Miyata T, Yamada N, Iida Y, Nishimura J, Takeda J, Kitani T, Kinoshita T
Department of Immunoregulation, Osaka University, Japan.
N Engl J Med. 1994 Jan 27;330(4):249-55. doi: 10.1056/NEJM199401273300404.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder in which there is a deficiency in the synthesis by hematopoietic cells of the glycosyl-phosphatidylinositol molecules that anchor proteins to the cell membrane. Recently, we demonstrated that a gene termed PIG-A (for phosphatidylinositol glycan class A), a component of glycosyl-phosphatidylinositol biosynthesis, was responsible for PNH in two patients. The present study was undertaken to elucidate whether PIG-A is the gene responsible for all cases of PNH and to characterize further the somatically acquired abnormalities of this gene.
We studied granulocytes from 15 patients with PNH. The cell content of CD55 and CD59 was assessed by fluorescence-activated flow cytometry. PIG-A transcripts were reverse-transcribed, amplified by the polymerase chain reaction, and cloned into plasmids. The structure of the cloned complementary DNA was analyzed by nucleotide sequencing, and its function was assessed on the basis of its ability to restore to normal the abnormal phenotype of a PIG-A-deficient cell line after transfection.
Three patients had size abnormalities of PIG-A transcripts with different patterns, and in one patient a very low level of the PIG-A transcript was found. Eleven patients had transcripts of normal size, but the transfection assay revealed that in each patient some of them were nonfunctional. The percentage of nonfunctional PIG-A transcripts was correlated with the percentage of affected granulocytes (P < 0.001). Sequence analysis demonstrated somatic mutations in two of the patients.
PIG-A is the gene responsible for PNH in all patients studied to date.
阵发性睡眠性血红蛋白尿症（PNH）是一种获得性疾病，造血细胞合成将蛋白质锚定到细胞膜的糖基磷脂酰肌醇分子存在缺陷。最近，我们证明了一个名为PIG - A（磷脂酰肌醇聚糖A类）的基因，作为糖基磷脂酰肌醇生物合成的一个组成部分，是两名患者PNH的病因。本研究旨在阐明PIG - A是否是所有PNH病例的致病基因，并进一步表征该基因体细胞获得性异常情况。
我们研究了15例PNH患者的粒细胞。通过荧光激活流式细胞术评估CD55和CD59的细胞含量。PIG - A转录本进行逆转录，通过聚合酶链反应扩增，并克隆到质粒中。通过核苷酸测序分析克隆的互补DNA的结构，并根据其转染后恢复PIG - A缺陷细胞系异常表型至正常的能力评估其功能。
3例患者的PIG - A转录本存在不同模式的大小异常，1例患者发现PIG - A转录本水平极低。11例患者的转录本大小正常，但转染试验显示，每位患者中一些转录本无功能。无功能PIG - A转录本的百分比与受影响粒细胞的百分比相关（P < 0.001）。序列分析显示2例患者存在体细胞突变。
PIG - A是迄今为止所有研究患者PNH的致病基因。
