Study On Associations Of Intestinal Flora And FUT2/3 Gene Polymorphisms With Ankylosing Spondylitis

Objective Ankylosing spondylitis(AS)usually has a great impact on the living quality and work capacity of the patients,and has become a serious public health problems.AS is closely related to genetic factors,especially HLA-B*27 gene.Up to now,at least 36 genes susceptible to AS have been found,which are involved in all aspects of human immunology.AS is closely associated with both intestinal mucosal lesions and gut inflammation,and 50%?60%of patients with AS are accompanied by intestinal inflammation.About two-thirds of the susceptibility genes of AS overlap with those susceptible to inflammatory bowel disease(IBD),and the same nucleotide polymorphisms(SNPs)have the same or similar effects in the two diseases.Dysbiosis of intestinal flora can induce IBD,and is closely related to many immune diseases,such as rheumatoid arthritis(RA).Several studies have revealed significant differences in the composition of intestinal flora between the patients with AS and healthy controls,suggesting that the dysbiosis of intestinal flora may play certain role in the occurrence of AS.Therefore,it is of special value to further search for the susceptible genes to AS in the related genes of intestinal inflammation and gut microbiota.Human FUT2 and FUT3 genes are two blood group genes,which jointly control the synthesis of human histo-blood group antigens(HBGAs).A number of studies have shown that FUT2 and FUT3 gene polymorphisms are related to intestinal flora and IBD,and it is hypothesized that these genes may also be associated with the susceptibility to AS and its symptoms.However,the number of studies on the correlation between intestinal flora and AS is still relatively small so far,with some inconsistent results for defferent populations,so their conclusions need to be further verified.Diet has a significant influence on intestinal flora,thus it may also affect the susceptibility to AS.But this factor has mostly been overlooked in previous studies,and their findings may be biased.In view of these reasons,this study aims to further verify the association between intestinal flora and AS in local population,and explore the influence of diet on gut microbiota and AS.On this basis,the study intends subsequently to explore the correlation between FUT2/FUT3 genes,HBGAs,and AS,so as to lay a foundation for further research on relevant mechanisms.Part I Alterations of Intestinal Flora in Patients with Ankylosing SpondylitisMethods A case-control study was designed,and patients with AS and age-and sex-matched healthy controls were recruited.Their dietary habits,living environment and health status were investigated.Fresh fecal specimens of the participants were collected,fecal flora DNA was extracted,and 16S r DNA was amplified.The amplified DNA fragments were purified using the Agencourt AMPure XP PCR purification beads.Specific tag sequences were added to the ends of the DNA fragments,which were purified again.The quality of the DNA library was evaluated and sequenced by a Mi Seq Benchtop sequencer.The sequencing data were screened and analyzed,the acquired operational taxonomic units(OTUs)were taxonomically annotated.Analyses on the alpha diversity and the beta diversity of the intestinal flora were performed to compare the differences between the cases and the controls,and among the groups with different dietary type.Analyses on the correlation between the intestinal flora and AS disease indexes were also conducted.The bacteria with significant change in the patients with AS were retrieved from the GMrepo database,to obtain their test-data in other diseases,and to analyze the change-features of relative abundance for these bacteria in different diseases.Results A total of 207 participants were collected in this study(including 103patients with AS and 104 healthy controls).There were no significant differences in the male/female ratio(85/18 vs.84/20,?²=0.106,P=0.744)and age(33.29±11.66 vs.33.66±12.53,t=0.221,P=0.825)between the cases and the controls.1.77x 10⁷ optimized reads were obtained in total from all specimens.After screening and analysis,a total of 1270 OTUs were acquired,of which 951/1270(74.9%)were shared by the cases and the controls.In terms of dominant bacteria and alpha diversity indexes,there were no significant differences between the two groups(all P>0.05),but significant differences were found in relative abundance of the 49/225 genera between the two groups(all P_corrected<0.05).Megamonas and Streptococcus were the top two genera with the greatest increase in relative abundance,and Lachnospira and Mycoplasma with the greatest decrease in the AS group.In the aspect of beta diversity,principal coordinate analysis(PCOA),principal component analysis(PCA),nonmetric multidimensional scaling(NMDS),and the Adonis analysis showed significant differences in intestinal flora between the two groups(P<0.05).According to the results of factor analysis,,the participants were divided into four dietary types:A,B,C and D.There were significant differences in dietary types between the case group and the control group(?²=9.540,P=0.023).The alpha diversity of intestinal flora was basically similar among different dietary types(all P>0.05),but the abundance of Odoribacter and Alistipes was significantly different(all P<0.05).Associations were also found between gut microbiota and exercise,smoking,etc.(all P<0.05)At different taxonomic levels,the composition of intestinal flora was associated with the disease indexes of AS(all P<0.05).Compared with the data obtained from the GMrepo database,most of the relative abundances of six genera and six species,which showed the greatest differences in abundance between the AS-cases and the healthy controls in this study,were similar to their levels in patients with IBD(including Crohn's disease and ulcerative colitis),psoriasis,Behcet's syndrome,and RA.But the relative abundance of a few subgroups varied significantly among these diseases.Conclusions The intestinal flora is correlated with the susceptibility and clinical manifestations of AS.Dietary type is associated with the composition of intestinal flora and the susceptibility to AS.Environmental factors such as smoking are also correlated with the structure of gut microbiota.Most of the bacteria with significant difference between the cases and the controls may play a similar role in AS and relevant diseases.This study suggests that the factors related to intestinal flora may be generally of some value in AS researches.Part?Study of Associations between FUT2 and FUT3 Gene Polymorphisms and Ankylosing SpondylitisMethods A case-control study was adopted,and confirmed patients with AS were selected from the outpatient service of the Rheumatology and Immunology Department of a local hospital,the age-and sex-matched healthy controls were recruited from the Physical Examination Center of the hospital and local community health-service-stations.All the participants were informed of the study in detail and voluntarily signed a written informed consent prior to their participation.A detailed questionnaire on health status was completed by each of the patients with AS,including their living and eating habits,history of diseases,the clinical symptoms and the disease activity indexes of AS.The peripheral blood samples of all the participants were collected and genotyped using the SNPscan method for 3 SNPs of FUT2 gene and 2 SNPs of FUT3.According to the genotyping results,the secretor status,Lewis status,and Lewis serotype were determined.The frequencies of the genotype,allele,haplotype,secretor status,Lewis status,and Lewis serotype were compared respectively between the cases and the controls.The interactions among these factors,age,and gender were evaluated.The correlations between these factors and the related disease indexes of AS were analyzed.The p values of multiple comparisons were reported as p'values after correction.Results 673 patients with AS(546 males,127 females;28.58±9.31 years)and 687healthy controls(560 males,127 females;28.62±7.76 years)were included in this study.There were no significant differences in sex ratio(4.30 vs.4.41,?²=0.856,P=0.890)and age(28.6±9.3 years vs.28.6±7.8 years,t=0.004,P=0.997)between the two groups.The difference in the rs28362459 allele frequencies between the cases and the controls was statistically significant(?²=7.515,P=0.006,P'=0.030;OR_G/T=0.782,95%CI,0.650–0.941).The frequency of rs28362459-G in the patients was significantly lower than that of healthy controls(20.3%vs.24.7%).However,in both male and female subgroups,there was no significant difference in allele frequencies between the cases and the healthy controls(both P'>0.05).No significant differences were found in allele frequencies of the remaining SNPs and genotype frequencies of all the SNPs between the cases and the controls(all P'>0.05).There were significant differences in the frequencies of haplotype TT(rs812936–rs28362459)(?²=5.663,P=0.017,P'=0.039)and TG(rs812936–rs28362459)between the two groups(?²=7.456,P=0.006,P'=0.013).In female participants,the difference in the frequency of haplotype TG(rs812936–rs28362459)between the case and the control group was also statistically significant(?²=5.624,P=0.018,P'=0.047).In addition,there were two-factor interactions between rs28362459 and age,rs28362459 and gender,rs28362459 and rs1047781,Lewis status and secretor status.However,no multifactor interactions were found among these factors.At the phenotypic level,there were no significant differences between the cases and the controls in the frequency of secretor status and Lewis status,either for the total participants or for both of the sex subgroups(all P'>0.05).Moreover,there were no significant differences between the patients with AS and healthy controls in the frequencies of Lewis serotype,which was jointly determined by the secretor status and Lewis status(all P'>0.05).No associations were found between FUT2 and FUT3gene polymorphisms and the clinical symptoms of AS,such as Bath ankylosing spondylitis functional index(BASFI),Bath ankylosing spondylitis disease activity index(BASDAI),ankylosing spondylitis disease activity score(ASDAS),degree of pain,and spinal mobility(all P'>0.05).However,rs28362459-G was correlated with some of single BASFI/BASDAI indices(all P'<0.05).Conclusions FUT3 gene polymorphisms are associated with human susceptibility to AS,but no correlation has been found between FUT2 gene and AS-susceptibility.Rs28362459-G is associated with a lower susceptibility to the disease,and may be a protective factor for AS.FUT2 and FUT3 gene polymorphisms may be related to some clinical symptoms of AS.The results of this study suggest that some human blood group genes,HBGAs,and gut microbiota may be associated with the onset and progression of AS,which needs to be further verified by expanding the sample size in different ethnic populations,and needs further researches on relevant mechanisms.