Clinical And Genetic Characteristics Of Steroid-Resistant Nephrotic Syndrome And Functional Study On A Novel Mutation Of INF2 In Children

Objective:To identify the genetic pathogenic factors of steroid-resistant nephrotic syndrome(SRNS)in children,and to reveal the genetic characteristics of SRNS pathogenicity and/or susceptibility in Southeast Chinese population on molecular basis.Methods:Genetic testing was performed on children diagnosed with SRNS according to the“Guidelines for the diagnosis and treatment of steroid-resistant nephrotic syndrome(2016)”,who were admitted to our hospital and clinically.Expand on the basis of related domestic research in Chinese patients,high-throughput sequencing was performed to target all encoding genes,and mutations or unknown SRNS-related genetic variants screening was conducted using efficient,comprehensive,and semi-automated methods for clinical phenotypes,genetic mutations,pathogenicity,and inheritance analyzing,followed by cross-disciplinary analysis of molecular biology and molecular/medical genetics.Using in vitro cell transfecting experiments,expression of INF2 affected by mutation c.1504C>T(p.P502S)was investigated,and Western Blot,cell immunofluorescence,cell migration and adhesion experiments were used to detectfunctional changes in podocyte carrying the mutation.Results:Clinical and genetic testing data of a total of 81 children were collected.Six patients with congenital kidney disease(<= 3 months old),2 from 3-12 months old,43 from 1-6years old,26 from 6-12 years old,and 4 from 12-18 years old groups.The diagnostic rate by genetic test was 28.4%(23/81),of which the positive rate of children with congenital kidney disease was 100%(6/6),and the positive rate of children with non-congenital kidney disease(> 3 months of age)was 24%(17/71),but no gene mutations were identified in any of the four patients from age group older than 12 years.Eleven positive genes were detected: NPHS1(in 5 patients),PAX2(in 3 patients),INF2(in 3 patients),ANLN(in 3 patients),NPHS2(in 2 patients),FAT1(in 2 patients),and ACTN4,ADCK4,PLCE1,TRPC6,and WT1(in 1 patient,respectively),of which only NPHS1(4/5)and PAX2(2/3)pathogenic mutations were detected in patients < 3months of age,while genes identified in children 1-12 years showed more variety except ADCK4 detected in one patient from 3 months to 1 year old group.Surprisingly,the same ANLN variant,NM?018685: c.2521G> A,p.E841 K was detected in 3unrelated patients and the father of one of them,and the rate of yield in this study was3.7%(3/81),while the MAF in the Southern China Population Database is 0.014(1,000 genomes database).In vitro cellular experiments on the INF2 gene mutation c.1504C>T(p.P502S)showed that the mutation caused defects in podocyte migration and adhesion function.Conclusion:This study emphasized the concept of "idiopathic" based on the existing SRNS diagnostic guidelines,and proposes corresponding solutions.The genetic factors of SRNS show both the same and different results from previous Chinses studies,suggesting the characteristics of the SRNS-related genes in the Han population in southeastern China.Specifically,it was suggested that although the phenotypesassociated with INF2 deficiency are not very clear at present,haploinsufficiency of INF2 may be a high-frequency event leading to the onset of NRNS in southeast Chinese population,which has the value of localized medical genetic consultant and clinical practice;the ANLN mutation,NM?018685: c.2521G> A,is a susceptible variant for SRNS in southern populations(detection rate 3.7% > MAF 0.014 x 2),though,its molecular mechanism needs to be further clarified.This study confirms the previous research results of SRNS at the molecular level,and provides materials and basis for future research and guidance of clinical diagnosis and treatment.