Improved real-time multiplex polymerase chain reaction detection of methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C polymorphisms using nearest neighbor model-based probe design.
ABSTRACT: The disorders of folate metabolism caused by methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms may lead to several disease states including coronary heart disease, venous thrombosis, and several types of cancer. We have developed a real-time multiplex single-tube polymerase chain reaction procedure on the LightCycler for the detection of the two most commonly occurring variants, 677C>T and 1298A>C, in the MTHFR gene. An improved probe design, based on the nearest neighbor model for nucleic acid-probe duplex stability, resulted in a better separation (DeltaTm approximately 10 degrees C) of melting peaks of the wild-type and mutant alleles than that by the existing method (DeltaTm approximately 3 degrees C) for specimens heterozygous for the 1298A>C polymorphism. Of the 333 blood specimens analyzed by this procedure, we did not find any samples that gave ambiguous results. The specimens with homozygous mutation for one polymorphism were of the wild type for the other variant. The assay was validated by the comparison of the genotyping results of 50 blood specimens from the LightCycler polymerase chain reaction with the conventional restriction fragment length polymorphism procedures. There was 100% concordance of the test results obtained by the two techniques. This assay is reliable, economical, and can be performed by less trained technologists compared with the procedure performed by the conventional restriction fragment length polymorphism technique.
Project description:Clinical assessment and prognostic stratification of primary varicose veins have remained controversial and the molecular pathogenesis is unknown. Previous data have suggested a contribution of the MTHFR (methylenetetrahydrofolate reductase) polymorphism c.677C>T.We collected blood and vein specimens from 159 consecutive patients undergoing varicose vein surgery, or autologous vein reconstruction for arterial occlusive disease as controls. We compared the frequencies of c.677C>T and another polymorphism of MTHFR, c.1298A>C, with morphology and types of complicated disease. Morphology was recorded as a trunk or perforator type and peripheral congestive complication was defined as chronic venous insufficiency (CEAP C3-6) associated with edema and skin manifestations.Multivariate analysis of genotypes for c.677C>T and c.1298A>C indicated that c.677C>T was associated significantly with the trunk phenotype (43/53 patients, 81%, p < 0.01), while c.1298A>C was associated significantly with the perforator phenotype (18/24 patients, 75%, p < 0.01) of primary varicose veins. Accordingly, when both c.677C>T and c.1298A>C displayed a heterozygous genotype, the patients were more likely to present with both phenotypes. Additionally, c.1298A>C was found to be strongly linked to the congestive complication (34/51 patients, 67%, p < 0.01).Both polymorphisms of MTHFR may be involved in the morphological specification of primary varicose veins and contribute to the development of complicated disease.None.
Project description:BACKGROUND: Non-syndromic cleft lip with or without cleft palate (nsCL/P) is one of the most common congenital abnormalities of the orofacial region with a multifactorial etiology. The present study aimed to investigate the association of two common polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene (c.677C>T and c.1298A>C) with the occurrence of nsCL/P in an Iranian population. METHODS: Forty-five nsCL/P patients, 43 mothers of patients, and 101 unrelated controls participated in the present study. Analysis of c.677C>T and c.1298A>C polymorphisms in MTHFR gene was conducted using polymerase chain reaction and restriction enzyme digestions. RESULTS: There was no statistical difference in genotype and allele frequencies for c.677C>T variants between patients or their mothers and the control group. However, differences in the frequencies of alleles and genotypes of c.1298A>C polymorphism were statistically significant between patients and control group (P=0.01 for alleles and P=0.005 for genotypes). The odds ratios (OR) for the CC versus AA homozygotes were 6.1 (95% CI 1.8-20.5) and 4.2 (95% CI 1.1-15.4), in patients and mothers, respectively. CONCLUSIONS: We found no association between genetic polymorphism of MTHFR c.677C>T and the risk of nsCL/P in the population studied. Yet the results suggested that c.1298A>C polymorphism of MTHFR gene may be a risk factor for the occurrence of nsCL/P in the Iranian population.
Project description:Background:The gene for 5,10-methylenetetrahydrofolate reductase (NAD(P)H) or MTHFR gene encodes protein methylenetetrahydrofolate reductase (MTHFR), an enzyme important in folate metabolism. Aim:The aim of this study was to determine the frequencies of 677C>T and 1298A>C polymorphisms in the MTHFR gene of healthy subjects from the population. Material and methods:The blood samples were collected from 164 unrelated and healthy donors from population consisted of 98 females and 66 males. Both the MTHFR 677C>T and 1298A>C single nucleotide polymorphisms (SNPs) were analyzed by Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Linkage disequilibrium (LD) between pair of SNPs was calculated through Haploview analysis. Results:The frequency of MTHFR 677T allele in the population (32.62%) was in agreement with the frequency of this allele in most other populations, however, the frequency of MTHFR 1298C allele (38.41%) was higher than that reported for most other populations in the world. Haploview analysis showed a relatively strong LD between 677C>T and 1298A>C SNPs with D' values of 0.87. Conclusion:Regarding the two MTHFR polymorphisms, three of the nine combined genotypes were present in 87.2% of the population. 33.54% subjects were complex heterozygous (677CT/1298AC genotype), 34.15% subjects had 677CC/1298AC and 19.51% of 677CT/1298AA genotype. The subjects with 677TT genotype had a 1298AA or 1298AC genotype while subjects with 1298CC genotype had only 677CC genotype. The subjects with 677CC/1298AA genotype were only 3.05%. We were not found triple 677CT/1298CC and quadruple 677TT/1298CC mutation suggesting decreased viability of embryos with increased numbers of mutant alleles.
Project description:BACKGROUND:Adverse pregnancy outcomes (APOs) are involved in a series of abnormal pregnancies like embryo growth arrest, spontaneous abortion, premature birth, stillbirth, fetal malformation, birth defects and other pathological pregnancy, and childbirth complications. Polymorphism of methylenetetrahydrofolate reductase gene (MTHFR, 607093) is one of the main genetic causes of APO. However, there is still debate on whether MTHFR 1298A>C, rs1801131, polymorphism is related to APO. For the lack of extensive research in the Chinese population at present, the study aim to investigate the relationship between MTHFR 1298A>C polymorphism with APO through a large number of data. METHODS:A total of 241 samples from patients with APO and 117 healthy controls in Yunnan province were used for MTHFR gene polymorphism analysis, with double fluorescence quantitative polymerase chain reaction (PCR). In consideration of the low frequency of MTHFR 1298C/C genotype, which might affect the statistic results, further datasets of MTHFR 1298A>C polymorphism were collected from literature and analyzed. RESULTS:No statistical difference was detected in the frequency of MTHFR1298A>C polymorphism between two groups in Yunnan. Our data showed that the frequency of MTHFR 1298A/A genotype had the decreasing tendency among Chinese population from northern to southern, as well as eastern to western of China. The frequency of MTHFR 1298A/C and 1298C/C genotypes had the adverse tendency. The frequency of MTHFR 1298C/C genotype was significantly different between two groups in Chinese populations. The significant difference was also observed in the frequency of MTHFR 1298C/C polymorphism between two groups from central China and southern China. CONCLUSION:In summary, our data showed that MTHFR 1298C/C genotype was one of the important genetic factors of APO in China.
Project description:Aim: Variants of the MTHFR gene have been associated with a wide range of diseases. Materials & methods: The present study analyzed data from clinical genotyping of MTHFR 677C>T and 1298A>C in 1405 patients in urban primary care settings. Results: Striking differences in ethnogeographic frequencies of MTHFR polymorphisms were observed. African-Americans appear to be protected from MTHFR deficiency. Hispanics and Caucasians may be at elevated risk due to increased frequencies of 677C>T and 1298A>C, respectively. Conclusion: Individuals carrying mutations for both genes were rare and doubly homozygous mutants were absent, suggesting the TTcc is extremely rare in the greater population. The results suggest multilocus MTHFR genotyping may yield deeper insight into the ethnogeographic association between MTHFR variants and disease.
Project description:BACKGROUND: The polymorphism 5,10-methylenetetrahydrofolate reductase (MTHFR) c.1298A>C is associated with various diseases. 45 DNA samples homozygous for the A allele and 40 DNA probes homozygous for the C allele were taken from healthy German subjects of white Caucasian origin to analyze the haplotype of the two MTHFR c.1298A>C alleles. Samples were genotyped for the polymorphism MTHFR c.677C>T and for the silent polymorphisms MTHFR c.129C>T, IVS2 533 G>A, c.1068C>T and IVS10 262C>G. FINDINGS: Haplotype construction revealed that the C-allele of MTHFR c.1298A>C was more frequently observed in cis with c.129T, IVS2 533A, c.677C, c.1068T, and IVS10 262 G than expected from normal distribution. Estimation of the most recent common ancestor with the DMLE + 2.3 program resulted in an estimated age of approximately 36,660 years of the MTHFR c.1298C allele. CONCLUSION: Given that the era from 30,000 to 40,000 years ago is characterised by the spread of modern humans in Europe and that the prevalence of the MTHFR c.1298C allele is significantly higher in Central Europe in comparison to African populations, a selective advantage of MTHFR c.1298C could be assumed, e. g. by adaption to changes in the nutritional environment. The known founder ancestry of the T allele of MTHFR c.677C>T allele, together with the present data suggests that the MTHFR mutant alleles c.677T and 1298C arose from two independent ancestral alleles, that both confer a selective advantage.
Project description:Congenital thrombophilia is associated with an increased intraventricular hemorrhage (IVH) risk among newborns, but it may also play a protective role. The role of genetic polymorphisms involved in the coagulation pathway of IVH pathogenesis is probably a consequence of an increased risk of thrombosis in the fine blood vessels in the germinal matrix region.The aim of this study was to evaluate the possible relationship between Factor V (FV) 1691G>A, Factor II (FII) 20210G>A mutations and methylenetetrahydrofolate reductase (MTHFR) 677C>T; 1298A>C and Factor XIII (FXIII) 103G>T gene polymorphisms and the occurrence of IVH in 100 infants born from 24 + 0 to 32 + 0 weeks of gestation, born from singleton pregnancy, before 32 + 0 weeks of gestation, exposed to antenatal steroid therapy, and without congenital abnormalities.IVH developed 45 (45%) infants, including 15 (33.33%) diagnosed with IVH stage I, 20 (42.22%) with stage II, 8 (17.77%) with stage III, and 3 (6.66%) with stage IV. Analysis showed a prevalence 4.5 times higher of IVH stages II to IV in infants with the genotype CC (OR 4511 (1147-17.75); p = 0.026) of MTHFR 1298A>C gene polymorphism. Our investigation did not confirm any significant prevalence of IVH development in other studied mutations/polymorphisms.This study confirmed that the MTHFR 1298A>C polymorphism is associated with the risk of IVH. IVH is a significant problem for preterm infants. In addition to little progress in preventing IVH in preterm babies, substantial research that is focused on understanding the etiology, mechanism, and risk factors for IVH is imperative. In the era of personalized medicine, identification of genetic risk factors creates opportunities to generate preventative strategies.
Project description:Background:5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in one-carbon metabolism that ensures the availability of methyl groups for methylation reactions. Two single-nucleotide polymorphisms (SNPs) in the MTHFR gene, 677C>T and 1298A>C, result in a thermolabile enzyme with reduced function. These variants, in both the maternal and/or fetal genes, have been associated with pregnancy complications including miscarriage, neural tube defects (NTDs), and preeclampsia (PE), perhaps due to altered capacity for DNA methylation (DNAm). In this study, we assessed the association between MTHFR 677TT and 1298CC genotypes and risk of NTDs, PE, or normotensive intrauterine growth restriction (nIUGR). Additionally, we assessed whether these high-risk genotypes are associated with altered DNAm in the placenta. Results:In 303 placentas screened for this study, we observed no significant association between the occurrence of NTDs (N?=?55), PE (early-onset: N?=?28, late-onset: N?=?20), or nIUGR (N?=?21) and placental (fetal) MTHFR 677TT or 1298CC genotypes compared to healthy pregnancies (N?=?179), though a trend of increased 677TT genotype in PE/IUGR together was observed (OR 2.53, p?=?0.048). DNAm was profiled in 10 high-risk 677 (677TT + 1298AA), 10 high-risk 1298 (677CC + 1298CC), and 10 reference (677CC + 1298AA) genotype placentas. Linear modeling identified no significantly differentially methylated sites between high-risk 677 or 1298 and reference placentas at a false discovery rate <?0.05 and ?? ??0.05 using the Illumina Infinium HumanMethylation450 BeadChip. Using a differentially methylated region analysis or separating cytosine-guanine dinucleotides (CpGs) by CpG density to reduce multiple comparisons also did not identify differential methylation. Additionally, there was no consistent evidence for altered methylation of repetitive DNA between high-risk and reference placentas. Conclusions:We conclude that large-scale, genome-wide disruption in DNAm does not occur in placentas with the high-risk MTHFR 677TT or 1298CC genotypes. Furthermore, there was no evidence for an association of the 1298CC genotype and only a tendency to higher 677TT in pregnancy complications of PE/IUGR. This may be due to small sample sizes or folate repletion in our Canadian population attenuating effects of the high-risk MTHFR variants. However, given our results and the conflicting results in the literature, investigations into alternative mechanisms that may explain the link between MTHFR variants and pregnancy complications, or in populations at risk of folate deficiencies, are warranted.
Project description:Congenital heart defect (CHD) is one of the most common birth defects in the world. The methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes are two of the most important candidate genes for fetal CHD. However, the correlations between the two genes and fetal CHD were inconsistent in various reports. Therefore, this study is aimed to evaluate the parental effects of the two genes on fetal CHD via three genetic polymorphisms, MTHFR 677C>T (rs1801133), MTHFR 1298 A>C (rs1801131), and MTRR 66A>G (rs1801394).Parents with pregnancy history of fetal CHD were divided into two subgroups: ventricular septal defect (VSD) (21) and non-VSD groups (78). VSD, non-VSD, and 114 control parents (controls) were analyzed in this study. Genotyping of these genetic polymorphisms was done by sequencing.The MTHFR 677C>T polymorphism of either mothers or fathers was independently associated with fetal non-VSD (P < 0.05) but not VSD, while the MTRR 66A>G polymorphism was independently associated with fetal VSD (P < 0.05) but not non-VSD. No significance was found for MTHFR 1298A>C polymorphism.In either maternal or paternal group, the MTHFR 677C>T polymorphism was independently related to fetal non-VSD, while the MTRR 66A>G polymorphism was independently related to fetal VSD.
Project description:The study was conducted to investigate potential association between MTHFR genotypes and diabetic peripheral neuropathy (DPN) in Puerto Ricans with type-2 diabetes mellitus (T2DM) treated with metformin. The prevalence of major MTHFR polymorphisms in this cohort was also ascertained.DNAs from 89 metformin-treated patients with T2DM and DPN were genotyped using the PCR-based RFLP assay for MTHFR677C>T and 1298A>C polymorphisms. Frequency distributions of these variants in the study cohort were compared to those reported for three reference populations (HapMap project) and controls (400 newborn specimens). Chi-square (or Fischer's exact) tests and odds ratios (OR) were used to assess association with DPN susceptibility risk (patients vs. controls) and biochemical markers (wild types vs. carriers).Sixty-seven percent (67%) of participants carry at least one of these MTHFR polymorphisms. No deviations from Hardy-Weinberg equilibrium were detected. The genotype and allele frequencies showed statistically significant differences between participants and controls (p<0.0001 and p=0.03, respectively). Results suggest that 1298A>C but not 677C>T is associated with DPN susceptibility in this cohort (p=0.018). Different patterns of allelic dissimilarities are observed when comparing our cohort vs. the three parental ancestries. After sorting individuals by their carrier status, no significant associations were observed between these genetic variants (independently or combined) and any of the biochemical markers (HbA1c, folate, vitamin B12, homocysteine).Prevalence of major MTHFR variants in Puerto Rican patients with T2DM is first time ever reported. The study provides further evidence on the use of this genetic marker as an independent risk factor for DPN.