Barbaux S, Kluijtmans L A, Whitehead A S
Department of Pharmacology and Center for Pharmacogenetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Clin Chem. 2000 Jul;46(7):907-12.
Hyperhomocysteinemia, which is often associated with low folate status, is an independent risk factor for cardiovascular diseases and several other pathologies. The four most common functional polymorphisms in genes involved in folate/homocysteine metabolism are methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, methionine synthase (MS) A2756G, and cystathionine beta-synthase (CBS) 844ins68. The pathogenic impact of these variants is under active investigation in many laboratories. However, conventional genotyping methods, mostly using PCR followed by restriction enzyme digestion, often are compromised by partial fragment digestion. There is, therefore, a need to develop more reliable approaches to genotyping the above polymorphisms that may be applied in large-scale studies.
Sequence-specific heteroduplex generators for each of the MTHFR and MS single nucleotide polymorphisms were generated by site-directed mutagenesis. These were subcloned into a single construct, pHcyHG-1, which could be multiplexed with a simple PCR amplification across the CBS 844ins68 polymorphic site to generate composite genotype-specific banding patterns from individual genomic DNA samples that could be electrophoretically resolved.
The "multiplex heteroduplexing" method yielded unambiguous MTHFR, MS, and CBS genotypes in a single-tube reaction that could be analyzed in a single gel run.
This method permits unambiguous genotyping of the four most common functional variants of enzymes involved in folate/homocysteine metabolism. It is rapid, reproducible, and inexpensive, and requires no special preparative or analytic facilities; consequently, it will facilitate large-scale studies of the genetic basis of hyperhomocysteinemia and the many pathologies that have been associated with this phenotype.
高同型半胱氨酸血症常与低叶酸状态相关，是心血管疾病和其他几种病症的独立危险因素。参与叶酸/同型半胱氨酸代谢的基因中，四种最常见的功能多态性为亚甲基四氢叶酸还原酶（MTHFR）C677T和A1298C、甲硫氨酸合成酶（MS）A2756G以及胱硫醚β-合成酶（CBS）844ins68。许多实验室正在积极研究这些变异的致病影响。然而，传统的基因分型方法大多采用聚合酶链反应（PCR）后进行限制性酶切，常因片段消化不完全而受到影响。因此，需要开发更可靠的方法来对上述多态性进行基因分型，以便应用于大规模研究。
通过定点诱变生成针对MTHFR和MS每个单核苷酸多态性的序列特异性异源双链体生成器。将它们亚克隆到单个构建体pHcyHG-1中，该构建体可与跨越CBS 844ins68多态性位点的简单PCR扩增进行多重反应，从个体基因组DNA样本中生成可通过电泳分辨的复合基因型特异性条带模式。
“多重异源双链化”方法在单管反应中产生明确的MTHFR、MS和CBS基因型，可在一次凝胶电泳中进行分析。
该方法能够对参与叶酸/同型半胱氨酸代谢的酶的四种最常见功能变异进行明确的基因分型。它快速、可重复且成本低廉，无需特殊的制备或分析设备；因此，它将有助于对高同型半胱氨酸血症的遗传基础以及与该表型相关的许多病症进行大规模研究。
