Genetic determinants of folate and vitamin B12 metabolism: a common pathway in neural tube defect and Down syndrome?

One-carbon metabolism is under the influence of folate, vitamin B12 and genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR 677 C --> T and 1298 A --> C), of methionine synthase (MTR 2756 C --> G), methionine synthase reductase (MTRR 66 A --> G) and transcobalamin (TCN 776 C --> G). The pathogenesis of neural tube defect (NTD) may be related to this metabolism. The influence of the MTHFR 677 C --> T polymorphism reported in The Netherlands and Ireland can be questioned in southern Italy, France and Great Britain. MTRR, combined with a low level of vitamin B12, increases the risk of NTD and of having a child with NTD in Canada, while TCN 776 GG and MTRR 66 GG mutated genotypes associated with the MTHFR 677 CC wild-type are predictors of NTD cases in Sicily. Down syndrome (DS) is due to a failure of normal chromosomal segregation during meiosis, possibly related to one-carbon metabolism. MTHFR 677 C --> T and MTRR 66 A --> G polymorphisms are associated with a greater risk of having a child with DS in North America, Ireland and The Netherlands. In contrast, MTHFR 677 C --> T has no influence on DS risk in France and Sicily, while homocysteine and MTR 2756 AG/GG genotypes are predictors of DS risk in Sicily. In conclusion, NTD and DS are influenced by the same genetic determinants of one-carbon metabolism. The distinct data produced in different geographical areas may be explained by differences in the nutritional environment and genetic characteristics of the populations.     

MTRR 66A>G polymorphism in relation to congenital heart defects.

BACKGROUND: Evidence is accumulating that periconceptional folic acid supplementation may prevent congenital heart defects (CHD). The methionine synthase reductase (MTRR) enzyme restores methionine synthase (MTR) enzyme activity and therefore plays an essential role in the folate- and vitamin B(12)-dependent remethylation of homocysteine to methionine. We studied the influence of the MTRR 66A>G polymorphism on CHD risk. In addition, possible interaction between this variant and plasma methylmalonic acid (MMA) concentrations, as an indicator of intracellular vitamin B(12) status, was investigated. METHODS: Case-control and case-parental studies were conducted to explore this association. In total, 169 CHD patients and 213 child controls, and 159 mothers with a CHD-affected child and 245 female controls were included. RESULTS: The maternal MTRR 66AG and GG vs. AA genotypes revealed an odds ratio (OR) of 1.3 (95% CI 0.72-2.20) and 1.3 (0.71-2.37), respectively. Family-based transmission disequilibrium analysis did not reveal a significant association of the foetal 66G allele with the development of a heart defect in children (chi(2)=2.94, p=0.086). Maternal 66GG genotype in combination with high MMA concentration (above the 80th percentile) was associated with a three-fold (OR 3.3, 95% CI 0.86-12.50) increased risk for all types of CHD in offspring. CONCLUSIONS: These data indicate that maternal MTRR 66A>G polymorphism is not a risk factor for CHD. Maternal MTRR 66GG genotype with compromised vitamin B(12) status may possibly result in increased CHD risk. In addition to folate, vitamin B(12) supplementation may contribute to the prevention of CHD.     

High-level multiplex genotyping of polymorphisms involved in folate or homocysteine metabolism by matrix-assisted laser desorption/ionization mass spectrometry

BACKGROUND: Increased plasma total homocysteine (tHcy), a risk factor for cardiovascular disease, is related to genetic, environmental, and nutritional factors, in particular folate status. Future large epidemiologic studies of the genetic basis of hyperhomocysteinemia will require high-throughput assays for polymorphisms of genes related to folate and Hcy metabolism. METHOD: We developed a high-level multiplex genotyping method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection of 12 polymorphisms in 8 genes involved in folate or Hcy metabolism. The assay includes methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C, methionine synthase (MTR) 2756A>G, methionine synthase reductase (MTRR) 66A>G, cystathionine beta-synthase (CBS) 844ins68 and 699C>T, transcobalamin II (TCII) 776C>G and 67A>G, reduced folate carrier-1 (RFC1) 80G>A, paraoxonase-1 (PON1) 575A>G and 163T>A, and betaine homocysteine methyltransferase (BHMT) 742G>A. RESULTS: The failure rate of the assay was < or = 1.7% and was attributable to unsuccessful DNA purification, nanoliter dispensing, and spectrum calibration. Most errors were related to identification of heterozygotes as homozygotes. The mean error rate was 0.26%, and error rates differed for the various single-nucleotide polymorphisms. Identification of CBS 844ins68 was carried out by a semiquantitative approach. The throughput of the MALDI-TOF MS assay was 1152 genotypes within 20 min. CONCLUSIONS: This high-level multiplex method is able to genotype 12 polymorphisms involved in folate or Hcy metabolism. The method is rapid and reproducible and could facilitate large-scale studies of the genetic basis of hyperhomocysteinemia and associated pathologies.     

Association between the 2756A> G variant in the gene encoding methionine synthase and myocardial infarction in Tunisian patients

BACKGROUND: Elevated plasma total homocysteine (tHcy), a risk factor for coronary artery disease (CAD), is due to defects in genes encoding for enzymes involved in tHcy metabolism or from inadequate status of vitamins involved in tHcy disposal. Methionine synthase (MS), a vitamin B(12)-dependent enzyme, catalyses the remethylation of homocysteine to methionine using a methyl group donated by 5-methyltetra-hydrofolate, which is the major circulating form of folate in the body. Functional genetic variants of the MS may alter tHcy as well as folate levels which are independent risk factors for CAD. The influence of a common genetic polymorphism 2756A>G of the MS gene (MTR) on plasma tHcy, folate and vitamin B(12) levels and its relation to the risk of myocardial infarction (MI) in a Tunisian case-control study was investigated. METHODS: A total of 321 Tunisian patients with MI and 343 healthy controls were included in the study. The 2756A>G variant of the MTR was determined by polymerase chain reaction-restriction fragment length polymorphism analysis. Plasma tHcy was assessed with a fluorescent polarising immunoassay method. Plasma vitamin B(12) and folate were determined by microparticular enzyme immunoassay and ion-capture, respectively. RESULTS: A significant difference in genotype distribution and allele frequency was observed between patients and controls. Patients with MI had a frequency of 1.9% for the GG genotype, 26.2% for the AG genotype and 72% for the AA genotype. Controls had a frequency of only 0.9% for the GG genotype, 18.7% for the AG genotype and 80.5% for the AA genotype (chi(2)=6.97, p=0.03). The MI patient group showed a significant higher frequency of the G allele compared to controls (0.149 vs. 0.101; OR 1.55; 95% CI 1.10-2.18; p=0.008). The association between the 2756A>G variant in the gene encoding MS and MI was no longer significant after adjustment for other well-established risk factors. When clinical and laboratory values were compared amongst genotypes in the study groups, no significant differences were noted. CONCLUSIONS: The present study showed a significant but not independent association between the 2756A>G polymorphism of the MTR (presence of G allele) and MI in the Tunisian population.     

影响亚甲基四氢叶酸还原酶和甲硫氨酸合成酶还原酶基因多态性的因素分析

目的了解影响女性亚甲基四氢叶酸还原酶(MTHFR)C677T、A1298C位点和甲硫氨酸合成酶还原酶(MTRR)A66G位点基因多态性的因素。方法采用横断面调查研究方法,以湖北省内1 902例女性为对象,采集口腔黏膜上皮细胞,提取DNA,用实时荧光定量PCR技术检测MTHFR和MTRR基因,统计分析不同民族MTHFR和MTRR基因多态性的差异,与其他地区人群的MTHFR和MTRR基因多态性进行比较,并分析不同位点基因多态性之间的关联关系。结果 (1)湖北省汉族女性和少数民族女性在MTHFR C677T和MTRR基因多态性构成差异无统计学意义(P>0.05),而MTHFR A1298C位点基因多态性构成差异有统计学意义(P<0.05)。(2)MTHFR C677T位点的纯合突变基因型TT的频率从南到北呈上升趋势,MTHFR A1298C位点的纯合突变基因型CC的频率从南到北呈下降趋势,而MTRR A66G位点基因多态性在不同地区差异不大。(3)MTHFR C677T位点的纯合突变基因型TT的频率在A1298C位点的正常型、杂合型、纯合突变型中分别为15.9%、0.1%、0.0%,差异有统计学意义(P<0.01);MTHFR C677T位点的纯合突变基因型TT的频率在MTRR A66G位点的正常型、杂合型、纯合突变型中分别为9.4%、5.9%、0.7%,差异无统计学意义(P>0.05)。结论 MTHFR A1298C位点基因多态分布存在民族差异;而MTRR C677T、A1298C位点基因多态分布存在地区差异;同一基因不同位点的基因多态性有关联。     

The heritability of plasma homocysteine, and the influence of genetic variation in the homocysteine methylation pathway

BACKGROUND: The extent of genetic influence on plasma homocysteine, a risk factor for ischaemic heart disease, is uncertain. Many association studies have investigated common polymorphisms and their role in hyperhomocysteinaemia, but only the thermolabile variant of methylene tetrahydrofolate reductase (MTHFR) has shown an association (small but robust). AIM: To estimate the heritability of plasma homocysteine and the contributions of well-studied common SNPs in the three main candidate genes in the homocysteine methylation pathway. DESIGN: Twin study. METHODS: We studied 216 monozygotic and 790 dizygotic pairs of twins; all were women. Blood was collected after overnight fasting for measurement of homocysteine, folate, vitamin B12, and extraction of DNA. Heritability was estimated by structural modelling, including correction for known environmental influences, particularly serum folate. The frequency of a common coding SNP in MTHFR and methionine synthase (MTR), and two coding SNPs in methionine synthase reductase (MTRR) were measured in dizygotic twins by ABI 7700 Sequence Detection, and the contribution of each to homocysteine variance was determined. RESULTS: The heritability of homocysteine was 57% (95%CI 51-63%). The highest contribution to homocysteine was serum folate, accounting for 10.13% of variance. This was twice the total genetic contribution of 4.56%, and only the C1763T SNP of MTRR showed significant association with homocysteine. DISCUSSION: Homocysteine has one of the highest heritabilities of common risk factors for ischaemic heart disease. This is not accounted for by the commonly studied SNPs in MTHFR, MTR and MTRR.     

育龄女性MTHFR、MTRR基因多态性分布以及与血清叶酸和同型半胱氨酸水平的相关性研究

目的探讨叶酸代谢关键酶基因5,10-亚甲基四氢叶酸还原酶(MTHFR)C677T、A1298C和甲硫氨酸合成酶还原酶(MTRR)A66G多态性在育龄女性中的分布特征,并分析不同遗传特征对外周血叶酸和同型半胱氨酸(Hcy)水平的影响。方法选取2018年3月至2020年1月在上海计生所医院妇科就诊的汉族育龄女性2 651例,根据知情同意原则,采集口腔黏膜上皮脱落细胞,抽提基因组DNA,使用荧光定量PCR方法检测MTHFR C677T、A1298C和MTRR A66G基因多态性,采用化学发光法检测外周血叶酸水平,循环酶法检测血清Hcy水平。结果 (1)MTHFR C677TCC、CT、TT的基因型频率分别为33.2%、47.9%、18.9%,C、T等位基因频率分别为57.1%、42.9%;MTHFR A1298C AA、AC、CC的基因型频率分别为66.6%、30.5%、2.9%,A、C等位基因频率分别为81.8%、18.2%;MTRR A66G AA、AG、GG的基因型频率分别为56.4%、36.9%、6.8%,A、G等位基因频率分别为74.8%、25.2%。(2)MTHFR C677T和A1298C两位点连锁有7种组合,频率最高的是CT/AA(31.9%),没有CT/CC和TT/CC组合。两位点间存在完全连锁不平衡(D′=0.984,R2=0.161)。(3)MTHFR C677T不同基因型的叶酸和Hcy水平差异有统计学意义(P<0.05)。TT基因型的叶酸水平低于CC基因型,TT基因型的Hcy水平高于CC基因型。经单因素Logistic回归分析发现,MTHFR C677T TT基因型发生高Hcy血症的危险性是CC基因型的9.97倍(95%CI:3.81~26.05)。MTHFR A1298C和MTRR A66G不同基因型与血清叶酸及Hcy水平差异无统计学意义(P>0.05)。(4)经单因素回归分析,Hcy水平与叶酸水平呈负相关(R2=0.061,P<0.05),叶酸水平可解释Hcy水平个体差异的6.1%。结论获取了上海市汉族育龄女性MTHFR和MTRR基因多态性的群体遗传学特征,血清Hcy水平与MTHFR C677T基因多态性以及血清叶酸水平有关。筛查MTHFR和MTRR基因多态性并监测Hcy水平对围生期保健有重要的指导意义。     

同型半胱氨酸及其代谢关键酶MTRR基因多态性与颅内动脉瘤的关系

目的:探讨同型半胱氨酸(Hcy)及其代谢关键酶蛋氨酸合成酶还原酶(methionine synthase reductase,MTRR)基因多态性与颅内动脉瘤的关系。方法:采用化学发光技术检测76例颅内动脉瘤患者(颅内动脉瘤组)和77例健康成人(对照组)的血浆Hcy水平,并利用聚合酶链反应-限制性内切酶片段长度多态性技术(PCR-RFLP)检测其代谢关键酶MTRR基因多态性。结果:颅内动脉瘤患者血浆Hcy水平[(16.14±6.72)μmol/L]明显高于对照组[(11.20±3.20)μmol/L],两组间血浆甘油三酯(TG)和总胆固醇(TC)水平差异无显著性。两组MTRRA66G基因型分布比较,颅内动脉瘤组GG基因型及G等位基因频率均明显高于对照组,患颅内动脉瘤的风险是对照组的7.30和2.41倍。MTRR A66G不同基因型颅内动脉瘤患者比较,血浆TG、TC浓度差异无显著性,GG基因型血浆Hcy浓度较其他基因型高;对照组不同基因型间血浆Hcy、TG、TC水平差异均无显著性。结论:高Hcy血症与颅内动脉瘤病发生有一定关系;MTRR A66G的AA基因型可能是颅内动脉瘤的保护性基因,发生变异时患病风险增加。     

Low frequency of mutated methylenetetrahydrofolate reductase 677C-->T and 1298A-->C genetics single nucleotide polymorphisms (SNPs) in Sub-Saharan populations.

5,10-Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) are two of the key enzymes in the folate/vitamin B12-dependent remethylation of homocysteine to methionine. The frequencies of MTHFR single nucleotide polymorphisms (SNPs), 677C-->T, 1298A-->C, 1317T-->C and of MTR, 2756A-->G, have been widely studied in Caucasians, but they have never been reported simultaneously in a large population from Sub-Saharan Africa. Presently, we report the prevalence of these SNPs and their relationship to homocysteine in 240 subjects recruited in West Africa. The frequencies of the mutant genotypes 677TT (0.8%) and 1298CC (2%) were lower than that usually observed in Caucasians, while the frequency of the mutant 1317CC was higher (16%). We formed a systematic association of the mutated MTHFR 677C-->T SNP with a 1298A/1317T common haplotype. The MTHFR mutant genotype 677TT was associated with an intermediate hyperhomocysteinemia (92.4 +/- 6.0 micromol/l) higher than that described in Caucasians. The 2756A-->G SNP in the MTR was similarly distributed in Africans compared to Caucasians. In conclusion, the MTHFR 677TTor 1298CC genotypes are much rarer in Africans than in Caucasians. The 677TT low frequency may be related to the high effect of this mutation on homocysteine metabolism in the environmental conditions of this African region.     

叶酸代谢相关酶基因多态性与唐氏综合征发生易感性的关系

目的:探讨参与叶酸代谢的亚甲基四氢叶酸还原酶(MTHFR)基因C667T和甲硫氨酸合成还原酶(MTRR)基因A66G基因多态性与中国汉族母亲生育唐氏综合征(Downsyndrome,DS)患儿易感性的关系。方法:生育过DS患儿的母亲30例,对照母亲70例,MTHFR、MTRR基因C667T及A66G基因座多态性采用聚合酶链反应———限制性片段长度多态性的方法检测。结果:携带MTHFR以及MTRR突变基因型的母亲并不增加孕DS的可能性,两个基因的联合作用分析也表明同时携带MTRR突变位点和MTHFR任一突变位点并不增加孕DS的可能性。MTHFR和MTRR突变基因型的分布在病例组和对照组间差异没有显著性(P>0.05)。结论:在本研究的人群中,MTRR基因A66G基因座多态性以及MTHFR基因C667T基因座多态性并不增加母亲孕DS的危险性,MTHFR以及MTRR基因突变位点并不是母亲生育DS患儿的危险因素。     

Association of MTHFR, MTR, and MTRR polymorphisms with Parkinson's disease among ethnic Chinese in Taiwan

BackgroundInfluence of folate/homocysteine conversion is considered to be important in the pathogenesis of Parkinson's disease (PD). However, association of the folate metabolic pathway gene polymorphisms with PD susceptibility remains unclear.MethodsTo test this possibility in PD, we conducted a hospital-based case-control study constituting 211 patients and 218 age- and sex-matched controls of ethnic Chinese in Taiwan. Genotyping assay was performed to screen for polymorphisms of the methylenetetrahydrofolate reductase (MTHFR C677T), methyltetrahydrofolate-homocysteine methyltransferase (MTR A2756G), and 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR A1049G and C1783T) genes and assess the association between these genotype polymorphisms and PD risk using logistic regression analysis.ResultsOf these four non-synonymous polymorphisms, the MTRR 1049GG variant was significantly associated with PD susceptibility (OR = 3.17, 95%CI = 1.08–9.35). Furthermore, we stratified our patients based on the MTHFR 677TT genotype in different strata, a significant association between the joint effect of polymorphisms and PD risk was observed in those patients whose genotypes were MTRR A1049G/MTR A2756G or MTRR C1783T/MTR A2756G (P < 0.05).ConclusionOur findings provide support for the synergistic effects of polymorphisms in the folate metabolic pathway genes in PD susceptibility; the increased PD risk would be more significant in carriers with the polymorphisms of MTHFR, MTR, and MTRR genes.     

Detection of MTRR 66A-->G polymorphism using the real-time polymerase chain reaction machine LightCycler for determination of composition of allele after restriction cleavage

The MTRR gene codes for methionine synthase reductase, one of the enzymes involved in the conversion of homocysteine to methionine. This conversion influences the overall level of total plasma homocysteine (tHcy) and mutations, which reduces the enzyme activity and results in an increased concentration of tHcy. A high homocysteine level is a well-documented independent risk factor for cardiovascular disease. A polymorphism in the gene for methionine synthase reductase (MTRR 66 A>G) has been shown to be associated with the risk of giving birth to a child with Down's syndrome, and the risk of having a foetus with neural tube defects. We have established a method for analysing MTRR 66A>G on DNA from dried blood spots using melting temperature analysis. The DNA was extracted from dried blood spots using a fast procedure by boiling only.     

Detection of MTRR 66A→G polymorphism using the real‐time polymerase chain reaction machine LightCycler for determination of composition of allele after restriction cleavage

The MTRR gene codes for methionine synthase reductase, one of the enzymes involved in the conversion of homocysteine to methionine. This conversion influences the overall level of total plasma homocysteine (tHcy) and mutations, which reduces the enzyme activity and results in an increased concentration of tHcy. A high homocysteine level is a well‐documented independent risk factor for cardiovascular disease. A polymorphism in the gene for methionine synthase reductase (MTRR 66 A>G) has been shown to be associated with the risk of giving birth to a child with Down's syndrome, and the risk of having a foetus with neural tube defects. We have established a method for analysing MTRR 66A>G on DNA from dried blood spots using melting temperature analysis. The DNA was extracted from dried blood spots using a fast procedure by boiling only.     

四川德阳地区汉族女性人群MTHFR和MTRR基因多态性研究

目的探讨四川德阳地区汉族女性人群编码叶酸代谢通路中关键酶的基因5,10-亚甲基四氢叶酸还原酶(MTHFR)C677T、A1298C和甲硫氨酸合成酶还原酶(MTRR)A66G位点的基因型及等位基因的频率分布,以指导孕妇的叶酸补充。方法采用Taqman-MGB方法对中国四川德阳地区2 573例妇女的MTHFR基因C677T、A1298C和MTRR基因A66G位点进行基因检测,然后根据检测结果进行大规模基因型频率分布统计,并参考山东、河南、海南三省相关数据进行统计学比较。结果 MTHFR基因C677T的等位基因频率分别为63.45和36.55,MTHFR基因A1298C的等位基因频率分别为78.20和21.80,MTRR基因A66G的等位基因频率分别为72.81和27.19。四川地区人群与其他地区人群基因多态性分布频率比较发现,MTHFR基因C677T、A1298C多态性分布地区差异有统计学意义(P值均<0.01)。结论本研究结果表明,MTHFR基因C677T、A1298C多态性分布具有地区差异,MTHFR多态性是孕妇生育神经管缺陷(neural tube defects,NTDs)后代的危险因素,因此基于MTHFR多态性检测的孕期叶酸补充指导和监测将是进一步降低新生儿出生缺陷的重要方法。     

Hyperhomocysteinemia is related to residual glomerular filtration and folate, but not to methylenetetrahydrofolate-reductase and methionine synthase polymorphisms, in supplemented end-stage renal disease patients undergoing hemodialysis.

Glomerular filtration is one of the major determinants of plasma total homocysteine (tHcy). To evaluate the respective roles of residual glomerular filtration (by measuring a specific protein marker, cystatin C), genetic polymorphisms and nutritional status in tHcy blood levels in end-stage renal disease patients (ESRD) under hemodialysis and supplemented with folate, we measured tHcy, folate, vitamin B12 (B12), creatinine, cystatin C, albumin and C-reactive protein and determined the polymorphism of methylenetetrahydrofolate reductase (MTHFR) (C677T and A1289C) and of methionine synthase (MS) (A2756G) in 114 ESRD patients before hemodialysis and 76 control subjects. All patients received a folate supplementation of 700 microg/day. Hyperhomocysteinemia was observed in all patients and exceeded the upper normal limit by 2-fold in 52.4% of the patients. Serum folate was significantly increased and the B12 level was not different from controls. Folate, Cystatin C and creatinine were significantly correlated to tHcy, while no correlation was found between tHcy, albumin and C-reactive protein. No difference in genotype frequency between ESRD patients and controls was found for MTHFR A1289C and MS A2756G. The MTHFR 677TT genotype was less frequent and was associated with a significantly higher tHcy level in patients. Folate and residual glomerular filtration estimated by cystatin C and creatinine levels were two independent determinants of tHcy in ESRD patients. These data suggest that hyperhomocysteinemia is a consequence as well as a complicating factor of renal failure.     

Genetic defects as important factors for moderate hyperhomocysteinemia.

The genes for the enzymes methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS), methionine synthase reductase (MSR) and cytathionine-beta-synthase (CBS) play an important role in homocysteine metabolism. Rare mutations in these genes cause severe hyperhomocysteinemia and clinical symptoms. Growing interest has focused on common mutations with moderate effects on homocysteine levels. We studied 280 subjects of different age groups for the following mutations: MTHFR677C-->T and 1298A-->C, MS2756A-->G, MSR66A-->G and the 68 bp insertion in the CBS gene. The median value for homocysteine increased significantly with age (median homocysteine levels: 7.5, 12.4 and 16.5 micromol/l in the age groups 20-43, 65-75 and 85-96 years, respectively). The genotypes of the MTHFR677C-->T mutation were associated with differences in plasma homocysteine levels, but without reaching significance. Individuals homozygous for the MTHFR677C-->T mutation had a 2.3 micromol/l higher median homocysteine level compared to individuals with the wild-type allele. This effect was pronounced in combination with low folate levels and abolished with higher folate in plasma. For the other three mutations no association with homocysteine values could be determined. The analysis of homocysteine metabolite cystathionine by backward regression analysis revealed a significant correlation of the MS2756A-->G mutation with cystathionine level. This increase could indicate a disturbed remethylation. In summary, larger and homogeneous study populations are necessary to quantify the small effects of common mutations on homocysteine levels. This may also be the reason that no effects of genetic interactions between two genotypes were observed.     

A CBS haplotype and a polymorphism at the MSR gene are associated with cardiovascular disease in a Spanish case-control study

OBJECTIVES: The aim of this study was to evaluate the association of polymorphisms present in genes related to homocysteine (Hcy) metabolism with coronary artery disease (CAD). DESIGN AND METHODS: We examined 8 polymorphisms in the cystathionine beta-synthase (CBS), glutamate carboxypeptidase II (GCPII), methionine synthase (MS), methionine synthase reductase (MSR) and methylenetetrahydrofolate reductase (MTHFR) genes in 140 CAD patients and 113 controls, by means of Chi-square, logistic regression, ANOVA and the Mann-Whitney U test. RESULTS: The c.66 G allele of MSR conferred an odds-ratio for CAD of 1.76 (95% CI 1.12-2.77), while a CBS haplotype [c.699C-c.844wt-c.1080C] was found over-represented in CAD [OR of 2.16 (1.29-3.63)]. CONCLUSIONS: Our results not only highlight the involvement of the MSR and CBS genes in the etiology of cardiovascular disease, but also emphasize the strength of haplotype analyses in association studies.     

Genetic and environmental influences on plasma homocysteine: results from a Danish twin study

BACKGROUND: Increased plasma homocysteine has been linked to many clinical conditions including atherosclerosis and ischemic stroke. We assessed the genetic and environmental influences on homocysteine in adult twins and tested the influence of 3 candidate polymorphisms. METHODS: Homocysteine was analyzed in 1206 healthy twins, who were genotyped for 3 polymorphisms: MTHFR 677C>T, MTR 2756A>G, and NNMT (dbSNP: rs694539). To perform quantitative trait linkage analysis of the MTHFR locus, the genotyping was supplemented with 2 genetic markers localized on each site of the MTHFR locus. The twin data were analyzed using biometric structural equation models as well as a combined association and linkage analysis in 2 age cohorts. RESULTS: Age, sex, and MTHFR genotype have a significant impact on homocysteine concentrations, whereas the other genotypes were not associated with homocysteine concentrations. The variance in homocysteine could be solely ascribed to additive genetic and nonshared environmental factors, with an estimated additive genetic proportion of total variation at age 18-39 years of 0.63 (95% CI, 0.53-0.71) and at age 40-65 years of 0.27 (95% CI, 0.10-0.41). The impact of the MTHFR locus is estimated to explain 53% (95% CI, 0.07-0.67) of the total phenotypic variation in persons 18-39 years old and 24% (95% CI, 0.00-0.39) in persons 40-65 years old, i.e., almost all additive genetic variance. CONCLUSIONS: Homocysteine concentrations have a high heritability that decreases with age. The MTHFR gene locus is responsible for almost all the variation attributable to genetic factors, leaving very little influence of other genetic variations.     

Evaluation of nutritional and genetic determinants of total homocysteine, methylmalonic acid and S-adenosylmethionine/S-adenosylhomocysteine values in Brazilian childbearing-age women

BACKGROUND: Cobalamin (Cbl) and folate deficiencies and gene polymorphism of key enzymes or carriers can impair homocysteine metabolism and may change the serum values of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). We investigated the nutritional and genetic determinants for total homocysteine (tHcy), methylmalonic acid (MMA) and SAM/SAH in healthy Brazilian childbearing-age women. METHODS: Serum concentrations of Cbl, folate, red blood cell folate, ferritin, tHcy, MMA, SAM, SAH and other metabolites were measured in 102 healthy unrelated women. The genotypes for MTHFR C677T, MTHFR A1298C, MTR A2756G, MTRR A66G, TC2 C776G, TC2 A67G and RFC1 A80G gene polymorphisms were identified by PCR-RFLP. RESULTS: Serum folate and Cbl were inversely correlated with tHcy and serum MMA, respectively. Cbl deficiency was associated with increased MMA and reduced alpha-aminobutyrate, serine and N-methylglycine concentrations. No variable was associated with SAM/SAH ratio. In addition, gene polymorphisms were not selected as determinants for tHcy, MMA and SAM/SAH ratio. Iron, Cbl and folate deficiencies were found respectively in 30.4%, 22.5% and 2.0% of individuals studied. CONCLUSIONS: There was a high frequency of Cbl and iron deficiency in this group of childbearing-age women. Serum folate and Cbl were the determinants of serum tHcy and MMA concentration, respectively.     

Molecular analysis of homocystinuria in Brazilian patients

BACKGROUND: Cystathionine beta-synthase (CBS) deficiency is the most common cause of homocystinuria. However, no data are available concerning the molecular basis of this disease in Brazilian populations. METHODS: We studied 14 Brazilian patients from 11 unrelated families using a combined screening approach, involving restriction analysis, single-strand conformational polymorphism (SSCP) scanning, and sequencing. RESULTS: All patients presented homocysteine levels higher than 200 mumol/l before the beginning of treatment. The most common CBS gene mutations, p.G307S (c.919G > A) and p.I278T (c.833T > C), were evaluated and the allele c.919A was not found. One allele with the c.844 ins68 (4.5%) in the CBS gene was found. Three families (6 patients) presented the allele c.833 C (13.6%), without the insertion in the heterozygous state. SSCP scanning and sequencing showed 3 alleles p.T191M (13.64%) in 2 families. One allele with a novel mutation was found in exon 4 (c.168T > A) of the CBS gene (4.5%). We also analyzed c.677C > T and c.1298A > C polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene and the 2756A > G polymorphism in the methionine synthase (MTR) gene. The frequencies of mutated alleles were: 50% c.677T and 18.2% c.1298C for MTHFR, and 27.3% c.2756G for MTR. CONCLUSION: In spite of the high level of racial mixing in the country, Brazilian homocystinuric patients did not present a high prevalence of the most common mutations described in the literature.