[Mutation analysis of the eda-A1 gene for hypohidrotic ectodermal dysplasia and construction of recombined eukaryotic expression vector].
OBJECTIVE
The purpose of this study was to clone and analyze mutation in the eda-A1 gene for hypohidrotic ectodermal dysplasia (HED), and to construct a new recombined eukaryotic expression vector (mutant M, wild W) as a basis for further study on the genetic function.


METHODS
After total mRNA was extracted from peripheral blood lymphocytes from the HED affect patient and control, eda-A1 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) with a pair of specific primers containing the constriction enzyme sites of BamH I and Hind III. When the vector pcDNA3.1(-) and eda-A1 (M/W) were digested by BamH I and Hind III respectively, eda-A1 (M/W) fragment was then ligated to vector pcDNA3.1 (-) and the new vector was named as pcDNA3.1 (-)-eda-A1-M/W.


RESULTS
eda-A1 gene was successfully cloned and a novel missence mutation was identified, which changes the codon 306 from glutamine to proline. PCR, restrictive endonuclease analysis and DNA sequencing were then performed to identify the recombinant eukaryotic expression vector pcDNA3.1 (-)-eda-A1-M/W, and the results were surely confirmed.


CONCLUSIONS
Our result indicates that the novel missense mutation in eda is associated with the isolated tooth agenesis and provide preliminary explanation for the abnormal clinical phenotype at a molecular structural level. And also, the recombinant eukaryotic expression vector pcDNA3.1 (-)-eda-A1-M/W was successfully constructed, which will be thereafter taken use of further study on eda gene in odontogenesis.
Lei
Ke
K
Dept. of Prosthodontics, School of Stomatology, Lanzhou University, Lanzhou 730000, China.
Che
Tuan-Jie
TJ
Wang
Jin-Ming
JM
Deng
Ni
N
Zhang
Lin
L
He
Xiang-Yi
XY
D053358:Ectodermal Dysplasia 1, Anhidrotic; D005822:Genetic Vectors; D006801:Humans; D009154:Mutation; D009805:Odontogenesis; D012333:RNA, Messenger; D017422:Sequence Analysis, DNA
D012333:RNA, Messenger