The Somatic Mutation Of PIG-A Gene And The Expressions Of EGR-1 And WT1 Genes In The Bone Marrow Cells Of The Patients With Paroxysmal Nocturnal Hemoglobinuria

Objective:To investigate the somatic mutations of PIG-A gene and the expressions of EGR-1 and WT1 genes in the bone marrow cells of the patients with Paroxysmal nocturnal hemoglobinuria(PNH), then expore the characteristics of PIG-A gene mutation in chinese patients with PNH and the possible mechanism of PNH clonal expansion related to EGR-1 and WT1 genes.Methods:Twenty patients(16 PNH and 4 AA-PNH patients) and 10 healthy controls were enrolled in this study. The quantities of CD55- cells and CD59" cells in both peripheral blood and bone marrow were examined with flow cytometric assay(FCM), and then sorting CD59- cells from bone marrow mononuclears(BMMNCs).The mutant segments of PIG-A gene were identified by DNA sequencing; The expressions of EGR-1 and WT1 mRNA in CD 59" and CD59+ BMMNC were measured with semiquantitive RT-PCR.Results:1. Nine in 16 patients with PNH and lin 4 patients with AA-PNH were measured for their PIG-A gene mutation. Five single base substitution were found, including A→G,G→T,A→T,T→G and G→C, resulting in one consense and seven missense mutations. Each of 3 PNH patients had more than two point mutations of PIG-A gene. Insert or deletion mutation of PIG-A gene were not found. The rates of PIG-A gene mutation above-mentioned was 30.0%(3/10),20.0%(2/10),10.0%(1/10),10.0%(1/10) and 10.0%(1/10), respectively. The proportion of CD59- cells is higher in the patients with PIG-A gene mutation in BMMNC than those without mutation, and the proportion of CD59- cells is also higher with A→G than those of patients with other mutations.2. The relative mRNA expressions of EGR-1 in CD59- BMMNC, CD59+ BMMNC of PNH or AA-PNH patient and BMMNC of normal controls were 1.00±0.11,0.86±0.14 and 0.85±0.06, the expression in CD59- BMMNC was significiantly higher than that in CD59+BMMNC or normal controls (p<0.05). There was no significant difference between the expression in CD59+BMMNC and normal controls (p>0.05).The relative mRNA expressions of WT1 in CD59- BMMNC, CD59+ BMMNC of PNH and AA-PNH patients and BMMNC of normal controls were 1.06±0.16,0.90±0.14 and 0.85±0.06, the expression of in CD59- BMMNC was significiantly higher than that in CD59+ or normal controls BMMNC (p<0.05), and there was no significant difference between CD59+ and normal controls BMMNC (p>0.05). The relative mRNA expressions of EGR-1 and WT1 in BMMNC of PNH and AA-PNH cases were positively correlated with the proportion of CD59-cells(coefficient correlation was 0.509 and 0.481 respectively, p<0.05), but not related to the proportion of CD59+ cells.Conclusions:The PIG-A gene mutation in some Chinese patients mainly manifested as single base substitution, occurred at random sites and without hot spot, the overexpression of EGR-1 and WT1 genes in PNH clone could promote the abnormal clone expansion.