Analyzing MEN2A By Whole Genome Sequencing, And Gene Mutation Analysis And Prenatal DNA Diagnosis Of6EPPK Pedigrees

Background:(1)Multiple endocrine neoplasia type2(MEN2) is an autosomal dominant hereditary cancer syndrome with major components of medullary thyroid carcinoma (MTC), pheochromocytoma and hyperparathyroidism. MEN2is classified into3subtypes:MEN2A, FMTC (familial medullary thyroid carcinoma), and MEN2B. So far, RET proto-oncogene is the only gene associated with the onset of MEN2. The point mutations of634codon account80%～90%of MEN2A, and p.Cys634Arg caused the most serious symptoms;(2)Epidermolytic palmoplantar keratoderma (EPPK) is a relative common autosomal dominant genodermatoses that often includes disabling diffuse hyperkeratosis of the palms and soles for which no satisfactory treatment is currently available. EPPK is mainly caused by dominant-negative mutations in keratin9(KRT9). Penetrance is100%. Mutations of keratin1(KRT9) were found in some mild EPPKs.Objectives:(1) By using the whole-genome sequencing (WES), we try to explore the molecular pathogenesis of MEN2A efficiencily and comprehensively, looking for the intron, the variable region variations occurred in MEN2A that affected the clinical signs of disease, the age of onset and the cancer metastasis;(2) To identify the causative mutations in EPPK pedigrees and to explore the DNA-based prenatal diagnosis for the fetus at risk of EPPK.Methods:(1) A MEN2A pedigree including11family members was collected. Whole genome sequencing (WES) was carried out in the proband, and all data was filtered dbSNP,1000Genome Project, HapMap and other databases. One of InDel mutation in the intron was selected to further subcloning sequencing. By PCR-DNA sequencing of11family members,20exons of RET gene was tested to verify the variations and the polymorphisms;(2) Six EPPK families were enrolled. To identify the causive mutation of EPPK, the whole coding regions of KRT9and KRT1were amplified and directly sequenced to detect the mutations, respectively. The variants were confirmed by AS-PCR and microsatellite DNA linkage analysis. Prenatal DNA diagnosis was performed in four EPPK families.Results:(1) Accorging to the WGS data of proband, there was a mutation of c.1900T>C (p.Cys634Arg) within exon11of RET. Sanger sequencing verified the mutation in Ⅱ:1, Ⅱ:7and Ⅲ:4among11family members, showing the accuracy and efficiency of WGS. Subcloning sequencing revealed that Ⅱ:1and11:7also had the InDel of exon2lies at the upstream71bp region (43595836nucleotide site, intron regions);(2) Three heterozygous missense mutations, c.487C>T (p.Arg163Trp) in2pedigrees, c.488G>A(p.Arg163Gln) in1pedigree, c.482A>G (p.Asn161Ser) in2pedigrees were detected in the affected individuals, with no disease-causing gene mutation was found in one pedigree. Fetal DNAs puried from amniotic fluid were tested to identify the known mutation existed in the family. Two of four fetuses predicted to be normal with the KRT9wild type, while two were found to be mutant.Conclusions:(1) With our knowledge, this study is the first research to apply WES technique into the causative genetic screening of MEN2A. InDel of exon2within RET gene lies at the upstream71bp region may affect the abnormal splicing of mRNA, thus it cannot produce the proper encoded amino acid sequences, which may influence the disease;(2) Precise knowledge of the KRT9mutation has facilitated DNA-based prenatal diagnosis in EPPK, which has proved to be of great benefit for individuals or couples at risk of having children with this disorder. Genetic heterogeneity may be affected by environmental factors, the nucleotide polymorphism site, or the unknown factors such as genetic modifications.