| Background and Aim:Ankylosing spondylitis(AS)is a chronic autoimmune disease characterized by chronic inflammatory response.Inflammatory low back pain is the main manifestation,usually insidious onset.AS has a high disability rate.Patients suffer from back pain,low back pain,and joint pain for a long time.The human body’s axial bone and the joint structure of the limbs are progressively destroyed,and eventually joint bone stiffness occurs,which leads to the movement of the neck,waist and lower limbs.Decreased ability,or even complete loss of mobility.Therefore,studying the pathogenesis of AS is of great significance for the early diagnosis and treatment of the disease.At present,the pathogenesis of AS is still unclear,and it is a chronic disease caused by a combination of genetic,environmental,and epigenetic factors.Previous studies have proved that AS has familial aggregation.Therefore,we selected an AS family to study the characteristics of its genetic susceptibility genes and intestinal flora,and look forward to finding new AS susceptibility genes and microbial markers.The epigenetic role of HDAC4 in AS is studied,which can ultimately guide the early diagnosis of AS and biological targeted therapy.Methods:1.WES sequencing and HLA-B27 typing detection:1)The study included a Chinese Han AS family with four generations of members.All linked family members were invited to participate in the study(18family members).Therefore,patients with AS were diagnosed according to the criteria of the Spondylo Arthritis International Society(ASAS).Nineteen ethnically matched subjects with unrelated or autoimmune diseases were recruited as healthy controls(HCs).2)Use the Fast Pure Cell/Tissue DNA Isolation Mini Kit to extract genomic DNA from the collected blood samples.3)Whole Exome Genome Sequencing(Whole Exome Genome Sequencing)was performed on the DNA of AS patients in 6 families to screen the susceptibility genes of AS.4)Single nucleotide polymorphism(SNP)sequencing was performed on the DNA of 18 AS members and 19 healthy control subjects to verify susceptibility genes.5)HLA-B27 typing was performed on the DNA of 19 AS members and 20 healthy control subjects.6)Data analysis.2.16 S r RNA sequencing:1)The study included a Chinese Han AS family with four generations of members.A total of 14 family members were invited to participate in the study.AS patients were diagnosed according to the criteria of the Spondylo Arthritis International Society(ASAS).The stool samples and saliva samples of each family member were collected and divided into stool-disease group(faeces-patients,F-Ps),stool-control group(faeces-controls,F-Cs),saliva-disease group(saliva)-patients,S-Ps),saliva-controls(saliva-controls,S-Cs).Thirty-four unrelated AS patients(AS group)and age-sex-matched healthy subjects were recruited as healthy controls(HCs).2)Total DNA was extracted from the collected stool samples and saliva samples,and DNA was quantified by Nanodrop,and the quality of DNA extraction was detected by 1.2% agarose gel electrophoresis.3)Design corresponding primers according to the conserved regions in the sequence,and add the sample-specific Barcode sequence,and then perform PCR amplification on the r RNA gene variable region(single or continuous multiple)or specific gene fragments.4)Use the Tru Seq Nano DNA LT Library Prep Kit from Illumina to prepare the sequencing library.5)Using Mi Seq sequencer for paired-end sequencing,the corresponding reagent is Mi Seq Reagent Kit V3(600 cycles);if using Nova Seq sequencer for paired-end sequencing,the corresponding reagent is Nova Seq 6000 SP Reagent Kit(500 cycles).6)Data analysis.3.Blood HDAC4 changes correlate with disease activity and response to TNF-αinhibitors:7)The study included 132 patients with active AS who were admitted to the Rheumatology and Immunology Department of the First Hospital of Jilin University from May 2019 to July 2021,and recruited 30 healthy subjects as a healthy control group(healthy controls,HCs).8)Collect clinical data and blood samples of patients with ankylosing spondylitis after admission to understand their clinical characteristics.The peripheral blood of 132 AS patients W0(before treatment),127 AS patients W4(4 weeks after treatment),116 AS patients W8(8 weeks after treatment)and 104 AS patients W12(12 weeks after treatment)were collected.Peripheral blood mononuclear cells(PBMC)and serum samples were isolated.For DCs and HCs,peripheral blood samples were collected after recruitment,and then PBMC samples were isolated.9)Collect PBMCs from all subjects,and detect the expression of HDAC4 by reverse transcription quantitative polymerase chain reaction(RT-q PCR).10)The percentages of helper T cells(T-helper 1,Th1)and helper T cells(T-helper 17,Th17)in the fresh peripheral blood of patients were detected by flow cytometry.Proinflammatory cytokines(eg,tumor necrosis factor(TNF),interleukin(IL)-1β,IL-6,and IL-6)were detected in serum of all patients by enzyme-linked immunosorbent assay(ELISA).],cytokines secreted by Th1 cells [interferon-gamma([interferon-gamma,IFN-γ)],cytokines secreted by Th17 cells(IL-17A)levels.11)54 patients with ankylosing spondylitis were treated with TNFi,at W4,W8 and W12,the treatment response was assessed according to the ASAS40 response criteria.1)Statistical analysis.Results:1.WES sequencing and HLA-B27 typing detection:1)A total of 246846 SNPs and 40211 indel sequences were generated from 6patients(Ⅲ-1,Ⅲ-3,Ⅳ-1,Ⅳ-3,Ⅱ-6,Ⅲ-19)and Ⅱ-1 of WES.2)On 1000 Genomes data(1000g_all),NHLBI ESP6500 data(esp6500siv2_all),gnom AD data(gnom AD_ALL and gnom AD_EAS)and in-house Novo-Chinese exome database from Novgene.Variations in exons or splice sites(splicing,about 10 bp)were preserved.Similar to recent studies,a prioritization scheme was applied to identify disease-causing mutations in patients.In addition,we excluded synonymous mutations and predicted functions for SIFT and Poly Phen-2mutations.Benign and likely benign variants were filtered according to American College of Medical Genetics(ACMG)guideline categories.Finally,three candidate genes(4 SNPs,gene names ZBTB22,IP6K3,KCNK5)were obtained.3)The frequency of ZBTB22 variant was 36.1% in AS familial cases and 0% in unrelated controls(P<0.001).Likewise,the frequency of IP6K3 variants was 36.1%in AS familial cases and 0% in unrelated controls(P<0.001).4)all patients in the family are HLA-B27 positive,50% of HCs are HLA-B27 positive,and all of them are of the same type,namely HLA-B2704.All non-family HCs were negative for HLA-B27.2.16 S rRNA sequencing:1)At the family classification level,the species differences between fecal samples and saliva samples are more obvious.In the bacterial flora distribution of stool samples,bacteroidaceae was the dominant species,and in the bacterial flora distribution of saliva samples,streptococcus(streptoidaceae)was the dominant species.In the family samples,the abundance of Bifidobacteriaceae in the F-Ps group was significantly higher than that in the other three groups,while the abundance of porphyromonadacee was slightly lower than that in the other three groups.In non-family fecal samples,the species distribution was not significantly different,while the species abundance of enterobacterianceae in the AS group was significantly higher than that in the HCs group.2)There was no significant difference in the six diversity indices(Observed,Chao1,ACE,Shannon,Simpson and Coverage)in the family sample.One possible reason is that the genetic heredity and living habits of members of the same family are relatively similar,resulting in insignificant differences in the diversity of flora species;it may also be that the number of cases in the family is slightly smaller,and it is not yet able to distinguish the few differences between groups In the non-family sample,except the Coverage index,the remaining 5 diversity indexes found significant differences(P <0.05).3)At the class level of the family samples,diarrhea and GN05 were positively correlated(P < 0.05),and no significant correlation was found between other species and environmental factors.At the family level,there was a negative correlation between Syntrophomonadaceae and inflammatory arthritis grades on CT(P < 0.05),and a strong positive correlation between Lactobacillaceae and diarrhea(P < 0.01),Prevotellaceae was positively correlated with Rhodospirillaceae and HLA-B27(P<0.05),Veillonellaceae and smoking history were strongly negatively correlated(P <0.01).There was a positive correlation between Thermaceae and inflammatory back pain(P < 0.05).3.Changes in blood HDAC4 correlate with disease activity and response to TNF-α inhibitors:1)Changes in blood HDAC4 correlate with disease activity and response to TNF-α inhibitors: 1.HDAC4 had a strong negative correlation with TNF(rs=-0.370,P<0.001),and a weak negative correlation with IL-1β(rs=-0.260,P=0.003).However,there was no correlation between HDAC4 and IL-6 in AS patients(Rs=0.134,P=0.125).2)HDAC4 had no correlation with Th1 cells(rs=-0.190,P=0.118)and its secreted cytokine IFN-γ(rs=-0.138,P=0.115),while AS patients had no correlation with Th17 cells(rs=0.316,P=0.008)and its secreted cytokine IL-17A(rs=-0.276,P=0.001)were negatively correlated.3)Compared with NSAID-treated patients(Fig.5A),TNFi-treated patients achieved a higher proportion of ASAS40 responses at W8(P=0.026)and W12(P=0.032),but not at W4(P=0.397)The proportions of the two are similar.Among all AS patients,responders had higher HDAC4 at W8(P=0.014)and W12(P=0.006)than non-responders.Responders had higher HDAC4 expression at W12 compared with non-responders after treatment(P=0.016).Conclusions:1.Three rare variants that may cause AS were identified(ZBTB22: c.C1672 T,p.R558 C and c.G1870 A,p.E624 K,IP6K3: c.G509 A,p.S170N).2.There were significant differences between the gut microbiome of AS patients and healthy controls,and the abundance of gut microbial communities decreased in AS patients;actinobacteria,Bifidobacteriaceae,and enterobacterianceae were found in Predominant bacterial species in AS patients may be related to the occurrence of AS;some environmental factors are usually also related to the pathogenesis of AS,and there is a certain correlation between them and the imbalance of microbiota.3.The expression level of HDAC4 in AS patients was negatively correlated with pro-inflammatory cytokines(TNF,IL-1β,IL-17A)and Th17 cells;after NFi treatment,the expression level of HDAC4 in AS patients increased;HDAC4 was negative Regulates Th17 polarization of na(?)ve CD4+ T cells,but not Th1 or Th2 cells. |
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