Mutation analysis of Pig-A gene in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by the expansion of an hemopoietic cell clone unable to produce the glycosyl-phosphatidyl inositol (GPI) anchor. A variety of mutations in the PIG-A gene has been so far detected in this disease. Although there is no specific treatment, recombinant human erythropoietin (rHu-EPO) has been reported to be effective in some cases. However, is not clear whether the possible beneficial effect of rHu-EPO is due to the stimulation of the normal or the GPI defective erythroid clone. The aim of this study was to investigate the molecular defect in 13 PNH patients, one of whom spontaneously recovered from the disease. Moreover, in one patient, the PIG-A negative clone was monitored by quantitative-competitive PCR during rHu-EPO treatment. The diagnosis of PNH was made by Ham's and sucrose hemolysis tests and by red cell CD55 and CD59 antigens determination. The study of the entire codifying region and flanking intronic sequences of PIG-A gene was done by SSCP analysis and sequencing. The DNA sequence revealed the presence of 9 new and one known mutations. In three patients no abnormalities were detected. We found 7 small deletions (del A231, del C243. del 258-264, del G342, del T689, del T774, del 1251-1254), one 7 nt duplication (dupl 604-610), and two missense mutations (T728C, new and C55T, already reported in PNH). This latter was found in the spontaneously recovered patient and in another case in combination with T728C. C55T was also detected in the mucous membrane cells and in one out of 50 normal controls, thus confirming its polymorphic nature. Whether it may represent a predisposing factor for PNH remains to be elucidated. Moreover, in one patient (del T689) undergoing rHu-EPO treatment, a specific primer for the mutation was prepared and used for quantitative-competitive PCR. The increase in hemoglobin levels (from 7.3 to 9 g/dL) was related with an increase of PIG-A negative molecules (from 102 to 5x 10' in 100 ng DNA) and of CD55'/CD59 cells (from 53 to 98%), suggesting that in this patient the efficacy of rHu-EPO therapy was connected with the stimulation of the abnormal clone.