The Use Of High Resolution Melting Approach In The Screening Of MTHFR Gene Polymorphism In Hypertension Patients

Objective: Hyperhomocysteinemia is a medical condition characterized by an abnormally high level of homocysteine in the blood, conventionally described as above 10 μmol/L. Elevated homocysteine is a known risk factor for atherosclerosis and thrombosis. It has also been shown to be associated with peripheral vascular diseases,cerebrovascular disease, hypertension,hypertensive heart disease, coronary artery occlusion and thrombosis.Methylene tetrahydrofolate reductase(MTHFR) is the rate-limiting enzyme in the methyl cycle, MTHFR catalyzes the conversion of5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate for homocysteine remethylation to methionine, this metabolic pathway is important to convert homocysteine(a potentially toxic amino acid) to methionine. There are genetic polymorphisms associated with this gene, the most investigated is C667 T, C at position 667(leading to an alanine at aminoacid 22) is the normal allele, the 667 T allele(leading to valine substitution at amino acid 222) encodes a thermolabile enzyme with reduced activity. This mutation may leads to decreased 5-methyltetrahydrofolate and increased homocysteine level, resulting in several illnesses including H-type hypertension. Hence, it is essential to screen MTHFR C667 T polymorphism in patients with hypertension.High Resolution Melt(HRM) analysis is a combined with saturated fluorescent dyes, no probe and real-time fluorescence quantification method,used to identify variations in nucleic acid sequences, the advantages of HRM include high throughput, low cost, simple, fast workflow, accuracy and real close-tube. All genetic analysis starts with the cllection of DNA samples,blood is the most common source, but with several disadvatages: it needs medical staff and medical equipment; it is painful invasive manipulation;increased possibility of infection; blood samples should be kept at low temperature. While, saliva DNA is non-invasive, user-friendly sample collection, especially useful for infants and ole patients, high quality DNA is suitable for sensitive downstream applications.Methods: 500 patients with hypertension and 500 normal individuals were collected in our vasculocardiology deparment during 2014.01 to 2014.07,and each of them were collected 2 m L saliva samples and 200 ul blood samples,we used Saliva DNA purification kit to extracte DNA from saliva samples and whole blood DNA purification kit to extract DNA form blood samples. The DNA purity was evaluated by OD260/280.According to MTHFR gene sequence from Genbank, we used Primer5 to design HRM primers and sequencing primers of C667 T, then optimal reaction conditions were explored in Bio-Rad CFX Manager, the result of genotype was recorded. Besides, all DNA samples were sequenced by Sanger sequencing, and the accuracy and specificity was compared between those two methods.4 m L limosis vein blood were collected by EDTA anticoagulation tube,which used to detect Hcy and folic acid level, according to MTHFR C667 T genotype, 500 patients were divided into 3 groups(CC、CT、TT groups), the Hcy and folic acid level were compared between those three groups, as a result, we can preliminary discusse the relationship between MTHFR C667 T gene polymorphisms and Hcy level.Results:1) DNA purity of saliva and blood samples: 500 patients’ saliva samples and500 normal saliva samples were extracted by saliva DNA extraction kit(Wuhan Charm Biotech, China), the DNA purity was evaluated using OD260/280, the mean OD260/280 is 1.809(95%CI 1.791-1.827), and can meet the demand of HRM detection. 500 patients’ blood samples and 500 normal blood samples were extracted by blood DNA extraction kit(Shanghai Finegene, China), the DNA purity was evaluated using OD260/280, the mean OD260/280 is 1.823(95%CI 1.720-1.8254), which was quite similar to theresult of saliva samples.2) a High Resolution Melting(HRM) to screen for C667 T mutations of MTHFR: Primer sequences used for HRM analysis was set as follows:Forward TGAGGCTGACCTGAAGCA; Reverse TGTCAGCCTCAAAG AAAA. reaction system includes primers(0.8umol/L), 2×Eva Green premix10 ul,DNA 5ng, double distilled water 4.2ul. The PCR protocol was set as follows: Pre-denaturation at 95℃for 1 minutes, denaturation at 95℃ for 5seconds, annealing at 60℃ for 20 seconds for a total 40 cycles. HRM analysis of all samples was undertaken postrun by incubation at 50°C for 20 s followed by ramping from 65 to 95°C, with fluorescence data acquisition at 0.2°C increments every cycle.3) the results of established HRM and direct sequence analysis: Among 1000 samples, 270(27.0%), 425(42.5%)and 305(30.5%) were detected as CC, CT and TT genotypy, respectively. To assess the sensitivity and the specificity of established HRM method, all the 1000 samples were re-screened by direct sequencing, 268(26.8%), 426(42.6%)and 306(30.6%) were detected as CC,CT and TT genotypy, respectively, and the results indicated 99.8%concordance between DNA sequence analysis and HRM.4) The Hcy and folic acid level of patients: 500 patients were divided into CC,CT and TT groups, the Hcy levels in TT group is significantly higher than CC and CT groups(P<0.01), the trend of folic acid level is just the opposite.Conclusion:1 Saliva harbours a wide spectrum of genetic data that can be used for HRM method with the main advantage of non-invasion.2 Our results strongly prove that established HRM approach is a reliable,sensitive simplicity and low-cost method for screening C667 T variant of MTHFR.3 MTHFR C667 T genetic variant may be the predictors of Hcy level.