Explore The Pathogenesis Of Genetic Diseases Through IPS Cell Models And Co-expression Network Analysis (WGCNA)

Genetic disease refers to a group of diseases caused by changes in genetic material or controlled by pathogenic genes.These diseases are of high incidence,various kinds and complex phenotypes.The pathogenesis of most genetic diseases remains unclear.As a result,symptomatic treatments have been adopted to delay disease progression.In the past,many animal models of genetic diseases have been established.These animal models have helped us learn a lot about the complex pathogenesis of genetic diseases.However,most animal models can not completely reproduce the development process of human diseases due to the difference of species between animal and humans.Induced pluripotent stem cells(iPSC)derive from patients and can retain the genetic defects of the patients.Therefore,it can be used as a perfect model for the study of all kinds of genetic diseases.And iPSC,as a kind of cell type derived from the patient,can also be used in regenerative medicine to bring possible cures for more genetic diseases.Professor Zhengfeng Xu and his group have been committed to applying iPS technology to the research of genetic disease.In previous work,we set up a platform for the reprogramming of peripheral blood mononuclear cells(PBMC)and amniotic fluid cells(AFC)to iPSC.iPSC disease model of mitochondrial genetic disease myoclonus and myoclonic epilepsy and ragged red fibers(MERRF)has been established.This study includes two parts.In the first part,iPSCs from MERRF patients obtained previous were differentiationed into cardiomyocytes.iPSC and cardiomyocytes were sequenced and then analysed through weighted correlation network analysis(WGCNA)to explore the new molecular mechanism of myocardial injury of MERRF syndrome.In the second part,cells of genetic diseases including Trisomy 21 syndrome(T21),Williams syndrome and phenylketonuria(PKU)were reprogrammed to establish the iPSC disease model.Genetic defects were detected of the three groups of iPSC.As well,sequencing and WGCNA were conducted to explore the pathogenesis of these genetic diseases.Objective MERRF is a kind of mitochondrial disease caused by an A to G mutation at mtDNA 8344 location.About 53% MERRF patients also suffer from cardiomyopathy except myoclonic epilepsy and cerebellar ataxia.This study aims to obtain MERRFspecific cardiomyocytes by in vitro differentiation from MERRF patient-derived iPSC and explore mechanism of MERRF myocardial damage by sequencing combined with WGCNA analysis.Methods MERRF-derived iPSC were differentiated into cardiomyocytes.These cardiomyocytes were identified and validated by immunofluorescence staining and RTPCR.Inner structures of cardiomyocytes with different mutation rates were examined by transmission electron microscopy.RNA was extracted of cell lines with the lowest and the highest mutation rates to performe sequencing.Important genes were selected for qPCR based on the results of sequencing.Results 4 group of MERRF-derived iPSCs were all differentiated into beating cardiomyocytes successfully.Cardiomyocytes expressed normal cardiac marker genes identified by Immunofluorescence staining and RT-PCR.Compared to cardiomyocytes with lower mutation rates,mitochondria of cardiomyocytes with higher mutation rates was broken and swelled under electron microscope,and the beating frequency of cardiomyocytes was relatively lower.The result of WGCNA analysis involved some genes and signaling pathways related to cardiac development,muscle differentiation,and myocardial cell contraction and so on.Expression of these genes were significantly different between M1-CMs and M4-CMs.The result of these important genes inspected by qPCR test was almost consistent with that of sequencing.Conclusion MERRF-derived iPSC was successfully differentiated into cardiomyocytes with analysis of structural and function,which indicated the cardiomyocytes we established were suitable model for disease research.The genes and signaling pathways found by WGCNA and qPCR were associated with heart development.They could provide information for exploring the molecular mechanism of MERRF myocardial injury and providing potential targets for the treatment of myocardial injury in the future.Objective To establish iPSC disease models of 3 common genetic diseases including PKU,Williams syndrome and T21.To explore the pathogenesis of these 3 kinds of genetic diseases through sequencing and WGCNA analysis so as to provide the basis for the early intervention and treatment of the genetic disease.Methods PBMC of PKU and Williams syndrome,and AFC of T21 were collected.4 transcription factors(Oct4,Sox2,Klf4 and c-Myc)were used to reprogrammed PBMC and AFC to iPSC.The pluripotency of iPS cell lines were identified by immunofluorescence staining,RT-PCR and embryoid differentiation.Mutation points of PKU-iPSC were detected by sanger sequencing.Copy number variation of WilliamsiPSCwas identified by chromosome chip analysis.Chromosome karyotype of T21-iPSC was detected.Sequencing and WGCNA were performed of these iPS cell lines.qPCR was used to identify important genes related to diseases.Results iPS cell lines of these genetic diseases were obtained successfully.Immunofluorescence staining,RT-PCR,and embryoid differentiation were used to identify the pluripotency.Gene defects of T21-iPS,PKU-iPS and Williams-iPS were all consistent with primary patients’.The module of T21-iPS expressed specially showed some items related to the differentiation of hematopoietic progenitor cells.While the special module of PKU-iPS and Williams-iPS indicated some items related to axon,neuron development and so on.The major results of qPCR validation were consistent with the results of sequencing.Conclusion iPS disease models of PKU,Williams syndrome and T21 were successfully established and verified.All the three lines of iPSCs could retain the genetic defects of the disease.GATA3 gene associated with leukemia was found highly expressed in T21-iPS by WGCNA and qPCR.ERBB2 and LEF1 genes were found highly expressed in Williams syndrome related to the process of neurodevelopment,indicating that the phenotype of the disease can be shown earlier at iPS level.These iPSC models will provide a good platform for further studying of molecular mechanism of these genetic diseases.