The Establishment Of Inherited Nephropathy Gene Diagnostic System And Clinical Application Research In Type Ⅳ Collagen Related Nephropathy

Obective Type Ⅳ collagen is the scaffold structure of glomerular basement membrane,which plays a vital role in the function of glomerular filtration barrier.The mutations in type Ⅳ collagen gene(COL4A3,COL4A4 and COL4A5)can result in a group of clinically,pathologically,and genetically heterogeneous kidney diseases.The disease spectrum including Alport syndrome(AS),benign familial hematuria(BFH)and focal segmental glomerulosclerosis(FSGS).The large sizes of these genes and the absence of mutation hot spots complicated mutational analysis by routine polymerase chain reaction(PCR)and Sanger sequencing,which is extremely inefficient and tedious.In order to clariy the correlations of genotype and phenotype,the COL4A3,COL4A4 and COL4A5 gene mutations and mode of inheritance in type Ⅳ collagen related nephropathy.We establish inheried nephropathy gene diagnostic system based on the targeted capture associated next generation sequencing.Furthermore,the relationship of these three diseases was determined by comparision of clinical phenotype,pathological features and genetic test results.It provides basis for the study of gene diagnosis,genetic counseling,and prenatal diagnosis.Methods Genomic DNA was isolated from whole blood of all available family members using the QIAamp DNA Blood MiNi Kit.The pipeline was followed to capture and enrich targeted sequences,prepare sequencing library,and next-generation sequencing.The control database used in the pipeline are 1000 genome database(http://www.1000 genomes.org),dbSNP database and a BGI in-house database which included 2087 normal subjects.Phylop(phyloP46wayPlacental)was used to calculate the conservation of each SNP.The functional impact of missense variation was analyzed with the PolyPhen-2(Polymorphism Phenotyping v2.2.5)and SIFT algorithm(Sorting Intolerant From Tolerant v5.1).The HSF(http://ud.be/HSF3)algorithm was used to predict the functional impacts of splicing variations identified.Specific primers were designed according to the detected mutations,and then specific fragments were amplified.Sanger sequencing verification and qPCR verification were performed for the detected point mutation and copy number variation respectively.Additionally We collected and analyzed clinical data,pathological results on the basis of gene mutation.Results 1.Using inheried nephropathy gene diagnostic system based on the targeted capture associated next generation sequencing,we detected 29 gene mutations,including 23 novel mutations in 21 Alport syndrome patients,2 benign familial hematuria patients,1 focal segmental glomerularsclerosis patient.Of these new mutations,4 were in the COL4A3 gene,7 were in the COL4A4 gene,and 12 were in the COL4A5 gene.2.Twenty-two point mutations and two fragments deletion were detected in 21 Alport patients.Among them,16 were missense mutations,1 were nonsense mutation,4 were frameshift mutation and 1 were 5 ’UTR mutation.16,(76 %,16/21)patients showed X-linked inheritance(COL4A5 gene mutations),4,(4/21,12 %)showed autosomal recessive inheritance(COL4A3 and COL4A4 homozygous mutations or compound heterozygous mutations),1(1/21,5 %)showed autosomal dominant inheritance(COL4A3 or COL4A4 heterozygous mutations).A comparison of the clinical manifestation caused by different types of mutations in COL4A5 suggested that nonsense mutations and glycine substitution by an acidic amino acid are more severe than the other missense mutations.3.COL4A4 c.1536 dupG,COL4A4 c.622G>C and COL4A4 c.4844C[2] heterozygous mutations can lead to ADAS,BFH,and FSGS respectively.Using bioinformatics analyses and pedigree verification,we show that these novel variants are pathogenetic and cosegregate with ADAS,BFH,and FSGS.4.Both COL4A4 c.622G>C monogenic heterozygous mutation and COL4A4 c.1471C>T,COL4A3 c.3418+1G>T digenic gene mutations in cis can result in benign family hematuria.Conclusion 1 The inheried nephropathy gene diagnostic system based on the targeted capture associated next generation sequencing can be highly effective,precise,and rapid for gene detection in type IV collagen related nephropathy.We have detected 23 novel mutations,these novel mutations can expand the genotypic spectrum of type Ⅳ collagen related nephropathy.The reasons for the clinical phenotypic heterogeneity of Alport syndrome are explained to some extent.This study provided the basis for the research of genetic diagnosis,genetic counseling,and prenatal diagnosis.2 By comparing the clinical,pathological and genetic testing results,we found that COL4A4 heterozygous mutations associated ADAS,BFH and FSGS are subtypes of type Ⅳ collagen related nephropathy that represent different phases of disease progression.It provided clues for further study of pathogenesis,precise treatment and prognosis of type Ⅳ collagen related nephropathy.3 Both COL4A3 or COL4A4 monogenic heterozygous mutation and COL4A3 and COL4A4 digenic gene mutations in cis can result in benign family hematuria.This study enriched the mode of digenic inheritance of the benign family hematuria.