Prognostic,Genetic Diagnosis Of FSGS And Functional Study Of ADCK4

Objective Focal segmental glomerulosclerosis(FSGS)is a morphological pathology diagnosis,characterized by podocyte injury,resulting in glomerular filtration barrier dysfunction.The reported prevalence of FSGS in China varies from 3.3%to 16%,which is one of the most common glomerulonephritis with unsatisfactory prognosis.Clinical prognosis analysis is vital in high-risk patient identification,treatment guidance and prognosis evaluation of primary FSGS.As for hereditary FSGS,establishing suitable gene diagnosis strategy warrants the genetic diagnosis.Knowing the precise molecular etiology of disease can help clinicians determine the exact therapeutic course,and counsel patients and their families about prognosis,steering transformation of the traditional intervention to personalized medicine.Methods In the clinical prognostic analysis,we reviewed retrospective clinical data and renal outcomes of 250 biopsy-diagnosed primary FSGS patients to identify independent predictive risk for progression to end-stage renal disease(ESRD).The association of baseline variables with the primary outcome-ESRD was tested by univariate and multivariate Cox regression model.In our genetic diagnostic study,FSGS in both children and adults were screened by different genetic testing(Sanger sequencing and Panel sequencing)with the purpose of establishing genetic diagnostic strategy in FSGS patients.In our mechanism study,we investigated the pathogenesis of ADCK4 mutation in the podocyte impairment by the lentinvirus overexpression model and the Crispr/Cas9 mutated model.Results In the clinical prognostic analysis,by univariate Cox analysis,severtity of tubulointerstitial lesion(TIL)and glomerulosclerosis,systolic blood pressure(SBP),estimated glomerular filtration rate(eGFR),serum UA,calcium and phosphorus were correlated with the degree of renal impairment at the time of biopsy.In the fully adjusted Cox proportional hazards model,there were four baseline variables with a significant independent effect on the risk of ESRD,including TIL grade(HR=3.493[2.01,6.07],P<0.01),SBP(HR=1.024[1.002,1.046],P=0.029),serum phosphorus(HR=3.15[1.838,5.398],P<0.01),eGFR(HR=0.973[0.957,0.99],P<0.01).Then we developed a risk score for disease progression based on the regression coefficients for the four independent predictors retained in the best model:RiskScore=-0.027(eGFR[ml/min/1.73m2])+0.024(SBP[mmHg])+1.147(P[mmol/L])+1.251TIL.Survival ROC analysis revealed that the risk score provided considerably improved discriminative power compared to individual predictors,with the AUC of 0.8(95%CI 0.722-0.891).To our knowledge,our study is the first risk score to predict the renal progression of FSGS patient efficiently.In our genetic diagnostic study,we designed and applicated a hereditary kidney disease gene panel containing 706 candidate genes.Through high-throughput sequencing in 26 adult(≥ 18 years)steroid resistant FSGS(SR-FSGS),the mutations in conserved coding region of WT1,TRPC6,ADCK4,NPHS2 and COL4A3 were detected in 7/26(27%)patients,indicating an essential role of podocyte-related mutation in SR-FSGS.In order to screen common disease-causing gene(NPHS2,WT1 exon8-9and ADCK4)in FSGS,Sanger sequencing was performed in 104 children and adolescent patients(<18 years)from four centres,giving a mutation detection rate of 2.9%(3/104),which is significantly lower than those reported from Caucasian cohorts,revealing a racial difference of genetic background.Base on the results above,we recommend to strengthen genetic testing in SR-FSGS,and cost-effective Panel sequencing is the first choice for clinical diagnosis currently,incorporating genomic diagnostics into precision medicine.In our mechanism study,we found that the content of CoQ10 was significantly decreased in three ADCK4 mutant stably transfected model compared with the wild type(WT)podocyte,suggesting that these three mutations could affect CoQ10 biosynthesis and then induced renal insufficiency.As for mus-Adck4 model,the mitochondria of mutant podoctye were observed to be swollen,while the ridge was shorter,decreased and peripheral compared with WT podocyte.The results of the Annexin V-PI assay by flow cytometry showed that more apoptotic cells were detected in the mutant models,while the wound scratch assay indicated that the migration ability of mutant cells were impaired compared with WT podocyte.Conclusions Our data indicate that primary FSGS patients with higher SBP,TIL grade,serum phosphorus and lower eGFR at baseline are at a higher risk of progression to ESRD.The new progression risk score calculated based on these four baseline variables offers a clinical guidance for risk stratification,treatment intervention and prognosis assessment.We performed Sanger sequencing and Panel sequencing respectively in children and adult SR-FSGS cohort,revealing a high detection rate of 27%in adult SR-FSGS patients,indicating an essential role of podocyte-related mutation in SR-FSGS.Certain gene mutation could provide potential treatment target.While in our children cohort,NPHS2,WT1(exon8-9)and ADCK4 is less detected due to diverse racial background.Vitro experiments demonstrated that three novel ADCK4 mutations could induce the decrease of CoQ level in podocyte.Lower CoQ level could affect mitochondrial morphology,induce apoptosis,and thus affect the normal function of podocyte.