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.
METHODS: A systematic review was performed for all the articles retrieved from multiple databases, up until March 2017. Data were extracted from all eligible studies, and meta-analysis was carried out using RevMan 5.3 and R package 3.2.1. The strength of association between each studied polymorphism and ischemic stroke risk was measured as odds ratios (ORs) and 95% confidence intervals (CIs), under fixed- and random-effect models.
RESULTS: A total of 79 studies reporting on the association between the studied polymorphisms and ischemic stroke risk were identified. The pooled data indicated that all genetic models of APOA5 rs662799 (ORs = 1.23-1.43), allelic and over-dominant models of APOA5 rs3135506 (ORs = 1.77-1.97), APOB rs1801701 (ORs = 1.72-2.13) and APOB rs1042031 (ORs = 1.66-1.88) as well as dominant model of ABCA1 rs2230806 (OR = 1.31) were significantly associated with higher risk of ischemic stroke. However, no significant associations were observed between ischemic stroke and the other five polymorphisms, namely ApoB (rs693) and APOC3 (rs4520, rs5128, rs2854116 and rs2854117), under any genetic model.
CONCLUSIONS: The present meta-analysis confirmed a significant association of APOA5 rs662799 CC, APOA5 rs3135506 CG, APOB rs1801701 GA, APOB rs1042031 GA and ABCA1 rs2230806 GG with increased risk of ischemic stroke.
METHODS: Polymerase chain reaction primers were designed and validated to specifically amplify the cytosine that is followed by guanine residues (CpGs) A and B regions. Prior epigenotyping on 110 Kelantanese Malays, the serial pyrosequencing assays for the CpGs A and B regions were validated using five validation controls. The mean values of the DNA methylation profiles of CpGs A and B were calculated.
RESULTS: The mean DNA methylation levels for CpGs A and B were 0.984 ± 0.582 and 2.456 ± 1.406, respectively. The CpGs 8 and 20 showed the highest (5.581 ± 4.497) and the lowest (0.414 ± 2.814) levels of DNA methylation at a single-base resolution.
CONCLUSION: We have successfully developed and validated a pyrosequencing assay that is fast and can yield high-quality pyrograms for DNA methylation analysis and is therefore applicable to high throughput study. Using this newly developed pyrosequencing assay, the MTHFR DNA methylation profiles of 110 Kelantanese Malays were successfully determined. It also validated our computational epigenetic research on MTHFR.
METHODS: All case-control studies published in different languages such as English, Japanese, Korean, Spanish, Chinese, Hungarian, Ukrainian, or Russian were identified from databases. The pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated via fixed- and random-effect models. Sensitivity analysis, heterogeneity test, Hardy Weinberg Equilibrium, and Egger's regression analyses were performed in this study.
RESULTS: A total of 490 case-control studies with 138,592 cases and 159,314 controls were included in this meta-analysis. Pooled ORs from all the genetic models indicated that MTHFR 677TT and 1298CC, eNOS +894TT and VNTR, PDE4D SNP 83, ACE DD, AGT 235TT, PON1 192RR, and ApoE ε4 polymorphisms were increasing the risks of ischemic stroke. Nevertheless, PDE4D SNP 87 and eNOS -786T>C polymorphisms are not associated with ischemic stroke risks.
CONCLUSIONS: Hence, the evidence from this meta-analysis concluded that MTHFR (677C>T and 1298A>C), eNOS (+894G>T and VNTR), PDE4D SNP 83, ACE I/D, AGT 235M>T, PON1 192Q>R, and ApoE ε2ε3ε4 polymorphisms predispose individuals to ischemic stroke.
MATERIALS AND METHODS: Study subjects including 216 ischemic stroke patients and 203 healthy controls were recruited upon obtaining ethical clearance. SNP genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism assays. Gene expression levels were quantified by real-time polymerase chain reaction assays. Statistical and genetic analyses were conducted with SPSS version 22.2, PLINK version 1.07 and multifactor dimensionality reduction software.
RESULTS: Study subjects with G allele, CG or GG genotypes of SLC17A3 rs9379800 demonstrated increased risk of ischemic stroke with the odds ratios ranging from 1.76-fold to 3.14-fold (p<0.05). When stratified study subjects according to the ethnicity, SLC17A3 rs9379800 G allele and CG genotype contributed to 2.14- and 2.96-fold of ischemic stroke risk among Malay population significantly, in the multivariate analysis (p<0.05). However, no significant associations were observed for PITX2, NINJ2, TWIST1, Rasip1, and MUT polymorphisms with ischemic stroke risk in the multivariate analysis for the pooled cases and controls as well as when stratified them according to the ethnicity. Lower mRNA expression levels of Rasip1, SLC17A3, MUT and FERD3L were observed among cases (p<0.05). After FDR adjustment, the mRNA level of SLC17A3 remained significantly associated with ischemic stroke among Malay population (q=0.034).
CONCLUSION: In conclusion, this study suggests that SLC17A3 rs9379800 polymorphism and its gene expression contribute to significant ischemic stroke risk among Malaysian population, particularly the Malay who resided at the Northern Region of the country. Our findings can provide useful information for the future diagnosis, management and treatment of ischemic stroke patients.