Evaluation of High Resolution Melting for MTHFR C677T Genotyping in Congenital Heart Disease.

Ying Wang,Yi Liu,Shuying Yue,Hui Wang,Kun Zhang,Haiyan Zhang,Yanhui Ma,Xiaomeng Yang,Rui Dong,Wolfgang Arthofer

doi:10.1371/journal.pone.0151140

Abstract

BackgroundHigh resolution melting (HRM) is a simple, flexible and low-cost mutation screening technique. The methylenetetrahydrofolate reductase (MTHFR) gene encoding a critical enzyme, potentially affects susceptibility to some congenital defects like congenital heart disease (CHD). We evaluate the performance of HRM for genotyping of the MTHFR gene C677T locus in CHD cases and healthy controls of Chinese Han population.MethodsA total of 315 blood samples from 147 CHD patients (male72, female 75) and 168 healthy controls (male 92, female 76) were enrolled in the study. HRM was utilized to genotype MTHFR C677T locus of all the samples. The results were compared to that of PCR-RFLP and Sanger sequencing. The association of the MTHFR C677T genotypes and the risk of CHD was analyzed using odds ratio with their 95% confidence interval (CIs) from unconditional logistic regression.ResultsAll the samples were successfully genotyped by HRM within 1 hour and 30 minutes while at least 6 hours were needed for PCR-RFLP and sequencing. The genotypes of MTHFR C677T CC, CT, and TT were 9.52%, 49.66%, and 40.82% in CHD group but 29.17%, 50% and 20.83% in control group, which were identical using both methods of HRM and PCR-RFLP, demonstrating the sensitivity and specificity of HRM were all 100%.ConclusionMTHFR C677T is a potential risk factor for CHD in our local residents of Shandong province in China. HRM is a fast, sensitive, specific and reliable method for clinical application of genotyping.

Highlights

Congenital heart disease (CHD) is the most common defects with 1% prevalence worldwide and leading non-infectious cause of morbidity and mortality in infant [1]
We evaluate the performance of High resolution melting (HRM) for genotyping of the methylenetetrahydrofolate reductase (MTHFR) gene C677T locus in congenital heart disease (CHD) cases and healthy controls of Chinese Han population
All the samples were successfully genotyped by HRM within 1 hour and 30 minutes while at least 6 hours were needed for PCR-RFLP and sequencing

Summary

Introduction

Congenital heart disease (CHD) is the most common defects with 1% prevalence worldwide and leading non-infectious cause of morbidity and mortality in infant [1]. The MTHFR gene rs1801131 (C677T) has been identified as a risk factor for CHD in Asian, especially in Chinese Han population by using PCR-RFLP or PCRsequencing [11,12,13,14,15,16]. High resolution melting (HRM) is a simple, rapid, flexible, sensitive, specific and low-cost mutation screening technique without post-PCR processing, has been strongly suggested as a standard approach for mutation scanning in clinical diagnosis [17,18]. The methylenetetrahydrofolate reductase (MTHFR) gene encoding a critical enzyme, potentially affects susceptibility to some congenital defects like congenital heart disease (CHD). We evaluate the performance of HRM for genotyping of the MTHFR gene C677T locus in CHD cases and healthy controls of Chinese Han population

Methods

Results

Conclusion