The Association Study Beween Intestinal Microorganisms And Genetic Variations In PRRC2A And TNFSF13B And Neuromyelitis Optica Spectrum Disorder

Neuromyelitis optica spectrum disorder(NMOSD)is a rare antibody-mediated demyelinating disease of the central nervous system(CNS),with a global incidence rate of about 0.3-4.4/10 million.It is estimated that there are 200,000-3 million NMOSD patients worldwide,and the incidence rate of Asian population is significantly higher than that of Caucasian population.Neuromyelitis optica(NMO)was previously thought to be a subtype of multiple sclerosis(MS),but the discovery of a specific marker in the serum-aquaporin-4 antibody(AQP4),not only confirmed NMO as an independent disease entity,but also helped to define NMOSD.The typical clinical manifestations of NMOSD include myelitis,severe optic neuritis,and/or refractory vomiting and hiccups(rear polar zone syndrome).Without timely treatment,approximately half of the patients will progress to irreversible blindness and severe disability,and one-third of patients will die within 5 years after the first attack.The high disability and mortality of the disease place a heavy burden on society and families.Therefore,early diagnosis and active treatment are essential for patients.NMOSD is a complex autoimmune disease,and its etiology and pathogenesis have not been fully elucidated.More and more research evidences showed environmental and and genetic factors work are involved in the occurrence and progress of the disease.In-depth study of the association between potential environmental factors and genetic predisposition factors and NMOSD is not only helpful for the early comprehensive risk assessment of the disease,but also helpful for the early diagnosis and timely treatment of the disease.Therefore,this thesis will study from the following three parts:In the first part,based on the environmental susceptibility factor-intestinal microorganisms as clues,in-depth study of the correlation between intestinal microorganisms and NMOSD,and explore the association between certain gut microbioata and clinical severity indicators.Further using the intestinal flora as biomarkers to build NMOSD disease prediction model,which lays a fundamental foundation for the gut micorbioa as a potentially non-invasive biomarker for the dynamic monitoring the severity of NMOSD.In the second part,we explore and verify the susceptibility association between genetic variations in the proline rich coiled-coil 2A(PRRC2A)and NMOSD.The third part we focus on the correlation between genetic variations in the TNF superfamily member13b(TNFSF13B)and NMOSD,which together with the second part expanding the susceptibility gene spectrum of NMOSD in Han Chinese population.Part 1 The association study between intestinal microorganismsand neuromyelitis optica spectrum disorderBackground and objective:Emerging evidence indicates that intestinal microorganisms play an essential role in the pathogenesis of NMOSD.However,the results are highly heterogeneous and mainly conducted in the AQP4~+NMOSD patients.Methods:In this study,16S ribosomal RNA gene sequencing targeting V3-V4hypervariable region sequencing technology was performed on fecal samples of 50subjects,including 14 AQP4~+NMOSD patients(P1,n=14),8 AQP4~-NMOSD patients(P2,n=8),and 28 normal controls(C,n=28).Results:There were significant differences in the intestinal microbial diversity and composition between AQP4~+NMOSD and AQP4~-NMOSD and normal controls.We found that some specific bacteria were closely correlated with clinical disease-specific parameters.Remarkably,this study also identified 9 genera-level microbial biomarkers that are related to NMOSD,wtih an area under the curve(AUC)of 0.97.Conclusions:This study is the first to characterize gut microbiota features in AQP4~+NMOSD and AQP4~-NMOSD.Both AQP4~+NMOSD and AQP4~-NMOSD have unique microbiota profiles and metabolic pathways.Taking together,these findings not only support for NMOSD to the growing list of diseases associated with gut microbial alterations,but also suggest that the intestinal microorganisms can be used as a non-invasive biomarker as a target for future individualized treatment in NMOSD.Part 2 Association study between genetic variations in PRRC2 A and neuromyelitis optica spectrum disorderBackground and objective: NMOSD and MS are both chronic inflammatory demyelinating diseases of CNS with unclear etiology.PRRC2A(also termed BAT2)is locates in the major histocompatibility complex III(MHC III)region.Previous studies have shown that PRRC2 A is a susceptible gene and increase the underlie risk of various autoimmune diseases.A recent research showed that prrc2 a protein as a modifier plays an important role in the process of myelination.Therefore,the purpose of this study is to explore the association between genetic variations in PRRC2 A and NMOSD in Chinese population.Methods: A total of 121 NMOSD patients,including 50 AQP4+NMOSD and71AQP4-NMOSD,and 75 MS were enrolled in this study.Based on the Chinese population data from the 1000 G database,we used Haploview 4.1 program to select candidate tag-SNPs,and a total of 11 tag-SNPs of PRRC2 A gene were selected.Finally,10 tag-SNPs loci were included for genotying for disease-related susceptibility analysis.Results: Genotype analysis found that 4 tag-SNPs(rs2736163,rs2736157,rs2242659,rs9366785),6 tag-SNPs(rs2736163,rs2736157,rs2242659,rs2736657,rs2261033,rs9366785)and one tag-SNPs(rs2736163)were significantly associated with AQP4+ NMOSD,AQP4-NMOSD and MS,respectively.In addition,further haplotype analysis founded that haplotype TCAGCAATAG increased the risk for NMOSD AQP4+ by about 15-folds.(p=0.001724,OR [95%CI]: 15.398 [1.590-149.120])and haplotype ACAATAGTAG increased the risk for NMOSD AQP4-by about1.8-folds(p=0.020270,OR [95%CI]:1.851 [1.095-3.129]).However,no significant haplotypes were found to increase the risk for MS.Conclusions: PRRC2 A,as a genetic susceptibility gene,can increase the risk for developing NMOSD and MS in Han Chinese population,especially more closely related to NMOSD.In summary,the results of our study from clinical perspective not only supported for the idea that the PRRC2 A gene may have an impact on demyelinating diseases pathogenesis from clinical perspective,but also further expands the spectrum of genetic susceptibility gene for NMOSD.Part 3 Association study between genetic variations in TNFSF13 B and neuromyelitis optica spectrum disorderBackground and objective: Recently,using case–control samples from Sardinia,Italy,Steri and colleagues conducted a genome-wide association study on MS and systemic lupus erythematosus(SLE).They reported a TNFSF13 B variant that was significantly associated with increased risk to both conditions.The variant is an insertion deletion that causes GCTGT in the 3’ untranslated region(UTR)changes into A(referred to by Steri and colleagues as BAFF-var),resulting in a truncated of the protein encoded by TNFSF13 B,which can escape the micro RNA inhibition and increase the production of soluble BAFF.The present study is the first to explore the susceptibility association between TNFSF13 B and NMOSD in Han Chinese population,and verify the above Steri and colleagues’ s important finding in Chinese MS patients.Methods: A Chinese cohort of 160 MS,181 NMOSD,and 504 matched healthy volunteers were included in this study.Sanger sequencing of TNFSF13 B gene exons was performed on blood samples of all these subjects.For the variants that may cause the change of protein function,the related plasmids were constructed and transfected into 293 T cells for functional study.Results: A heterozygous variant c.97C>T(p.R33W)was found in one MS and a3’UTR variant(c.*14T>C,rs373756878)was found in one NMOSD.In addition,the variant c.313G>A(p.A105 T,rs201543678)was detected in MS,NMOSD and healthy controls,but there was no significant difference between the three groups.Online prediction tools,Mutation Taster,Poly Phen2 and SIFT were used to predict these three variants.The prediction results showed that the two variants c.97C>T(p.R33W)and c.313G>A(p.A105 T,rs201543678)may possible benign.Of note,there was no prediction information about C.* 14 T > C site.Considering that the C.* 14 T > C variant is also located in the 3’UTR region of TNFSF13 B,it might increase the risk of disease susceptibility through a biological mechanism similar to that of BAFF-var.Therefore,we constructed the pmir Glo 3’ UTR luciferase reporter plasmid to conduct reporter assay.The result showed that the transcriptional activity of plasmid containing pmir Glo 3’UTR C.* 14 T > C was increased,and the fluorescence value was significantly higher than that of pmir Glo 3’UTR wild-type plasmid.Conclusions: We did not detect the BAFF-var in MS and NMOSD,suggesting that the BAFF-var might be race-specific.However,we identified a new TNFSF13 B variant in NMOSD patients and the biological mechanism of the identified new variant is similar to that of BAFF-var,suggesting that TNFSF13 B can increase the risk of NMOSD in Han Chinese population.