High-throughput Sequencing-based Exploration Of Pathogenic Genes Associated With Paroxysmal Nocturnal Hemoglobinuria

ObjectivesThis study intended to analyze the incidence of mutations in myeloid cancer candidate genes in Paroxysmal nocturnal hemoglobinuria(PNH)and explore the mechanism concerning the clonal expansion and hypercoagulability of PNH from the aspect of genetics.MethodsWe conducted whole-exome sequencing(WES)of the peripheral blood samples collected from 41 patients diagnosed with PNH,6 of which conducted further WES after separating PNH+and PNH-fractions with magnetic beads to explore if the mutations concurred with PIGA.The demographic information and laboratory results were collected from the Electronic Medical Record System.1.178 genes commonly mutated in myeloid cancer were analyzed in our study.The correlation of mutation with clinical presentation and results of laboratory test were examined.Furthermore,the existence of mutations was checked in the WES data from PNH+and PNH-fractions.2.We analyzed the existence of known mutation related to thrombosis.And we compared the difference of mutation between subjects with and without thrombosis.Sequencing following cell sorting was conducted to see the distribution of mutation in subgroup of cells.Results1.The average mutation load is 3.85.The mutations in myeloid cancer candidate genes in PNH+and PNH-fractions are the same except for PIGA.The most commonly mutated genes are MAP3K4 and CSMD1(12.5%).Patients with RUNX1T1 mutation were found to have larger PNH clone,severer hemolysis and lower hemoglobin level.The mutation rate is negatively correlated with PNH clone size of genes associated with worse outcome in AA(DNMT3A?RUNX1?JAK2?JAK3?CSMD1).Sample from six patients went through WES after cell sorting.Mutations with potential selective advantage are more prevalent in PNH+fractions compared with PNH-fractions.2.13 out of the 41 PNH patients experienced thrombosis events,4 of which had more than one event.Coronary artery,visceral vein and deep vein are the most common sites of thrombosis.Common inherited hypercoagulable states(Factor V Leiden mutation,prothrombin gene mutation,deficiency of protein S/protein C/antithrombin)do not happen more frequently in PNH patients.A total of 55 candidate genes were found to have a significant higher mutation rate in thrombosis group compared with non-thrombosis group.These genes participate in several pathways such as signaling,transcription,transportation,immune reaction,ubiquitination,and metabolism.These mutations exist both in CD59+ and CD59-cells.Further analysis revealed that SRRD and EGR4 mutation is associated with visceral thrombosis and myocardial infarction,respectively.The mutation load of thrombosis candidate genes is negatively correlated with PNH clone size.Conclusion1.Mutations in myeloid cancer candidate genes also exist in PNH.The mutation rate of genes associated with worse outcome in AA is negatively correlated with PNH clone size.Additional events like second mutation with selective advantage may be the driver event of the expansion of PNH clone.2.PNH patients do have a higher frequency of mutation in some thrombosis candidate genes.SRRD and EGR4 mutation is associated with visceral thrombosis and myocardial infarction.Patients with small PNH clone who suffered from thrombosis events may have a heavier mutation load of thrombosis candidate genes.