Association Study Of UBASH3A And UBASH3B Gene Single Nucleotide Polymorphisms, MRNA Expression Levels And Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus(SLE) is a complex and systemic autoimmune disease, characterized by producing a variety of autoantibodies, immune complexes, and acute or chronic inflammation manifestations in various organ of the body. The clinical manifestations varying in different individuals,which ranging from mild clinical signs(such as a rash or non-erosive arthritis) to the life-threatening of systemic organs involvement(such as lupus nephritis, neuropsychiatric disorders, etc.).Nowdays, the cause of the disease is not clear, and may be related to the genetic factors, environmental factors and their interactions, which causing a series of abnormal immune reactions.Overall, the prevalence of SLE patients varies with different regions and ethnic populations.The prevalence of SLE is approximately 15-144/100, 000 in Amercia, 46-85/100, 000 in the Swedish population, 18.8-21.7/100, 000 in Korea, about 70/10, 000 in Chinese population, while 60/10, 000 in Hong Kong.Previous studies showed that genetic factors may play an important role in the pathogenesis of SLE. Genome-wide association(GWA) and candidate association studies have made progress in identifying SLE risk loci, and provided evidences for the exploration of pathogenesis of polygenic diseases and drug therapy targets.Currently, revealing the pathogenesis of SLE from the genetic deletions perspective has become a hot topic area.Protein tyrosine phosphatase(PTPs) is a key enzyme in regulating cell signaling pathways. Protein tyrosine phosphorylation plays an important role in many aspects, such as cell growth, differentiation, migration, sugar metabolism, synaptic transmission and immune response,etc.The first PTP sequence was found about 26 years ago; At present, there are about more than 100 kinds of genes in humans; Protein tyrosine phosphorylation is mainly controled by PTPs and protein tyrosinekinase(PTK).The regulation of PTPs activity in vivo are accompanied by a variety of mechanisms, including alternative splicing of the messenger RNA(m RNA), regulation of protein steady-state levels, post-translational modifications, dimerization and subcellular localization.Regulation of PTPs is involved in cell signal transduction function, which is based on the tyrosine phosphorylation and play a role in the intracellular protein dephosphorylation of the tyrosine residues.PTPs is a natural antagonist of PTK. PTPs plays a role in regulating T cell function and immune homeostasis, through inhibition of T cell receptor signaling pathway. It has been proved that the related gene in PTPs family participate in a variety of cell signal pathway; Meanwhile, the imbalance of PTPs activity may be involved in human autoimmune disease, diabetes, cancer, and pulmonary tuberculosis, etc. The PTPs carried single nucleotide polymorphisms(SNPs) which associated with diseases might become good candidate genes for ADs, and involved in the suppression of spontaneous T cell activation;This might be related to the dephosphorylation, reaction inhibition through inactivation of T cell related kinases and their substrates.UBASH3A(also known as TULA or STS-2) and UBASH3B(also known as TULA-2 or STS-1) were newly discovered members of the PTPs family, and may become new regulators with a variety of cell functions.They encodes TULA family protein, and downregulate receptor-mediated signaling pathway in T-cells and platelets. The two proteins share 40% sequence identity; UBASH3 B gene was widely expressed in many tissues, and the UBASH3 A gene was mainly in the spleen,kidney, lung, bone marrow, peripheral blood lymphocytes and hematopoietic cells.Protein amino acid sequences of UBASH3 A and UBASH3 B contain N-terminal and ubiquitin associated structure; intermediate Src homology 3(SH3) domains, mediate the combination of proline-rich CBL, and involve in protein dimerization through the phosphoglycerate mutase(PGM) domain of C-terminal(Figure 1).Recently, GWAS and candidate gene genetic association studies have suggested that UBASH3 A and UBASH3 B gene polymorphisms may be associated with different autoimmune disorders, such as type 1 diabetes, vitiligo, rheumatoid arthritis,systemic lupus erythematosus,celiac disease(CD), experimental autoimmune meningitis(EAE) and Behcet’s disease,etc. But the conclusions were not consistent. For instance, UBASH3 A gene polymorphism was associated with type I diabetes in the UK and Denmark populations;Unfortunately,the UBASH3 A gene was failed to prove as a causative gene in patients with RA from Denmark, France and Germany. This may be due to different autoimmune diseases have complicated, clinical heterogeneity, and the sample size of different studies were varied, but they provide valuable clues for the study of etiology on autoimmune diseases. In addition, several genes in the PTPs family may arouse alteration of m RNA level or PTPs family protein steady-state expression level.These results have been observed in the changement of the amino acid sequence in non-coding regions and thus, influence the expression levels of the protein.At present, the studies which concerning the relationship between UBASH3 A and UBASH3 B genes and SLE susceptibility are limited, especially in the Chinese Han population. According to the latest progress in this research area, our research group put forward the following hypotheses:(1) the expression of UBASH3 A and UBASH3 B gene level changes may be associated with SLE patients; The alteration of individual clinical phenotype may be related to the variations in the UBASH3 A gene and UBASH3 B gene;(2) UBASH3 A and UBASH3 B gene single nucleotide polymorphisms may be associated with SLE and lupus nephritis(LN), which are important functional gene in TCR signaling pathway.Thus, studies on expression level alteration of UBASH3 A and UBASH3 B in patients with SLE will provide reference for biological diagnosis on SLE disease subtype markers, but also help to elucidate the pathogenic mechanisms and reveal new therapeutic targets for SLE.Therefore, we explored the relationship between UBASH3 A and UBASH3 B gene single nucleotide polymorphisms and m RNA levels and SLE. The research contents are as follows:Firstly, through the method of genotyping, we analyzed the roles of UBASH3 A and UBASH3 B SNPs in the pathogenesis of SLE and LN susceptibility; Moreover, we explored the relationship between SNPs and clinical symptoms and laboratory parameters; In addition, we also analyze the effect of haplotypes in UBASH3 A gene of SLE cases and potentially disease risk; and explore the mechanism of polymorphisms in genetic susceptibility in SLE patients.Secondly,Vii ATM 7 high throughput real-time fluorescence quantitative polymerase chain reaction(RT-q PCR) was adopted for analyzing UBASH3 A and UBASH3 B gene m RNA expression level from peripheral blood mononuclear cells(PBMCs) in SLE patients and controls, and explore its association with clinical symptoms, disease activity, and laboratory parameters.In summary, we explored the relationship between UBASH3 A and UBASH3 B genes and SLE in a Chinese Han population, by analyzing SNPs and the expressions levels of m RNA in patients with SLE; We assumed that the expressions of UBASH3 A and UBASH3 B levels were different in SLE patients under different clinical characteristics, and speculated that UBASH3 A and UBASH3 B genes may be the therapeutic targets of gene therapy, so as to to provide reference for the early detection of SLE and clinical decision-making.Objective The genotype and allele frequencies in UBASH3 A SNPs(rs3788013, rs2277798, rs1893592, and rs11203203) and UBASH3 B rs4936742 loci were compared between SLE patients and healthy controls;The relationship between UBASH3 A and UBASH3 B and with the susceptibility to SLE in a Chinese Han population were analyzed, haplotype of UBASH3 A gene in the Chinese population was also evaluated;Meanwhile, we also exlpored the distributions between the genotype and allele frequencies of the main clinical features in SLE patients and healthy controls. The m RNA expression levels of UBASH3 A and UBASH3 B were between SLE patients and healthy controls, lupus nephritis and SLE without nephritis, active stage of cases and inactive stage of cases in PBMC.We analyzed their associations with disease activity, the main clinical features and laboratory index, we also explored the role of them in the occurrence and development in SLE.Method A total of 792 cases of SLE patients, including outpatient and inpatient SLE cases from department of Rheumatology at first affiliated hospital of Anhui medical university and Anhui provincial hospital;They were diagnosed by two associate chief physician level or above specialists, and in accordance with the classification criteria criteria of SLE patients(ACR, revised in 1997).These SLE patients were identified with malignant reticuloendothelial cell histiocytosis,hemolytic anemia, thrombocytopenia and connective tissue disease,thus, non-SLE cases were excluded. The collection period of subjects were from September 2012 to October 2014. It lasted more than two years, and patients got further consultation and refused cooperation were excluded. A total of 777 healthy volunteers were come from the medical center and volunteer blood donors of Anhui Medical University. Persons who are the risk of SLE and other related autoimmune diseases were excluded; Without use of immunosuppressive agents and hormone drugs, no major disease history and keep health in a month before the investigation.RNA samples of 32 SLE cases and 30healthy controls were obtained from the two hospitals listed above, and the diagnosis and exclusion criteria of SLE patients and healthy controls were the same as above. Age and gender of healthy controls were comparable with SLE patients. The content of questionnaire included basic information, clinical and laboratory parameters of SLE patients; The content of questionnaire were mainly obtained by face-to-face interview with SLE patients, and combined with the supplementation from the hospital electronic medical record information system.Meanwhile, 5ml of PBMC were extracted; We also using Fluidigm® 192.24 Dynamic Array and Taq Man probe genotyping detection methods to analysis five loci of UBASH3 A and UBASH3 B gene; Genotype and allele frequencies distribution between SLE patients and healthy controls, LN and patients without LN were compared in our study. Dominant and recessive models were also analyzed. The association of UBASH3 A and UBASH3 B gene SNPs and clinical features and experimental parameters were analyzed in this study. RT-q PCR was used to analyze the m RNA expression levels of UBASH3 A and UBASH3 B from PBMC. The statistically differences between different groups were compared and analyzed using the Mann-Whitney U test; Spearman rank correlation analysis was used to analyze the association between UBASH3 A and UBASH3 B m RNA expression levels and clinical feaures and SLEDAI.Statistical analysis was performed using SPSS11.5 software, estimated and adjusted odds ratios(ORs) and 95% confidence intervals(95% CIs) were calculated.Statistical power and sample size were calculated by the free-download Power and Sample Size Calculation Software. Haplotype analysis was calculated by online SHEsis software. P value lower than 0.05 was viewed as statistically significant.Results(1) In this study, rs3788013, rs2277798, rs1893592, rs11203203 and rs4936742 genotype frequencies in the patient group and healthy controls were reached HWE(P>0.05).(2) Genotyping of UBASH3 A and UBASH3 B For rs3788013 SNP, the allelic and genotypic frequencies were statistically significant different between cases and controls(A vs. C:χ2=9.016,P=0.003,OR=0.795,95% CI 0.684-0.924;AA vs. CC:χ2=5.965,P=0.015,OR=0.632,95% CI 0.437-0.913;CA+AA vs. CC:χ2=5.736,P=0.017,OR=0.770,95% CI 0.622-0.954); While the distribution of allele and genotype frequencies were not statistically significant between non-LN and LN patients(P>0.05). For rs2277798, rs1893592 and rs11203203 SNP, the allele and genotype frequencies of between cases and controls were not statistically significant(P>0.05). For UBASH3 B rs4936742 SNP, the distribution of allelic and genotypic frequencies were not reached statistical significance(P>0.05).(3) Haplotypes of UBASH3 A The haplotypes constructed by UBASH3 A rs2277798, rs11203203, rs3788013, and rs1893592 polymorphisms formed 7 kinds of common haplotype structure in a Chinese Han population. The results showed that the frequency of haplotype GGCA SLE of patient group was significantly higher than that of the control group(χ2=10.155, P=0.001, OR=1.61, 95% CI 1.20-2.17); In addition, the GGAA haplotype frequencies in the patient group was significantly lower than those of healthy controls(χ2=6.221,P=0.012, OR=0.81,95% CI 0.68-0.96).(4) The association of UBASH3 A and UBASH3 B SNPs and clinical features The distribution of AC genotype for rs3788013 was significantly increased in patients with vasculitis as compared with patients without this feature(χ2=8.837, P=0.012). The AG genotype frequency of rs2277798 SNP in patients with hematuria was increased when compared to patients without this feature(χ2=11.569, P=0.003).The C allele of rs1893592 demonstrated an increased risk of developing SLE in patients with skin rash, oral ulcer and arthritis when compared to those without these features(χ2=5.301, P=0.021, OR=0.671, 95% CI 0.477-0.944,; χ2=4.803, P=0.028, OR=0.765, 95% CI 0.602-0.972). The allele frequency of C was higher in SLE patients with rash than those without rash(χ2=4.130, P=0.042, OR=0.776, 95% CI 0.608-0.991).There was also an increased frequency of TCgenotype in patients with anti ds-DNA positive patients in UBASH3 B rs4936742 polymorphism(χ2=7.410, P=0.025).(5) Analysis of UBASH3 A m RNA expression levels in PBMC and their correlation with the main clinical symptoms Our results showed that the UBASH3 A m RNA expression level from PBMC was lower in SLE patients than that of healthy controls(P=0.002), lower in active stage of cases than inactive stage of patient group(P=0.000); But there was no statistically difference between lupus nephritis and those without nephritis(P>0.05).The expression of UBASH3 A m RNA expression levels were negative associated with decrease of white blood cell, positive anti-ds DNA antibody, positive anti-SSB antibody(rs=-0.380, P=0.032; rs=-0.385, P=0.030; rs=0.433, P=0.013); But similar results were not found in other clinical symptoms such as fever, arthritis, hair loss,etc(P>0.05). The expression of UBASH3 A m RNA expression levels were associated with the first onset age of SLE patients(rs=-0.358,P=0.044); Our results also suggested that UBASH3 A m RNA expression level was also negatively correlated with SLEDAI(rs=-0.350, P=0.049).(6) The UBASH3 B m RNA expression levels in PBMC were comparable between the patient group and control group, active and inactive group, LN and SLE without LN group;We did not find the correlation between the UBASH3 B m RNA expression levels and clinical manifestations or SLEDAI(P>0.05).Conclusion UBASH3 A gene rs3788013 polymorphism is associate with the susceptibility to SLE in a Chinese Han population; GGCA halpotype formed by UBASH3 A gene polymorphisms may increase the risk of SLE, while GGAA may reduce the risk of SLE; Rs3788013 polymorphism may be associated with SLE patients who merged with vasculitis, rs1893592 polymorphism may be associated with SLE patients who combined with oral ulcers, arthritis and rash. rs2277798 SNP may be associated with SLE patients who merged with hematuria; Rs4936742 SNP may be correlated with SLE patients those combined with positivity for anti-ds-DNA.The expression of UBASH3 A m RNA levels were lower in SLE patients than the control group, and were associated with some clinical symptoms, such as decrease of white blood cell, positivity for anti-ds DNA antibody and anti-SSB antibody.The m RNA expression level is lower in active stage group than inactive ones. Furthermore, the expression level was negative associated with SLEDAI and age of first onset.The findings suggest that UBASH3 A gene might contribute to SLE susceptibility and influence the clinical phenotype of the disease.The dysregulation of UBASH3 A m RNA levels in SLE patients and their correlations with experimental parameters suggested that UBASH3 A may involve in the pathogenesis of SLE.