Effect of cystathionine beta-synthase variant 844ins68bp and methylenetetrahydrofolate reductase A1298C polymorphisms in xenografts on 5-FU efficacy and doubling time

The association of MTHFR and CBS variants with the doubling time and responsiveness to several chemodrugs was analyzed in 26 human cancer xenografts. The tumors homozygous for the absence of insertion (NN) for the CBS 844ins68bp were more chemosensitive than those with insertion (NI) to TS-1 (P=0.0048), suggesting a potential effect of this variant on fluoropyrimidine efficacy. Furthermore, the doubling time of tumors with a variant C allele (AC or CC) in MTHFR-A1298C was significantly longer than that of tumors with a normal allele (AA) (P=0.0008). Twenty-nine cellular proliferation-related genes were associated with MTHFR-A1298C genotyping and with the doubling time.     

Interaction of folate and homocysteine pathway genotypes evaluated in susceptibility to neural tube defects (NTD) in a German population

Neural tube defects (NTD) are likely to result from an interaction of several genes and environmental factors. Because periconceptional folate intake reduces the NTD risk in the fetus, and because mothers of children with NTD showed elevated plasma homocysteine levels, gene polymorphisms of the folate and homocysteine pathway, such as 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C→T, MTHFR 1298A→C and cystathionine β-synthase (CBS) 844ins68, have been implicated in the etiology of NTD. Several studies have demonstrated that these polymorphisms may indeed be associated with NTD in some populations. In order to evaluate the role of these polymorphisms and their interaction in NTD, we genotyped 417 individuals for case-control studies and 129 families for transmission disequilibrium tests. We are the first to present detailed data on MTHFR haploid genotypes in combination with CBS 844ins68. The MTHFR risk genotype 677CT/1298AC, known to be associated with decreased enzyme activity and increased homocysteine, was found significantly more often in patients than in controls (P = 0.02). A CBS insertion allele in addition to MTHFR 677CT/1298AC heterozygosity or MTHFR 677TT/1298AA homozygosity did not result in an increased risk for NTD. This is in agreement with the recently reported homocysteine-lowering effect of the CBS 844ins68 allele in carriers of MTHFR variants. Received: August 28, 2000 / Accepted: December 4, 2000     

Polymorphisms in <Emphasis Type="Italic">methionine synthase</Emphasis> (A2756G) and <Emphasis Type="Italic">cystathionine β-synthase</Emphasis> (844ins68) and susceptibility to carcinomas of the upper gastrointestinal tract

Purpose

Folate deficiency is considered to increase the risk for the development of malignant tumors such as prostate and colorectal cancer. Methionine synthase (MTR) and cystathionine ß-synthase (CBS) are enzymes that play a central role in folate metabolism, thereby affecting DNA methylation and synthesis. A single A→G substitution at nucleotide 2756 of the MTR and a 68 bp CBS insertion polymorphism in exon 8 have been associated with decreased enzyme activity. The purpose of this study is to compare the association of the MTR A2756G polymorphism and CBS insertion polymorphism with susceptibility to carcinomas of the upper gastrointestinal tract.

Methods

Using the restriction fragment length polymorphism (RFLP)-PCR, the prevalence of MTR A2756G and CBS insertion polymorphism was determined in healthy controls (n = 257) and in patients with esophageal squamous cell carcinoma (ESCC) (n = 263), Barrett’s esophagus-associated esophageal adenocarcinoma (BC) (n = 89), cardiac carcinoma (CC) (n = 144), or gastric carcinoma (GC) (n = 221) from German Caucasian subjects.

Results

No significant difference in MTR A2756G genotype distribution was observed between controls (A/A 66.9%, A/G 29.8%, G/G 3.3%) and patients with ESCC (A/A 61.7%, A/G 36.3%, G/G 2.1%), BC (A/A 69.2%, A/G 26.9%, G/G 3.9%), CC (A/A 51.8%, A/G 44.6%, G/G 3.6%), or GC (A/A 73.4%, A/G 20.9%, G/G 5.7%). Similarly, the CBS genotype (I: allele with 68 bp insertion; N: allele without insertion) distribution among German patients with ESCC (N/N 86.8%, I/N 13.2%), BC (N/N 90.2%, I/N 9.8%), CC (N/N 90.1%, I/N 9.9%) or GC (N/N 91.3%, I/N 8.7%) was not different from healthy controls (N/N 90.4%, I/N 9.6%). The gene allele constellation I/I was not present.

Conclusions

The current study suggests that there is no association between MTR A2756G polymorphism and the CBS (844ins68) insertion polymorphism and cancer of the upper gastrointestinal tract.

     

miR-30a-5p启动子区DNA甲基化在同型半胱氨酸致肝损伤中的作用

目的:探讨微小RNA-30a-5p(miR-30a-5p)启动子区DNA甲基化在肝损伤中的作用。方法:随机选取4周龄胱硫醚β-合成酶(CBS)基因正常(CBS+/+)小鼠(n=12)及单基因敲除(CBS+/-)小鼠(n=12),均给予高蛋氨酸饮食8周。HL-7702细胞体外常规培养,分为对照(control)组、同型半胱氨酸(Hcy)组和Hcy+5-氮杂胞苷(AZC)组。全自动生化分析仪检测小鼠血清Hcy、丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)水平;微板法测定肝细胞ALT和AST水平;小鼠肝脏石蜡切片行HE染色观察肝脏损伤情况;细胞活力染色检测肝细胞活力;RT-qPCR法检测小鼠肝脏组织和肝细胞中miR-30a-5p的表达;Pearson相关性分析肝脏miR-30a-5p表达与血清ALT和AST水平的相关性;巢式降落式甲基化特异性PCR(nMS-PCR)检测小鼠肝脏组织以及肝细胞中miR-30a-5p启动子区DNA甲基化水平的变化。结果:与CBS+/+对照组相比,CBS+/-组小鼠血清的Hcy、ALT和AST水平显著升高(P<0.05);HE染色显示肝细胞肿胀,可见核碎裂、溶解;肝脏miR-30a-5p表达明显降低(P<0.01);小鼠肝脏组织中miR-30a-5p的表达与血清ALT和AST的水平呈负相关(r2=0.4557,P=0.0003;r2=0.4626,P=0.0003);miR-30a-5p启动子区DNA的甲基化水平升高(P<0.01)。在细胞水平,与control组相比,Hcy组的ALT和AST水平升高(P<0.05,P<0.01),细胞活力显著降低,肝细胞miR-30a-5p启动子区DNA的甲基化水平升高(P<0.01),而使用AZC后miR-30a-5p启动子区DNA的甲基化水平降低(P<0.05),miR-30a-5p的表达上调(P<0.05)。结论:miR-30a-5p启动子区DNA高甲基化可能在肝损伤中发挥重要作用。     

内源性H2S与消化系统疾病

近年研究发现,内源性硫化氢（H2S）作为体内第三种具有生物学效应的气体信号分子,广泛参与了多种消化系统疾病的病理生理过程：继续探索内源性H2S的作用具有重要的理论和临床意义.本文对内源性H2S与消化系统疾病的关系作一综述。     

阿尔茨海默病患者同型半胱氨酸代谢相关酶基因突变频度的研究

目的　探讨胱硫醚β合成酶 (CBS)基因 84 4ins6 8、甲硫氨酸合成酶 (MS)基因A2 75 6G、亚甲基四氢叶酸还原酶 (MTHFR)基因C6 77T三种同型半胱氨酸代谢相关酶基因突变在阿尔茨海默病 (AD)发病中的意义 .方法　PCR扩增AD 6 6例及 14 3例对照者的CBS、MS、MTHFR基因突变点 ,直接或经限制性内切酶消化后行凝胶电泳确定其基因型 .结果　AD病人MTHFR基因中 ,基因型C/T占 5 6 .0 6 % ,明显高于对照组 (p <0 .0 1) ,C/C明显低于对照组 (p <0 .0 1) ,T/T与对照组无明显差异 (p>0 .0 5 ) .AD病人中MTHFR基因等位基因C的频率相对危险率 (RR) ,T的频率与对照组的差异有显著性 (p<0 .0 5 ) .CBS 84 4ins6 8、MSA2 75 6G各种基因型频率在AD组与对照组之间无差异 .结论　AD病人中MTHFR T频率明显高于正常人 ;而MTHFR C频率则明显低于正常人 ,而CBS 84 4ins6 8、MSA2 75 6G突变可能不足以构成阿尔茨海默病的独立遗传性危险因子     

胱硫醚-β-合成酶/硫化氢在非酒精性脂肪肝中的变化及意义

目的：观察脂肪肝中CBS/H2S的变化及其意义。方法用高脂饮食喂养SD 大鼠,建立非酒精性脂肪性肝模型,6周后处死动物,测其血清中的硫化氢,肝酶和血脂变化情况。取肝脏进行病理检查,ELISA法检测肝脏CBS及亚甲基蓝法测H2S的含量。结果模型组血清中TG、TC较对照组明显增加[（2.28±0.51）vs（1.18±0.85）]、[（4.5±1.25）vs （1.61±0.33）],（P<0.05）、H2S明显减少[（16.12±7.21）vs（20.91±10.30）],（P<0.05）;S-腺苷蛋氨酸干预组血清中TG、TC较模型组明显好转[（1.31±0.62）vs（2.28±0.51）]、[（3.2±0.66）vs（4.5±1.25）],（P<0.05）、H2S明显增加[（32.3±3.52）vs（16.12±7.21）],（P<0.01）;羟基胺干预组血清中TG、TC、H2S较模型组显著减少[（3.87±2.63）vs（2.28±0.51）]、[（5.0±1.01）vs（4.5±1.25）]、[（10.3±1.63）vs（16.12±7.21）],（P<0.01）。模型中CBS含量较对照组明显减少[（1.92±0.31）vs（4.12±0.21）],（P<0.05）、过氧化脂质明显增加[（78.2±7.56）vs（28.2±11.9）],（P<0.05）;与模型组比较S-腺苷蛋氨酸干预组CBS含量增加,过氧化脂质减少[CBS ：（2.96±0.78）、过氧化脂质：（34.2±3.68）],羟基胺干预组则CBS减少（1.32±0.17）、过氧化脂质增加（90.13±16.0）。肝脏脂肪变性与硫化氢的量呈反比。结论细胞膜损伤导致CBS外泄可能是非酒精性脂肪肝硫化氢减少的主要原因之一;激活CBS增加硫化氢可以改善肝内脂肪积聚。     

高分辨率熔解曲线法检测CBS基因SNP的技术优化

目的：探索高分辨率熔解曲线法（highresolutionmelting,HRM）检测胱硫醚β-合酶（CBS）基因SNP位点所需的最佳反应体系和条件,建立快速高效的对CBS基因分型的方法。方法通过普通PCR初步确立引物的退火温度范围,在实时荧光定量PCR（qPCR）-HRM反应中调整确定最佳退火温度。对影响qPCR-HRM的因素包括反应程序、模板DNA、MgCl2再分别进行优化,确立最佳反应体系和反应条件,对在此基础上得出的基因分型结果通过测序验证其准确性,从而建立系统的HRM检测CBS基因SNP的方法。结果HRM检测CBS基因该片段最佳退火温度为62℃,qPCR-HRM最佳反应体系为20μl,引物浓度为0．2μmol/L,Mg2＋为2．5μmol/L,模板DNA为60ng。在优化的体系条件下筛选出的突变型样本经测序验证与实验结果一致。结论HRM可以作为检测SNP准确、经济、高效的方法。     

胱硫醚-γ-裂解酶和胱硫醚-β-合成酶在高血压大鼠阴茎海绵体中的表达

目的:探讨高血压对大鼠阴茎海绵体组织胱硫醚-γ-裂解酶(CSE)和胱硫醚-β-合成酶(CBS)表达的影响及与大鼠勃起功能的关系。方法:健康雄性SPF级自发性高血压大鼠(SHR)与对照组WKY(Wistar-Kyoto)大鼠各10只,于12周龄时测定大鼠血清睾酮、阴茎海绵体内压/平均动脉压(ICP/MAP)、血浆和阴茎海绵体内源性H2S的含量,采用免疫组化和Western印迹分析CSE和CBS在阴茎海绵体内的表达。结果:SHR与WKY大鼠的血清睾酮值没有显著差别,SHR的ICP/MAP在0、3和5 V电刺激盆腔神经节(MPG)时均显著低于WKY组(n=10,P0.05)。SHR血浆H2S含量显著低于WKY大鼠[(10.49±1.35)μmol/Lvs(21.92±2.75)μmol/L,P0.05],SHR阴茎海绵体组织内源性H2S含量显著低于WKY大鼠[(52.60±3.44)nmol/mg prot vs(87.67±2.12)nmol/mg prot,P0.05],SHR阴茎海绵体组织内源性H2S生成率也较WKY大鼠显著降低[(1.14±0.07)nmol/(mg·min)vs(4.35±0.32)nmol/(mg·min),P0.05]。CSE及CBS主要表达在SHR和WKY大鼠阴茎海绵体平滑肌细胞和血管内皮细胞的胞质,SHR的CSE及CBS蛋白表达量均显著低于WKY大鼠(P0.05)。ICP/MAP与CSE和CBS表达呈高度正相关(r=0.955、0.977,P均0.05)。结论:高血压大鼠阴茎海绵体组织中CBS和CSE的表达下降,导致阴茎海绵体内H2S合成减少,可能是高血压引起勃起功能下降的机制之一。