[Rapid detection of common ATP7B mutations in Wilson disease by high resolution melting analysis].
OBJECTIVE
To detect the most prevalent mutations, R778L and P992L of ATP8B gene, in Chinese Wilson disease(WD) patients by high resolution melting (HRM) analysis after polymerase chain reaction (PCR).


METHODS
Genomic DNA was extracted from peripheral blood samples obtained from 30 cases of WD by the standard phenol/chloroform method. DNA fragments encompassing ATP7B exons 8 and 13 were produced by PCR amplification. The amplicons containing the R778L or P992L mutations were then generated by nested PCR. The nested PCR products were subjected to HRM analysis using the HR-1 instrument. Mutations detected in HRM analysis were verified by restriction analysis using restriction enzyme (MspI or AluI or AfaI) or DNA sequencing.


RESULTS
HRM analysis of the fragments encompassing ATP7B exon 8 showed four curve patterns. Subsequent restriction analysis and DNA sequencing proved that the four different curves represent four different genotypes: the wild type, the R778L/R778L homozygote, the R778L heterozygote, and the R778L/752.33delG compound heterozygote. Three HRM curve patterns were observed for the fragments encompassing ATP7B exon 13, representing the wild type, the P992L heterozygote, and the P992L/S975Y compound heterozygote. In our studied samples, allele frequencies of the R778L, P992L and S975Y mutations were 25%, 15% and 1.67%, respectively.


CONCLUSIONS
HRM analysis is a simple, accurate and sensitive approach for rapid detection of the ATP7B mutations and could be used as an optimized method for genetic testing in WD.
McKusick-Zhang Center for Genetic Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, P.R. China.
Xiuli
X
Zhao
Yanshan
Y
Liu
Shangzhi
S
Huang
Yan
Y
Meng
Miao
M
Sun
Wei
W
Yang
Xue
X
Zhang
D000251:Adenosine Triphosphatases / Q000235:genetics*; D001483:Base Sequence; D027682:Cation Transport Proteins / Q000235:genetics*; D000073840:Copper-Transporting ATPases; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D005091:Exons / Q000235:genetics; D005615:Freezing; D005787:Gene Frequency; D005838:Genotype; D006527:Hepatolenticular Degeneration / Q000235:genetics*; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D013997:Time Factors
D027682:Cation Transport Proteins; D004247:DNA; D004262:DNA Restriction Enzymes; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases