HETEROGENEOUS PIG-A MUTATIONS IN DIFFERENT CELL LINEAGES IN PAROXYSMAL-NOCTURNAL HEMOGLOBINURIA

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect of hematopoietic stem cells in which affected cells are characterized by the lack of glycosylphosphatidylinositol (GPI)-anchored proteins. The lesion in PNH lies in the defective synthesis of N-acetyl-D-glucos aminyl-phosphatidylinositol (GlcNAc-PI), the first intermediate in GPI biosynthesis. Reintroduction of the PIG-A gene into GPI-patient cells reportedly complements this defect. We have analyzed here PIG-A trans cripts of six PNH patients. GPI(+) and GPI(-) cell lines from each ind ividual were used, ie, Epstein-Parr virus-transformed B-lymphoblastoid cell lines, T-cell lines, and natural killer cell clones. Reverse tra nscriptase polymerase chain reaction and sequencing showed three diffe rent PIG-A splicing variants in GPI(+) cell lines, in which the larges t transcript contained the wild-type PIG-A coding region sequence. GPI -deficient cell lines showed abnormal splicing variants. Sequencing of PIG-A complementary DNA and genomic DNA showed heterogeneous mutation s ranging from different point mutations to small deletions. Two lymph ocyte cell lines (T- and B-cell lines) of one patient presented with t he same mutation. For another patient, two different mutations were de tected in one natural killer cell line. Therefore, different cell line ages have somatic mutations in PIG-A that lead to PNH. (C) 1995 by The American Society of Hematology.