Identification Of New Pathogenic Mutation For Alport Syndrome And Establishment Of Patient-derived Induced Pluripotent Stem Cells

BackgroundAlport syndrome is a kind of hereditary nephropathy with hematuria as the most common clinical manifestation,accompanied by proteinuria and progressive renal decline,often with or without sensorineural deafness and anterior cone lens and other extrarenal symptoms.The glomerular basement membrane(GBM)is abnormal.It was found that about 85%of Alport syndrome is X-linked dominant disease caused by COL4A5 gene mutation,and about 15%is autosomal recessive and autosomal dominant disease caused by COL4A3 and/or COL4A4 gene mutation.At present,the diagnosis of Alport syndrome is mainly based on clinical symptoms,family history,laboratory examination,immunofluorescence examination of tissue basal membrane Ⅳ collagen a chain and electron microscopy of renal tissue biopsy.Although electron microscopy of renal tissue biopsy and collagen Ⅳ staining are important indicators for the diagnosis of Alport syndrome,gene sequencing is necessary for the diagnosis of some suspected Alport syndrome patients with atypical pathological changes.At present,most research on genetic diseases are based on cell or animal models.It is difficult to simulate the pathogenesis of gene mutation in human body.Induced pluripotent stem cells(iPSC)have become a new model for studying genetic diseases as they preserve the genetic background of the parental somatic cells.hiPSCs have been used in studies of Long QT syndrome and sickle cell anemia.Aims(1)To use high throughput sequencing to diagnose a patient with clinical syptoms of Alport syndrome and identify the disease-causing mutation(2)To establish hiPSCs from peripheral blood mononuclear cells(PBMCs)from the patient Methods and results(1)The patient was a 25-year-old female with urine occultation blood 3+,urine protein 3+,24-hour urine protein quantification 3.2g/24h,accompanied by cleft lip and palate and nonsensorineural hearing loss.Alport syndrome was suspected based on clinical findings and renal pathology.(2)We extracted the DNA from the patient and sequenced the gene loci associated with hereditary nephropathy.We identified two heterozygous mutations in COL4A3 gene,c.4243G>C(p.G1415R)and c.4216G>A(p.G1406R).We adopted Sanger sequencing to analyze the two loci in the patient and her family members,and found that the c.4243G>C mutation was inherited from her father and the c.4216G>A mutation was inherited from her mother.The family conforms to autosomal recessive Alport syndrome.(3)To establish the patient-derived hiPSCs,we collected venous blood from the patient,and isolated and cultured PBMCs.PBMCs were reprogrammed into hiPSCs by transferring plasmids expressing transcription factors OCT4,SOX2,KLF4,MYC and LIN28.Through immunofluorescence staining,RT-qPCR,detection of exogenous transgenes,karyotype analysis and mycoplasma detection,we found the established hiPSCs expressed surface antigens TRA-1-60,TRA-1-81,SSEA4 and transcription factors OCT4 and SOX2.The mRNA level of endogenous multipotent genes OCT4,SOX2 and NANOG was similar to embryonic stem cells.The established hiPSCs maintained normal karyotype and COL4A3 c.4243G>C and c.4216G>A mutations.ConclusionsIn this study,gene sequencing was used to confirm the diagnosis of Alport syndrome in patients with clinical symptoms and renal pathology.A novel likely pathogenic gene mutation of the COL4A3 gene was identified.Through induced pluripotent stem cell technology,patient-derived hiPSCs were established,laying a foundation for further research on the pathogenesis and treatment of Alport syndrome.