[Rapid screening for MTHFR gene 677C>T polymorphism in Down syndrome using high resolution melting curve and pyrosequencing].
OBJECTIVE
To establish a rapid method for detecting MTHFR gene 677C>T polymorphisms with high-resolution melting curve method (HRM) and pyrosequencing.


METHODS
Peripheral blood samples were collected from 155 Down syndrome patients and 182 normal controls from Children's Hospital of Shanghai. The accuracy of three methods including regular HRM, internal control HRM and artificial heterozygosity HRM was compared. Meanwhile, allele frequencies in 10, 30 and 50 mixed samples were measured with pyrosequencing, and the results were compared with that of HRM.


RESULTS
Heterozygosity of 677C>T polymorphism could be distinguished by various HRM methods. However, homozygotes CC and TT were only identifiable by internal control HRM and artificial heterozygosity HRM. The accuracy of pyrosequencing for allele frequency has improved with increased sample number. When the number of mixed samples has exceeded 30, the difference between pyrosequencing results and actual values became less than 4%. TT genotype was more frequent in Down syndrome patients than controls (25.2% vs. 14.3%). No significant difference was found in T allele frequency between the two groups (44.9% vs. 40.1%).


CONCLUSIONS
Respectively, internal control HRM and pyrosequencing may be ideal methods for determination of genotypic and allelic frequencies.
Department of Neonatolgy, Children's Hospital of Shanghai, Affiliated Children Hospital of Shanghai Jiaotong University, Shanghai 200040, P. R. China. Email: xiaohui_gong@hotmail.com.
Jing-jing
JJ
Sun
Yun-lin
YL
Shen
Chong-bing
CB
Yan
Yi-huan
YH
Chen
Xiao-hui
XH
Gong
D002648:Child; D002675:Child, Preschool; D004314:Down Syndrome / Q000175:diagnosis / Q000201:enzymology / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics*; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D044366:Transition Temperature
C573423:MTHFR protein, human; D042965:Methylenetetrahydrofolate Reductase (NADPH2)