The Effects Of The COL4A5 Gene Mutation In XLAS On Podocyte

Alport Syndrome(AS)is a congenital/ hereditary kidney disease with clinical features of eye,ear,and kidney lesions.It is one of the leading causes of end-stage renal disease(ESRD)in children.For children,because of the occult onset,diversified clinical manifestations,and untypical pathological features,it is easy to be misdiagnosed or missed diagnosed.Genetic diagnosis plays an important role in the early diagnosis of AS.So far,COL4A5 gene mutation has been found to be the main cause of XLAS,and podocytes play an important role in the development of the disease.However,the effect of different COL4A5 gene mutations on the morphology and function of podocytes remains unclear.Therefore,this study will analyze the clinical characteristics and gene mutation characteristics of children with COL4A5 gene mutations in order to reduce the incidence of clinical missed diagnosis;simultaneously from the COL4A5 gene mutation on human podocyte function,provides new experimental evidence of AS genetic diagnosis and Pathogenesis.Part I: Clinical analysis of COL4A5 mutant Alport syndrome in children ObjectivesThe genotypes and clinical phenotypes of XLAS children with COL4A5 gene mutations were summarized in order to increase awareness of Alport syndrome.MethodsA retrospective analysis was conducted from June 2011 to June 2016 in 31 cases of XLAS with COL4A5 gene mutation confirmed in Renal Rheumatology Department of Shanghai Children’s Hospital Affiliated Children’s Hospital(Shanghai Children’s Hospital),through the collection of clinical features,laboratory tests,pathological diagnosis,and other data.At the same time,SPSS software was used for statistical analysis of the above data.Results(1)Among the 31 children with XLAS who had a COL4A5 gene mutation,12(38.7%)were females and 19(61.2%)were males with an average age of 2.6 years.Clinically hematuria proteinuria as the first performance of more boys than girls,the group of 8 males(8/19,accounting for 42.1%),only 2 females(2/10,accounting for 20%)(P<0.05).There was no significant difference in the age of onset,positive family history,predisposing factors,and ear and ear lesions between male and female children(P>0.05).(2)Twenty-six patients underwent percutaneous renal biopsy under ultrasound guidance.Renal histopathological light microscopy showed that 20 cases showed glomerular minor lesion,5 cases had mesangial glomerulosclerosis,and 1 case had focal segmental glomerulosclerosis;electron microscopy showed only 4 cases(26.7%)of typical AS basement membrane changes occurred;type IV collagen alpha chain fluorescence staining,60% of children(a total of 15 cases)α3,α5 were negative.(3)A total of 31 mutations were detected in this group,of which 19 were missense mutations,2 were large deletions,4 were mutations at the splice sites,and 6 were frameshift mutations.Among the 19 missense mutations,16 mutations were Gly-X-Y mutations.Among the 31 patients with mutations,11 were found to have novel mutations,accounting for 35.4%.ConclusionsChildren’s XLAS onset of occult,mostly hematuria or hematuria with proteinuria,the clinical phenotype has no obvious specificity,renal pathological manifestations are atypical(mostly glomerular minor lesion).COL4A5 gene missense mutations are the most common,and with highly novel mutations,which make it difficult to diagnose AS.And genetic testing can help increase the detection rate of AS.Part II: Effects of COL4A5 Gene Mutation on Podocyte FunctionThrough the first part of the study,we found that COL4A5 gene mutation detection is one of the new diagnostic criteria of XLAS,which is helpful for improving AS because the clinical manifestations of children with XLAS have no obvious specificity at the time of onset,and the pathological tissue morphology is not typical and usually cause clinical missed diagnosis.The detection rate,and how to determine the pathogenicity of the mutant gene,is also one of the hot issues that plague clinicians.More than 800 mutations in the COL4A5 gene have been identified,but few functionally verified mutations have been reported.To further clarify the pathogenicity of the COL4A5 gene mutation and its effect on podocyte function,a COL4A5 shear was used in children with a definite family history.Take the mutant-c.232-1G>A mutation as an example to try to investigate the effect of mutant genes on the expression of podocyte-specific molecules and type IV collagen,so as to provide new clues for clinical validation of gene mutation.ObjectivesTo investigate the effect of cleaved site mutation C.232-1G>A of COLA45 gene on the expression of type IV collagen and cell function in podocytes,so as to provide a new basis for clinical diagnosis and provide new clues for the mechanism of disease.Methods(1)Subject: Human glomerular podocyte cell,courtesy of Prof.Moin Saleem.Experimental conditions were podocytes differentiated at 37°C for 12-14 days.Cells were plated within 24 hours before transfection.(2)Experimental grouping: randomly divided into four groups,blank control group,empty vector group,normal plasmid group and mutant plasmid group.The plasmid was designed with pcDNA3.1 as the vector.Except for the c.232-1G>A mutation site of the COL4A5 mutant plasmid,the rest of the COL4A5 gene sequences were identical to the normal COL4A5 plasmid.Experimental contents: 1)Human nephrin and synaptopodin expression was confirmed by in vitro immunofluorescence staining of human glomerular podocytes.2)The expression of COL4A5/COL4A3/COL4A1 mRNA and protein in podocytes of each group was detected by real-time quantitative PCR and Western blot.3)The expression of podocin and α-actinin-4 were detected by Western blot.(3)Statistical methods: SPSS statistical software was applied.All measurement data were expressed as mean±standard deviation(x±s).Between the two groups,the first test data were in accordance with the positive distribution and analyzed by single factor analysis of variance LSD method,P<0.05 was considered statistically significant.Results(1)Human podocytes were successfully cultured and positive for the expression of human podocyte-specific markers(nephrin,synaptopodin).(2)The expression of COL4A5 protein and mRNA in the podocyte after transfection of the COL4A5 gene c.232-1G>A mutant plasmid was significantly lower(P value was less than 0.01 and 0.05 compared with the control group)(Western blot,indirect immunofluorescence,qPCR);And on the COL4A3,COL4A1 mRNA and protein expression levels have no effect(P>0.05).The expression of podocin and α-actinin-4 were detected by Western blot at 36 hrs after transfection.The results showed that the expression level of podocin protein in the mutant plasmid group was significantly lower than that in other experimental groups,P<0.05;α-actinin-4.There was no significant difference between the groups.(3)After transfection of the COL4A5 gene c.232-1G>A mutant plasmid,the expression of specific molecules podocin in podocytes was significantly reduced(P<0.05)(Western blot method)without affecting the expression level of α-actinin-4(P>0.05).ConclusionsThe COL4A5 gene c.232-1G>A mutation resulted in the down-regulation of COL4A5 protein and podocin,which may be related to the podocyte function,but the exact mechanism remains to be elucidated.