The Mechanism Of Second Gene Mutation Promoting Clonal Proliferation In Paroxysmal Nocturnal Hemoglobinuria

Objective:To evaluate the clinical data of patients with Paroxysmal nocturnal hemoglobinuria(PNH)and evaluate the therapeutic efficacy and prognostic factors of glucocorticoids and chemotherapy.To explore the PIG-A gene mutation and other gene mutations beyond the PIG-A gene by whole exon sequencing technology,screening possible pathogenic mutations and studying its role in the proliferation of PNH clones,and preliminarily explore the possible mechanism of PNH clone to gain the proliferation advantage.Methods:1.The clinical data of 92 PNH patients in Department of Hematology of Tianjin Medical University General Hospital were analyzed retrospectively.The clinical features and common complications of PNH patients were summarized.The therapeutic effects of glucocorticoid and chemotherapy were observed and the risk factors affecting survival and prognosis were analyzed,including the correlation between the LDH level,the PNH clones size,combination of thrombus,combination of bone marrow failure,the frequency of hemolytic seizures and the prognosis.2.A total of 13 patients were enrolled,immunomagnetic beads were used to separate CD59~-cells from peripheral blood granulocytes,genomic DNA of CD59~-cells was extracted and sequenced by whole genome exon sequencing technology,and biological information processing,analysising and screening of pathogenic mutation genes were performed on the original sequencing data.The mutation sites,mutation patterns and mutation rates of PIG-A gene were analyzed;Cluster analysis and mutation cluster analysis were performed for other than PIG-A genes and thrombus related genes selected from above.Meanwhile,KEGG signal pathway enrichment analysis was performed.3.K562 cells were cultured in vitro and the proportion of PNH clones in K562cells and PNH patients was detected by flow cytometry,the expression level of the pathogenic mutation gene mRNA screened in the above cells was detected by qPCR,The expression of RBPJ with high expression and high abundance was screened out.The expression of RBPJ gene was silenced by siRNA technology,and the cell proliferation level before and after silencing was detected by CCK-8,cell cycle and apoptosis rate were detected by flow cytometry.Results:1.The clinical data of 92 patients with PNH were summarized:the clinical manifestations of PNH included hemoglobinuria(61.73%),anemia(88.89%),hemorrhage(14.81%)and pancytopenia(20.99%).Complications included infection(35.87%),thromboembolism(13.58%)and renal function impairment(16.05%).The total one year effective rate of glucocorticoid treatment for PNH was 70.37%.After 2to 4 weeks of treatment,the hemoglobin level increased significantly,while the levels of RET,LDH and TBIL decreased,showing a good treatment response.Patients with relapsed or refractory PNH or severe hormone dependent and severe complications were treated with DA or HA regimen,the hemolysis index of PNH patients was obviously improved and the PNH clones decreased significantly.All the patients were divorced from blood transfusion and the dosage of adrenocorticosteroids was reduced by more than half of those before chemotherapy.The total survival period of 10 years after diagnosis was 70.09%.The significant increase of LDH,complication of thrombosis and combined with bone marrow failure were all adverse factors of lower10 year survival rate.2.Detection of gene mutation in PNH cloned cells:(1)The whole exon was sequenced in 7 PNH and 6 PNH-AA patients.The average depth was 500×,about95%of the exon area was over 100×.The results of the raw data included 191059single nucleotide variants and 24404 insertion/deletion mutations,among them including 29364 nonsynonymous mutations,7936 truncated mutations,893 frameshift mutations,and 465 shear site mutations.After screening,analyzing and screening the raw datas,1652 coding regions and 1074 splicing sites were screened out,including1074 non synonymous mutations and 578 shear site mutations among them.These significant variants that may cause changes in protein function include 378 nonsense mutations,139 shear site mutations,151 frameshift mutations,and 821 non synonymous mutations in highly conserved sequence regions.There were 10009mutation genes of grade 1 to 3 levels in 13 cases,of which 4027 mutation genes of grade 1 and 2,and 5982 mutation of grade 3.(2)61.54%PNH patients(8/13)detected PIG-A gene mutation.The mutagenesis mainly included code shift,truncation,shear site and un-synonymous mutation.The mutation level of the shear site,the code and the truncation were all grade one,and the non synonymous mutation was grade two.(3)In addition to the PIG-A gene,we analyzed the above 1-3 level mutation genes by somatic cell gene cluster analysis,that is,all samples on the same gene were mutated(not limited to the same locus).The total number of samples with a total number of more than 5 cases is 43,and the mutation forms include non-code,non-synonymous,code-shifting and non-coding region mutation.(4)We analyzed the KEGG signal pathway of 5717 1-3 level mutation genes screened by bioinformatics,the enriched signal pathways include:Apoptosis signaling pathway,peanut four acid metabolism signal pathway,antigen processing and expression signal transduction pathway,AMPK signaling pathway and adherens junction signaling pathway,the difference is not statistically significant(p>0.05).A total of 112 tumor related genes were found in the COSMIC tumor cell mutation related gene pool.KEGG pathway enrichment analysis showed that the difference of cancer pathway was significant(p=0.00),suggesting that somatic mutation of tumor pathway gene recurred in PNH patients.(5)After filtering and screening the raw sequencing datas,the total mutations were 105994,and the thrombus related genes were 529.The total mutagenesis of each database(dbSNP,Chinese,ExAC)was 73,and the total number of grade 1-3mutation was 20.The 1-2 level mutation genes include BMPR2,ZFPM2,F5,F8,F12,F13A1,MCL1,MMRN1,NBEAL2,NOS1 and PC,and the 3 level mutation genes include ETV7?F2R?GP1BB?ITGA2B?NOS2?PF4?SERPINC1?THBD and THBS1.KEGG pathway enrichment analysis showed that thrombus related mutation genes were significantly enriched in Notch signaling pathway,Wnt signaling pathway and peanut four acid metabolism signal pathway,and the above signaling pathways were statistically significant(p<0.05).3.The expression silencing and cell function detection of the pathogenic mutation gene RBPJ:(1)Further screening 18 highly expressed target genes:RYR1,MUC2,FLG,WASH1,ZNF717,UBXN11,TCHH,CTBP2,ANAHK2,ANAHK,MUC12,LILRB3,POTEH,HRNR,MECOM,RBPJ,DNAH14 and NBPF1.The results of 22 cases of PNH and K562 cell qPCR showed that the RBPJ was highly expressed,after transfection of siRNA targeting gene RBPJ by K562 cells,the RBPJ gene was silenced,and the expression level of mRNA decreased by more than 50%.(2)Cell proliferation assay showed that there was no significant change in cell proliferation after transfection of 24h by siRNA-RBPJ.With the prolongation of transfection time,cell proliferation activity decreased gradually.After transfection of72h with siRNA-RBPJ,the OD values of control group,siRNA-scr group and siRNA-RBPJ group were 1.9690±0.2062,1.2660±0.3878 and 0.4947±0.0992,respectively.The result of comparison between groups was:siRNA-RBPJ group and siRNA-scr group,siRNA-RBPJ group and control group were significantly different(p=0.0289 vs 0.0004),control group and siRNA-scr group had no significant difference(p=0.0502),There were significant differences between the three groups(p=0.0014).(3)The results of apoptosis experiment showed that the apoptosis rate of cell apoptosis increased after siRNA-RBPJ transfection,and the apoptosis rate was gradually increasing with the transfection times.After transfection of siRNA-RBPJ,the apoptosis rates of the cells in the control group,the siRNA-scr group and the siRNA RBPJ group were(4.271±0.3881)%,(5.000±0.5237)%,(25.71±3.9490)%.The results of the comparison between the groups were as follows:there was significant difference between siRNA-RBPJ group and siRNA-scr group,siRNA-RBPJ group and control group(p=0.0008vs 0.0007).There was no significant difference between the control group and the siRNA-scr group(p=0.1059),but there was a significant difference between the three groups(p<0.0001).(4)The results of cell cycle test showed that after siRNA RBPJ transfection,the G0/G1 phase of siRNA-RBPJ group was significantly different from that of control group and siRNA-scr group(p=0.0285vs 0.0150).There was no significant difference between the control group and the siRNA-scr group(p=0.0.9550),and there was a significant difference between the three groups(p=0.0198).The S phase of siRNA-RBPJ group was significantly different from that of the control group and siRNA-scr(p=0.0028 vs 0.0112).There was no significant difference between the control group and siRNA-scr(p=0.7652).There was a significant difference between the three groups(p=0.0006).After silencing RBPJ gene expression,the proportion of cells in G0/G1 phase increased,the proportion of S phase cells decreased,the cells were blocked in G0/G1 phase,and the cell proliferation ability decreased.Conclusion:1.The clinical manifestations of PNH include hemoglobinuria,anemia,hemorrhage and pancytopenia.Infection,thromboembolism and renal dysfunction are common complications of PNH.Glucocorticoids are the first line drugs for the treatment of hemolytic seizures in PNH,with a definite effect,and the total effective rate is 70.37%.Reduced doses of DA or HA regimen can achieve satisfactory results for PNH patients with relapsed or refractory or hormone dependent and no reduction in severe complications.The total survival time of PNH in 10 years after diagnosis was 70.09%,LDH increased significantly,complication of thrombosis and bone marrow failure were all unfavorable factors for 10 year survival rate reduction.2.The whole genome exon sequencing technique has been used to discover mutant genes other than PIGA,including ZNF717,SUZ12,RBPJ,MUC4,CUX1,MLL2,MAGEC1,TET2 et al,the functions of above-mentioned genes are closely related to the regulation of cell proliferation,differentiation,anti-apoptosis,and the promotion of tumor cell invasion and progression.It is confirmed that the second gene mutation is involved in the proliferation of PNH clone.3.F5,F8,F12,ZFPM2,THBD,ITGA2B and other thrombus related gene mutations were detected by whole exon sequencing technology,and the enrichment signal pathway included Notch,Wnt and peanut four enoic acid metabolic signaling pathway.Gene mutation may be one of the genetic risk factors for the formation of thrombus in PNH patients4.Screening of highly expressed and first class mutation gene RBPJ by next generation sequencing of PNH patients,after verifying its high expression in K562cells,siRNA-technology silenced the RBPJ gene.After the expression of RBPJ gene silenced by siRNA-technology,the results showed that the proportion of cells in the G0/G1 phase increased,the proportion of S cells decreased,the cells blocked in the G0/G1 stage,the apoptosis rate increased and the proliferation ability weakened.It is suggested that the high expression of RBPJ gene may be involved in the proliferation of PNH abnormal clones.