Mutation In GJB6 Gene In A Large Chinese Han Family With Hidrotic Ectodermal Dysplasia

BackgroundThe skin of human is developed from two germ layers:epidermis is developed from ectoderm and connective tissue is developed from mesoblastema. Ectodermal dysplasias is genetic disorders resulting in structural or functional abnormalities in two or more of the major structures derived from ectoderm. The structures affected include hair, sweat glands, teeth, nails, sebaceous glands and mucous glands. These disorders contain several isoforms. Differentiation among the different isoforms is based on the types of ectodermal defects, associated on ectodermal anomalies and mode of inheritance. Hidrotic ectodermal dysplasia (HED) (OMIM 129500;), also named as Clouston syndrome. This disorder was first found and reported in a French-Canadian kindred. It is a rare autosomal dominant disease which occurs with a frequency of 1:100.000. The clinical characteristics of this disorder include nail dystrophy, alopecia and hyperkeratosis of the palms and soles. Sweat glands and teeth of patients with hidrotic ectodermal dysplasia are usually normal. This disease is caused by GJB6 gene, which located at chromosomes 13 and encodes the connexin 30 protein.We collected a Chinese Han family with hidrotic ectodermal dysplasia consisting of 252 individuals including 45 HED patients (26 males and 19 females), which is the biggest pedigree reported in literature so far. We collected and analyzed the clinical information of these patients and blood samples of the individuals from this family were obtained (25 affected persons and 14 unaffected persons).ObjectiveAnalyze the typicality and specificity of hidrotic ectodermal dysplasia in this family through collecting the clinical information and determine the mode of inheritance. The purposes of this study were to make sure the pathological mutation of this disease in this Chinese family through gene test of GJB6 gene. Discuss the relationship between genotype and phenotype.MethodsClinical information of this large Chinese family with hidrotic ectodermaldysplasia was collected and analyzed. Blood samples and pictures of the individuals from this family were obtained (25 affected persons and 14 unaffected persons). We also collected 218 normal healthy Chinese Han people as controls. The genomic DNAs were isolated using a commercial kit (QIAGEN) according to the manufacturer s protocol. The coding region of GJB6 was amplified by polymerase chain reaction (PCR) using the specific primers and the PCR products were sequenced bidirectionally. Total mRNA was extracted from the proband’s scalp skin which constitutively expresses Cx30 gene using TRIZOL reagent (Invitrogen,China) according to the manufacturer’s guidelines. cDNA was obtained by reverse transcription using a corresponding kit (Takara PrimeScriptTM 1st Strand cDNA Synthesis Kit,Dalian,China). Total RNA from the proband’s scalp skin was reverse transcribed using a Takara PrimeScript TM 1st Strand cDNA Synthesis Kit (Dalian,China). A 309-bp fragment of the Cx30 gene mRNA was amplified by polymerase chain reaction (PCR) from cDNA using specific primers. PCR products were sequenced bidirectionally. The GJB6 gene from 218 normal healthy Chinese Han people was also tested to eliminate the universality of the mutation.ResultsA heterozygous missense mutation 263Câ†'T in GJB6 gene was identified by bi-direct sequencing of PCR products derived from genomic DNA of the patients. This substitution leads to the predicted amino acid change Ala88-to-Val in 25 affected individuals. This mutation was not founded in 14 unaffected individuals and 218 unrelated, population matched control individuals, indicating that the substitution do not represent a common polymorphisms. The p.A88V mutation in GJB6 gene was also confirmed in cDNA from the proband’s scalp skin source. The results suggested that the A88V variant played a pathologic role in affected individuals from this Chinese family. We found a novel phenotype of this variant in this Chinese HED family.Conclusion:our data reveals that a recurrent mutation A88V in GJB6 played a pathogenic role in a large Chinese Han family and emphasizes the importance of gene test in this congenital disorder.