The Study Of Hereditary Susceptibility And Serum Markers In Chronic Spontaneous Urticaria

Background and objective:Chronic spontaneous urticaria(CSU)is defined as recurrent development of transient wheals,angioedema(AE),or both for>6 weeks due to known or unknown causes.CSU affects around 0.5-1%of the population at any time and occurs more commonly in women.Although any age group can be affected,CSU mostly occur at 20-40 years.Around 15%of patients with CSU were symptomatic for more than 5 years.Mast cells are the major effector cell type that release histamine,cytokines and chemical mediators involved in the pathogenic mechanisms of CSU.IgE or IgG autoantibodies play important roles in the pathogenesis of CSU.The existence of autoantibodies against IgE and high-affinity IgE receptors(FcεRI)on cutaneous mast cells(MCs),characterized by their ability to cause a direct mast cell degranulation.Mast cells are the major effector cell type that release histamine,cytokines and chemical mediators involved in the pathogenic mechanisms of CSU.Moreover,CSU is thought to have an autoimmune background because of various types of serum autoantibody,such IgGs against thyroglobulin(TG)and thyroid peroxidase(TPO),which were elevated in sera from CU patients,although it is unknown how these autoantibodies are involved in the pathogenic mechanism.Although anti-thyroid IgG autoantibodies seem not to be directly involved in the degranulation of MCs,they may enhance MC susceptibility to other activating signals.Chronic inflammation,caused by anti-thyroid IgG autoantibodies,disrupts the normal architecture of the thyroid gland and leads to the release of sequestered autoantigens,which induce a low-grade autoimmune response.There is a strong link between CSU and elevated levels of anti-thyroid IgG autoantibodies(anti-TPO,anti-TG).A study in a large population of patients with chronic urticaria(CU)showed an increased prevalence of the disorder among first-degree relatives compared to the general population and indicated that genetic factors doubtless make important contributions to susceptibility to CU development and progression.To date,large numbers of genetic association studies using the candidate gene approach for CSU have been conducted in different populations.Genetic polymorphisms of CSU in histamine-related genes,including TGFβ1,CRTH2,C5AR1,ORAI1,CACNA1C were suggested to be involved in mast cell activation and histamine metabolism.Major histocompatibility complex(MHC)alleles were suggested to be candidate markers in different ethnic of CSU.Human leukocyte antigen(HLA)class Ⅰ/class Ⅱ alleles and CSU have been investigated.A genome-wide association study(GWAS)usually screens 500000 to 1 000 000 SNPs by comparing the allele/genotype frequency between subjects affected by disease and those not affected.In the past decade,GWAS have led to a wealth of new information on the physiological and pathophysiological mechanisms that mediate many human diseases,for example asthma,atopic dermatitis,rheumatoid arthritis,systemic lupus erythematosus,type 1 diabetes.Urticaria Activity Score(UAS),Angioedema Activity Score,Chronic Urticaria Quality of Life,Angioedema Quality of Life and Urticaria Control Test are validated clinical tools for assessment of disease activity of CSU.However,these instruments are subjective and retrospective.MicroRNAs(miRNAs)decrease targeted mRNA levels and/or reduce expression of their cognate target proteins.They can accomplish these effects through a number of different mechanisms including RNA degradation,induced decapping/deadenylation,altered cap protein binding,reduced ribosome occupancy,and/or sequestration of mRNA from translational machinery.MiRNAs,either alone or in combination with other known biomarkers,have been used as diagnostic tools in many studies.As secreted miRNAs remain stable in body fluids,they are more readily available for assay and less invasive than biopsies.These advantages make them a very appealing diagnostic instrument.Th2-mediated immune pathways induce mast cell activation and degranulation via IgE antibody production and its binding to the FcεR.Th17 cells show both inhibitory and stimulatory effects on antibody production by mast cells.Of particular relevance to the current study are the recent findings revealing that Th2/Th17 cytokines,transcription factors and Th2 chemokines are upregulated or elevated in CSU patients’ skin lesions,plasma and serum,respectively.Accordingly,Th2 and Th17 pathways may be involved in the pathogenesis of CSU,and CCL17 and IL-17 can serve as serum markers of Th2 and Th 17 inflammation,respectively.The first part,to explore CSU associated SNPs in whole genome and identify susceptibility genes/loci for CSU by using two-stage GWAS approach and to build the GWAS database of CSU cases and controls in Chinese Han population.The second part,to investigated expression profiles of miRNAs,CCL17 and IL-17 in sera of CSU patients and identify the biomarkers that can assess the severity of the CSU and the treatment response.The third part,to measured the serum sIgE for 20 allergens,serum levels of tIgE,anti-TPO and anti-TG in a Chinese CSU population and identify the association between these serum paramenters and CSU.Methods:The first part,samples in the first stage GWAS were genotyped by Illumina HumanOmniZhongHua-8 BeadChips.A total of 941 subjects,443 CSU patients and 498 healthy controls from Chinese Han population,were genotyped by this BeadChips.After quality controls,we conducted genome wide association analysis,and selected 247 potentially associated SNPs for replication study in independent cohort(828 CSU and 1018 healthy controls)using Fluidigm SNP genetype.The second part,a quantitative PCR(qPCR)-based array was generated from sera as obtained from 20 active CSU patients and 20 healthy controls.Upregulated or downregulated miRNAs were validated by reverse transcription qPCR in sera from 59 active CSU patients and 58 healthy controls.The third part,1366 CSU patients and 1424 healthy volunteers were recruited from 15 medical centers in China.Serum sIgE,tIgE,anti-TPO and anti-TG were detected in all patients and healthy controls using immunoblotting,enzyme-linked immunosorbent assays(ELISA)and chemiluminescence microparticle immunoassay,respectively.Results:The first part,built the GWAS database of 753,603 autosomal SNPs,including total 443 CSU patients and 498 healthy controls in Chinese Han population.After investigating replication study,we identified two genes,RUNX2 and MICA located within region of SNP rs72862937 and rs1882 associatied with CSU,respectively.The second part,serum relative expression of miR-125a-5p(P<0.0001)and serum CCL17 level(P<0.0001)were significantly elevated in CSU patients in comparison with healthy controls.Serum IL-17 was nondetectable in 77 CSU patients and 76 healthy controls.Only 2 CSU patients and 2 healthy controls showed detectable levels of IL-17 of approximately 20 pg/mL.MiR-125a-5p expression was positively correlated with CCL17(Spearman rho=0.394,p=0.002).Serum relative expression of miR-125a-5p(P=0.012)was even higher in refractory CSU cases(n=10).In 12 CSU patients who reached remission,serum relative expression of miR-125a-5p(P=0.018)and serum CCL17 level(P=0.001)of remission phase decreased significantly in comparison with those of active phase.The third part,1366 CSU patients with a mean age of 37.9 years(range 12-78 years)were enrolled and occurred in women more than wen(M:F 1:2.06).The mean age of the first CSU attack was 33.7 years(range 12-77 years).CSU mostly occur at 20-40 years.Nocturnal urticarial attacks were reported by 68.2%of patients(n=932).The mean UASday was 4.4 ± 1.3.Of the 1366 CSU patients,22.6%had concurrent angioedema.Three hundred and sixty(26.4%)CSU patients had personal history of atopic diseases and 377(27.6%)patients had urticaria/atopic diseases family history.Of the 1366 CSU patients,the percentage of sensitized to any allergen(74.7%)was significantly higher compared with healthy controls(62.6%,P<0.0001).Male CSU patients were more susceptible to sensitization.The prevalence of allergic sensitization decreased with increasing age in CSU patients.The tIgE level(P<0.0001)was significantly higher in CSU patients than those in healthy conrols.The tIgE level was associated with male gender,AE,CSU disease duration over 24 months,sensitizeding to willow/poplar/elm,mugwort,mite,cockroach,egg white,codfish/lobster/scallop,shrimp and crab.Of the 1366 CSU patients,18.2%,30.5%and 32.7%had positive anti-TPO,positive anti-TG and positive anti-thyroid IgG antibodies(anti-TG/anti-TPO),respectively.In the 1424 healthy controls,13.3%,21.4%and 23.9 had positive anti-TPO,positive anti-TG and positive anti-thyroid IgG antibodies,respectively.There were statistically significant differences between CSU patients and healthy controls as to the percentage of anti-TPO(P=0.001),anti-TG(P<0.0001)and positive anti-thyroid IgG antibodies(P<0.0001).The percentage of positive anti-thyroid autoantibodies was associated with female gender,higher age,serum lower tIgE level in CSU patients.Conclusion:The first part,RUNX2 and MICA located within region of SNP rs72862937 and rs1882 associatied with CSU.The second part,serum expression of miR-125a-5p and CCL17 are significantly upregulated in patients with active CSU and significantly decreased in the remission phase.Moreover,a further upregulation in serum miR-125a-5p expression was observed in refractory CSU cases.MiR-125a-5p and CCL17 as potential serum biomarkers for CSU.MiR-125a-5p and CCL17 may function synergistically in CSU.The third part,the percentage of sensitized to any allergen,tIgE level and percentage of positive anti-thyroid IgG autoantibodies were significantly higher in CSU patients than healthy conrols.Allergic sensitization decreased with increasing age.Male gender was more susceptible to allergic sensitization in CSU patients.The tIgE level was associated with male gender,AE,CSU duration over 24 months,sensitized to any allergen.The percentage of positive anti-thyroid autoantibodies was associated with gender,age,serum tIgE in CSU patients.