Genome-wide Association Study And HLA Region Fine Mapping Study Of Syphilis

Background.Syphilis is a sexually transmitted disease(STD)caused by Treponema pallidum(T.pallidum/TP).TP is an ancient pathogen that is highly invasive and invisible.One can be infected with syphilis when the skin or mucous membranes are exposed to as few as ten TP.TP can invade any tissues or organs of the human body.After proliferating and replicating for a period of time after entering the human body,it can cause clinical symptoms.If left untreated,it can lead to very serious complications,mainly neurosyphilis,cardiovascular and gum syphilis.If the treatment delays or treatment is not complete,pregnant women with syphilis can lead to stillbirth,premature birth,neonatal death,low birth weight or congenital syphilis.TP has strong pathogenicity and immune evasion characteristics,and the mechanism involved is complex.In addition,since TP is not easily cultivated in vitro,there is no suitable and mature animal research model,resulting in unclear pathogenic mechanism,by which the human body clears TP and TP escaping the host’s immune system(causing its long-term persistence in the human body).The human body’s innate and adaptive immune system plays an important role in the clearance of TP.Early syphilis carries the highest possibility of infection,and sexual contact with an individual who has early syphilis leads to a 50%-60% risk of syphilis infection.A retrospective study also showed that about 38.3% of the sexual partners remained seronegative after having unprotected sex with syphilis patients,suggesting that the host’s unkown factors may affect susceptibility to TP.Genome-wide association studies(GWAS)have been widely used to study genetic susceptibility to complex diseases and many disease-related susceptibility genes have been discovered.GWAS also has outstanding achievements in the study of the susceptibility to infectious diseases such as the detection of susceptible genes for infectious diseases such as tuberculosis,leprosy,hepatitis B / C virus infection and related liver cancer.Human leukocyte antigen(HLA)system is one of the most complex antigen systems known so far.It has been a hot research field,because it involves in autoimmune disease,infectious disease,inflammatory disease and so on.Several studies have reported that HLA is associated with the HIV,HBV infection,HCV infection,malaria or EBV.Previous studies have also reported that HLA alleles(HLA-A02,Aw32,B07,B18,C1,HLA-DRB1*14)may be associated with susceptibility / resistance to syphilis.Imputation is to use reference groups’ complete genotypic information linked to each other to create a marker haplotype information,so that the target population’s genotype missing sites can be filled with complete information.Imputation can increase the coverage rate of genome-wide genetic markers and research efficiency,and increase the success rate of screening.It is inferred that the imputation of untyped SNPs can increase the number of SNPs analyzed.It is useful in further analysis of SNPs data from different types of platforms.Direct sequencing of HLA regions is expensive,time-consuming and labor-intensive.Traditional imputation methods cannot impute classic HLA alleles and amino acid polymorphisms.SNP2 HLA is a good tool for imputation studies of HLA regions.SNP2 HLA has been used successfully in a many diseases’ studies.It not only imputes the classic HLA alleles but also imputes amino acid polymorphisms of these classic alleles,therefore allows direct correlation analysis of amino acid polymorphisms.Objectives.Using GWAS and HLA fine mapping study to search for genetic or functional variations associated with syphilis,and to evaluate the relationship between mutations and syphilis through functional annotation,and to explore the correlation between host’s genome factors and susceptibility/resistance to syphilis.Methods.Whole genome genotyping was performed in 2,530 Chinese Han people(1,499 syphilis patients and 1,031 controls)using the Illumina Human Omni Zhong Hua-8 Bead Chip microarray.Association analysis was performed after quality control and candidate SNPs associated with syphilis were selected and replicated in 6,254 independent samples(3,086 syphilis cases and 3,168 controls)using the Sequenom Mass Array system.Meta analysis of the two-stage experimental data was conducted to search for genetic variations associated with syphilis.At the same time we conducted a genotype-phenotype analysis in order to find polymorphisms / loci associated with syphilis phenotypes.The cases were divided into different groups according to their clinical phenotypes,including the stage of syphilis(primary,secondary,tertiary,latent,neurosyphilis,gestational),gender(male and female),prognosis(cured and not cured),concomitant(with and without concomitant).In addition,the genotyping data of the HLA region(29-34 Mb)in the genome-wide genotyping data of 2,530 samples were extracted.Then we use SNP2 HLA software for HLA imputation,with the MHC region sequencing information of 10,689 Han individuals(Han-MHC)obtained by the previous sequencing as the reference panel.Results.After quality control and statistical analysis of the genotyping data in the discovery stage,forty-four candidate SNPs(P <1.0 × 10-4)were selected for replication.The genotyping data and replication data of the whole genome were analyzed using meta-analysis.One SNP was found to achieve genome-wide significance: rs74858962 SNP at 3p13(PMeta < 5 × 10-8).Stratified analysis did not find any statistical correlation between phenotype and genotype.Fine mapping of HLA regions revealed that the HLA-DQB1*05 allele and polymorphisms of HLA-DQβ1 were associated with syphilis(P < 2.11 × 10-6 after Bonferroni correction).Conclusions.This study was the first GWAS study of syphilis.One syphilis susceptible region(3p13),one HLA allele(HLA-DQB1*05)and polymorphisms of HLA-DQβ1 were identified to be related to syphilis.This study enriches the genetic variation of syphilis in Han Chinese and provides new insight into the study of susceptibility / resistance to syphilis,which helps to improve the understanding of the pathogenesis of syphilis and also provide a theoretical basis for prevention and vaccine discovery of syphilis.