title: A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays.
abstract: BACKGROUND
  Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS -786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS -786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS -786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS -786 T > C were calculated using the Hardy Weinberg equation.
  
  
  METHODS
  The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing.
  
  
  RESULTS
  The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS -786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele).
  
  
  CONCLUSIONS
  Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS -786 T > C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS -786 T > C variants.
authors.affiliation: Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. wynnelkw@gmail.com
authors.forename: Keat Wei
authors.initials: KW
authors.lastname: Loo
authors.forename: Lyn Robyn
authors.initials: LR
authors.lastname: Griffiths
authors.forename: Siew Hua
authors.initials: SH
authors.lastname: Gan
mesh_terms: D004587:Electrophoresis, Agar Gel; D005787:Gene Frequency; D005828:Genetics, Population; D006801:Humans; D008296:Malaysia; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide
chemical_list: D052250:Nitric Oxide Synthase Type III; D042965:Methylenetetrahydrofolate Reductase (NADPH2)