MTHFR C677T Genotype Frequency in Patients of Middle Eastern Descent as Determined by Real-Time PCR and Melting Curve Analysis

摘要

The 5,10-methylenetetrahydrofolate reductase gene (MTHFR) 677C>T polymorphism produces an elevation in plasma homocysteine concentrations when present in the homozygous state. Increased homocysteine levels have been associated with a greater risk for vascular diseases, including cardiovascular disease and ischemic stroke. In this study, we genotyped 42 nucleic acid samples for the C677T allele from our database of Middle Eastern patients as routine validation of the MTHFR 677C>T assay. Our study is the first to evaluate MTHFR C677T genotype frequency in a population of Middle Eastern patients residing in the United States. Among the patients, 47.6% were wild type, 40.5% were heterozygous, and 11.9% were homozygous for the C677T variant. Although C677T genotype frequency in our patient population is slightly higher than that reported by Golbahar et al, (2005), statistical analysis showed no statistically significant difference beyond chance in genotype profiles (x~2 = l.54, df = 2, p =0.1675). However, our findings implicate the need for a larger sample size to explore the need to implement standard clinical screening of MTHFR 677C>T. We also highlight the robust, reliable, and reproducible assay afforded by the use of anchor and sensor hybridization probes within the LightCycler~® platform to perform amplification and melting curve analysis protocols. Melting curve profiles that are produced display distinct and robust T_m peaks based on the degree of anchor and sensor hybridization to amplicons produced from template DNA that is either wild-type, heterozygous, or a homozygous variant at the MTHFR 677C>T locus. A 10℃ gap between T_m peaks allows for rapid and accurate qualitative identification of genotype.