Single nucleotide polymorphism C677T in the methylenetetrahydrofolate reductase gene might be a genetic risk factor for infertility for Chinese men with azoospermia or severe oligozoospermia

Aim： To analyze the distribution of the single nucleotide polymorphism （SNP） C677T in the methylenetetrahydrofolate reductase （MTHFR） gene in 355 infertile Chinese patients with idiopathic azoospermia or severe oligozoospermia and 252 fertile Chinese men as controls to explore the possible association of the SNP and male infertility. Methods： Using the polymerase chain reaction （PCR）-restriction fragment length polymorphism technique, the allele and genotype distribution of SNP C677T in the MTHFR gene were investigated in both patients and controls. Results： The frequencies of allele T （40.9% vs 30.4%, P = 0.002, odds ration [OR] = 1.58, 95% confidence interval [CI]： 1.24-2.02） and mutant homozygote （TT） （18.3% vs. 11.5%, P = 0.023, OR = 1.72, 95% CI： 1.07-2.76） as well as carrier with allele （TT ＋ CT） （63.4% vs. 49.2%, P = 0.0005, OR = 1.79, 95% CI： 1.29-2.48） in infertile patients were significantly higher than those in controls. After patient stratification, the significant differences in distribution of the SNP between each patient subgroup and control group still remained. Conclusion： Our findings indicate that there is an association of SNP C677T in the MTHFR gene with male infertility, suggesting that this polymorphism might be a genetic risk factor for male infertility in Chinese men.

Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and gastric cancer susceptibility

AIM: To identify the association between methylenetetrahydrofolate reductase (MTHFR) polymorphisms and gastric cancer (GC) susceptibility.

Association Between Homocysteine Level and Methylenetetrahydrofolate Reductase Gene Polymorphisms in Type 2 Diabetes Accompanied by Dyslipidemia

Objective To investigate the association between total homocysteine(tHcy)level in plasma and methylenetetrahydrofblate reductase(MTHFR)C677T and A1298C genetic polymorphisms in a Chinese Han nationality population with type 2 diabetes mellitus(T2DM)accompanied by dyslipidemia.Methods This case-control study enrolled T2DM patients with dyslipidemia and without dyslipidemia respectively.Sanger dideoxy-mediated chain-termination method was used to detect the gene polymorphisms of MTHFR C677T and A1298C.Plasma tHcy and lipid levels were measured as well.The genotype frequency and allele frequency between the dyslipidemia and non-dyslipidemia groups were compared by using Chi-square test.Plasma tHcy level ofT2DM patients who carried the different genotypes was compared by Student's t test.Results Finally,82 T2DM patients with dyslipidemia and 94 ones without dyslipidemia were included in this study.There was a significant correlation between tHcy level and MTHFR C677T gene polymorphism inT2DM patients(t=2.27,P=0.02).Moreover,the plasma tHcy level in the dyslipidemia patients who carried MTHFR 677TT genotype was significantly higher than that in those with CT+CC genotype(13.62+6.97 vs.10.95+3.62pmol/L,t=2.2O,P=0.03);while for patients without dyslipidemia,comparison of the tHcy level between those who carried the above two alleles showed no significantly difference(13.34±6.03 vs.12.04±5.09μmol/L,t=1.08,P=0.29).Conclusion MTHFR 677TT genotype might associate with higher tHcy level in T2DM patients with dyslipidemia.

Folate levels in mucosal tissue but not methylenetetrahydrofolate reductase polymorphisms are associated with gastric carcinogenesis

AIM: To evaluate whether folate levels in mucosal tissue and some common methylenetetrahydrofolate reductase (MTHFR) variants are associated with the risk of gastric cancer through DNA methylation. METHODS: Real-time PCR was used to study the expression of tumor related genes in 76 mucosal tissue samples from 38 patients with gastric cancer. Samples from the gastroscopic biopsy tissues of 34 patients with chronic superficial gastritis (CSG) were used as controls. Folate concentrations in these tissues were detected by the FOL ACS: 180 automated chemiluminescence system. MTHFR polymorphisms were analyzed by PCR-RFLP, and the promoter methylation of tumor-related genes was determined by methylation-specific PCR (MSP). RESULTS: Folate concentrations were significantly higher in CSG than in cancerous tissues. Decreased expression and methylation of c-myc accompanied higher folate concentrations. Promoter hypermethylation and loss of p16INK4A in samples with MTHFR 677CC were more frequent than in samples with the 677TT or 677CT genotype. And the promoter hypermethylation and loss of p21WAF1 in samples with MTHFR 677CT were more frequent than when 677CC or 677TT was present. The 677CT genotype showed a non-significant higher risk for gastric cancer as compared with the 677CC genotype. CONCLUSION: Lower folate levels in gastric mucosal tissue may confer a higher risk of gastric carcinogenesisthrough hypomethylation and overexpression of c-myc.

Methylenetetrahydrofolate Reductase Gene Polymorphism C677T is Associated with Increased Risk of Coronary Heart Disease in Chinese Type 2 Diabetic Patients

Objective Chronic cardiovascular diseases induced by long-term poor blood glucose control are the main cause of death in patients with type 2 diabetes mellitus(T2DM).Previous researches report that methylenetetrahydrofolate reductase gene(MTHFR)polymorphisms might influence the occurrence of coronary heart disease(CHD)in T2DM patients.The purpose of this study was to evaluate whether MTHFR C677T and A1298C mutations are associated with the risk of CHD inT2DM patients.Methods A total of 197 subjects with T2DM were studied,of which 95 patients with CHD.The genotypes of MTHFR C677T and A1298C were analyzed by using dideoxy chain-termination method,and compared between patients with CHD and those without CHD.Results We found that the frequency of the 677T allele was significantly higher in T2DM patients with CHD than those without CHD(P=0.011).However,there was no significant difference in any of the examined haplotypes between T2DM patients with and without CHD.Furthermore,the 677T allele was associated with a higher risk of CHD development in diabetic patients with lower homocysteine(Hey)levels(≤15μmol/L)(P=0.006),while no effect of MTHFR gene polymorphism on the incidence of CHD was found in patients with higher Hey levels(>15 μmol/L)(P=0.491).Conclusion The MTHFR C677T gene polymorphism is associated with the risk of CHD of diabetic patients and could be used as an effective marker for CHD in Chinese diabetic populations with normal Hey levels.

Is the C677T polymorphism in methylenetetrahydrofolate reductase gene or plasma homocysteine a risk factor for diabetic peripheral neuropathy in Chinese individuals?

The present study enrolled 251 diabetic patients, including 101 with neuropathy and 150 without neuropathy. Of the 150 patients, 100 had no complications, such as retinopathy, nephropathy, or neuropathy. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to identify methylenetetrahydrofolate reductase gene variants. Plasma homocysteine levels were also measured. Homocysteine levels and the frequency of hyperhomocysteinemia were significantly higher in patients with diabetic peripheral neuropathy compared with diabetic patients without neuropathy （P 〈 0.05）. In logistic regression analysis with neuropathy as the dependent variable, the frequency of C677T in methylenetetrahydrofolate reductase was significantly higher in patients with diabetic peripheral neuropathy compared with patients without diabetic complications. Homocysteine levels were significantly higher in patients with diabetic peripheral neuropathy carrying the 677T allele and low folic acid levels. In conclusion, hyperhomocysteinemia is an independent risk factor for diabetic neuropathy in Chinese patients with diabetes. The C677T polymorphism in methylenetetrahydrofolate reductase and low folic acid levels may be risk factors for diabetic peripheral neuropathy in Chinese patients with diabetes.

Association of methylenetetrahydrofolate reductase C677T polymorphism and serum lipid levels in the Guangxi Bai Ku Yao and Han populations

Objectives The association of methylenetetrahy-drofolate reductase(MTHFR) gene polymorphism and serum lipid profiles is still controversial in diverse ethnics.Bai Ku Yao is an isolated subgroup of the Yao minority in China. The aim of the present study was to eveluate the association of MTHFR C677Tpolymorphism and several environmental factors with serum lipid levels in the Guangxi Bai Ku Yao and Han populations.Methods A total of 780 subjects of Bai Ku Yao and 686 participants of Han Chinese were randomly selected from our previous stratified randomized cluster samples.Genotyping of the MTHFR C677T was performed by polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis,and then confirmed by direct sequencing.Results The levels of serum total cholesterol(TC),high-density lipoprotein cholesterol (HDL-C),low-density lipoprotein cholesterol(LDL-C), apolipoprotein(Apo) AI and ApoB were lower in Bai Ku Yao than in Han(P【0.05-0.001).The frequency of C and T alleles was 77.4%and 22.6%in Bai Ku Yao,and 60.9%and 39.1%in Han(P【0.001);respectively.The frequency of CC,CT and TT genotypes was 58.7%,37.3%and 4.0%in Bai Ku Yao,and 32.6%,56.4%and 11.0%in Han(P【 0.001);respectively.The levels of TC and LDL-C in both ethnic groups were significant differences among the three genotypes(P【0.05-0.01).The T allele carriers had higher serum TC and LDL-C levels than the T allele noncarriers. The levels of ApoB in Han were significant differences among the three genotypes(P【0.05).The T allele carriers had higher serum ApoB levels as compared with the T allele noncarriers. The levels of TC,TG and LDL-C in Bai Ku Yao were correlated with genotypes(P【0.05-0.001),whereas the levels of LDL-C in Han were associated with genotypes(P【 0.001).Serum lipid parameters were also correlated with sex, age,body massindex,alcohol consumption,cigarette smoking, and blood pressure in the both ethnic groups.Conclusions The differences in serum TC,TG,LDL-C and ApoB levels between the two ethnic groups might partly result from different genotypic and allelic frequencies of the MTHFR C677Tor differentMTHFR gene-enviromental interactions.

Polymorphisms in methylenetetrahydrofolate reductase gene: Their impact on liver steatosis and fibrosis of chronic hepatitis c patients

Aim & Background: The mechanism of steatosis in Hepatitis C virus infection is multifactorial;therefore, it is complex and unclear. The aim of this study was to investigate the effects of methylentetrahydrofolate reductase (MTHFR) gene polymorphisms on the course of chronic hepatitis C virus infection and the development of steatosis due to hepatitis C virus. Methods: This study included 109 patients with chronic hepatitis C virus infection. Necroinflammatory activity, degrees of fibrosis and steatosis and MTHFR gene polymorphisms were investigated. Polymerase chain reaction-restriction fragment length polymorphism was used to determine MTHFR C677T and A1298C polymorphisms. Results: Fibrosis was correlated with age (r = 0.336, p = 0.002), platelet (r = ?0.448, p < 0.0001), ALT (r = 0.241, p = 0.026), AST (r = 0.361) and GGT (r = 0.224, p = 0.039). Steatosis was only correlated with fibrosis. MTHFR C677T and A1298C polymorphisms did not have a significant effect on the degree of steatosis (p = 0.857, p = 0.202 respectively). There was a relation between MTHFR C677T and the degree of fibrosis but not A1298C (p = 0.014, p = 0.187 respectively). Conclusion: We found that MTHFR C677T polymorphism contributed to the development of fibrosis in patients with chronic hepatitis C virus infection.

A review of methylenetetrahydrofolate reductase in one-carbon metabolism and psychiatric disorders

Methylenetetrahydrofolate reductase(MTHFR)is a key enzyme for the critical process of one-carbon circulation,which convert5,10-methylenetetrahydrofolate to5-methyltetrahydrofolate and participate in folate and homocysteine conversion correlated to methyl group supply.The enzyme activity decline depends on the gene polymorphism.MTHFR impacts on the methylation process which is related to psychiatric diseases.Studies have shown association between MTHFR gene polymorphisms and mental disorders,some of which stratified by folate and cobalamin levels.In this review,we will summarize the testimony on the relationship between methylation and MTHFR polymorphism as well as the implication on psychiatric diseases by MTHFR mutation.

Women with Methylenetetrahydrofolate Reductase Gene Polymorphism and the Need for Proper Periconceptional Folate Supplementation

Maternal folate supplementation is critical for fetal development. Women with MTHFR （methylenetetrahydrofolate reductase） gene polymorphisms may not be getting the proper folate form to support fetal development. The objectives of this review were to： （1） undertake a comprehensive review on the association of MTHFR polymorphisms with the risk for various congenital diseases and other adverse pregnancy outcomes, （2） assess the efficacy and safety of current folic acid and other supplementations in women with the MTHFR polymorphism, and （3） provide guidance on the appropriate supplementation for women of childbearing potential with the MTHFR gene polymorphism in order to decrease these adverse pregnancy outcomes. Our assessments show that women with MTHFR gene polymorphism cannot efficiently convert folic acid to L-5-methyl-tetrahydofolate, the predominant active form of folic acid, due to reduced MTHFR enzymatic activity. L-5-methyl-tetrahydrofolate is currently commercially available under several brand names. Based on our comprehensive review and knowledge of the biochemistry of the folates, we recommend that L-5-methyltetrahydrofolate be given in combination with folic acid to women with MTHFR polymorphism that are pregnant or planning to become pregnant. Further study is needed to determine the optimal dose.

Correlation between methylenetetrahydrofolate reductase gene C677T polymorphism and preeclampsia in pregnant women

Objective: To study the correlation between methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism and preeclampsia in pregnant women. Methods: Pregnant women who were diagnosed with preeclampsia in Jianshi People's Hospital between July 2014 and March 2017 were selected as the PE group of the research, and healthy pregnant women who received antenatal care and gave birth in Jianshi People's Hospital during the same period were selected as the control group of the research. The MTHFR gene C677T polymorphism in peripheral blood, the contents of homocysteine (Hcy) metabolism indexes and the expression of apoptosis genes and invasion genes were determined. Results: The proportion of MTHFR gene C677T locus TT genotype in peripheral blood of PE group was significantly higher than that of control group while the proportion of CT and CC genotypes were significantly lower than those of control group;Hcy levels in serum and placenta as well as FasL, Caspase-8, Bax, Caspase-9 and Caspase-3 mRNA expression in placenta of PE women with TT genotype were significantly higher than those of PE women with CT genotype and CC genotype while folic acid levels in serum and placenta as well as Notch-1, N-cadherin, Vimentin, CatL and CatB mRNA expression in placenta were significantly lower than those of PE women with CT genotype and CC genotype. Conclusion: MTHFR gene C677T locus mutation can participate in the occurrence of preeclampsia by affecting the Hcy metabolism as well as the expression of apoptosis genes and invasion genes.

Correlation of methylenetetrahydrofolate reductase polymorphism with Hcy metabolism and inflammatory response in patients with recurrent cerebral infarction

Objective:To study the correlation of methylenetetrahydrofolate reductase (MTHFR) polymorphism with Hcy metabolism and inflammatory response in patients with recurrent cerebral infarction.Methods: 40 patients with recurrent cerebral infarction who were treated in Yulin Third Hospital between December 2013 and December 2016 were selected as recurrent group, 58 patients with primary cerebral infarction were selected as primary group, and 60 healthy volunteers were selected as control group. Peripheral blood MTHFR gene C677T polymorphism and serum levels of Hcy metabolism indexes and inflammatory response indicators were determined.Results: CC genotype constituent ratio of recurrent group was significantly lower than that of primary group and control group while CT genotype and TT genotype constituent ratio were significantly higher than those of primary group and control group;serum Hcy, HMGB1, sCD40L, YKL-40, Lp-PLA2 and MMP-9 levels in recurrent group and primary group were significantly higher than those in control group while VitB12 and FA levels were significantly lower than those in control group;serum Hcy, HMGB1, sCD40L, YKL-40, Lp-PLA2 and MMP-9 levels in recurrent group were significantly higher than those in primary group while VitB12 and FA levels were significantly lower than those in primary group. Serum Hcy, HMGB1, sCD40L, YKL-40, Lp-PLA2 and MMP-9 levels in patients with CC genotype were significantly lower than those in patients with CT genotype and TT genotype while VitB12 and FA levels were significantly higher than those in patients with CT genotype and TT genotype;serum Hcy, HMGB1, sCD40L, YKL-40, Lp-PLA2 and MMP-9 levels in patients with CT genotype were significantly lower than those in patients with TT genotype while VitB12 and FA levels were significantly higher than those in patients with TT genotype.Conclusion: MTHFR gene C677T polymorphism is closely related to the recurrence of cerebral infarction, and allele C mutation to T will affect Hcy metabolism and aggravate inflammatory response.

Method for Detecting NADPH-Cytochrome P450 Reductase in Liver Microsomal Fractions by Using Native Polyacrylamide Gel Electrophoresis and NADPH-Diaphorase Staining

By combining native polyacrylamide gel electrophoresis (PAGE) and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, a simple method for detecting NADPH-cytochrome P450 reductase in tissue samples was established. When rat liver microsomal fractions were examined by this method, several bands with different mobility were visualized. Western blot analysis indicated that the band which appeared in the most anodal position among them represented NADPH-cytochrome P450 reductase. SDS-PAGE/Western blot analysis revealed that the molecular weight of the protein forming the band was around 80 kDa, which was identical to that of rat NADPH-cytochrome P450 reductase. The intensity level of NADPH-diaphorase staining assigned to this enzyme estimated by this method increased four times in microsomal fractions prepared from rat fed ethanol chronically compared to that from controls. When a dilution series of a rat liver microsomal fraction was examined by this method and SDS-PAGE/Western blot analysis, their staining intensities representing this enzyme were significantly correlated with each other. Using the naive PAGE/NADPH-diaphorase staining method, NADPH-cytochrome P450 reductase is detected in rat liver microsomes. This method is beneficial because compared with the conventional SDS-PAGE/Western blot analysis, the quantification of NADPH-cytochrome P450 reductase in tissue samples is allowed to be more easily done.

家蚕氟中毒后中肠NADPH-细胞色素P450还原酶和NADPH-细胞色素C还原酶活性的变化

为了探究家蚕对NaF的代谢机制,以家蚕耐氟品种T6和氟化物敏感品种734为材料,从5龄起蚕开始分别添食50、100、200、400 mg/kg NaF溶液处理后的桑叶,检测蚕体中肠微粒体酶液中的黄素蛋白NADPH-细胞色素P450还原酶(CPR)和NADPH-细胞色素C还原酶(CR)的活性变化。氟物化敏感品种734的4个NaF处理组第3天的中肠CPR活性低于对照组,其余时间几乎都高于对照组,400 mg/kg NaF处理组在第4天的CPR活性最高,且各NaF处理组的CPR活性差异显著(P<0.05);耐氟品种T6 NaF处理组和对照组的中肠CPR活性整体上呈先升后降的趋势,几乎都在第2天达到最高值,各组之间的差异不显著(P>0.05)。氟化物敏感品种734的4个NaF处理组的中肠CR活性呈现先升后降的趋势,而对照组呈下降趋势;耐氟品种T6的50、100、400 mg/kg NaF处理组在第1～2天的中肠CR活性呈明显下降趋势,之后的变化相对较小,对照组的CR活性仅在第3～4天略高于NaF处理组,而其余时间NaF处理组的CR活性较高。2个家蚕品种添食不同浓度NaF后的中肠CR活性差异均不显著(P>0.05)。试验结果显示,耐氟品种T6在NaF作用下中肠的CPR和CR活性变化范围(分别为对照组的0.4～2.0倍和0.3～2.9倍)远小于氟化物敏感品种734这2种酶的活性变化范围(分别为对照组的0.6～9.3倍和0.4～4.6倍)。初步推测CPR和CR与家蚕对氟化物代谢具有一定的关联。

NADPH-细胞色素P-450还原酶的制备及在外来化合物代谢中的作用

ＮＡＤＰＨ－细胞色素Ｐ－４５０还原酶是肝微粒体酶系的主要组成成分。本文报道了一种简单经济制备还原酶的方法，并通过ＮＡＤＰＨ的氧化证明还原酶及细胞色素Ｐ－４５０２Ｂ１重组酶系能代谢苯、二氯乙烯和石棉。但代谢过程中的中间产物未能引起质粒ＤＮＡ电泳图谱的变化。

微粒体细胞色素P-450、NADPH-细胞色素P-450还原酶的纯化与重组活性

经苯巴比妥钠诱导的雄性大白鼠的肝微粒体纯化的细胞色素P-450同功酶组份,经SDS-PAGE鉴定呈电泳纯,分子量为55kD。部分纯化的NADPH-细胞色素P-450还原酶,含72和77kD两个蛋白质组分。上述细胞色素P-450和NADPH-细胞色素P-450还原酶与卵磷脂制备的脂质体重组后的活性试验表明,对艾氏剂有环氧化作用,对环已烷有羟化作用,对溴氰菊酯的羟化作用微弱。当重组系统中缺少细胞色素P-450组份时,对环已烷不再起作用。同时还研究了纯化的细胞色素P-450的光谱特性。

NADPH-细胞色素C还原酶对碘有机化的影响

目的 :探讨NADPH 细胞色素C还原酶 (CR)在碘有机化中的作用。方法 :应用荧光分析和同位素技术测定甲状腺H2 O2 浓度、CR活性和蛋白结合碘 (PBI) ,观察CR所引起的H2 O2 变化对PBI形成的影响。结果 :Graves病甲状腺CR活性高于正常 ,其H2 O2 水平和所形成的PBI亦明显高于正常 ;加CR抑制剂后 ,Graves病和正常甲状腺CR活性降低近 84 % ,同时H2 O2 下降近4 5% ,PBI形成随CR和H2 O2 水平降低而减少 ;而葡萄糖 /葡萄糖氧化酶系统可使PBI恢复正常。结论 :CR通过其所产生的H2 O2影响PBI的形成 。

重组NADPH-细胞色素P450还原酶在大肠杆菌中的表达和纯化

目的 在大肠杆菌中表达重组NADPH -细胞色素P4 5 0还原酶 (CPR)并纯化获得有活性的CPR蛋白。方法 将已构建的NADPH -细胞色素P4 5 0还原酶重组质粒pMW1 72a CPR采用温和转化法转入大肠杆菌BL 2 1中 ,依次使用超速离心、DE5 2纤维素阴离子交换层析和 2′ 5′ADP亲和层析的方法纯化CPR ,并检测酶的活性。结果 获得了纯度达 99%以上的可溶性CPR蛋白 ,纯化倍数 5 .2 0倍 ,检测酶的比活性为每分钟 1 0 4 5 .4 9nmol mg。结论 获得了纯度高、有活性的CPR蛋白 ,可作为工具酶用于某些相关酶的研究 。

NADPH依赖的乙酰乙酰辅酶A还原酶基因phbB的克隆和在大肠杆菌中的表达

目的 :在大肠杆菌中克隆和表达NADPH依赖的乙酰乙酰辅酶A还原酶基因。方法 :通过PCR方法克隆来源于Alcali geneseutrophus菌的NADPH依赖性的乙酰乙酰辅酶A还原酶基因phbB ,将其克隆在大肠杆菌表达质粒pET2 8a中 ,进行PCR和DNA序列测定验证 ,并通过IPTG诱导表达。结果 :通过PCR和DNA核酸序列分析 ,证实获得的基因片段为phbB全编码序列 ,phbB被克隆在大肠杆菌表达质粒pET2 8a中 ,重组表达质粒在大肠杆菌BL2 1(DE3 )中经IPTG诱导获得表达。结论 :获得了Al caligeneseutrophus菌的NADPH依赖的乙酰乙酰辅酶A还原酶基因 ,将其克隆在pET2 8a中 ,经诱导获得PhbB的表达 。

雷斯青霉NADPH-细胞色素P450还原酶基因的克隆及生物信息学分析

工业上利用丝状真菌雷斯青霉(Penicillium raistrickii)转化生产高效避孕药孕二烯酮的关键中间体15α–羟基左旋乙基甾烯双酮.已知参与雷斯青霉甾体转化反应的关键酶为P450羟化酶,该羟化酶体系由细胞色素P450羟化酶和NADPH–细胞色素P450还原酶(CPR)组成,但有关其15α–羟基化反应的分子基础尚不清楚.根据转录组测序数据库,通过RT-PCR扩增克隆了一个雷斯青霉NADPH-细胞色素P450还原酶基因.该基因的开放阅读框为2,082,bp,编码694个氨基酸的多肽链,预测的蛋白相对分子质量为7.63×104,具有CPR蛋白的典型结构域(FMN结合域、FAD和NADPH结合域).NCBI BLAST结果显示雷斯青霉CPR与意大利青霉(P.,italicum)NADPH–细胞色素P450还原酶具有较高的同源性,一致性为93%.