| Background:Paroxysmal nocturnal hemoglobinuria(PNH)is a super rare disease.There lackes researches in characters of PNH with large population in China,especially analysis of PNH subtypes.And the molecular mechanism of PNH clonal amplification and thrombosis of PNH is still inconclusive.Objective:This research aimed to summarize and analyze the clinical characteristics of PNH between different subtypes without complement inhibitor therapy in China,and to explore the mechanism of PNH clonal proliferation and thrombosis of PNH.Methods:1.PNH patients’clinical data,who were treated in Hospital from 2009 to 2019 and followed up for more than 2 years was cllected,including the current situation of domestic PNH patients from the aspects of laboratory and clinical characteristics,therapy,clonal evolution,prognosis and pregnancy and delivery of female PNH patients.2.Peripheral blood of 6 PNH patients were collected for sorting out CD59 positive and CD59 negetive cells,and sequenceed by whole exon sequencing(WES)and whole transcriptome sequence respectively.At the same time,peripheral blood of 6 matched healthy controls was selected as transcriptome control.According to the transcriptome upregulation and down-regulation of the genome and its association with WES,genes with simultaneous abnormal expression of transcriptome and abnormal WES were first selected.Selected genes were enriched by Gene Ontology(GO),and then genes related to proliferation,immunity and thrombosis were selected.3.Peripheral blood of another 40 PNH patients was collected for WES,and the target genes that simultaneously meet the frequency less than 0.001 in 1000 Genomes and the incidence more than 10%in 40 patients were selected.Another 40 samples were sequenced by Sanger to verify the final target genes.4.And 7 of 40 samples sequenced by Sanger method,whose peripheral blood were collected and sorted by flow cytometry to verify the target gene through quantitative polymerase chain reaction(Q-PCR),Western Blot,flow cytometry and mass spectrometry.Results:1.512 patients were included in total,including 288 males(56.3%)and 224 females(43.7%).Median age was 33(9~80)years old.The proportions of patients with classic PNH,combined with bone marrow failure(BMF/PNH)and subclinical PNH were 46.8%,51.6%and 1.6%,respectively.Symptoms in patients with classic PNH are more likely to be associated with hemolysis,while bleeding is more common in patients with BMF/PNH.Compared with BMF/PNH patients,patients with classic PNH had higher PNH clone,lactate dehydrogenase(LDH)level and lower ferritin level.Although the incidence of thrombosis was similar in patients with classic PNH and BMF/PNH(P=0.66),the incidence of renal deficiency was higher in BMF/PNH(P<0.05).BMF/PNH were more often treated with immunosuppresive therapy(P<0.05),while classic PNH were more treated with glucocorticoids,iron supplements and anticoagulants(P<0.05).Moreover,compared with patients who were effective in immunosuppresive therapy,PNH clones were larger in those of patients who were ineffective(66.89 ± 35.01%vs 38.64±30.47%,P=0.001).For patients with continuous effect(at diagnosis vs last follow-up:38.64±30.47%vs 39.50±43.13%,P=0.829)and without effect(at diagnosis vs last follow-up:68.89±35.01%vs 96.00±1.00%,P=0.133),PNH clone was stable during follow-up.For patients who were effective at first but relapsed during follow-up,PNH clone after relapse were significantly higher than before therapy(43.53±30.96%vs 93.33±3.22%,P<0.01).Compared with BMF/PNH,classic PNH had lower incidence of myeloid malignancy(P=0.02).The main causes of death in patients with PNH were thrombosis(29.6%),bleeding(18.5%)and infection(18.5%).There was no difference between classic PNH and BMF/PNH.Compared with BMF/PNH,classic PNH have better overall survival,and the prognosis of MDS/PNH is the worst.2.Results of GO enrichment indicated that T cell activation related genes were up-regulated,while platelet degranulation,coagulation and hemostasis,leukocyte proliferation,and platelet activation related genes down-regulated in PNH patients when compared with healthy control.Genes related to leukocyte proliferation,apoptosis regulation,immune activation,platelet activation and coagulation regulation were down regulated in CD59 positive cells compared with CD59 negative cells.3.Then 416 proliferation/apoptosis related genes,716 immune related genes and 141 thrombus related genes were first distinguished.After screening these genes in WES sequencing results of 40 PNH cases,10 target genes were then obtained.After Sanger sequencing verification,6 target genes were finally determined,which were SELP(mutation incidence 12.5%),NRP1(10%);SLC15A4(5%),vWF(2.5%),ABCA13(2.5%)and FLT1(2.5%).In addition,out of target base mutations SWAP70(62.5%)and EpHB2(10%)were also included.The abnormal frequencies of SWAP70,EpHB2,ABCA13 and SLC15A4 in thrombotic group were significantly higher than patients without thrombotic group(P<0.05).4.Some target genes were verified by Q-PCR,Western Blot,flow cytometry respectively.It was found that NRP1 and EphB2 in PNH were different from healthy controls in both RNA and protein levels;SELP in PNH was different from healthy controls only in RNA level;FLT1 and vWF in PNH were different from healthy controls only in protein level;SWAP70 in PNH were similar in both RNA and protein levels to healthy controls,but SWAP70 in CD59 positive was lower than CD59 negative in protein expression level.Among the above differential genes,founction of FLT1,EpHB2 and NRP1 are related to proliferation and apoptosis;SELP,vWF and EpHB2 were associated with thrombosis;SWAP70 was associated with immunity.What’s more,FLT1,EpHB2,NRP1 and SWAP70 were also differentially expressed between CD59 positive and CD59 negative cells.Mass spectrometry analysis showed that anti proliferative protein(BTG1)was expressed in CD59 positive cells,while cycle related protein(INCA1,NPEPPS)was expressed in CD59 negative cells.Conclusions:1.Symptoms in patients with classic PNH are more likely to be associated with hemolysis,and PNH clone was larger.Classic PNH is mainly treated with supportive therapy,while BMF/PNH is mainly with immunosuppressive therapy,and the response to immunosuppressive therapy was related to size of PNH clone.BMF/PNH is easier to have clone evolution than classic PNH.2.Compared with healthy controls,genes related to immune function such as T cell activation were up regulated,while leukocyte proliferation,platelet degranulation,coagulation,hemostasis,and platelet activation related genes were down regulated in PNH.Among them,down-regulation of proliferation and thrombolism related genes in CD59 positive cells were more obvious.3.The selected high-frequency genes in PNH are also different from normal people in RNA and/or protein levels,and there are differences in the expression between CD59 positive and CD59 negetive cells,and some of them are also more frequent in PNH thrombosis group.These genes may be closely related to thrombosis of PNH and proliferation of CD59 negative cells.4.Antiproliferative proteins expressed in CD59 positive cells and cell cycle related proteins expressed in CD59 negative cells.Full text conclusion:Age at diagnosis of PNH is young.Incidence of thrombosis is high and indicates poor prognosis.Effect of immunosuppressive therapy is related to PNH clone.The genome characteres of PNH display downregulation of proliferation and coagulation,up-regulation of immune activation.What’s more,abnormal expression of genes and proteins also are different between CD59 positive and CD59 negative cells.The abnormal expression of some genes except for PIGA may be closely related to mechanism of clone proliferation and thrombosis. |
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