Possible Risk Factors of Thrombosis in Chinese Patients with Paroxysmal Nocturnal Hemoglobinuria

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease caused by an acquired mutation of the X-linked PIGA gene on the hematopoietic stem cell. Thrombosis is one of the most common causes of mortality in PNH, but the predisposing factors for thrombosis are yet to be defined. In this study, we outline the clinical characters of thrombotic PNH and detect the susceptible genes which may lead to thrombotic formation.

Methods: There were totally 94 patients with PNH diagnosed between 2011 and 2015 enrolled in the study. Clinical data like sex, age, hemoglobin level, reticular cell percentage, white blood cell and platelet count, LDH level, CD59- and FLAER granulocytes percentage, thrombophilia risk factors like level of protein C, protein S, antithrombin III, APC resistance, blood fat, phospholipid antibody were evaluated. Samples from patients were genotyped for the reported 33 alleles in 21 genes including MTHFR, PROC, PROS, F2, F5, ABO, prothrombin and other genes which are reported as high risk factors for venous thromboembolism (VTE) by polymerase chain reaction fragment length polymorphism methods (PCR-RFLP). Furthermore, we detected plasma VIII factor and vWF levels which were affect by ABO polymorphism.

Results:Of the 94 PNH patients, 16 (17%) patients had at least 1 episode of thrombotic event. Only 2 patients had arterial thrombosis and 14 patients had venous thrombosis. The medium age of patients with thrombosis was 42-year old, similar to those without (43-year-old, p=0.199). Male : female ratio was 1.29 in thrombosis group and 1.16 in non-thrombosis group (p=1.000). Although there was no difference in level of hemoglobin, white blood cell count, platelet count, reticular cell count, LDH, protein C, protein S, antithrombin III, APC resistance, blood fat, phospholipid antibody level (p>0.05) between patients with thrombosis and those without, the percentage of CD59- erythrocytes( p=0.001 ), CD59- granulocytes ( p=0.004 ) and FLAER- granulocytes (p=0.003) was higher in thrombotic patients. Patients with the TT genotype (rs495828 in the ABO gene) were approximately 3.03 folds prone to thrombus formation than those with the GG genotype (p=0.0204 ), and patients with the TC genotype (rs2519093 in the ABO gene) were approximately 4.24 folds prone to thrombus formation than those with the CC genotype ( p=0.0352 ). In addition, minor allele frequencies of rs495828 or rs2519093 and CD59- erythrocytes were independent risk factor for thrombosis in PNH patients. No association was detected between other SNPs and risk to thrombosis. There was no difference in level of vWF-Ag (156% vs 152%, p=0.870) and VIII factor (190% vs 156%, p=0.198) between patients with or without thrombosis, although the level was much higher in PNH patients than normal controls (178% vs 107%, p=0.005 and 203% vs 128%, p=0.003 respectively).

Conclusion: Compared with non-thrombotic patients, thrombotic PNH patients have have bigger PNH clone. And for the first time, our results suggested that the rs495828/rs2519093 in the ABO gene confers risk to thrombosis in PNH. Therefore, rs495828/rs2519093 polymorphism may represent a potential genetic biomarker in PNH patients for thrombus formation.