Gjb3 Gene Mutations in Non-Syndromic Hearing Loss of Bloch, Kurd, and Turkmen Ethnicities in Iran.

BACKGROUND: Hearing loss (HL) is one of the most common heterogeneous congenital disabilities worldwide. Gap junction protein ß-3 (GJB3) gene encodes Connexin31 protein (Cx31). The hereditary type of hearing impairment in this gene are known to cause both autosomal recessive and autosomal dominant form. In addition, GJB3 mutations have been involved in sensorineural deafness, erythrokeratodermia variabilis (EKV), and neuropathy diseases. We aimed to investigate GJB3 mutations in people suffering from HL among three different ethnicities of Iranian population (Baloch, Kurd, and Turkmen). METHODS: In this descriptive study, 50 GJB2-negative non-syndromic hearing loss (NSHL) Iranian individuals from 3 ethnic groups of Baloch (n=17), Kurd (n =15) and Turkmen (n=18) were enrolled. DNA extractions, PCR, and mutation detection was carried out for the two large deletions of the GJB6, del (GJB6 -D13S1830,) and del (GJB6 -D13S1854) followed by direct DNA sequencing method for the GJB3. RESULTS: DNA sequencing of GJB3 was shown a missense heterozygous mutation rs199689484 (NM_024009.3) GJB3: c.340G>A (p.Ala114Thr) in a Baloch patient, and a polymorphism rs35983826 (NM_024009.3) GJB3: c.798C>T (p.Asn266=) in a Turkman patient, in coding region of the GJB3. We did not detect del (GJB6 -D13S1830) and del (GJB6 -D13S1854) among these three ethnicities in Iran. CONCLUSION: Deafness is a heterogeneous disorder. Specific genes and mutations contribute to hearing loss that varies from locus to locus as well as from population to population.

Whole-exome sequencing identifies a novel missense variant within LOXHD1 causing rare hearing loss in a Chinese family.

BACKGROUND: Deafness, autosomal recessive 77 (DFNB77) is a rare non-syndromic hearing loss (NSHL) worldwide, which is caused by deleterious variants within lipoxygenase homology domains 1 (LOXHD1). Here we identified that a novel missense variant of LOXHD1 was associated with NSHL in a Chinese family under consanguineous marriage. CASE PRESENTATION: A 28-year-old woman suffered a bilateral profound NSHL. Impedance audiometry, temporal bone computerized tomography (TBCT) scans and magnetic resonance imaging-inner ear hydrography (MRI-IEH) did not find any obvious abnormality of middle or inner ear. Routine genetic detection did not find pathogenic variants in common HL-associated genes. Therefore, we performed a whole-exome sequencing (WES) in this family. By trio-WES, co-segregation validation and bioinformatics analysis, we revealed that a novel homozygous variant in this patient, LOXHD1: c.5948C > T (p.S1983F), might be the pathogenic factor. Her parents (heterozygotes) and brother (wild-type) were asymptomatic. CONCLUSIONS: We successfully identified a novel variant of LOXHD1 associated with a rare NSHL from a Chinese family. Our finds highlight the effectiveness of trio-WES for molecular diagnosis of rare NHSL, and expand the genotypic spectrum of DFNB77.

Investigation of the mtDNA mutations in Syrian families with non-syndromic sensorineural hearing loss.

OBJECTIVE: Hearing loss is a common sensory disorder, and at least 50% of cases are due to a genetic etiology. Several mitochondrial DNA mutations (mtDNA) have been reported to be associated with nonsyndromic hearing loss (NSHL) in different population. However, There is no previous available data about the frequency of mtDNA mutations as etiology for deafness in Syrian. The aim of present study is to investigate the incidence of common mt DNA mutations in our families with congenital hearing loss and not related to the ototoxicity or aminoglycosides. METHODS: A total of 50 deaf families were enrolled in the present study. Direct sequencing and PCR-RFLP methods were employed to detect seven mt DNA mutations, including A1555G, A3243G, C1494T, G3316A, T7510C, A7445G, and 7472insC. RESULTS: Our results revealed a high prevalence of mt DNA mutation (10%) in deaf families (5/50). In surprising, the unexpected mutations were observed. The G3316A mutation was found in 2 families as homoplasmic genotype. Also, we found the homoplasmic and heteroplasmic genotype for the C1494T mutation in two families. In one family the heteroplasmic genotype for T7510C mutation was observed; this family harbor 35delG mutation in GJB2 gene. None of the common mtDNA mutations (A1555G, A3243G) and other mutations (A7445G, 7472insC) were detected here. CONCLUSION: Our findings indicate to significant contribution of the mt DNA mutations in our families with NSHL. The presented data is the first report about mt DNA and it will improve the genetic counseling of hearing impaired in Syrian families.

Connexin 26 (GJB2) Mutations Associated with Non-Syndromic Hearing Loss (NSHL).

OBJECTIVE: To determine the prevalence and spectrum of Connexin 26 (GJB2) mutations in pre-lingual non-syndromic hearing loss (NSHL) patients in authors' centre and to review the data of Indian patients from the literature. METHODS: Sanger sequencing of entire coding region contained in single exon (Exon 2) of GJB2 gene in 15 patients of NSHL. RESULTS: GJB2 mutations were found in 40% (6/15) of NSHL patients, out of which mono-allelic were 33.3% (2/6). Bi-allelic GJB2 mutations were identified in 4 of 6 patients. Most common GJB2 mutation identified was c.71G > A(p.W24X), comprising 30% of the total GJB2 mutant alleles. Six studies involving 1119 patients with NSHL were reviewed and 4 of them have reported c.71G > A(p.W24X) as the commonest mutation while 2 studies found c.35delG as the commonest. GJB2 mutations accounted for 10.9%-36% cases of NSHL. Sixteen other mutations in GJB2 gene were reported in Indian patients out of which 6 mutations other than c.71G > A(p.W24X) viz., c.35delG, c.1A > G(p.M1V), c.127G > A(p.V43 M), c.204C > G(p.Y86X), c.231G > A(p.W77X) and c.439G > A(p.E147K) were identified in the present study. CONCLUSIONS: Connexin 26 (GJB2) mutations are responsible for 19.4% of NSHL in Indian population. The c.71G > A(W24X) and c.35delG were the most prevalent GJB2 mutations accounting for 72.2% (234 of 324 total mutated alleles from 7 studies) and 15.4% (50 of 324 total mutated alleles from 7 studies) respectively. Thus, screening of these two common mutations in GJB2 gene by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) would greatly help in providing easy genetic diagnosis and help in genetic counseling of the families with NSHL.

First report of prevalence c.IVS1+1G>A and del (GJB6-13S1854) mutations in Syrian families with non-syndromic sensorineural hearing loss.

OBJECTIVE: Mutations in GJB2 and GJB6 genes are a frequent cause of congenital non-syndromic hearing loss (NSHL). Mutational screening has usually focused on coding region of GJB2 gene. A few studies have been conducted on the non-coding region and exon 1. c.IVS1+1G>A (a splice site mutation in GJB2 gene have been detected as disruptive mutation. Del (GJB6 D13S1830) is found in many populations, but del (GJB6 D13S1854) is reported from a few restricted countries. This study was carried out to investigate the prevalence of splice site mutation c.IVS1+1G>A and two common deletions in GJB6 gene as the genetic etiology of hearing impairment in 70 Syrian families. METHODS: The frequency of the c.IVS1+1G>A mutation and two deletions were determined by PCR-RFLP and A multiplex PCR assay. RESULT: Our results showed a high prevalence of IVS1+1G>A mutation (20%) and del(GJB6-D13S1854) (15.7%) in deaf families. The homozygous genotype (c.IVS1+1G>A/c.IVS1+1G>A) was observed in one family and the compound heterozygous genotypes (c.35delG/c.IVS1+1G>A) and (c.IVS1+1G>A/V153I) were observed in 7 families and one family respectively. Also, the heterozygous state (c.IVS1+1G>A/unknown) was detected in 5 families. The study of del((GJB6-D13S1854) was showed a compound heterozygous genotype del((GJB6-D13S1854)/c.IVS1+1G>A) in the same families (5 families) having heterozygous genotype of c.IVS1+1G>A mutation. Also, del(GJB6-D13S1854) is combined with c.35delG mutation in 2 families and it was observed in the heterozygous state del(GJB6-D13S1854)/unknown) in 4 families. In contrast, the del(GJB6-D13S1830) described in many population was absent in our patients. CONCLUSION: Our findings indicate to significant contribution of the splice site mutation and del(GJB6-D13S1854) in our deaf families and these mutations were important causes of hearing impairment.

DFNB16 is a frequent cause of congenital hearing impairment: implementation of STRC mutation analysis in routine diagnostics.

Increasing attention has been directed toward assessing mutational fallout of stereocilin (STRC), the gene underlying DFNB16. A major challenge is due to a closely linked pseudogene with 99.6% coding sequence identity. In 94 GJB2/GJB6-mutation negative individuals with non-syndromic sensorineural hearing loss (NSHL), we identified two homozygous and six heterozygous deletions, encompassing the STRC region by microarray and/or quantitative polymerase chain reaction (qPCR) analysis. To detect smaller mutations, we developed a Sanger sequencing method for pseudogene exclusion. Three heterozygous deletion carriers exhibited hemizygous mutations predicted as negatively impacting the protein. In 30 NSHL individuals without deletion, we detected one with compound heterozygous and two with heterozygous pathogenic mutations. Of 36 total patients undergoing STRC sequencing, two showed the c.3893A>G variant in conjunction with a heterozygous deletion or mutation and three exhibited the variant in a heterozygous state. Although this variant affects a highly conserved amino acid and is predicted as deleterious, comparable minor allele frequencies (MAFs) (around 10%) in NSHL individuals and controls and homozygous variant carriers without NSHL argue against its pathogenicity. Collectively, six (6%) of 94 NSHL individuals were diagnosed with homozygous or compound heterozygous mutations causing DFNB16 and five (5%) as heterozygous mutation carriers. Besides GJB2/GJB6 (DFNB1), STRC is a major contributor to congenital hearing impairment.

Non-syndromic hearing loss gene identification: A brief history and glimpse into the future.

From the first identified non-syndromic hearing loss gene in 1995, to those discovered in present day, the field of human genetics has witnessed an unparalleled revolution that includes the completion of the Human Genome Project in 2003 to the $1000 genome in 2014. This review highlights the classical and cutting-edge strategies for non-syndromic hearing loss gene identification that have been used throughout the twenty year history with a special emphasis on how the innovative breakthroughs in next generation sequencing technology have forever changed candidate gene approaches. The simplified approach afforded by next generation sequencing technology provides a second chance for the many linked loci in large and well characterized families that have been identified by linkage analysis but have presently failed to identify a causative gene. It also discusses some complexities that may restrict eventual candidate gene discovery and calls for novel approaches to answer some of the questions that make this simple Mendelian disorder so intriguing.

A systematic review and meta-analysis of common mutations of SLC26A4 gene in Asian populations.

OBJECTIVES: The IVS7-2A>G (c.919-2A>G) and p.H723R (c.2168A>G) mutations of SLC26A4 gene are recognized as a risk factor for the non-syndromic hearing loss. To elucidate the variable results, a meta-analysis and systematic review was performed from all case-control studies by pooling data on them. METHODS: The case-control studies were assessed with a modification of the Newcastle-Ottawa Scale (NOS). The strength of association between c.919-2A>G, c.2168A>G and hearing loss risk was measured by odds ratios (ORs) with 95% confidence intervals (CIs). RESULTS: We included 14 case-control studies and 16 case series studies in present study. There was a higher prevalence of the c.919-2A>G mutation in the case group than that in the control group (12.4% vs 0.9%; OR = 13.05, 95% CI: 8.41-20.23, Z = 11.47, P<0.00001). CONCLUSIONS: In conclusion, the results from this meta-analysis suggest that NSHL patients have an increased risk of the c.919-2A>G mutation of SLC26A4 gene in Asians, especially in Chinese.

Prevalence Study of GJB2,SLC26A4 and mtDNA 12S rRNA1555A>G Mutations in Hui Ethnic Group Patients with Non-Syndromic Hearing Loss from Northwest China / 听力学及言语疾病杂志

Objective To investigate the prevalence of GJB2 ,SLC26A4 and mitochondrial DNA 12S rRNA m .1555A>G(mtDNA 1555A>G) mutations in Hui ethic group patients with nonsyndromic hearing loss (NSHL) from Northwest China .Methods A total of 420 peripheral blood samples were collected from unrelated Hui ethic group probands with NSHL in Northwest China .Amplified the target gene by polymerase chain reaction (PCR) af-ter extracting genomic DNA from whole blood .The mtDNA 1555A>G mutation was detected by PCR -Alw26I di-gestion ,then direct sequencing was used to the positive samples of mtDNA 1555A> G ,the coding region of GJB2 gene ,exon 8 and 19 of SLC26A4 gene .Results There were 11(2 .62% ) cases caused by mtDNA 1555A>G homo-zygous mutation in 420 patients with NSHL .There were 41(9 .76% ) cases including homozygote and compound het-erozygote ,caused by GJB2 gene mutation ,which was the most frequent deafness -related gene .The allel frequency of c .235delC accounted for 6 .90% ,as well as the most frequent(51 .33% ) mutational pattern in GJB2 gene .There were 20 patients(4 .76% ) were found carring two allel mutations in SLC26A4 gene .The allel frequency of c .919 -2A>G was 5 .0% ,accounting for a total of 68 .85% in all base alterations of SLC26A4 gene ,which was the major mutant form of SLC26A4 gene .Conclusion GJB2 gene is the most common deafness -gene in Hui ethnic group pa-tients with NSHL from Northwest China ,while c .235delC is the main mutant form ,and c .919-2A>G is the hot-spot mutation of SLC26A4 gene .Through this study we can provide the molecular epidemiology basis for Hui ethnic group patients with NSHL from Northwest China in genetic diagnosis ,genetic counseling and therapy by associated testing of three frequent hearing loss genes .

Analysis of the Mutations in SLC26A4 Gene in Children with Non-syndromic Hearing Loss / 听力学及言语疾病杂志

Objective To study the SLC26A4 mutations in children with non -syndromic hearing loss by ge-netic testing method ,for the purpose of investigating etiology and mutation regularity of hearing loss ,and to provide basic information for the molecular diagnosis of hearing loss .Methods Blood samples and clinical data of 137 spo-radic cases with non -syndromic hearing loss and 126 normal controls were collected .The SLC26A4 gene of the pa-tients and normal controls were amplified by polymerase chain reaction (PCR) ,then subjected to automatic DNA se-quencing .Results Pathologic SLC26A4 mutations were identified in 23 out of 137 patients ,and in 23 out of 119 bi-lateral deafness ,mutate rate were 16 .79% and 19 .33% ,respectively .SLC26A4 mutations were identified in 19 out of 20(95% ) patients with bilateral LVA .A total of 11 mutations were identified in the present study ,including 4 novel mutations (E29K(c .85G>A) ,R79X(c .235C> T) ,C282G(c .844T>G) ,V285I(c .853G>A) )and 7 repor-ted mutations .In the present study ,IVS7-2A>G was the most common mutation ,and was detected in 19 out of 23(82 .61% ) patients with SLC26A4 mutations .Conclusion SLC26A4 mutations ,the common reason for non -syndromic hearing loss ,were closely related with LVA .IVS7-2A>G was the most common mutation in SLC26A4 mutant .

Genetics of non syndromic hearing loss in the republic of macedonia.

Hearing impairment is the most common sensory deficit in humans affecting 1 in 1000 newborns. When present in an infant, deafness may have dramatic effects on language acquisition, seriously compromising the quality of their life. Deafness is influenced by both genetic and environmental factors, with inherited causes as the most prominent etiological factor in deafness in developed countries. The genetic basis of hearing loss is complex with numerous loci and genes underlying hereditary sensoryneural non syndromic hearing loss (NSHL) in humans. Despite the wide functional heterogeneity of the genes, mutations in the GJB2 gene are found to be the most common cause of sporadic and recessive NSHL in many populations worldwide. Molecular characterization of deafness in the Republic of Macedonia was performed in 130 NSHL profoundly deaf children from different ethnic origins. Molecular studies included direct sequencing of the GJB2 gene and specific polymerase chain reaction (PCR) analyses for the del(GJB6-D13S1830) mutation. Five common mitochondrial DNA (mtDNA) mutations [A1555G, 961delT+ C(n), T1095C, C1494T and A827G] were also analyzed using the SNaPShot method. In preliminary studies, GJB2 gene mutations were found in 36.4% of analyzed patients, with predominance of 35delG in Macedonian and Albanian patients and W24X in Gypsy patients, respectively. No del(GJB6-D13S1830) mutation was found. None of the analyzed deafness-associated mutations in mtDNA were identified in the studied patients.