Molecular Genetics Analysis Of Chinese X-linked Hypohidrotic Ectodermal Dysplasia

Ectodermal dysplasias (EDs) is a complicated disorder comprising more than 200 clinical combinations. Most EDs have family history, inherited as autosomal dominant, autosomal recessive and X-linked modes, although there are still some sporadics. X-linked hypohidrotic ectodermal dysplasias(XLHED,OMIM# 305100), also called Christ-Siemens-Touraine syndrome, is the most common type. It is a rare X chromosome linked disorder mainly affecting males and is inherited through female carriers. The incidence is about less than one in per 100,000. Epithelial-and mesenchymal-derived organs including hair, tooth, nails, and sweat glands are mainly affected. Patients have specific clinical features:sparse hair, eyelashes and eyebrows, small, misshapen or missing teeth, diminished sweating with a history of high fevers in hot weather, decreased salivary secretions and a special facial appearance. Inability to sweat caused by abnormal glands can bring life-threatening hyperthermia which was responsible for the 30% mortality rate in the first two years of life. Facial characteristics include prominent forehead, narrow and short maxillary regions, small palatal depth, small cranial length, and depressed nasal root and bridge of the nose. While most heterozygous carriers only show minor to moderate degrees of these abnormalities.Previous studies confirmed XLHED is due to aberrant of EDA gene, a member of TNF-related ligand family, located close to the X inactivation centre. The EDA undergoes extensive alternative splicing. As a result, it has at least eight transcripts. EDA-A1, the longest isoform, encodes a 391-amino acid protein with a domain similar to TNF at the C terminus. EDAR which binds exclusively to EDA-A1 is activated by it and uses EDARADD as an adaptor to build an intracellular signal-transducing complex. Similarly, XEDAR is only bind to EDA-A2 which deletes two amino acids, glu308 and val309 compared with EDA-A1. EDA-A1 and EDA-A2 activate NF-kappa-B-promoted transcription after binding to their receptors.Heretofore, over one hundred EDA mutations have been found in XLHED, most of which are missense mutations. They centralize in four clusters:the junction of the transmembrane and the extracellular domain, the protease recognition site, the trimerizing collagen-like domain, and the TNF homology domain. But there is still no explicit mutuality between genotype and phenotype.EDA contains a large CpG island in its promoter. CpG islands located in gene promoters represent a major target for DNA hypermethylation, which impairs transcription related to regional or specific methylation events. Promoter CpG island's hypermethylation contributes to gene silencing by inhibiting the binding of certain transcription factors to their recognition sequence, attracting methylated DNA-binding proteins, and/or through chromatin remodeling. However, whether aberrant methylation was related to XLHED carriers'phenotype or not was controversial.In the present study, we want to further underscore the function of EDA in the development of teeth, ectoderm and appendages by analyzing Chinese XLHED pedigrees, direct sequencing and to study the correlations between XLHED carriers' phenotype and EDA promoter's methylation state by quantatively investigating the EDA promoter's methylation.Objects:1. Ascertain some Chinese XLHED families through proband confirmation2. Enlarge Chinese mutation spectrum3. Evaluate the relationship between the genotype and phenotype of these patients 4. Estimate the conjunction between alleviating carriers'phenotype and EDA prompter's methylation levelMethods:1. After getting informed consents, we carefully inquired the illness histroy, checked their symptoms and collected 5-10 ml peripheral blood samples of each member in families. Genomic DNA was extracted by modified salting out procedure2. Screened EDA sequence of members of these pedigrees by direct sequencing3. Quantitatively analyzed the EDA promoter's methylation level of XLHED carriers by using Prosequencing systemResults:1. We ascertained eight Chinese XLHED families. Male patients exhibited full features of XLHED while female carriers only showed mild symptoms except the proband of family six.2. After sequencing all EDA exons, seven mutations, three missence mutations, two frameshift mutations and two exon deletion, were confirmed.3. Compared to normal controls,78.26% of XLHED carriers in this study were hypermethylated in foure sites of EDA's promoter.Conclusions:1. We reported seven Chinese XLHED families and one sporadic patient.2. EDA gene is also responsible for most Chinese XLHED patients.3. EDA promoter's hypermethylation might be responsible for XLHED carriers' alleviated phenotype.