GENETIC RISK FACTORS FOR HYPERHOMOCYSTEINEMIA

高同型半胱氨酸血症的遗传风险因素

基本信息

批准号：
6043997
负责人：
Roy A GRAVEL
金额：
$ 17.5万
依托单位：
UNIVERSITY OF CALGARY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-08-01 至 2001-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (Adapted from applicant's abstract) A common amino acid polymorphism of methylenetetrahy-drofolate reductase is associated with hyperhomocysteinemia and predisposition to cardiovascular disease. It is hypothesized that mutations of other enzymes of homocysteine metabolism may have similar impact. The focus of this study will be on two enzyme systems of homocysteine remethylation. One is methionine synthase (MS) which requires two gene products: the MS apoenzyme (deficient in cblG disorder) and a reducing system associated with MS function (cbIE disorder). The second is betaine-homocysteine methyltransferase (BHMT). The investigators have cloned cDNAs for human MS and BHMT and identified mutations and a polymorphism of the MS apoenzyme. Their specific aims are: 1) Identify amino acid polymorphisms in the MS (cblG, cbIE) and BHMT genes (cbIE to be cloned). This will be performed by SSCP analysis for the detection of mutations from RT-PCR or genomic DNA PCR products. 2) Clone the cbIE cDNA by functional complementation or homology-based RT-PCR (used successfully for the MS cbIG cDNA cloning). The complementation method will involve transformation of an E. coli methionine auxotroph, already expressing human MS, with a human cDNA library. Successful complementation will require the cblE gene product to permit growth in methionine-free medium. The homology method makes use of conserved sequences identified between flavodoxin/flavodoxin reductase of prokaryotes and eukaryotic flavoproteins to specify degenerate oligonucleotides for RT-PCR based cloning. 3) Express polymorphisms in human cell or E. coli expression systems to assess impact of mutations. Mutant fibroblasts will be the recipient for cDNA-mediated complementation assays. Expression of cDNAs in E. coli will permit biochemical analysis of purified protein. These studies will include introducing mutations into E. coli MS for analysis in conjunction with the flavodoxin system. 4) Examine impact of polymorphisms on homocysteine and nutrient levels. Identified polymorphisms will be screened in human subjects to determine if mutations are associated with abnormal homocysteine or nutrient (folate, B12) levels. Evaluate impact of polymorphisms on cardiovascular disease. Coronary artery disease patients will be evaluated for genotype status for polymorphisms shown to be associated with altered metabolities to determine if genotype frequencies vary from control groups. These studies, will determine the importance of genotype in the metabolic causes of hyperhomocysteinemia and risk of cardiovascular disease. (End of Abstract)

描述（改编自申请人的摘要）甲基环甲基二酸酯还原酶与高脑膜半胱氨酸血症有关心血管疾病的易感性。假设同型半胱氨酸代谢的其他酶的突变可能具有相似的影响。这项研究的重点将放在两个酶系统上同型半胱氨酸二甲基化。一个是需要蛋氨酸合酶（MS）两个基因产物：MS载酶（缺乏CBLG疾病）和A 减少与MS功能相关的系统（CBIE障碍）。第二个是甜菜碱 - 荷囊苷甲基转移酶（BHMT）。调查人员有克隆的人类MS和BHMT的CDNA，并确定了突变和A MS载酶的多态性。他们的具体目的是：1）确定 MS（CBLG，CBIE）和BHMT基因（CBIE）中的氨基酸多态性克隆）。这将通过SSCP分析来检测来自RT-PCR或基因组DNA PCR产物的突变。 2）克隆CBIE cDNA 通过功能互补或基于同源的RT-PCR（成功使用对于MS CBIG cDNA克隆）。互补方法将涉及大肠杆菌蛋氨酸型菌群的转化，已经表达了人类 MS，带有人类cDNA库。成功的补充将需要 CBLE基因产物允许无蛋氨酸培养基生长。同源性方法利用了确定的保守序列原核生物和真核生物黄蛋蛋白的黄素毒素/黄霉素还原酶为基于RT-PCR的克隆指定退化寡核苷酸。 3）表达人类细胞或大肠杆菌表达系统中的多态性以评估影响突变。突变的成纤维细胞将成为cDNA介导的接受者互补分析。大肠杆菌中cDNA的表达将允许纯化蛋白质的生化分析。这些研究将包括将突变引入大肠杆菌MS，以进行分析黄霉素系统。 4）检查多态性对同型半胱氨酸和营养水平。确定的多态性将在人类中进行筛查受试者确定突变是否与异常同型半胱氨酸有关或营养（叶酸，B12）水平。评估多态性对心血管疾病。将评估冠状动脉疾病患者用于与改变有关的多态性的基因型状态确定基因型频率的代谢是否因对照组而异。这些研究将确定基因型在代谢中的重要性高层结晶血症的原因和心血管疾病的风险。（结束抽象的）