Diagnosis and molecular analysis of red cell enzyme anomalies and analysis of red cell deformabilities

通过对117例遗传性非球形溶血性贫血患者的酶学分析，发现葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症16例，丙酮酸激酶（PK）缺乏症7例，磷酸果糖激酶缺乏症1例，磷酸甘油激酶缺乏症1例。由于糖酵解酶缺陷，PK缺乏症是与遗传性非球型溶血性贫血相关的最常见的红酶病。通过对5个日本缺钾家族的分析，我们确定了三个突变（664- 6in GAC, 1468T, 1436A）。此外，我们在一名韩国男孩中发现了新的纯合突变（1231A），这是韩国首次在基因水平上确定PK缺乏分子异常。G6PD A是非洲人常见的G6PD变异，可引起急性溶血，由感染和某些药物以及蚕豆引起。这种表型可以由常见的376G突变和另外3种突变中的任何一种组合引起：202A、680T或968T。我们发现了一个日本男孩G6PD缺乏症与急性溶血。利用PCR-SSCP分析结合直接测序，我们发现了一个错义突变202A，但未能找到对应的376G突变或任何其他突变。我们的结论是，人类G6PD基因的单个突变202A可自行引起急性溶血。我们使用微通道阵列在单个细胞水平上检测了正常和缺钾条件下红细胞的变形能力。我们无法获得小于6 μm的微通道。因此，不可能在生理条件下证明atp缺乏状态下红细胞变形能力降低的明显结果。

We discovered 16 cases of glucose-6-phosphate dehydrogenase (G6PD) deficiency, 7 cases of pyruvate kinase (PK) deficiency, a case of phosphofructokinase deficiency, and a case of phosphoglycerate kinase deficiency by the enzymatic analysis of 117 cases associated with hereditary non-spherocytic hemolytic anemia.PK deficiency is the most common erythroenzymopathy associated with hereditary non-spherocytic hemolytic anemia due to a glycolytic enzyme defect. We determined three mutations (664-6 ins GAC, 1468T, 1436A) by the analysis of 5 Japanese PK-deficient families. In addition, we elucidated the new homozygous mutation (1231A) among a Korean boy, which is the first Korean PK deficiency determined the moleculer abnormalities at the gene level.G6PD A is a common G6PD variant among Africans that may cause acute hemolysis triggered by infections and certain drugs, as well as by fava beans. This phenotype can be caused by a combination of the common 376G mutation and either of 3 additional mutations: 202A, 680T, or 968T. We discovered a Japanese boy of G6PD deficiency associated with acute hemolysis. Using PCR-SSCP analysis combined with direct sequencing, we identified a missense mutation 202A, but failed to find the counterpart 376G mutation or any additional mutation. We concluded that a single mutation 202A in the human G6PD gene can cause acute hemolysis by itself.We examined the red cell deformabilities between normal and PK-deficient conditions at a single cell level using the microchannel array. We could not obtain the microchannel less than 6 μm. Therefore, it was impossible to demonstrate the apparent result of the reduced red cell deformabilities under the ATP-deficient state at the physiological condition.