Abnormalities of PIG-A Transcripts in Granulocytes from Patients with Paroxysmal Nocturnal Hemoglobinuria

Toshio Miyata, Norio Yamada, Yoshiyasu Iida, Junichi Nishimura, Junji Takeda, Teruo Kitani, Taroh Kinoshita

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203 Citations (Scopus)


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., Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder of blood cells in which the affected cells originate from an abnormal hematopoietic stem cell; the condition is characterized by nocturnal hemoglobinuria, chronic hemolytic anemia, and thrombosis1,2. Complement-mediated hemolysis is a prominent feature of the disease and is due to deficient cell-surface expression of decay-accelerating factor (CD55) and CD59, which protect blood cells from the action of complement3. The fundamental cause of these deficiencies is defective synthesis of the molecules of glycosyl-phosphatidylinositol that anchor these and many other proteins to the cell membrane4,5. We have shown that…

Original languageEnglish
Pages (from-to)249-255
Number of pages7
JournalNew England Journal of Medicine
Issue number4
Publication statusPublished - 1994 Jan 27


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