[Whole blood allele-specific PCR, a simple method to detect four ATP7B gene mutations in Wilson disease].
OBJECTIVE
To establish a simple method to detect four ATP7B gene mutations in Wilson disease using allele-specific PCR(AS-PCR) with whole blood polymerase chain reaction.


METHODS
Four allele-specific PCR primers specific for the mutations(G2333T, C2850T, G2855A, G2975T) were designed, and PCR was optimized to screen the whole blood samples. The amplified gene products with mutation were separated with agarose gel electrophoresis to detect the pattern of point mutation and allele types. Exons 8, 12 and 13 of the ATP7B gene were amplified with PCR, and the amplification products were sequenced to confirm the mutation.


RESULTS
The detection of four ATP7B gene mutations by AS-PCR with whole blood was accomplished with 100% accuracy. In the 27 healthy subjects, the mutation rate of G2855A was 51.8%. No mutation was detected for G2333T, C2850T and G2975T. Among the 22 patients, 11 were mutated for G2333T, C2850T or G2975T. The mutation rate was therefore 50%.


CONCLUSIONS
Our experiment has established an AS-PCR based method for detecting four ATP7B gene mutations using whole blood samples, which has provided a simple and effective means for the early diagnosis of Wilson disease. This method is rapid, convenient, accurate and economical for detecting point mutations of the ATP7B gene.
Department of Pharmacology, Suzhou Key Laboratory of Aging and Nervous Diseases, School of Pharmaceutical Science, Soochow University, Suzhou, Jiangsu 215123, P. R. China. zhqin5@mail.com.
Wei
W
Sun
Junjie
J
Guan
Jin
J
Wang
Zhenghong
Z
Qin
D000251:Adenosine Triphosphatases / Q000235:genetics*; D000293:Adolescent; D000328:Adult; D000483:Alleles; D027682:Cation Transport Proteins / Q000235:genetics*; D002648:Child; D000073840:Copper-Transporting ATPases; D005260:Female; D006527:Hepatolenticular Degeneration / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
D027682:Cation Transport Proteins; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases