A novel variant in the GJB6 gene in a large Chinese family with a unique phenotype of Clouston syndrome / 医学前沿

Clouston syndrome (OMIM #129500), also known as hidrotic ectodermal dysplasia type 2, is a rare autosomal dominant skin disorder. To date, four mutations in the GJB6 gene, G11R, V37E, A88V, and D50N, have been confirmed to cause this condition. In previous studies, the focus has been mainly on gene sequencing, and there has been a lack of research on clinical manifestations and pathogenesis. To confirm the diagnosis of this pedigree at the molecular level and summarize and analyse the clinical phenotype of patients and to provide a basis for further study of the pathogenesis of the disease, we performed whole-exome and Sanger sequencing on a large Chinese Clouston syndrome pedigree. Detailed clinical examination included histopathology, hair microscopy, and scanning electron microscopy. We found a novel heterozygous missense variant (c.134G>C:p.G45A) for Clouston syndrome. We identified a new clinical phenotype involving all nail needling pain in all patients and found a special honeycomb hole structure in the patients' hair under scanning electron microscopy. Our data reveal that a novel variant (c.134G>C:p.G45A) plays a likely pathogenic role in this pedigree and highlight that genetic testing is necessary for the diagnosis of Clouston syndrome.

Hipoacusia neurosensorial no sindrómica: caracterización molecular, desempeño auditivo y tratamiento en pacientes con mutaciones en las conexinas 26 y 30 / Non-syndromic sensorineural hearing loss: molecular characterization, auditory performance, and treatment in patients with connexin 26 and 30 mutations

La hipoacusia congénita o de aparición temprana es un trastorno sensorial muy frecuente en niños. Las causas son diversas, pueden intervenir factores genéticos y/o ambientales. El 80% de la sordera hereditaria es no sindrómica y de herencia autosómica recesiva. Hasta un 50% de estos casos se deben a mutaciones en el locus DFNB1 donde están localizados los genes GJB2 y GJB6, que codifican las conexinas 26 y 30, dos proteínas que se expresan predominantemente en la cóclea. Se han reportado más de 100 mutaciones en el gen GJB2, con una mutación muy frecuente, 35delG, que representa hasta un 85% de los alelos mutados. Una deleción en el gen GJB6, (delGJB6-D13S1830), surge como la segunda mutación más frecuente. La hipoacusia debida a mutaciones en estos genes es de inicio prelocutivo, con un grado de severidad que varía de moderado a profundo, existiendo casos leves en menor proporción, con variaciones inter e intrafamiliares. Es generalmente estable, bilateral, y afecta a todas las frecuencias. El conocimiento de las causas genéticas de la hipoacusia ha permitido contar con nuevas herramientas para el diagnóstico, y como consecuencia, se ha optimizado el asesoramiento genético y facilitado el diagnóstico precoz de los pacientes, incluso en el período prenatal. La detección precoz tiene un impacto inmediato en la implementación de terapias que permiten una estimulación auditiva temprana. En esta revisión se describe el papel de las conexinas en la fisiología auditiva, así como también las características moleculares y audiológicas y el desempeño auditivo con audífonos e implante coclear en pacientes que presentan mutaciones en las conexinas 26 y 30.

Congenital or early appearing hearing loss is a very common sensory disorder in children. The causes for the disorder are diverse and genetic as well as environmental factors may be involved. Overall, 80% of the hereditary deafness is non-syndromic and of autosomal recessive inheritance. Up to 50% of the cases are associated with mutations in the DFNB1 locus that contains the GJB2 and the GJB6 genes encoding connexins 26 and 30, two proteins that are predominantly expressed in the cochlea. More than 100 mutations of the GJB2 have been reported. The 35delG is a common mutation accounting for up to 85% of the mutated alleles. A deletion in the GJB6 gene, (delGJB6-D13S1830), is the second most frequent mutation found. Hearing loss due to mutations in these genes has an onset before speech develops and degree of severity varies from moderate to severe, with a lower incidence of mild cases and inter- and intrafamily variations. The condition is usually stable, bilateral, and affecting all frequencies. Increased knowledge on the genetic causes of hearing loss has allowed for the development of new diagnostic tools and consequently, improvement of genetic counseling and early, even prenatal, diagnosis. Early detection has an immediate impact with implementation of early auditory stimulation therapies. In this review the role of connexins in auditory physiology described, as well as molecular and audiological features and auditory performance with hearing aids and cochlear implants in patients with connexins 26 and 30 mutations.

Mechanistic effect of the human GJB6 gene and its mutations in HaCaT cell proliferation and apoptosis

We constructed lentiviral vectors containing the human wild-type GJB6 gene and the mutant variants A88V and G11R. The three proteins were stably expressed by the Tet-on system in the HaCaT cell line and used to study the functional effect of the variants. The CCK-8 assay and flow cytometric analyses were used to determine the levels of cell proliferation and apoptosis. Western blot analyses were performed to analyze the relevant clinical indicators of hidrotic ectodermal dysplasia and markers of apoptosis in transfected HaCaT cells. The CCK8 assay and the flow cytometry results showed a significant increase (P<0.05) in the apoptosis of HaCaT cells expressing the A88V and G11R mutants. In addition, we demonstrated that the A88V and G11R mutants induced the apoptosis of transfected HaCaT cells via the activation of caspase-3, -8, -9, and PARA. No change was observed in the activity of BAX compared with the control. This study provides further clarification on the mechanisms underlying the effect of the mutant variants A88V and G11R of the GJB6 gene on the induction of HaCaT cell apoptosis.

A gene study of a family with hidrotic ectodermal dysplasia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the clinical features and molecular mechanism of hidrotic ectodermal dysplasia (HED).</p><p><b>METHODS</b>A clinical and gene study was performed for five generations (91 people) in the family of one proband with HED. GJB6 gene detection was performed for 7 patients and 3 normal people in this family.</p><p><b>RESULTS</b>Among the 91 people in this family, there were 17 HED patients, who were manifested as having dysplasia of the fingernails and toenails and sparse or absent hair or body hair. The male patients had a greater degree of sparse hair compared with female patients. In the younger generations, damage to the fingernails and toenails was gradually alleviated. There were patients in each generation, the patient's mother or father definitely had this disease. Both males and females developed this disease, and the inheritance pattern was autosomal dominant inheritance. A heterozygous missense mutation, 31G→A, in GJB6 gene was detected in all patients in this family, but this mutation was not detected in family members without the clinical manifestations of HED.</p><p><b>CONCLUSIONS</b>HED is a hereditary disease with autosomal dominant inheritance and has the clinical features of dysplasia of the fingernails and toenails, hyperkeratosis of palms and soles, and sparse or absent hair or body hair. Male patients have a greater degree of sparse hair. In the younger generations, damage to the fingernails and toenails is gradually alleviated. The missense mutation 31G→A in the GJB6 gene may be one of the molecular mechanisms for HED.</p>

Etiologic analysis of severe to profound hearing loss patients from Chifeng city in Inner Mongolia / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the etiology of patients with severe to profound hearing loss and to identify the ratio of hereditary hearing loss in Chifeng area in Northern China.</p><p><b>METHODS</b>DNA were extracted from peripheral blood of 134 deaf patients from Chifeng special educational school and 100 normal hearing controls in Northern China. Audiology examinations showed that all patients had severe to profound bilateral sensorineural hearing impairment. Sequence analysis of the whole coding areas of GJB2, GJB3, GJB6, SLC26A4, mtDNA12SrRNA and mtDNAtRNASer(UCN) were performed. Individuals carrying SLC26A4 mutation were given further temporal bone CT scan.</p><p><b>RESULTS</b>The ratio of hearing loss related to genetic factors in this population was 60.45% (81/134). About 33.58% (45/134) of the patients were given accurate genetic diagnosis. GJB2 mutations were responsible for approximately 17.16% of the cases in ChiFeng area. By screening SLC26A4 followed by temporal bone CT scan, we diagnosed 20 cases of enlarged vestibular aqueduct (EVA) and/or other inner ear malformation. SLC26A4 mutations account for about 14.93% of the cases. The aminoglycoside-related mtDNA 1555A>G mutation accounted for 0.76% of the cases in Chifeng area. In addition, another 13.43% (18/134) of the cases carried heterozygous GJB2 mutation and their hearing loss may be related to GJB2. 6.72% (9/134) of the cases carried heterozygous SLC26A4 mutation who were not found EVA by temporal bone CT or not took CT examination for some reasons. However, their hearing loss may also be SLC26A4-related. About 2.24% (3/134) of the cases carried mtDNA 12SrRNA 1095 T>C which may also be an aminoglycoside-related mutation and very likely be the cause of hearing loss. GJB3 might participate in the pathomechanism of hearing loss in 1.49% (2/134) of the patients. GJB6 mutation was not detected in this population.</p><p><b>CONCLUSIONS</b>The ratio of hearing loss related to genetic factors in the sample drawing population from Chifeng was 60.45% (81 cases). GJB2 is the most common gene and SLC26A4 is the second common gene next to GJB2 that cause deafness in this area.</p>

Screening and identification interaction proteins of connexin 30 / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore interaction proteins affect functions of connexin 30 (Cx30) by screening and identification interaction proteins of Cx30.</p><p><b>METHODS</b>The fusion expression vecto of CX30-C-terminal functional domain-pGEX-4T-2-GST was constructed, and then, fusion protein and GST were purified. They were incubated with the proteins of the foetus brain tissue disruption to pull down interaction proteins. The interaction proteins were separated by SDS-PAGE. Differential straps were cut to enzymolysis to prepare for mass chromatographic analysis, and then to index and screen interaction proteins in NCBInr database. The interaction proteins were identified by immunolocalization.</p><p><b>RESULTS</b>The four interaction proteins of Cx30 were screened in the foetus brain tissue, as follow, Keratin 16, Camk2b, Tubulin beta-3 and alpha-tubulin. Cx30 was proved to coexist with Keratin 16 and Tubulin beta-3.</p><p><b>CONCLUSIONS</b>Keratin 16, Camk2b, Tubulin beta-3 and alpha-tubulin are the interaction proteins of Cx30. The interaction proteins affect the assembly, intracellular transport, and channel switch of Cx30.</p>

Single nucleotide polymorphisms and haplotypes analysis of DFNB1 locus in Chinese sporadic hearing impairment population / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The DFNB1 locus, which contains the gap junction beta-2 (GJB2) and gap junction beta-6 (GJB6) genes, plays a key role in the nonsyndromic and sporadic hearing impairment. Mutations of DFNB1 result in autosomal recessive nonsyndromic hearing impairment (ARNSHI). Previous researches have identified mutations in GJB2 and GJB6, but single nucleotide polymorphisms (SNPs) of DFNB1 locus have not been studied. So we chose five SNPs to evaluate whether there is difference between deafness people and normal-hearing people in Han Chinese.</p><p><b>METHODS</b>Five SNPs in the DFNB1 region were examined using a case-control association study between cases with sporadic hearing impairment and controls with normal hearing. The HWEsoft and SHEsis softwares were used to analyze the results.</p><p><b>RESULTS</b>Single-locus association analysis showed a positive association for three SNPs: rs9315400, rs2274084 and 235delC. When we compared the distributions of the haplotypes, we also found significant differences between cases and controls in the haplotype combination of rs2274084 and rs2274083 (chi(2) = 12.978, df = 3, global P = 0.004719).</p><p><b>CONCLUSIONS</b>The haplotypes composed of rs2274084 and rs2274083 suggested that C-C may be a risk haplotype for the sporadic hearing impairment while T-T may be protective against hearing impairment. From that point of view, we can conclude that the SNPs of DFNB1 locus also plays an important role in sporadic hearing impairment cases.</p>

Mutation analysis of GJB2, GJB3 and GJB6 gene in deaf population from special educational school of Chifeng city / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the genetic causes of nonsyndromic deaf patients in special educational school of Chifeng city. Inner Mongolia by genetic screening testing method. This study focused on analyzing mutations of coding sequence of GJB2, GJB3 and GJB6 gene.@*METHOD@#DNA were extracted out from peripheral blood of 134 nonsyndromic deaf probands of Chifeng special educational school and 100 normal hearing controls in northern China. First, GJB2 gene mutation was analyzed by direct sequencing for its only exon in the open reading frame. Individuals found with heterozygous GJB2 mutation were given further testing for GJB6 del(GJB6-D13S1830) and direct sequencing for its exon. In 91 probands with unknown genetic cause (excluding probands who carried mtDNA A1555G mutation and GJB2 gene bi allele mutation and probands who were diagnosed as enlarged vestibular aqueduct by temporal CT), GJB3 gene mutation was analyzed by direct sequencing for its exon.@*RESULT@#The sequencing results revealed that forty-one cases carried GJB2 mutation. of which twenty-two were homozygous or compound heterozygous and nineteen were heterozygous. Further testing for GJB6 del(GJB6-D13S1830) and analysis of its coding sequence in GJB2 heterozygous cases showed no positive result. Four subjects in control group carried pathogenetic mutation of GJB2 gene. Six types of novel variants of GJB2 gene were detected. Of the 91 deaf probands with unknown etiology. two probands were found carrying heterozygous pathogenetic mutation of GJB3 gene. one of whom also carried GJB2 235delC heterozygous mutation. One subjects in the control group carried pathogenetic mutation of GJB3 gene. Three types of novel variants of GJB3 gene were found.@*CONCLUSION@#By screening GJB2.GJB3 and GJB6 gene, we found 32.1% probands carrying GJB2, GJB3, and GJB6 mutations and we are able to determine genetic cause related to these three genes from one family for 16.42 percent of nonsyndromic deaf probands in special educational school of Chifeng city. The discovery of novel variants of GJB2 and GJB3 gene makes the mutational and polymorphic spectrum more plentiful in Chinese population.

GJB6 gene mutation analysis in Chinese nonsyndromic deaf population / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the contribution of GJB6 gene (encoding connexin 30) mutation in Chinese population with sporadic non-syndromic hearing impairment.@*METHOD@#Three hundred and seventy-two nonsyndromic hearing impairment patients and 182 normal controls were first tested for GJB6 del(GJB6 > D13S1830) using specific PCR primers. Then PCR was performed with a pair of primer flanking the whole coding sequence of GJB6 gene. Sequencing of GJB6 whole coding sequence PCR products was subsequently applied in all subjects with hearing loss and normal controls.@*RESULT@#None of the patients and normal controls carried GJB6 del (GJB6 > D13S1830). Two single base pair changes were detected , one in the patient group and the other in the control group. The mutation found in the patient group was not detected in the control subjects.@*CONCLUSION@#Mutation of GJB6 gene is not frequent in Chinese non-syndromic hearing-loss population. Screening for GJB6 gene can be ranked as unconventional deaf gene test in China temporarily.

Mutation screening and prenatal diagnosis of hidrotic ectodermal dysplasia in a Chinese family / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the mutations in Cx30 gene in a Chinese family with hidrotic ectodermal dysplasia (HED) and to make prenatal diagnosis on the embryo which has been pregnant for 5 months.</p><p><b>METHODS</b>A family including 2 affected and 4 unaffected individuals was collected, and their informed consents were obtained. The affected woman had a five-month pregnancy. An 884 bp fragment containing the whole GJB6 coding sequence was amplified by PCR and the products were bi-direction sequenced directly. The mutation was further confirmed with restriction endoenzyme digesting. On the base of successful gene diagnosis, the following detection procedure on the pregnant baby was performed. First the whole coding region of Cx30 was amplified using primers Cx30-F and Cx30-R and the PCR products were digested by Hae II. Then the PCR products were cloned into pUCm-T vector. Blue-white blot screening method and PCR-restriction endoenzyme digesting technique were used to identify the correct clones. The mutant allele clone was sequenced to confirmed the mutation.</p><p><b>RESULTS</b>A heterozygous missense mutation 263C --> T in the Cx30 gene was detected in the affected little girl and her affected mother, which led to an amino acid substitution (A88V) in the second transmembrane domain of GJB6. The mutation was confirmed by Hae II digestion. A88V mutant allele cannot be cut while the wild normal allele can be cut into two fragments, 520 and 278 bp. The result of analyse on the five-month pregnancy show the embryo carried the A88V mutation too. So the embryo will be a patient.</p><p><b>CONCLUSION</b>An A88V missense mutation in the Cx30 gene can also cause HED in Chinese Han population. Based on the gene diagnosis, prenatal diagnosis can be played using bi-direction sequencing and confirmed with restriction endoenzyme digesting.</p>

Deafness genes for nonsyndromic hearing loss and current studies in China / 中华医学杂志(英文版)

<p><b>OBJECTIVES</b>To review the identified deafness genes related to nonsyndromic hearing loss (NSHL) and summarize their expressions and functions in the cochlea and to introduce the current studies of molecular genetics on NSHL in China.</p><p><b>METHODS</b>The presented data are based on a review of the literature as well as the author' s experience with NSHL and communications with other researchers in China over the past 3 years.</p><p><b>RESULTS</b>Currently, 23 deafness genes related to NSHL have been cloned and identified. Some genes are associated with both NSHL and syndromic hearing loss (SHL), in both dominant and recessive deafness. Deafness genes have a highly specific expression pattern in the inner ear. Some functional categories are starting to emerge from a characterization of deafness genes. There are interacting genes in the genetic background that influence the extent of hearing impairment. The GJB3 gene, which is associated with high-frequency hearing impairment, was cloned in a Chinese laboratory. Mutations in some genes, such as GJB2 and mitochondrial 12S rRNA, have been screened in Chinese patients with NSHL. Mapping new deafness gene loci as well as identifying new genes and their functions is an active area of study in China.</p><p><b>CONCLUSIONS</b>It is challenging for us to continue identifying new deafness genes and analyze gene functions. By identifying genes responsible for monogenic hearing impairment, more insight may be gained into the molecular process of hearing and the pathology of hearing loss.</p>