Screening Of Blood Diagnostic Markers Of Idiopathic Membranous Nephropathy And Its Correlation With PLA2R1 Gene Polymorphism

ObjectiveIdiopathic membranous nephropathy（IMN）is an autoimmune disease characterized as deposition of immune complex and the increasing of urinary protein.The pathogenesis is related to environmental pollution and genetics.The incidence rate varies in different country and has a trend of increasing in China.The diagnosis of IMN is based on renal biopsy,but because of the invasive examination,it can not be accepted by the majority of patients.Therefor,it is urgent to find a sensitive new biomarker to diagnose IMN.It is reported that anti-PLA2R-Ab and anti-THSD7A-Ab antibodies exist in the circulation of IMN patients.Circular RNA participates in the occurrence and development of autoimmune diseases and is expected to become a new biomarker of IMN.Therefore,the aims of this article are to explore the trend of IMN incidence rate in recent years in Guangzhou,the clinical value of plasma anti-PLA2R-Ab,anti-THSD7A-Ab and hsa_circRNA₁01022 in IMN patient,and the correlation between IMN and PLA2R1 gene polymorphism,so as to provide evidence for the diagnosis,treatment and pathogenesis of IMN.Methods（1）Group A（166 IMN patients,38 secondary membranous nephropathy（SMN）patients,52 patients with other kidney disease）are from Nanfang hospital,Southern Medical University.Group B（212 IMN patients,118 SMN patients,84 patients with other kidney disease）are from Shiyan,Hubei province.The plasma anti-PLA2R-Ab and anti-THSD7A-Ab were detected.A case-control study was conducted to screen differentially expressed circRNAs in plasma using gene chip technology by collecting plasma samples from 6 paired anti-PLA2R-Ab negative IMN patients and normal healthy controls,then the target circRNAs were verified in 20 paired plasma samples by qRT-PCR.To further expand the sample size,50 normal controls,89 IMN patients and 66 non IMN patients were selected to detect the relative expression of hsa_circRNA₁01022 in plasma,and to evaluate the efficacy of hsa_circRNA₁01022 in the diagnosis of IMN.（2）The Outcomes in 49 IMN patients in Group B[anti-PLA2R（+）,n=27;anti-THSD7A（+）,n=6;anti-PLA2R（-）and anti-THSD7A（-）dual negative,n=16]who received immunosuppressive therapy was observed for 12 months.The 24-hour urine protein,anti-PLA2R-Ab,anti-THSD7A-Ab and hsa_circRNA₁01022 were detected at the time of diagnosis and at 3,6 and 12 months after immunosuppressive therapy.（3）PLA2R1 gene polymorphisms of（rs3749119,rs3749117,rs35771982,rs3828323,rs4664308）were directly sequenced after gene amplification.Results（1）The anti-PLA2R-Ab positive rate（60.8%）in IMN patients of group A was significantly higher than that in SMN patients（13.8%）（P<0.001）.The anti-PLA2R-Ab positive rate（71.7%）in IMN patients of group B was significantly higher than that in SMN patients（9.3%）（P<0.001）.The results of priliminary verification showed that hsa_circRNA₁01022,hsa_circRNA₁03668 and hsa_circRNA₁00789 were up-regulated,hsa_circRNA₁01145 was down-regulated,consistent with gene chip results.The area under ROC curve of anti-PLA2R-Ab was 0.860,the area under ROC curve of hsa circRNA 101022 was 0.729,and the combined AUC of hsa_circRNA₁01022 and anti-PLA2R-Ab was 0.925.（2）After treatment with prednisone and cyclophosphamide,the plasma anti-PLA2R-Ab and hsa_circRNA₁01022 were gradually reduced.The rate of partial remission was lower in anti-PLA2R（+）patients than in anti-PLA2R（-）patients 3 months（P=0.045）and 6 months（P=0.006）after immunosuppressive therapy.The rate of complete remission was lower in anti-PLA2R（+）patients than in anti-PLA2R（-）patients 12 months（P=0.037）after immunosuppressive therapy.There was no statistically significant difference in the recurrence rate between the anti-PLA2R（+）group,the anti-THSD7A（+）group and the anti-PLA2R（-）group at 12 months of follow-up.（3）The genotype and allele distributions of rs35771982,rs3749117,and rs4664308 differed significantly between IMN patients and controls in Group A and in Group B.The genotype and allele distributions of rs4664308 also differed significantly between SMN patients and controls in Group B.The genotype and allele distribution of rs35771982 and rs4664308 differed significantly between P.LA2R-Ab（+）and PLA2R-Ab（-）IMN patients in Group B.Conclusion（1）Anti-PLA2R-Ab and hsa_circRNA₁01022 can be used as a diagnostic markers of IMN.Combined detection of plasma anti-PLA2R-Ab and hsa_circRNA₁01022 can improve the diagnostic sensitivity.Anti-PLA2R（-）IMN patients have shorter duration to get partial remission for immunosuppressive therapy.（2）PLA2R1 gene polymorphism rs35771982,rs3749117 and rs4664308 are associated with IMN,PLA2R1 gene polymorphism rs4664308 is correlated with SMN.PLA2R1 gene polymorphism rs4664308 and rs35771982 could affect the concentration of anti-PLA2R-Ab.