Screening Susceptibility Genes In The Genome HLA Region Of SLE By Using MHC Genes Capturing And Sequencing

Background:Systemic lupus erythematosus (SLE) is a complex autoimmune disease, its basic features is producing variety of autoantibodies, immune complex deposition and complement activation, resulting in multiple systems and organs damaged, including the skin, joints, kidneys and central nervous system, ultimately inducing renal failure, lupus encephalopathy and severe secondary infection, and even death in patients with life-threatening cause serious harm to human health. The etiology of SLE is still not been full understanding, but several genetic, environmental factors and immune system disorders have been implicated. The contribution of genetic factors to SLE predisposition is evident. Human leukocyte antigen (HLA) genes, located on 6p21.3 of human chromosome 6 short arm, are closely linked gene complex composed by a number of loci. So far, they are the highest polymorphism complexes in the known genes. HLA genes code and generate proteins that were related to immune recognition and immune response respectively as well as involved in the process of antigen presentation and the induction of immune response. At present, the abnormality of HLA genes is one of the main factors leading to the morbidity of SLE through the genome-wide association study with large sample. In recent years, studies have shown that whole genome sequencing and PCR+ Sanger were the main means to identify causative genes in SLE, but there were high costs with the former and low coverage with the latter, they were not the ideal methods. At present, studies have shown that capturing and sequencing with the target area has obvious advantages. Major histocompatibility complex (MHC) genes capturing technology may be the new way to effective analyze the pathogenesis of SLE with HLA genes.Objective:1. To screen possible variations associated with SLE in HLA region by using MHC genes capturing technology and high-throughput sequencing technology and discuss the correlation between variations and SLE.2. To obtain susceptibility genes of SLE and provide a new way to explore the nosogenesis of SLE as well as for early diagnosis and treatment in the long run.Methods:1. After rigorous screening,15 SLE patients and 15 healthy volunteers were selected as disease group and control group of our study, respectively. Peripheral blood was collected from patients and volunteers.2. Peripheral blood genomic DNA was extracted from all subjects and their yield and quality were assessed by UV absorbance.3. HLA genes were captured by using chip hybridization with NimbleGen Human MHC Kit and were amplified by PCR. Then we used Illumina HiSeq 2000 to sequence the genes that have been captured.4. Sequencing data were analysis via bioinformatics and compared with public databases, including dbSNP132, 1000 genome project and control group, to screen out possible causative mutation. SIFT and Polyphen-2 were used to predict possible impact of protein in secondary structure and functional according to these variants.Results:1. In disease group and normal control group, the coverage of high-throughput capturing and sequencing were 97.9% and 98.0% respectively, with the corresponding mapping rate were 98.9% and 98.36%, the average sequencing depth were 91.10 and 90.99 and the sequencing depth greater than 10×were 95.60% and 95.50%. We obtain total 27065 and 27058 SNPs in SLE disease group and the control group respectively after bioinformatics analysis.2. After comparing with public databases, including dbSNP132,1000 genome project, and control group to filter out common mutations and using SIFT and Polyphen-2 to predict the secondary structure and functional impact of protein, we found that there were 3 possible causative mutations, which were DRB1*15:02:01, DQA1*01:02:02 and DQB1*03:03:02 respectively, may be associated with SLE.Conclusion:1. HLA genes associated with SLE, especially HLA-II closely associated with SLE. Research on HLA-II may contribute to reveal the pathogenesis of SLE, and provide new ideas for the treatment of SLE.2. DRB1* 15:02:01, DQA1* 01:02:02 and DQB1* 03:03:02 are important susceptibility genes associated with SLE, may be used as therapeutic targets and provide new evidence for SLE diagnosis and treatment.