The Role And Mechanism Of SMARCAL1 Mutation In Schimke Immune-bone Dysplasia Renal Injury

Research backgroundGenetic metabolic diseases are a group of diseases with corresponding pathological changes and clinical manifestations due to the obstruction of normal material metabolic pathway,substrate accumulation,lack of products and increase of bypass metabolites due to gene mutation.Most of them are autosomal recessive diseases,and a few are autosomal dominant inheritance,X-linked sex inheritance and so on.The clinical manifestations of most genetic metabolic diseases are not specific,which can lead to dysfunction of multiple organ system,disability and death in severe cases.If genetic metabolic diseases are not found in advance,most of them have no effective treatment.Therefore,the occurrence of such diseases not only affects the quality of life of children,but also causes a large burden on families and society.Schinke immune osteodysplasia(SIOD)is a rare autosomal recessive disease,which is mainly characterized by hormone resistant nephrotic syndrome,spinal epiphyseal dysplasia,T cell immune deficiency and special face.In 2002,foreign scholars identified smarcall(MIM:20606622,SWI/SNF related,matrix associated,actin dependent regulator of chromium,subfamily A.like 1)as the pathogenic gene of the disease.SMARCAL1 is located in 2q34-q36 and contains 18 exons encoding 954 amino acids.As of February 2014,100 related mutations of SMARCAL1 had been reported in the human genome mutation database.At present,there are mainly two hypotheses about the pathogenesis of SIOD in the world:(1)it may be caused by the instability of replication fork in the process of cell proliferation.It was found that in endogenous SMARCAL1 silent cells,SIOD related mutations could not prevent replication related DNA damage;However,after reintroducing wild-type SMARCAL1,the DNA damage was repaired.(2)It may be caused by cell cycle arrest and premature apoptosis.Ciccia et al.[7]found that SMARCAL1 failed cells are very sensitive to DNA damage agents that induce replication fork breakage.Based on the above research results,SMARCAL1 mutation aggravates DNA damage related to replication,and is closely related to the regulation failure of cell cycle,which eventually leads to the occurrence of multisystem damage.The clinical manifestations of SIOD are diverse,but the most prominent are spinal epiphyseal dysplasia and renal injury.Most patients with SIOD have been treated in different hospitals and departments with primary nephrotic syndrome,short stature and severe infection,causing great confusion to the diagnosis,and the disability and death rate of the disease is high,At present,all known reports on SIOD show that patients will eventually develop into renal failure,which is manifested as the progress of proteinuria in the early stage.Progressive nephropathy is ineffective to hormone therapy,and eventually develop into irreversible renal failure,which is also an important reason for the death of children with SIOD.Most of the previous diagnosis of SIOD was based on the clinical manifestations of patients.With the development of high-throughput sequencing technology,it provides an effective guarantee for the refinement of clinical disease diagnosis.If we use accurate gene diagnosis in the early stage of proteinuria,we can reduce the production of proteinuria through clinical active intervention,so as to delay the process of renal failure,It is very meaningful for the survival rate of children and the quality of life of their families.In this study,the frameshift mutation c.1071delT of SIOD disease was found for the first time;p.(phe357fs)was searched by using multiple public databases,but the mutation was not reported.It was a new mutation,and polyphen₂ was applied at the same time,SIFT,Align GVGD,MutationTaster and other bioinformatics analysis software analyzed and predicted the test results,which indicated that the mutation was harmful,so it was predicted to be a pathogenic mutation.The previous studies of our research group confirmed for the first time that the new mutation led to the reduction of SMARCAL1 protein expression,so as to clarify its pathogenicity.In this study,siRNA technology will be further used to silence SMARCAL1 gene and transfect HEK293 embryonic kidney cells.The expression changes of SMARCAL1 mRNA and protein in HEK293 embryonic kidney cells after siRNA transfection will be detected by real-time fluorescence quantitative PCR and Western blot,and the cell proliferation and apoptosis will be observed;Finally,the differential genes of SMARCAL1 gene silencing cell model were screened by transcriptome sequencing,and the correlation was analyzed by bioinformatics;So as to further explore the possible pathogenesis of renal injury caused by SMARCAL1 mutation.The purpose of this experiment is to enrich the gene spectrum of SIOD diseases in the human mutant gene database,and to explore the role and mechanism of smarcall gene mutation in renal injury in SIOD diseases,so as to provide new targets and new ideas for clinical treatment.This topic will be studied from the following two aspects.Part ?:case characteristics and detection of new mutant genes in children with Schimke immune bone dysplasiaObjective:to collect the data of a short stature child,clarify the specific site of gene mutation,whether it is new and the preliminary prediction of pathogenicity,and further enrich the gene spectrum of SIOD disease in the human mutation gene database.Methods:detailed medical history collection and comprehensive physical examination were carried out for outpatients with short stature,and relevant auxiliary examinations such as three routine examinations,liver and kidney function,electrolyte,blood lipid analysis,cellular immunity and humoral immunity,insulin-like growth factor,thyroid function,thoracolumbar and lateral films and pelvic films were improved.Full exon sequencing was used,Obtain the new mutation site of SMARCAL1 gene and evaluate its function.Results:1.Auxiliary examination:abnormal cellular immune function(auxiliary T lymphocytes:320.25/UL,auxiliary T lymphocytes:12.21,CD4+T lymphocytes/CD8+T lymphocytes:0.33);Massive proteinuria(24-hour urinary protein:978.11mg,urine routine:urinary protein+++);The anteroposterior and lateral films of thoracolumbar spine and the anteroposterior and lateral films of pelvis all suggest skeletal dysplasia;There are abnormal functions of skeletal system,immune system and urinary system.2.Full exon sequencing showed that the frameshift mutation c.1071delt was detected in SMARCAL1 gene;p.(phe357fs),found through the public database,this mutation has not been reported.It is a new mutation,and polyphen₂ is applied at the same time,SIFT,Align GVGD,MutationTaster and other bioinformatics analysis software analyzed and predicted the test results,which indicated that the mutation was harmful,so it was predicted to be a pathogenic mutation.Conclusion:1.A new frameshift mutation SIOD in SMARCAL1 was found,and the mutation type was c.1071delT.2;p.(Phe357fs),which is preliminarily predicted to be a new pathogenic mutation.2.Children with new frameshift mutation SIOD have skeletal dysplasia,abnormal cellular immune function and massive proteinuria.Part ?:the effect of SMARCAL1 gene silencing on the function of HEK293 embryonic kidney cells and its mechanismObjective:To investigate the effect of SMARCAL1 gene silencing on the function of HEK293 embryonic kidney cells and its mechanism.Methods:1.Sirna-1,sima-2,sirna-3 and a negative control sequence were designed and synthesized according to the mRNA sequence of human SMARCAL1 gene.2.40 nm was selected as the final transfection concentration of siRNA.The expression of SMARCAL1 mRNA and smarcal protein were detected after transfection.2.CCK-8 assay and Edu staining were used to observe the proliferation of HEK293 embryonic kidney cells in Blank control group,si-SMARCAL1 group and Negative control group,and the apoptosis of HEK293 embryonic kidney cells in the three groups were observed by flow cytometry.3.The differentially expressed genes in HEK293 embryonic kidney cells of si-SMARCAL1 group and negative control group were detected by transcriptome sequencing,and the differentially expressed genes were analyzed and screened by go function analysis and KEGG enrichment analysis;The differential genes were verified by real-time fluorescence quantitative PCR.4.The expression levels of KLF4,Nephrin,P53,Bax,Caspase3 and Bcl-2 mRNA in HEK293 embryonic kidney cells of negative control group and Si smarcall group were detected by fluorescence quantitative PCR.5.The expression levels of KLF4,Nephrin,P53,Bax,Caspase3 and Bcl-2 in HEK293 embryonic kidney cells of negative control group and Si smarcall group were detected by Western blot.Results:1.Negative control(NC)group and three siRNAs targeting smarcall,sirna-1,sirna-2 and sima-3,were transfected into HEK293 embryonic kidney cells.After 72 hours,more than 60%of HEK293 embryonic kidney cells in the four groups showed fluorescence,indicating that the transfection was successful.2.The relative expression level of smarcall mRNA suggests that the silencing efficiency of SMARCAL1 in siRNA-1 group is the best.At the same time,the expression levels of SMARCALI protein in HEK293 embryonic kidney cells of the four groups were detected respectively.Similarly,the siRNA-1 group had the best interference effect and the protein level decreased most significantly.2.At 24h,48h,72h and 96h of cell culture in blank control group,negative control group and si-SMARCAL1 group,the light absorption value of CCK-8 method at each time point of si-SMARCAL1 was significantly lower than that in blank control group and negative control group(P<0.001).3.After 3 days of cell culture in blank control group,negative control group and si-SMARCAL1 group,edu staining showed that the cell proliferation rate and number in si-SMARCAL1 group were significantly lower than those in blank control group and negative control group(P<0.001).4.Flow cytometry showed that the apoptosis rate of si-SMARCAL1 group was significantly higher than that of blank control group and negative control group(P<0.001).5.Transcriptome sequencing screened the differential genes after SMARCAL1 gene silencing,and a total of 7398 differentially expressed genes were screened.Among them,401(5.4%)genes were up-regulated and 6997(94.6%)genes were down regulated.6.Through the go(gene ontology)function of differentially expressed genes and the enrichment analysis of KEGG signal pathway,the volcano map of differentially expressed genes was drawn,showing that most differentially expressed genes were down regulated,including epithelial differentiation related core receptors and core transcription factors GLI3,GLI4,SOX 13 and KLF4,suggesting that SMARCAL1 may participate in the pathogenesis through the above ways.Based on the comprehensive analysis of FPKM value and change multiple,KLF4 may be one of the key factors in the pathogenesis of SMARCAL1.7.KEGG pathway enriched and analyzed the differential genes.The results showed that the differential gene expression mediated by si-SMARCAL1 group,the pluripotent regulatory signal pathway related to stem cell differentiation potential,the notch,Wnt pathways related to cell proliferation,differentiation and apoptosis,and the Hippo pathway related to organ development,proliferation and differentiation were significantly negatively enriched.The results show that SMARCAL1 may be related to renal epithelial differentiation,and its loss of function may hinder the differentiation and proliferation of renal function related cells,increase apoptosis,and cause renal function injury.8.The expression of KLF4,Nephrin,P53,Caspase3,Bax and Bcl-2 mRNA in HEK293 embryonic kidney cells in si-SMARCALl group and negative control group showed that the expression of KLF4,nephrin and bcl-2 mRNA in si-SMARCAL1 group was significantly lower than that in negative control group(P<0.001,P<0.001,P=0.005),and the expression of P53,caspase-3 and Bax mRNA in si-SMARCAL1 group was significantly higher than that in negative control group(P=0.013,P<0.001).9.The expression of KLF4,Nephrin,P53,Caspase3,Bax and bcl-2 protein in HEK293 embryonic kidney cells in si-SMARCALl group and negative control group showed that the expression of KLF4,Nephrin and Bcl-2 protein in si-SMARCAL1 group was significantly lower than that in negative control group(P<0.001,P<0.001,P=0.005),and the expression of P53,Caspase-3 and Bax protein in si-SMARCAL1 group was significantly higher than that in negative control group(P=0.013,P<0.001).Conclusion:1.Silencing SMARCAL1 gene in siRNA-1 group resulted in the most significant reduction of smrcall protein level.Therefore,embryonic kidney cell lines in siRNA-1 group were selected for follow-up experiments.2.The decrease of SMARCAL1 gene expression can lead to the increase of apoptosis and inhibition of proliferation of HEK293 embryonic kidney cells.3.Through bioinformatics analysis,KLF4 gene is one of the key factors of SMARCAL1 gene mutation leading to disease,and is highly related to the mechanism of apoptosis and renal injury.4.It is confirmed for the first time that the decreased expression of SMARCAL1 leads to the decreased expression of KLF4.The decreased expression of Nephrin is one of the mechanisms of renal injury in children with SIOD.5.It is confirmed for the first time that the decrease of smarcall expression leads to the decrease of KLF4 expression and the increase of P53 expression,which directly or indirectly promotes the expression of Caspase-3 and Bax protein and inhibits the expression of Bcl-2 protein,which is an important mechanism to mediate the increase of apoptosis and decrease of proliferation of HEK293 embryonic kidney cells.