Study On Gene Diagnosis,Novel Pathogenic Gene Mutation And Gene Methylation Of Alport Syndrome

Background: Alport syndrome（as）is one of the diseases of syndrome deafness.Its clinical manifestations are repeated microscopic or naked hematuria,chronic renal dysfunction,often accompanied by sensorineural deafness and eye lesions.It is a rare genetic disease.Mutations of COL4A3-A4-A5 genes are the main causes AS.At present,the diagnosis of AS is mainly based on the disease phenotype.The early clinical phenotype of AS is not obvious,so it is often missed diagnosis or misdiagnosis,and renal biopsy is sometimes confused with Benign familial hematuria（BFH）,Focal segmental glomerulosclerosis（FSGS）and other kidney diseases.Since the development of gene detection,because COL4A3-A4-A5 genes have 52,48 and 53 exons respectively,the traditional PCR and Sanger sequencing methods are inefficient and heavy workload.WES has a unique advantage in the discovery of novel mutation sites and even novel genes in susceptible genes,complex genetic diseases and single gene genetic diseases.However,the detection flux of ectopic sites in Mendelian single gene diseases is relatively high,the data analysis is complex,and there is a serious waste of resources.The latest international AS conference guidelines suggest that for patients with suspected AS,high-throughput target gene capture combined with NGS technology should be used to test the pathogenic variation of COL4A3-A4-A5 genes.High-throughput target gene capture combined with NGS technology has three advantages: 1.Reliability and low cost;2.It can provide the DNA sequence information of all three COL4 A genes at the same time;3.It has the potential to detect single nucleotide polymorphism（SNP）and insertion / deletion of various sizes.These techniques can identify up to 95% of the pathogenic COL4 A mutations more efficiently,and complete the gene diagnosis of AS more quickly and efficiently.Accurate genetic diagnosis can understand the risk of other members of the family and whether the disease will recur in future generations,which is helpful for cascade testing of other family members.Testing can prove whether a family member has a pathogenic mutation and whether he can be a kidney donor for an affected relative.In addition,prenatal diagnosis requires the identification of pathogenic mutations,and the understanding of pathogenic mutations often indicates a possible clinical process.Therefore,it is very important to diagnose and find new pathogenic gene mutations in AS patients and families.In addition,because there is no effective treatment for AS,the vast majority of patients will eventually progress to end stage kidney disease（ESKD）.At present,most treatments are not aimed at the repair of basement membrane,but at the associated inflammation and fibrosis.Although some breakthroughs have been made in gene therapy,the therapeutic effect is still not ideal because the key molecular and cellular signaling pathways regulating the progression of AS renal dysfunction have yet to be clarified.The potential genetic susceptibility,environmental factors and epigenetic mechanism of hereditary diseases contribute to the development of diseases.Recent studies have found that gene methylation is closely related to the occurrence and development of a variety of genetic diseases,membranous nephropathy,type I diabetic nephropathy and other kidney diseases.As is a genetic disease with renal phenotype.The development of AS may be closely related to gene methylation.Therefore,we conducted gene methylation research on AS,aiming to explore the key genes and signaling pathways in the development of disease.Therefore,this study intends to efficiently complete the gene diagnosis of AS through high-throughput target gene capture combined with NGS technology,find novel pathogenic gene mutations,summarize the gene diagnosis strategies of AS,deeply analyze the genotype phenotype relationship between novel pathogenic genes and known pathogenic gene mutations,and Illumina 850 k Methylation chip detection technology was used for genome-wide methylation difference analysis and pyrosequencing technology for verification,to explore the correlation between abnormal gene methylation and the development of AS disease,in order to screen out relevant genes and signaling pathways,and provide new ideas and clues for the research of AS disease.Objectives:1.Through high-throughput target gene capture combined with NGS technology,gene diagnosis of suspected AS patients and clinically diagnosed patients can be completed efficiently,and the genetic mode is clear,and novel pathogenic gene mutations are found.2.To analyze and summarize the genetic diagnosis strategies of AS and improve the efficiency of clinical diagnosis.3.By analyzing the genotype phenotype relationship between novel pathogenic genes and known pathogenic gene mutations in AS,we can expand the database of AS genotypes,and play a greater role in the diagnosis,prognosis and treatment of AS.4.Illumina 850 k Methylation chip detection technology was used for genome-wide methylation difference analysis and pyrosequencing technology for verification,to understand the methylation difference between AS group and control group,and to explore the relationship between abnormal gene methylation and the development of AS,in order to screen out the related genes and signaling pathways,and provide new ideas and clues for the research of AS disease.Methods:Part1 Study on gene diagnosis and novel pathogenic gene mutation of Alport syndromeThe clinical phenotype data（including asking all the subjects about the history,filling in the information registration form of deaf patients,drawing family map,routine physical examination,hearing test,fundus and eye fissure lamp examination,urine routine examination,renal function examination）and blood samples were collected for 3 AS family members and family members.DNA was extracted and the pathogenic gene COL4A3-A4-A5 of AS was screened by high-throughput target gene capture and BGS technology.Bioinformatics analysis was performed on the sequencing data,and the mutation sites were verified by PCR amplification and Sanger sequencing technology.Combined with the verification of family members,coseparation analysis of family diseases,clinical data and pathological results,the pathogenic mutation sites of the three families were identified.Summarize the gene diagnosis strategies of AS,the phenotypic characteristics,genotype characteristics,genotype phenotype relationship of novel and known pathogenic gene mutations and the characteristics of X-linked dominant Alport syndrome（XLAS）and Autosomal recessive Alport syndrome（ARAS）were comprehensively analyzed.Part2 Study on gene methylation of Alport syndromeUsing Illumina 850 k methylation chip detection and genome-wide methylation difference analysis,the methylation differences between AS group and control group were compared.The obtained data were analyzed by quality control analysis,differential methylation site screening,GO function enrichment analysis and KEGG analysis,and the abnormal methylation genes related to AS were preliminarily identified.According to the screening results,pyrosequencing technology was used to detect the methylation level of core genes,and the consistency of methylation chip and pyrosequencing results was analyzed to screen out the abnormal methylation genes related to AS disease.Results:Part1 Study on gene diagnosis and novel pathogenic gene mutation of Alport syndrome1.The pathogenic mutation was successfully identified in all three pedigrees,which was a known COL4A5 NM₀00495.3: c.697G>A（p.G233S）mutation,a novel COL4A5 NM₀00495.3:c.133 del G（p.E46Kfs*109）mutation and a rare COL4A4 gene complex heterozygous mutation NM₀00092.4: c.4758₄760del CCC,c.4333+3A>G,and both mutations were new.2.All members of a family with familial hematuria and individuals with hematuria and lamellar glomerular basement membrane or hearing loss,lens or macular retinopathy may have AS and should be tested for all three Alport gene（COL4A3-A4-A5）mutations;3.The discovery of novel pathogenic gene mutation and known pathogenic gene mutation further expanded the variation spectrum of XLAS caused by COL4A5 gene mutation and ARAS caused by COL4A4 gene mutation.The genotype and phenotypic characteristics can help the diagnosis,prognosis and treatment of AS;4.In XLAS,the male hearing curve is groove type.Groove type audiometry curve is rare in clinic,which is helpful to the diagnosis of AS;5.Hearing loss and eye changes are important components in the evaluation of XLAS and ARAS patients.They are not only valuable diagnostic tools,but also predictive factors for the overall prognosis of renal function.Part2 Study on gene methylation of Alport syndromeIn this part,18 differential methylation sites related to AS disease were screened by using the characteristics of high sensitivity and high throughput of methylated chip.The core genes were sequenced by pyrophosphate sequencing.These sites may be closely related to the development of AS disease.Among them,the differential methylation of cg05698732 in COL4A1 and cg03239936,cg11438287 in COL4A2 may affect the structural adjustment of basement membrane,which may be related to the development of AS;the abnormal methylation of the cg00086383 in IL17 RE and HLA may be related to the development of AS kidney disease.Conclusions:1.High-throughput target gene capture combined with NGS technology plays an important role in the etiological diagnosis of AS,which helps to diagnose AS patients and their relatives more efficiently and find novel pathogenic gene mutations.In our study,we found three novel pathogenic gene mutations,namely,COL4A5 gene c.133 del G,COL4A4 gene c.4758₄760del CCC and c.4333+3A>G,and COL4A4: c.4758₄760del CCC,C.4333+3A>G were rare complex heterozygous mutations2.All members of a family with familial hematuria and Individuals with hematuria and lamellar glomerular basement membrane or hearing loss,lens or macular retinopathy may have AS and should be tested for all three Alport gene（COL4A3-A4-A5）mutations;3.The discovery of novel pathogenic gene mutation and known pathogenic gene mutation further expanded the variation spectrum of XLAS caused by COL4A5 gene mutation and ARAS caused by COL4A4 gene mutation.The genotype and phenotypic characteristics can help the diagnosis,prognosis and treatment of AS;4.The differential methylation of cg05698732 in COL4A1,cg03239936 and cg11438287 in COL4A2,cg00086383 in IL17 RE and HLA may be closely related to the development of AS.