Cloning of a GPI-anchor synthsis gene, PIG-A and characterization of its clinical significanece

点击翻译按钮获取中文摘要

1.Molecular basis of paroxysmal nocturnal hemoglobinuria (PNH). Two events are essential for PNH ; 1) Somatic mutation of PIG-A in a hematopoietic stem cell (s) and 2) Clonal expansion of the mutant cells. We analyzed 63 patients and found PIG-A abnormalities in all, establishing that PIG-A is responsible for GPI deficiency in most if not all patients. The mutations were distributed widely within the gene, indicating random occurrence. About 20% of patients bore two or more mutant clones. So, PNH is an oligoclonal disease. PIG-A is located on the X-chromosome. This would be a basis of the uniformity of the responsible gene. In fact, we demonstrated that PIG-F and -B are autosomal. To test if PIG-A mutation causes the clonal expansion as well, we knocked out mouse Pig-a in the male embryonic stem cells and generated chimeric mice using these GPI deficient cells. Six were chimeric in hematopoietic cells, having 0.5-4% of GPI deficient erythrocytes. Proportions of the mutant erythrocytes did not increase for five to ten months, suggesting that other factor (s) may be required for clonal expansion. The mutant erythrocytes began to increase in one chimera at twelve months of age and reached to 30% at seventeen months. We are going to generate more chimeras to reproduce this.2.Structure and function of GPI-anchor synthesis genes. We cloned six new mammalian genes. With three previously cloned ones, nine genes are now available. We found that PIG-A and -H proteins both involved in the first step form a complex on the cytoplasmic side of the ER,and that PIG-B protein that is involved in transfer of the third mannose functions on the luminal side of the ER.These results partially clarified membrane topology of GPI synthesis.