| Objective:Alport syndrome(AS)is a rare monogenic hereditary kidney disease caused by mutations in COL4A3,COL4A4 and COL4A5 genes encoding type IV collagen α3α4α5 trimer network in the basement membrane.Genetic testing is the gold standard method for its diagnosis.In this study,the genetic analysis of patients with AS was carried out by next generation sequencing(NGS)and bioinformatics techniques,in order to improve the etiological diagnosis of patients,enrich the mutation spectrum of AS related genes,and analyze the genotype-phenoty correlations combined with the clinical characteristics and pathological examination of patients,so as to evaluate the prognosis of patients and provide reference for choosing appropriate time and method of clinical intervention.Methods:1.3 unrelated patients with AS diagnosed by renal biopsy in the affiliated Hospital of Inner Mongolia Medical University from January 2014 to November 2020 were selected as the subjects of the study.2.The age at onset,clinical symptoms,laboratory examination and family history of the patients were collected,the clinical and renal pathological data of the patients were collected,and the disease conditions of the family members were investigated.3.Peripheral blood samples were collected for whole exome sequencing(WES),and specific primers were designed for type IV collagen gene mutation to be verified by Sanger sequencing.Search for the inclusion of gene mutations in public databases such as db SNV,Clin Var and genome AD,and whether there are related literature reports.,and evaluate the pathogenicity of variants by bioinformatics software such as SIFT and Human splicing Finder.Results:1.Hematuria with proteinuria was the first symptom of 3 patients with AS.2 patients had renal insufficiency at the time of onset,and one of them developed to ESRD with peritoneal dialysis at the age of 18 years.Of the 3 patients with AS,1 was accompanied with mild sensorineural deafness,and the other patients had no extrarenal manifestations such as eyes and ears.2.Of the 3 patients,2 had a family history of AS.3.The renal pathological characteristics of the 3 patients were roughly the same.Light microscopy showed focal segmental glomerulosclerosis changes,electron microscopy showed thickening of the basement membrane,dense layer delamination and tearing in a basket-like shape;2 patients performed Indirect immunofluorescence staining of type IV collagen alpha chain showed negative results.4.Whole exome sequencing was performed on 3 patients,and 4 mutations of COL4A5 and 1mutation of COL4A4 were detected,of which one patient had a double mutation of COL4A5 and COL4A4.COL4A5 mutations include 2 newly discovered splicing mutations and 1frameshift deletion mutation,which are all pathogenic mutations predicted by bioinformatic software;1 benign missense mutation that has been included in public databases;COL4A4mutation is a newly discovered heterozygous missense mutation,its pathogenic significance is unknown.Conclusion:AS is caused by mutations in COL4A3,COL4A4 and COL4A5 genes responsible for encoding α3,α4 and α5 chains of type IV collagen.The severity of clinical phenotype depends on the type of mutation.Gene detection is the gold standard method for the diagnosis of AS.NGS is an efficient strategy for genomic research.Using this technology to detect genes in patients with AS can find most AS-related type IV collagen gene mutations,which not only completes the etiological diagnosis of AS,but also enriches the spectrum of the disease-related mutations,and the predictive analysis of changes in the structure and function of the transcript is also helpful for the prognosis of patients with AS,and provides support for clinical decisionmaking. |
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