Clinical Data Analysis Of Children With Alport Syndrome In A Single Center And Induction Of Pluripotent Stem Cells In A Child With COL4A5 Gene Mutation

Part one Clinical data analysis of children with Alport syndrome in single centerObjective To analyze the clinical characteristics of COL4A3,COL4A4,COL4A5 gene mutations in children of different genders.Methods Collected 45 children with Alport syndrome who were diagnosed at Guangzhou Women and Children′s Medical Center from March 2016 to December 2019.According to different genders,different mutant genes,different pathological types,etc,the children were grouped,and the children’s gender,age at diagnosis,renal function,and treatment status were statistically analyzed.Results 45 children with Alport syndrome（AS）were collected and analyzed statistically:（1）There were 31 male children（68.89%）,14 female children（31.11%）,and the male-to-female ratio was 2.21 :1;（2）The average age of male children at diagnosis was6.25±3.62 years old,and the average age of female children at diagnosis was 5.61±3.21 years old,and there was no statistical difference（P>0.05）;in the COL4A5 mutant group,the average age at diagnosis of children was 5.43±2.96 years old In the COL4A4 mutant group,the average age of children is 10.38±3.47 years.The average age of children with COL4A3 mutant group was 4.47±2.02 years old,and there was statistically significant among the groups（P<0.05）.（3）A total of 44 children with hematuria,12 male children with simple hematuria,and 10 female children.Among them,there were 17 children with COL4A5 gene mutation in glomerular hematuria,5 children with COL4A4 gene mutation,and 1 case with COL4A3.Among the children with nephrotic proteinuria,3 were male and 1 was female,including 2 children with COL4A5 gene mutation and 2 children with COL4a4 gene mutation.（4）There were two children with glomerular filtration rate（GFR）lower than 60 ml /（min · 1.73 m2）,one male and one female,and all of them had COL4a4 gene mutation.（5）There were 14 cases with family history,including 8 male cases（57%）and 6 female cases（43%）.Among them,10 cases had COL4A5 gene mutation,2 cases had COL4a4 and 2 cases had Col4a3 gene mutation.Among children with renal disease level proteinuria,there are 3male children and 1 female child,including 2 children with COL4A5 gene mutation and 2children with COL4A4 gene mutation.（4）There were 2 children with glomerular filtration rate（GFR）lower than 60 ml/（min·1.73 m2）,one case in each male and one case,and both had mutations in the COL4A4 gene.（5）There are 14 cases of children with family history,8cases of male children（57%）,6 cases of female children（43%）,of which 10 have COL4A5 gene mutations,and 2 have COL4A4 and COL4A3 gene mutations..（6）A total of 22 people have clear pathological types,which are divided into the following 4 types: mild glomerular disease,partial glomerular sclerosis,mesangial proliferative glomerulonephritis,and thin basement membrane.There are 11 children with 2 or more pathological types.Among these11 children,6 cases（55%）have COL4A5 gene mutations,2 cases（18%）have COL4A4 gene mutations,and COL4A3 gene mutations There are 3 cases（27%）.Conclusion Almost all children with AS have hematuria.Therefore,children with hematuria should be diagnosed as early as possible and their family members should be screened.When the children develop proteinuria,they should be treated in time to delay the progression to renal failure as much as possible.Part two COL4A5 gene mutation Alport syndrome family status and IPS establishmentObjective Used peripheral blood mononuclear cell PBMC（Peripheral blood mononuclear cell PBMC）of a male child with Alport syndrome with COL4A5 gene mutation to establish a hi PS（human-derived induced pluripotent stem cell）cell line,which provides experimental basis for the disease model of Alport syndrome in vitro.Methods Firstly,mononuclear cells were isolated from the peripheral blood of the child and cultured in PBMC medium.Then five transcription factors,OCT4,SOX2,MYC,KLF4,and BCL-XL,were introduced into the cultured cells using plasmids to make them Dedifferentiation,followed by disease mutation verification,alkaline phosphatase staining（AP staining）,karyotype analysis,RT-q PCR detection of pluripotency gene expression,cellular immunofluorescence detection,three germ layer directed differentiation experiments,integration analysis experiments,Mycoplasma detection.Results The stem cells induced by PBMC cells showed embryonic stem cell（ES）-like clonal shape.The pluripotency test showed that the induced stem cells were positive for alkaline phosphatase,the expression of Sox2,Oct4,Nanog,Lin28 and rex1 pluripotent genes was positive in the cells.the three-germ layer directional differentiation experiments show that they have the potential to differentiate into three different germ layers.Integration analysis experiments and mycoplasma detection showed that the established hi PS cells did not have foreign plasmid vector integration and mycoplasma infection.The disease mutation verification proved that the established i PS cells carry the COL4A5: NM₀00495.4:c.1517-1G>T mutation（hemizygous）.Conclusion Using somatic cells to establish an Alport syndrome stem cell disease model is a powerful experimental support for the development of Alport syndrome research,and creates a research platform for further clarification of the disease mechanism and drug screening.