[Application of PCR-DGGE technique in G-6-PD deficiency].
OBJECTIVE
To detect gene mutations of children with glucose-6-phosphorate dehydrogenase (G-6-PD) deficiency and of carriers of G-6-PD deficiency gene with the technique of polymerase chain reaction and denatured gradient gel electrophoresis (PCR-DGGE), and to explore the value of the technique in the diagnosis of G-6-PD deficiency and G-6-PD deficiency gene carrying.


METHODS
cDNAs were harvested by reverse transcription method after RNAs had been extracted from peripheral blood of 43 children with G-6-PD deficiency and of their family members (36 lineages). Electrophoresis behaviors of the fragment from exons 11-12 of G-6-PD cDNA were detected with the technique of PCR-DGGE. Gene sequencing was then performed for the abnormal electrophoresis bands.


RESULTS
Abnormal electrophoresis bands were found in the 1304-1520 fragment of G-6-PD cDNA in 33 out of 36 family lineages. The G-6-PD/6-PGD ratio was below 1.00 in 9 mothers of patients. Three of them had the G-6-PD/6-PGD ratio lower than 0.50. The PCR-DGGE bands were the same in the 3 mothers. Gene sequencing showed double heterozygote in the 3 mothers, but the maternal carriers of G-6-PD deficiency gene who had normal G-6-PD/6-PGD ratio showed mono-heterozygote in gene sequencing. Three mutational sites were found in the 1304-1520 fragment, i.e., C1311TG1376T and G1388A. The electrophoresis behaviors were different among the 3 gene mutational sites.


CONCLUSIONS
PCR-DGGE is a sensitive and reliable technique in the screening of gene mutations. It is useful in the diagnosis of G-6-PD deficiency, especially in the diagnosis of female G-6-PD deficiency gene carrying.
Li
Chang-Gan
CG
Children's Hospital of Shenzhen, Shenzhen, Guangdong, China. licg6336@sina.com
Chen
Xiao-Wen
XW
Chen
Yun-Sheng
YS
Wang
Ying
Y
Zhao
Wei-Ling
WL
Shi
Hong-Song
HS
Li
Cheng-Rong
CR
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005955:Glucosephosphate Dehydrogenase Deficiency / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA