loss. Detailed clinical histories, three-generation pedigrees, and comprehensive audiological assessments (tympanometry, auditory brainstem response, and/or pure-tone audiometry) were performed. The A1555G mutation was screened using the PCR–RFLP method.ResultsThe current study’s findings showed that only one patient (3.3%) is positive for the A1555G mutation. The ages of selected children ranged between 9 months and 12 years with a mean age of 5.9 years. All cases had bilateral severe or profound sensorineural hearing loss at the high frequencies (2–4 kHz).ConclusionThis pilot study identified the mitochondrial A1555G mutation in only 1 case among 30 Egyptian children with non-syndromic sensorineural hearing loss, suggesting a potential, though limited, role of this mutation in the studied population. Broader studies are recommended to better understand its prevalence and clinical significance.

AUDIOME: a tiered exome sequencing–based comprehensive gene panel for the diagnosis of heterogeneous nonsyndromic sensorineural hearing loss

The etiology of hereditary hearing loss is extraordinarily complex. More than 400 genetic syndromes are associated with hearing loss and more than 140 genetic loci associated with nonsyndromic hearing loss have been mapped, with more than 60 genes identified to date. Hereditary hearing loss can be inherited as an autosomal dominant, autosomal recessive, X-linked or mitochondrial (maternally inherited) condition. The overlapping audiologic phenotypes associated with many genes and the variability and/or reduced, sometimes age-related, penetrance of some phenotypic features of syndromic hearing loss can complicate the distinction between various genetic causes of nonsyndromic hearing loss and between nonsyndromic and syndromic hearing loss, especially in childhood. Testing for individual genes associated with nonsyndromic hearing loss, beyond GJB2 which encodes Connexin 26, can become expensive and, without specific phenotypic features to guide selection of genes for testing (such as enlarged vestibular aqueducts, low frequency hearing loss or auditory neuropathy), it is not likely to yield an etiology. Advances in DNA sequencing and the rapid decline in the cost of sequencing presage the availability of testing that can identify the etiology in the majority of cases of genetic hearing loss. However, until comprehensive genetic testing of hearing loss is clinically available and cost-effective, thorough phenotypic and audiologic evaluation and careful documentation of risk factors, infectious exposures and patient and family medical history will continue to be important to efforts directed toward etiologic diagnosis. The complexities associated with interpretation of genetic test results, genetic counseling and genetic risk assessment make consultation with medical geneticists important for many patients.

Background/Objectives: The OTOG gene is responsible for autosomal recessive non-syndromic sensorineural hearing loss and is assigned as DFNB18B. To date, 44 causative OTOG variants have been reported to cause non-syndromic hearing loss. However, the detailed clinical features for OTOG-associated hearing loss remain unclear. Methods: In this study, we analyzed 7065 patients with non-syndromic hearing loss (mean age 26.4 ± 22.9 years, 2988 male, 3855 female, and 222 without gender information) using massively parallel DNA sequencing for 158 target deafness genes. We identified the patients with biallelic OTOG variants and summarized the clinical characteristics. Results: Among the 7065 patients, we identified 14 possibly disease-causing OTOG variants in 26 probands, with 13 of the 14 variants regarded as novel. Patients with OTOG-associated hearing loss mostly showed congenital or childhood-onset hearing loss. They were considered to show non-progressive, mild-to-moderate hearing loss. There were no symptoms that accompanied the hearing loss in OTOG-associated hearing loss patients. Conclusions: We confirmed non-progressive, mild-to-moderate hearing loss as the clinical characteristics of OTOG-associated hearing loss. These findings will contribute to a better understanding of the clinical features of OTOG-associated HL and will be useful in clinical practice.

Recent genetic and genomic studies have greatly advanced our knowledge of the structure and function of genes involved in hearing loss. We are starting to recognize, however, that many of these genes do not appear to follow traditional Mendelian expression patterns and are subject to the effects of allelism and modifier genes. This review presents two genes illustrative of this concept that have varied expression pattern such that they may produce either syndromic or nonsyndromic hearing loss. One of these genes, cadherin 23, produces a spectrum of phenotypic traits, including presbycusis, nonsyndromic prelingual hearing loss (DFNB12), and syndromic hearing loss as part of Usher syndrome (Usher 1D). Missense mutations in CDH23 have been associated with presbycusis and DFNB12, whereas null alleles cause the majority of Usher 1D. Modifier gene products that interact with cadherin 23 also affect the phenotypic spectrum. Similarly, allelsim in the gene encoding wolframin (WFS1) causes either a nonsyndromic dominant low-frequency hearing loss (DFNA6/14/38) or Wolfram syndrome. Missense mutations within a defined region are associated with DFNA6/14/38, while more severe mutations spanning WFS1 are found in Wolfram syndrome patients. The phenotypic spectrum of Wolfram syndrome is also hypothesized to be influenced by modifier genes products. These studies provide increasing evidence for the importance of modifier genes in elucidating the functional pathways of primary hearing loss genes. Characterizing modifier genes may result in better treatment options for patients with hearing loss and define new diagnostic and therapeutic targets.

Pathogenic variants of MYH14 are known to be associated (in either a syndromic or nonsyndromic manner) with hearing loss. Interestingly, all reported cases to date of MYH14-related nonsyndromic hearing loss with detailed phenotypes have demonstrated mild-to-moderate progressive hearing loss with postlingual onset. In the present study, targeted resequencing (TRS) of known deafness genes was performed to identify the causative variant in two multiplex families segregating autosomal dominant (AD) inherited hearing loss. TRS uncovered two novel variants of MYH14 (c.A572G: p.Asp191Gly in the myosin head domain and c.C73T:p.Gln25* in exon 2) from two multiplex deafness Korean families. Notably, both probands showed phenotypes of congenital or prelingual severe hearing loss. It is remarkably uncommon to encounter such a severe-to-profound, prelingual, AD hearing loss. Given that the first variant, p. Asp191Gly, was the first documented missense allele discovered in the myosin head domain of this gene related to either congenital or prelingual severe nonsyndromic hearing loss, and also that the second variant, p. Gln25*, lead to a null allele, more severe phenotypes from our probands may have been the result of either genotype-phenotype correlation or genetic backgrounds, or both. In the present study, we report that MYH14 can manifest as nonsyndromic prelingual severe sensorineural hearing loss in an AD fashion in Koreans. The results of the present study suggest that further genetic studies of similar patients should consider MYH14 as a causative gene, and cochlear implantation during infant or early childhood should be indicated for those patients with certain MYH14 pathogenic variants.