Genetic Analysis of Lactate Dehydrogenase (LDH)-H(B) Subunit Variant

在人乳酸脱氢酶（LDH）H（B）亚基中发现了9种基因突变，包括2种转换、4种颠换和3种缺失/重复。采用聚合酶链反应（PCR）-DNA构象多态性（DCP）分析LDH突变基因。我们使用聚丙烯酰胺梯度凝胶和银染程序进行DCP分析，并观察到LDH-H变异体在个体中的异常迁移模式。突变等位基因的进一步序列测定一致地导致碱基置换和缺失/重复的检测。用错配PCR方法或扩增难治突变系统（ARMS）检测点突变。这些错配PCR方法和ARMS可以方便、准确地检测这些错义突变。这些方法不需要放射性同位素或繁琐的杂交程序，可以轻松进行任何分子诊断和家族分析。导致LDH缺陷突变的四个错义突变位于对LDH分子功能不可或缺的保守序列上，例如P轴稳定性、NAD结合和底物结合。1例电泳变异体经鉴定为第6位密码子的错义突变。该残基不埋在亚基分子中，而是位于蛋白质表面。我们应用蛋白质数据库的计算机模型预测了导致LDH基因突变的点突变的位置和功能，其中一个无义突变、两个重复突变（8bp和4 bp）和一个1bp缺失突变导致了LDH的提前终止。由于提前终止诱导四聚体形成的严重扰动和不稳定性，酶缺乏可能主要是由于更快的体内变性和降解的变体酶。除3例LDH-H基因第4外显子有错义突变外，其余突变均不同。

We demonstrated 9 kinds of genetic mutations including 2 transitions, 4 transversions and 3 deletion/duplications, in human lactate dehydrogenase(LDH) H(B) subunit. We analyzed LDH mutant genes by polymerase chain reaction(PCR)-DNA conformation polymrphism(DCP). We used polyacrylamide gradient gel and silver staining procedures for DCP analysis and observed abnormal migration patterns in individuals for LDH-H variant. Further sequence determination of mutant alleles consistently resulted in detection of base substitutions and deletion/duplications. Then, we used mismatched PCR method or amplification refractory mutation system (ARMS) for detection of the point mutations. These mismatched PCR methods and ARMS could be easily and clearly detect these missense mutations. These methods do not require radioisotopes or a cumbersome hybridization procedure and can be easily performed any molecular diagnosis and family analysis.Four missense mutations causing LDH deficient mutations are located at conserved sequences which are indispensable for the function of LDH molecules, such as stability of P-axis, NAD-binding and substrate-binding. A case of electrophoretic variant was identified to be owing to missense mutation located at codon 6. This residue is not buried in the subunit molecules but located at protein surface. We applied a computer model based upon Protein Data Bank to predict the location and function of point mutations causing genetic mutations of LDH.A nonsense mutation, two duplication mutations(8bp and 4bp) and a 1bp deletion mutation resulted in premature terminations. Because the premature termination induces serious perturbation and instability in tetramer formation, the enzyme deficiency may be due principally to more rapid in vivo denaturation and degradation of the variant enzyme. These mutations are different from each other except for a missense mutation in exon 4 of LDH-H gene (3 cases).