Identification of novel MYH14 variants in families with autosomal dominant sensorineural hearing loss.

Autosomal dominant sensorineural hearing loss (ADSNHL) is a genetically heterogeneous disorder caused by pathogenic variants in various genes, including MYH14. However, the interpretation of pathogenicity for MYH14 variants remains a challenge due to incomplete penetrance and the lack of functional studies and large families. In this study, we performed exome sequencing in six unrelated families with ADSNHL and identified five MYH14 variants, including three novel variants. Two of the novel variants, c.571G > C (p.Asp191His) and c.571G > A (p.Asp191Asn), were classified as likely pathogenic using ACMG and Hearing Loss Expert panel guidelines. In silico modeling demonstrated that these variants, along with p.Gly1794Arg, can alter protein stability and interactions among neighboring molecules. Our findings suggest that MYH14 causative variants may be more contributory and emphasize the importance of considering this gene in patients with nonsyndromic mainly post-lingual severe form of hearing loss. However, further functional studies are needed to confirm the pathogenicity of these variants.

Genetic heterogeneity in hereditary hearing loss: Potential role of kinociliary protein TOGARAM2.

Hearing loss (HL) is a heterogenous trait with pathogenic variants in more than 200 genes that have been discovered in studies involving small and large HL families. Over one-third of families with hereditary HL remain etiologically undiagnosed after screening for mutations in the recognized genes. Genetic heterogeneity complicates the analysis in multiplex families where variants in more than one gene can be causal in different individuals even in the same sibship. We employed exome or genome sequencing in at least two affected individuals with congenital or prelingual-onset, severe to profound, non-syndromic, bilateral sensorineural HL from four multiplex families. Bioinformatic analysis was performed to identify variants in known and candidate deafness genes. Our results show that in these four families, variants in a single HL gene do not explain HL in all affected family members, and variants in another known or candidate HL gene were detected to clarify HL in the entire family. We also present a variant in TOGARAM2 as a potential cause underlying autosomal recessive non-syndromic HL by showing its presence in a family with HL, its expression in the cochlea and the localization of the protein to cochlear hair cells. Conclusively, analyzing all affected family members separately can serve as a good source for the identification of variants in known and novel candidate genes for HL.

Dispersed DNA variants underlie hearing loss in South Florida's minority population.

BACKGROUND: We analyzed the genetic causes of sensorineural hearing loss in racial and ethnic minorities of South Florida by reviewing demographic, phenotypic, and genetic data on 136 patients presenting to the Hereditary Hearing Loss Clinic at the University of Miami. In our retrospective chart review, of these patients, half self-identified as Hispanic, and the self-identified racial distribution was 115 (86%) White, 15 (11%) Black, and 6 (4%) Asian. Our analysis helps to reduce the gap in understanding the prevalence, impact, and genetic factors related to hearing loss among diverse populations. RESULTS: The causative gene variant or variants were identified in 54 (40%) patients, with no significant difference in the molecular diagnostic rate between Hispanics and Non-Hispanics. However, the total solve rate based on race was 40%, 47%, and 17% in Whites, Blacks, and Asians, respectively. In Non-Hispanic Whites, 16 different variants were identified in 13 genes, with GJB2 (32%), MYO7A (11%), and SLC26A4 (11%) being the most frequently implicated genes. In White Hispanics, 34 variants were identified in 20 genes, with GJB2 (22%), MYO7A (7%), and STRC-CATSPER2 (7%) being the most common. In the Non-Hispanic Black cohort, the gene distribution was evenly dispersed, with 11 variants occurring in 7 genes, and no variant was identified in 3 Hispanic Black probands. For the Asian cohort, only one gene variant was found out of 6 patients. CONCLUSION: This study demonstrates that the diagnostic rate of genetic studies in hearing loss varies according to race in South Florida, with more heterogeneity in racial and ethnic minorities. Further studies to delineate deafness gene variants in underrepresented populations, such as African Americans/Blacks from Hispanic groups, are much needed to reduce racial and ethnic disparities in genetic diagnoses.

Novel GPR156 variants confirm its role in moderate sensorineural hearing loss.

Hereditary hearing loss (HL) is a genetically heterogeneous disorder affecting people worldwide. The implementation of advanced sequencing technologies has significantly contributed to the identification of novel genes involved in HL. In this study, probands of two Turkish families with non-syndromic moderate HL were subjected to exome sequencing. The data analysis identified the c.600G > A (p.Thr200Thr) and c.1863dupG (p.His622fs) variants in GPR156, which co-segregated with the phenotype as an autosomal recessive trait in the respective families. The in silico predictions and a minigene assay showed that the c.600G > A variant disrupts mRNA splicing. This gene belongs to the family of G protein-coupled receptors whose function is not well established in the inner ear. GPR156 variants have very recently been reported to cause HL in three families. Our study from a different ethnic background confirms GPR156 as a bona fide gene involved in HL in humans. Further investigation towards the understanding of the role of GPCRs in the inner ear is warranted.

Genome sequencing identifies coding and non-coding variants for non-syndromic hearing loss.

Hearing loss (HL) is a common heterogeneous trait that involves variants in more than 200 genes. In this study, we utilized exome (ES) and genome sequencing (GS) to effectively identify the genetic cause of presumably non-syndromic HL in 322 families from South and West Asia and Latin America. Biallelic GJB2 variants were identified in 58 probands at the time of enrollment these probands were excluded. In addition, upon review of phenotypic findings, 38/322 probands were excluded based on syndromic findings at the time of ascertainment and no further evaluation was performed on those samples. We performed ES as a primary diagnostic tool on one or two affected individuals from 212/226 families. Via ES we detected a total of 78 variants in 30 genes and showed their co-segregation with HL in 71 affected families. Most of the variants were frameshift or missense and affected individuals were either homozygous or compound heterozygous in their respective families. We employed GS as a primary test on a subset of 14 families and a secondary tool on 22 families which were unsolved by ES. Although the cumulative detection rate of causal variants by ES and GS is 40% (89/226), GS alone has led to a molecular diagnosis in 7 of 14 families as the primary tool and 5 of 22 families as the secondary test. GS successfully identified variants present in deep intronic or complex regions not detectable by ES.

Mutations in MINAR2 encoding membrane integral NOTCH2-associated receptor 2 cause deafness in humans and mice.

Discovery of deafness genes and elucidating their functions have substantially contributed to our understanding of hearing physiology and its pathologies. Here we report on DNA variants in MINAR2, encoding membrane integral NOTCH2-associated receptor 2, in four families underlying autosomal recessive nonsyndromic deafness. Neurologic evaluation of affected individuals at ages ranging from 4 to 80 y old does not show additional abnormalities. MINAR2 is a recently annotated gene with limited functional understanding. We detected three MINAR2 variants, c.144G > A (p.Trp48*), c.412_419delCGGTTTTG (p.Arg138Valfs*10), and c.393G > T, in 13 individuals with congenital- or prelingual-onset severe-to-profound sensorineural hearing loss (HL). The c.393G > T variant is shown to disrupt a splice donor site. We show that Minar2 is expressed in the mouse inner ear, with the protein localizing mainly in the hair cells, spiral ganglia, the spiral limbus, and the stria vascularis. Mice with loss of function of the Minar2 protein (Minar2tm1b/tm1b) present with rapidly progressive sensorineural HL associated with a reduction in outer hair cell stereocilia in the shortest row and degeneration of hair cells at a later age. We conclude that MINAR2 is essential for hearing in humans and mice and its disruption leads to sensorineural HL. Progressive HL observed in mice and in some affected individuals and as well as relative preservation of hair cells provides an opportunity to interfere with HL using genetic therapies.

Whole Mitochondrial Genome Analysis in Turkish Patients with Mitochondrial Diseases

Background: Mitochondrial diseases are a clinically heterogeneous group of rare hereditary disorders that are defined by a genetic defect predominantly affecting mitochondrial oxidative phosphorylation. Mitochondrial diseases are caused by mutations of genes encoded by either nuclear DNA or mitochondrial DNA. Hundreds of different mitochondrial DNA point mutations and large-scale mitochondrial DNA rearrangements have been shown to cause mitochondrial diseases including Kearns­Sayre syndrome, Leber's hereditary optic neuropathy, Leigh syndrome, myoclonic epilepsy with ragged-red fibers, mitochondrial encephalopathy lactic acidosis stroke. Aims: To investigate new variants that could be associated with mitochondrial diseases and to determine the effect of mitochondrial DNA mutations on the clinical spectrum. Study Design: Cross-sectional study. Methods: We screened whole mitochondrial DNA genome using next-generation sequencing in 16 patients who are considered to have mitochondrial disease. CentoGene and Mikrogen Genetic Diseases Diagnostic Center's database were used to investigate sequence variants. Detected variants were evaluated in bioinformatic databases to determine pathogenicity and were classified as class 1 (pathogenic), class 2 (likely pathogenic), and class 3 (variant of uncertain significance) according to CentoGene-ACMG database. Results: As a result of the study, 2 patients were diagnosed with Leigh syndrome as previously reported class 1 mutations in MT-ATP6 and MT-ND5 genes. Four variants were identified for the first time in literature and 2 variants, previously reported but with uncertain pathogenic effect, are thought to be associated with mitochondrial disease. Conclusion: Mitochondrial DNA screening should be among the primary clinical tests in patients with suspected mitochondrial disease to rule out DNA-associated mutations.

Long-range cis-regulatory elements controlling GDF6 expression are essential for ear development.

Molecular mechanisms governing the development of the mammalian cochlea, the hearing organ, remain largely unknown. Through genome sequencing in 3 subjects from 2 families with nonsyndromic cochlear aplasia, we identified homozygous 221-kb and 338-kb deletions in a noncoding region on chromosome 8 with an approximately 200-kb overlapping section. Genomic location of the overlapping deleted region started from approximately 350 kb downstream of GDF6, which codes for growth and differentiation factor 6. Otic lineage cells differentiated from induced pluripotent stem cells derived from an affected individual showed reduced expression of GDF6 compared with control cells. Knockout of Gdf6 in a mouse model resulted in cochlear aplasia, closely resembling the human phenotype. We conclude that GDF6 plays a necessary role in early cochlear development controlled by cis-regulatory elements located within an approximately 500-kb region of the genome in humans and that its disruption leads to deafness due to cochlear aplasia.

Adams-Oliver syndrome caused by mutations of the EOGT gene.

Adams-Oliver syndrome (AOS) is a rare congenital disease characterized by aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). It shows significant genetic heterogeneity and can be transmitted by autosomal dominant or recessive inheritance. Recessive inheritance is associated with mutations of DOCK6 or EOGT; however, only few cases have been published so far. We present two families with EOGT-associated AOS. Due to pseudodominance in one family, the recognition of the recessive inheritance pattern was difficult. We identified two novel AOS-causing mutations (c.404G>A/p.Cys135Tyr and c.311+1G>T). The phenotype in the presented families was dominated by large ACC, whereas TTLD were mostly subtle or even absent and no major malformations occured. Our observations along with the previously published cases indicate that the two types of recessive AOS (EOGT- vs. DOCK6-associated) differ significanty regarding the frequency of neurologic or ocular deficits.

Novel variant p.E269K confirms causative role of PLS1 mutations in autosomal dominant hearing loss.

Auditory reception relies on the perception of mechanical stimuli by stereocilia and its conversion to electrochemical signal. Mechanosensory stereocilia are abundant in actin, which provides them with structural conformity necessary for perception of auditory stimuli. Out of three major classes of actin-bundling proteins, plastin 1 encoded by PLS1, is highly expressed in stereocilia and is necessary for their regular maintenance. A missense PLS1 variant associated with autosomal dominant hearing loss (HL) in a small family has recently been reported. Here, we present another PLS1 missense variant, c.805G > A (p.E269K), in a Turkish family with autosomal dominant non-syndromic HL confirming the causative role of PLS1 mutations in HL. We propose that HL due to the p.E269K variant is from the loss of a stable PLS1-ACTB interaction.

A truncating CLDN9 variant is associated with autosomal recessive nonsyndromic hearing loss.

While the importance of tight junctions in hearing is well established, the role of Claudin- 9 (CLDN9), a tight junction protein, in human hearing and deafness has not been explored. Through whole-genome sequencing, we identified a one base pair deletion (c.86delT) in CLDN9 in a consanguineous family from Turkey with autosomal recessive nonsyndromic hearing loss. Three affected members of the family had sensorineural hearing loss (SNHL) ranging from moderate to profound in severity. The variant is predicted to cause a frameshift and produce a truncated protein (p.Leu29ArgfsTer4) in this single-exon gene. It is absent in public databases as well as in over 1000 Turkish individuals, and co-segregates with SNHL in the family. Our in vitro studies demonstrate that the mutant protein does not localize to cell membrane as demonstrated for the wild-type protein. Mice-lacking Cldn9 have been shown to develop SNHL. We conclude that CLDN9 is essential for proper audition in humans and its disruption leads to SNHL in humans.

Genetic Causes of Inner Ear Anomalies: a Review from the Turkish Study Group for Inner Ear Anomalies

Inner ear anomalies diagnosed using a radiological study are detected in almost 30% of cases with congenital or prelingual-onset sensorineural hearing loss. Inner ear anomalies can be isolated or occur along with a part of a syndrome involving other systems. Although astonishing progress has been made in research aimed at revealing the genetic causes of hearing loss in the past few decades, only a few genes have been linked to inner ear anomalies. The aim of this review is to discuss the known genetic causes of inner ear anomalies. Identifying the genetic causes of inner ear anomalies is important for guiding clinical care that includes empowered reproductive decisions provided to the affected individuals. Furthermore, understanding the molecular underpinnings of the development of the inner ear in humans is important to develop novel treatment strategies for people with hearing loss.

Dysfunction of GRAP, encoding the GRB2-related adaptor protein, is linked to sensorineural hearing loss.

We have identified a GRAP variant (c.311A>T; p.Gln104Leu) cosegregating with autosomal recessive nonsyndromic deafness in two unrelated families. GRAP encodes a member of the highly conserved growth factor receptor-bound protein 2 (GRB2)/Sem-5/drk family of proteins, which are involved in Ras signaling; however, the function of the growth factor receptor-bound protein 2 (GRB2)-related adaptor protein (GRAP) in the auditory system is not known. Here, we show that, in mouse, Grap is expressed in the inner ear and the protein localizes to the neuronal fibers innervating cochlear and utricular auditory hair cells. Downstream of receptor kinase (drk), the Drosophila homolog of human GRAP, is expressed in Johnston's organ (JO), the fly hearing organ, and the loss of drk in JO causes scolopidium abnormalities. drk mutant flies present deficits in negative geotaxis behavior, which can be suppressed by human wild-type but not mutant GRAP. Furthermore, drk specifically colocalizes with synapsin at synapses, suggesting a potential role of such adaptor proteins in regulating actin cytoskeleton dynamics in the nervous system. Our findings establish a causative link between GRAP mutation and nonsyndromic deafness and suggest a function of GRAP/drk in hearing.

FOXF2 is required for cochlear development in humans and mice.

Molecular mechanisms governing the development of the human cochlea remain largely unknown. Through genome sequencing, we identified a homozygous FOXF2 variant c.325A>T (p.I109F) in a child with profound sensorineural hearing loss (SNHL) associated with incomplete partition type I anomaly of the cochlea. This variant is not found in public databases or in over 1000 ethnicity-matched control individuals. I109 is a highly conserved residue in the forkhead box (Fox) domain of FOXF2, a member of the Fox protein family of transcription factors that regulate the expression of genes involved in embryogenic development as well as adult life. Our in vitro studies show that the half-life of mutant FOXF2 is reduced compared to that of wild type. Foxf2 is expressed in the cochlea of developing and adult mice. The mouse knockout of Foxf2 shows shortened and malformed cochleae, in addition to altered shape of hair cells with innervation and planar cell polarity defects. Expressions of Eya1 and Pax3, genes essential for cochlear development, are reduced in the cochleae of Foxf2 knockout mice. We conclude that FOXF2 plays a major role in cochlear development and its dysfunction leads to SNHL and developmental anomalies of the cochlea in humans and mice.

Identification of candidate gene FAM183A and novel pathogenic variants in known genes: High genetic heterogeneity for autosomal recessive intellectual disability.

The etiology of intellectual disability (ID) is heterogeneous including a variety of genetic and environmental causes. Historically, most research has not focused on autosomal recessive ID (ARID), which is a significant cause of ID, particularly in areas where parental consanguinity is common. Identification of genetic causes allows for precision diagnosis and improved genetic counseling. We performed whole exome sequencing to 21 Turkish families, seven multiplex and 14 simplex, with nonsyndromic ID. Based on the presence of multiple affected siblings born to unaffected parents and/or shared ancestry, we consider all families as ARID. We revealed the underlying causative variants in seven families in MCPH1 (c.427dupA, p.T143Nfs*5), WDR62 (c.3406C>T, p.R1136*), ASPM (c.5219_5225delGAGGATA, p.R1740Tfs*7), RARS (c.1588A>G, p.T530A), CC2D1A (c.811delG, p.A271Pfs*30), TUSC3 (c.793C>T, p.Q265*) and ZNF335 (c.808C>T, p.R270C and c.3715C>A, p.Q1239K) previously linked with ARID. Besides ARID genes, in one family, affected male siblings were hemizygous for PQBP1 (c.459_462delAGAG, p.R153Sfs*41) and in one family the proband was female and heterozygous for X-chromosomal SLC9A6 (c.1631+1G>A) variant. Each of these variants, except for those in MCPH1 and PQBP1, have not been previously published. Additionally in one family, two affected children were homozygous for the c.377G>A (p.W126*) variant in the FAM183A, a gene not previously associated with ARID. No causative variants were found in the remaining 11 families. A wide variety of variants explain half of families with ARID. FAM183A is a promising novel candidate gene for ARID.

MPZL2 is a novel gene associated with autosomal recessive nonsyndromic moderate hearing loss.

While recent studies have revealed a substantial portion of the genes underlying human hearing loss, the extensive genetic landscape has not been completely explored. Here, we report a loss-of-function variant (c.72delA) in MPZL2 in three unrelated multiplex families from Turkey and Iran with autosomal recessive nonsyndromic hearing loss. The variant co-segregates with moderate sensorineural hearing loss in all three families. We show a shared haplotype flanking the variant in our families implicating a single founder. While rare in other populations, the allele frequency of the variant is ~ 0.004 in Ashkenazi Jews, suggesting that it may be an important cause of moderate hearing loss in that population. We show that Mpzl2 is expressed in mouse inner ear, and the protein localizes in the auditory inner and outer hair cells, with an asymmetric subcellular localization. We thus present MPZL2 as a novel gene associated with sensorineural hearing loss.

Elucidating the genetic architecture of Adams-Oliver syndrome in a large European cohort.

Adams-Oliver syndrome (AOS) is a rare developmental disorder, characterized by scalp aplasia cutis congenita (ACC) and transverse terminal limb defects (TTLD). Autosomal dominant forms of AOS are linked to mutations in ARHGAP31, DLL4, NOTCH1 or RBPJ, while DOCK6 and EOGT underlie autosomal recessive inheritance. Data on the frequency and distribution of mutations in large cohorts are currently limited. The purpose of this study was therefore to comprehensively examine the genetic architecture of AOS in an extensive cohort. Molecular diagnostic screening of 194 AOS/ACC/TTLD probands/families was conducted using next-generation and/or capillary sequencing analyses. In total, we identified 63 (likely) pathogenic mutations, comprising 56 distinct and 22 novel mutations, providing a molecular diagnosis in 30% of patients. Taken together with previous reports, these findings bring the total number of reported disease variants to 63, with a diagnostic yield of 36% in familial cases. NOTCH1 is the major contributor, underlying 10% of AOS/ACC/TTLD cases, with DLL4 (6%), DOCK6 (6%), ARHGAP31 (3%), EOGT (3%), and RBPJ (2%) representing additional causality in this cohort. We confirm the relevance of genetic screening across the AOS/ACC/TTLD spectrum, highlighting preliminary but important genotype-phenotype correlations. This cohort offers potential for further gene identification to address missing heritability.

Research of genetic bases of hereditary non-syndromic hearing loss.

AIM: Hearing loss is the most common sensory disorder that affects approximately one per 1000 live births. With this project, we aimed to identify gene variants that were common causes of hearing loss in Turkey to contribute to the planning of genetic screening programs for hearing loss, as well as to improve genetic counseling to affected families. MATERIAL AND METHODS: Twenty-one families with at least two affected individuals and parental consanguinity who presented with non-syndromic severe-to-profound sensorineural hearing loss were included in this study. We first screened for mutations in GJB2 and mitochondrial DNA 12S RNA genes. Subsequently, we genotyped the TMIE c.250C>T and SNP markers flanking the SLC26A4, MYO7A, MYO15A, OTOF, CDH23, TMIE, TECTA, PCDH15, TMC1, TMPRSS3, TMHS genes in the remaining twelve families without mutations in GJB2. RESULTS: Screening for mutations in GJB2 gene showed c.[35delG];[35delG] mutation in four families, c.[35delG];[507C>A] mutation in two families, c.[35delG];[-23+1G>A] mutation in one family, and c.457G>A heterozygous mutation in one family. Genotyping SNP markers showed the c.[250C>T];[250C>T] mutation in TMIE in one family. A homozygous region with SNP genotypes was detected with the OTOF gene in one family, the TMPRSS3 gene in another family, and also a homozygous region was detected with TMHS, OTOF, and TMPRSS3 genes in another family. CONCLUSIONS: Further research will be required to determine the genetic bases of hearing loss in families with non-syndromic hearing loss.

Novel pathogenic variants underlie SLC26A4-related hearing loss in a multiethnic cohort.

OBJECTIVES: The genetics of sensorineural hearing loss is characterized by a high degree of heterogeneity. Despite this heterogeneity, DNA variants found within SLC26A4 have been reported to be the second most common contributor after those of GJB2 in many populations. METHODS: Whole exome sequencing and/or Sanger sequencing of SLC26A4 in 117 individuals with sensorineural hearing loss with or without inner ear anomalies but not with goiter from Turkey, Iran, and Mexico were performed. RESULTS: We identified 27 unique SLC26A4 variants in 31 probands. The variants c.1673A > G (p.N558S), c.1708-1G > A, c.1952C > T (p.P651L), and c.2090-1G > A have not been previously reported. The p.N558S variant was detected in two unrelated Mexican families. CONCLUSION: A range of SLC26A4 variants without a common recurrent mutation underlies SLC26A4-related hearing loss in Turkey, Iran, and Mexico.

Novel EYA1 variants causing Branchio-oto-renal syndrome.

INTRODUCTION: Branchio-oto-renal (BOR) syndrome is an autosomal dominant genetic disorder characterized by second branchial arch anomalies, hearing impairment, and renal malformations. Pathogenic mutations have been discovered in several genes such as EYA1, SIX5, and SIX1. However, nearly half of those affected reveal no pathogenic variant by traditional genetic testing. METHODS AND MATERIALS: Whole Exome sequencing and/or Sanger sequencing performed in 10 unrelated families from Turkey, Iran, Ecuador, and USA with BOR syndrome in this study. RESULTS: We identified causative DNA variants in six families including novel c.525delT, c.979T > C, and c.1768delG and a previously reported c.1779A > T variants in EYA1. Two large heterozygous deletions involving EYA1 were detected in additional two families. Whole exome sequencing did not reveal a causative variant in the remaining four families. CONCLUSIONS: A variety of DNA changes including large deletions underlie BOR syndrome in different populations, which can be detected with comprehensive genetic testing.