Screening Of Gout-related Genes Based On Total Exome Sequencing

Gout is a common arthritis caused by deposition of monosodium urate crystals within joints after chronic hyperuricaemia.Gout is the most frequent inflammatory joint disease with a strong genetic involvement.Especially,gout has become the second largest metabolic disease in China.It is the first time we use whole exome sequencing(WES)to explore the new susceptibility loci of Gout.This study recruited 33 Chinese adults.The gout group consists of 11 adult patients who were clinically diagnosed by endocrinologists as gout referred to the division of rheumatology of First Affiliated Hospital of Kunming Medical University.The healthy group which serves as control consists of 22 healthy adults from the medical center.Genomic DNA was extracted from whole blood samples,collected from the patients and the healthy people.We performed exome capture by Agilent SureSelect Human All Exon Kit followed by whole exome sequencing(Hiseq 3000)in subjects in this study.The advantages and disadvantages of various sequencing data analysis software were comparative analyzed,besides the features and operations were discussed,and also we have carried out BLASTs for the their exomes automatically and analyzed SNPs for subjects by SeqMule in this study.The variants were functionally annotated using an in-house pipeline.Based on these annotations,variants were filtered first for the nonsysnonymous(NS).Exome sequence data were analyzed using bioinformatics and statistical analysis methods to narrow down the variants by R software:filtering out synonymous mutations that do not affect the function of the genes,only retaining the mutation that affect the function of the genes such as nonsynonymous mutations;filtering out common mutations carrying out by normal people in the public genetic mutation database(dbSNP),the healthy individual mutations that have been published,and the mutations carrying out by the control who subject exome sequencing in this study;then making intersection of the mutation set of the patients,screening for single nucleotide variants(P<0.05)with statistical significance by fisher exact test,we reduce the number of candidate pathogenic mutations.After whole exome sequencing,bioinformatics analysis,statistical analysis and function prediction of the protein,we found 23 rare mutation genes,which include 24 rare single nucleotide variants.Through the screening in HGMD,Pubmed,snp138 and other public databases,we identified genetic variants in two SNPs(single nucleotide polymorphisms)of rs3419537 and rs73281920 that located in exonic regions of the KLRC2(c.56C>G,P = 0.00417)and NCOR1(c.59 T>G,P =0.00000143),which were found to be significantly associated with the development of gout.The mutations were predicted to be "damaging" by SIFT and PolyPhen-2.KLRC2(c.56C>G)was found to be located within the linkage region on 12p13.2,which was detected in 6 patients and not detected in healthy control group.NCOR1(c.59 T>G)was found to be located within the linkage region on 17p12-p11.2,which was detected in 9 patients and not detected in healthy control group.After bioinformatics analysis and statistical analysis by the R program self-made,WES revealed two mutation gene loci in 11 Chinese gout patients.With the discovery of two SNVs of gout,the researchers gain a rapid and accurate method to diagnosis gout.By comparing with several analysis softwares,SeqMule has resolved some disadvantages present in many softwares.It also can analyze the data from the whole genomes and the exomes automatically in a comprehensive,flexible and efficient way,and can better analyze the data from high throughput sequencing,finally improve the consistency and accuracy of the data analysis.