Exome-wide Association Study And Metagenomic Analysis Of Intestinal Microbiota In Patients Of Chronic Prostatitis/chronic Pelvic Pain Syndrome

BACKGROUND: Chronic prostatitis/chronic pelvic pain syndrome(CP/CPPS)is the most common disease of urology and andrology clinic,with an estimated prevalence of 2-16% in the male population whom were mainly affected around 35-45 years.CP/CPPS is a highly heterogeneous disease,and the cause is not yet clear.The involved biological mechanisms are very complex,and there is no identified single cause can explain the pathogenesis.Results from familial aggregation researches and twin studies suggest that genetic factors might play an important role in the occurrence of CP/CPPS.Besides,CP/CPPS is considered as a complex disease that occurs and develops under multiple environmental factors on the basis of genetic susceptibility,and the pathogenesis involves infection,immunity,neuropsychiatric functions and other problems.Recent studies have shown that there is a significant correlation between CP/CPPS and the changes of structure and abundance of intestinal flora.The exome array covers the putative functional genetic variations of exon,with the advantages of high flux,wide coverage,high sensitivity,high accuracy,low cost and high benefit,providing a powerful tool for the study of the genetic mechanism of complex disease.Target sequence capture and sequencing is a high-throughput research method based on the second-generation sequencing technology,which can obtain ultra-high sensitivity and accuracy by using a small amount of data and rapidly screen the genetic variants.The macro-genome sequencing of intestinal microbiota can analyze not only the microflora structure of intestinal bacteria but also their gene functions.Combined with these research strategies,we can make a comprehensive study of the genetic background of human body and the possible role of the structure and function of intestinal microbiota in the occurrence and development of CP/CPPS.OBJECTIVE: To investigate the role of host genetic susceptibility and intestinal microbiota in the pathogenesis of CP/CPPS,we perform exome-wide association study and metagenomic research on intestinal microbiota,combining with the target area capture and sequencing,gene pathway enrichment and hierarchical clustering analysis.The performance of these studies will provide theoretical foundations for diagnostic model that based on genetic susceptibility loci and disease associated intestinal flora structure,as well as giving support to individualized diagnosis and treatment.METHOD: Exome-wide association study of 1699 CP/CPPS patients and 1647 healthy controls was conducted applying the Illumina Human Exome Bead Chip.Then the target region capture and sequencing was carried out for the chromosomal area which might have correlation with disease.Combined with public database(Roadmap Epigenomics,ENCODE the projects,KEGG,Bio Grid etc.)data,the relevant functions of the genes found by the exome-wide association analysis and the target region capture and sequencing were further analyzed using quantitative trait loci(e QTL)analysis,pathway enrichment analysis,gene pathway clustering analysis and protein-protein interaction(PPI)analysis.RESULTS: Single variant tests revealed a significant association between CP/CPPS and rs7453920(MAF=0.08,P=8.78×10-9)on the intron of HLA-DQB2 and rs527369852(MAF=0.263,P=6.01×10-8)in the intergenic region.Target region capture and sequencing was performed on chromosome 6p21.32(chr6: 32427000-32454000)where the genetic variation loci of P<10-5 were concentrated on.It was found that the frequency distributions of three Indel(rs566122716,rs571840751,rs76318940)located in the upstream of HLA-DRB9 and 32 SNPS located between HLA-DRB9 and HLA-DRB5 genes were different between patients and controls,and the differences were statistically significant(all P<0.05).Among 240 loci with P<10-3,a total of 128 loci with e QTL effect were detected,most of which were located on the HLA region.The enriched genes included HLA-C,HLA-DRB1,HLA-DRB5,HLA-DQA1,HLA-DQB1,IL6 R,and TNFSF11 A.The pathways of these genes were closely related to autoimmune function(e.g.allograft rejection).PPI enrichment analysis indicated that PPI existed among HLA-DQB1,HLA-DQB2,HLA-DQA1,HLA-DRB1 and HLADRB5,because they could be clustered together.The pathways involved in this cluster include translocation of zap-70 to immune synapses,phosphorylation of CD3 and TCR zeta chains,and PD-1 signaling pathways.We found that HLA-DQB2 was associated with CP/CPPS susceptibility.Target region sequencing showed that 3 Indel,HLA-DRB5,and HLA-DRB9 were correlated to CP/CPPS.These findings might profit to enrich the results of genetic susceptibility to CP/CPPS in Chinese population and improve the understanding of the pathogenesis of CP/CPPS.METHOD: Metagenomic study on intestinal microbiota was conducted on 34 CP/CPPS patients and 32 healthy controls,adopting the whole genome shotgun sequencing method.The structural diversity of intestinal microbiota and the relationship between the relative abundance of species and disease were investigated through the analysis of the genome sequence assembly,gene prediction,metagenomic species positioning analysis,metagenomic population difference analysis,and biological pathway annotations and difference analysis.Using(partitioning around medoids with estimation of the number of clusters)PAMK algorithm and(multidimensional scaling)MDS analysis,the metabolic pathways corresponding to the genes of the microbiota were clustered,and the functional patterns were further studied.The random forest model in the machine learning algorithm was applied to screen the intestinal microbiota that could distinguish the clustering of metabolic pathways and the disease status,and construct the diagnostic model.RESULTS: The microbiota structure of CP/CPPS patients and the healthy control showed a different distribution.In CP/CPPS patients,the intestinal microbiota was dominated by Escherichia,a genus subordinate to Proteobacteria,while that of the healthy control was dominated by Erysipelotrichaceae,a genus subordinate to Actinobacteria.The relative abundance of the Escherichia in CP/CPPS patients were significantly higher than that of control group(P<0.05).Thirty-three metabolic pathways corresponding to bacteria genes were differentially expressed,including pathways that affect biochemical metabolism and neurologic disorders(e.g.the tricarboxylic acid cycle pathway,alzheimer's disease pathway).Pathway clustering analysis showed that these pathways could be divided into two clusters,among which the metabolic pathways of healthy controls were more consistent.In addition to participating in similar biological pathways in healthy controls,the intestinal microbiota of CP/CPPS patients also took part in several biological pathways that significantly differed from those in healthy controls,such as drug metabolism-other enzymes,clavulanic acid biosynthesis,cytochrome P450 to exogenous organisms and other biological pathways associated with drug metabolism.Using the combination of two genus of intestinal bacteria(Prevotella_copri and Escherichia_coli),the diagnosis model based on machine learning algorithm could distinguish the disease status of CP/CPPS and healthy controls,of which the prediction accuracy was 77.3%.Through metagenomic research,we found that there were significant differences in intestinal microbiota structure between CP/CPPS patients and the control group,and the relative abundance of Escherichia in CP/CPPS patients was significantly increased.The structural changes of microbiota in further influenced the progression of disease by affecting different metabolic pathways.The diagnostic model that constructed by using intestinal bacteria could distinguish patients into sub types,which therefore,providing a theoretical basis for accurate clinical classification and guiding individual clinical diagnosis and treatment.CONCLUSION: Applied exome-wide association analysis and intestinal microbiota metagenomic analysis,we identified a number of genetic variants associated with CP/CPPS and found disease-associated structural difference of intestinal microbiota.These findings will provide evidence for further studies on the effects of host heredity and intestinal microbes on the occurrence and development of CP/CPPS.