Mutation Analysis Of Eda Gene For Hypohidrotic Ectodermal Dysplasia And Initial Researchon Its Function

Objective:To clone and analyze mutation in the eda-A1 gene for hypohidrotic ectodermal dysplasia(HED), to construct a new recombined eukaryotic expression vector(mutant M,wild W) and transfect ECV cells,and to investigate its effects on cell cycle.Methods:After total mRNA was extracted from peripheral blood lymphocytes from the HED affect patient and the control,eda gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) with a pair of specific primers containing constriction enzyme sites of BamHâ… and Hindâ…¢.When vector pcDNA3.1(-) and the eda-A1(M/W) fragment were digested by BamHâ… and Hindâ…¢respectively,the liner eda-A1(M/W) fragment was then ligated to vector pcDNA3.1(-) and the new vector was named as pcDNA3.1(-)-eda-A1.And the recombined vector was sent out for DNA sequencing.Then cultured ECV cells were transiently transfected with the recombined vectors to over press eda gene,and the cell cycle was investigated by flow cytometry.Results:Eda gene was successfully cloned and a novel missence mutation(A907C) was identified,which changes the codon 306 from glutamine to proline;PCR,restrictive endonuclease analysis were then performed to identify the recombinant eukaryotic expression vector pcDNA3.1(-)-eda-A1, and the results were surely confirmed.Flow cytometry results showed that:in contrast with empty plasmid group(control group,C),more cells of wild group entered S phase,whereas a smaller number entered G₂/M phase(P＜0.01).And the mutant group had more cells in G₀/G₁ phase,but fewer in S phase and G₂/M phase(P＜0.01).In addition,compared with the wild group,the cells of mutant group in G₀/G₁ phase increased,but with a significant decrease in S phase(P＜0.01). Conclusion:Wild type of eda gene may have some impact on cell cycle,and it may be involved in process of DNA synthesis.As the proportion of G₀/G₁ phase cells increased,it is speculated that eda gene may have some role in cell proliferation.And the mutant and wild eda gene may have distinct features of molecular biology.This will be thereafter taken use of in further study on eda gene in odontogenesis.