DNA diagnosis of pyruvate dehydrogenase deficiency by PCR-SSCP analysis

丙酮酸脱氢酶（PDH）复合物缺陷是导致先天性乳酸血症的主要原因。大多数PDH复合物缺乏的病例是由PDH a亚基（Ela）突变引起的。Ela的基因位于X染色体上。Ela缺乏的诊断通常是通过测量培养细胞的PDH复合物活性来建立的。然而，当培养细胞中正常X染色体主要外露时，杂合型Ela缺乏症女性患者被误诊。因此，为了可靠地诊断女性患者Ela缺乏症，有必要确定潜在的基因突变。然后，我们采用PCR-SSCP和直接测序的方法对Ela缺乏症进行DNA诊断。我们检查了11例患有先天性乳酸血症的女性患者，通过测定培养细胞的酶活性，我们没有发现任何缺陷。与对照组相比，11名女性患者中有3名有异常的迁移模式。3例患者中有2例发生错义突变，导致Ela亚基（G89S和G291R）氨基酸残基改变。因此，我们认为PCR-SSCP分析和直接测序在女性乳酸血症患者中是诊断Ela缺乏症的有效方法。

Defect in pyruvate dehydrogenase(PDH) complex is a major cause of congenital lactic acidemia. Most cases with PDH complex deficiency result from a mutation in the PDH a-subunit (Ela). The gene for Ela is located on the X chromosome. The diagnosis for Ela deficiency is usually estabrished by the measurement of PDH complex activity of the cultured cells. However, the heterozygous female patients with Ela deficiency are misdiagnosed, when the normal X chromosome is predominantly exoressed in the cultured cells. Therefore, for reliable diagnosis of Ela deficiency in female patients, it is essential to define the underlying gene mutation. Then, we used the method of PCR-SSCP and direct sequencing for DNA diagnosis of Ela deficiency. We examined 11 female patients with congenital lactic acidemia, in whom we could not find any defect by the measurement of the enzyme activity of their cultured cells. Three of the 11 female patients had abnormal migration patterns compared with controls. Two of the 3 patients had missense mutations resulting in a changed amino acid residue in the Ela subunit (G89S and G291R). Then, we concluded that PCR-SSCP analysis followed by direct sequencing in female lactic acidemic patients was a useful method for the diagnosis of Ela deficiency.