Association Study Of Single Nucleotide Polymorphisms In Pre-miRNA With Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus (SLE) is a typical systemic autoimmune disease,which has different clinical manifestation, affects multiple organ systems such as kidneyand has a complex process. The outstanding performance of SLE patients was theemergence of variety autoantibodies such as anti-nuclear antibody in serum. At present,the pathogenesis of SLE remains unclear and no specific treatment aimed at controllingsymptoms. The patients have a poor quality of life and high mortality.Studies have shown that the disease concordance rate was significantly different inmonozygotic and dizygotic twins. It suggests that genetic factors play an important rolein the pathogenesis of SLE. Many SLE susceptibility genes or regions have beendiscovered and confirmed by different studies, such as BANK1, BLK, IRF5, ITGAM,STATA4, HLA,1q23,1q25-31,2q35-37,6q21,6p11-21,12q24,16q12and22q11.21.Environmental factors can’t be ignored in the pathogenesis of SLE. The combinedeffects of genetic and environmental lead to the innate and adaptive immune disorders,and eventually lead to the occurrence of SLE.In recent years, more researches have been focused on the role of miRNA(microRNA) in disease development. MiRNAs are approximately22nt, having aregulatory function, highly conserved and endogenous non-coding RNA. They aremainly through incomplete complementary pairing with the target mRNA3’untranslated region (3’UTR) to inhibit mRNA’s translation, mediate post-transcriptionalgene regulation, and have a wide range of biological functions.Several studies have shown that the expressions of miRNAs were significantdifference in SLE patients and normal controls and miRNAs play an important role in the occurrence and development of SLE. The limitation of miRNAs’ maturation canlead to malignant transformation of cells. The genetic mutation in pre-miRNA can leadto the limitation of miRNAs conversion from pre-miRNA to mature miRNA, affect theexpression of miRNAs and regulation of post-transcriptional gene, and ultimately affectthe incidence, development and prognosis of the diseaseGiven the importance of miRNA function, single nucleotide polymorphisms (SNPs)in miRNA gene region were considered to affect the function of miRNAs and diseasesusceptibility. Several studies have found that the genetic mutations in pre-miRNAswere associated with Crohn’s disease, ulcerative colitis, liver cancer, lung cancer,colorectal cancer, asthma, congenital heart disease and so on.Therefore, in this research, we carried out a case-control association study toexamine the single nucleotide polymorphisms in pre-miRNAs by using the SequenomMassArray SNP detection technology and investigate whether or not allele andgenotype frequencies of pre-miRNAs gene confer susceptibility to SLE in the Chinesepopulation. In addition, we analyzed whether SLE patients with nephritis (LN) and SLEinitial symptoms were associated with SNPs or not. The completion of this study wouldhelp to clarify the role of pre-miRNA gene mutations in the pathogenesis of SLE, andprovide a scientific basis for looking for new diagnostic and therapeutic targets in SLE.ObjectivesTo compare the allele distributions and genotype frequencies about SNPs(rs2910164, rs2292832and rs11614913) in the precursor region of hsa-miRNA-146,hsa-miRNA-149and hsa-miRNA-196a2gene between SLE patients and normal controls,lupus nephritis (lupus nephritis, LN) patients and SLE patients without nephritis, SLEpatients with an initial symptom and those without it, and to explore the association ofpre-miRNA gene polymorphisms with SLE susceptibility. MethodsOne thousand four hundred and seventy-nine SLE patients were recruited fromDepartment of Rheumatology and Immunology of the First Affiliated Hospital of AnhuiMedical University and Anhui Provincial Hospital. All SLE patients were according tothe1997revised American College of Rheumatology (ACR) classification criteria anddiagnosed by the two vice-director level and above specialists. LN patients werediagnosed as SLE and have renal impairment for2weeks or more, such as hematuria,pyuria, proteinuria and azotemia. Two thousand two hundred and sixty-eight normalcontrols were recruited from healthy volunteers, all of them were fulfilled these criteria:I without one of the diagnostic criteria for SLE; II themselves and their immediatefamily members without a history of autoimmune disease; III health in the last monthand no use of hormones and immunosuppressant drugs; IV no history of major diseases;After obtaining the informed consent, we collected data by self-designed questionnaireand5ml EDTA anti-coagulated venous blood samples from all studied subjects.Sequenom MassArray SNP detection technology was used to study SNPs genotypingin pre-miRNA.Epi Data3.0software was used for the database establishment, double entry anderror detection. SPSS10.01software was used to analyze data. The chi-square test wasused to count data analyses; t-test was used for the data analysis of the measurementdata; Logistic regression analysis was used to calculate odds ratios (ORs) and P valueswith adjustment for gender, age etc. Stata10.0software was used to calculate theHardy-Weinberg equilibrium (HWE). Haplotype analysis was assessed by using onlineSHEsis software. The powers of SNPs were calculated by Power and Sample Sizecalculation3.0software. The statistical significance defined as P value <0.05andcalculated based on two-sided tests. Results(1) HWE test There were no deviations from HWE observed in both SLE patients andnormal controls in each polymorphism (rs2910164controls:2=1.179, P=0.278; SLEpatients:2=0.392, P=0.531; rs2292832controls:2=0.096, P=0.757; SLE patients:2=0.652, P=0.419; rs11614913controls:2=0.080, P=0.777; SLE patients:2=0.450,P=0.502).(2) Association analysis of pre-miRNA gene SNPs and SLE susceptibility To SNPrs2910164of hsa-miRNA-146a, genotype frequencies for GG, GC and CC were17.7%,47.8%and34.5%in the SLE patients and18.3%,47.7%and34.0%in the normalcontrols, and no significant differences were exhibited either in the allele distributionsor in the genotype frequencies between SLE patients and normal controls (P>0.05); ToSNP rs2292832of hsa-miRNA-149, genotype frequencies for CC, CT and TT were10.8%,42.7%and46.6%in the SLE patients and10.3%,43.2%and46.5%in thenormal controls, and significant differences were existed neither in the alleledistributions nor in the genotype frequencies between SLE patients and normal controls(P>0.05); To SNP rs11614913of hsa-miRNA-196a2, genotype frequencies for CC, CTand TT were20.9%,48.7%and30.4%in the SLE patients and21.4%,50.0%and28.5%in the normal controls, and there were no significant differences existed in alleleand genotype frequencies between SLE patients and normal controls (P>0.05).(3) Association analysis of pre-miRNA gene SNPs and LN The present study included1392SLE patients with LN information,557patients of whom were LN patients(40.0%). To SNP rs2910164of hsa-miRNA-146a, genotype frequencies for GG, GC andCC were14.9%,46.9%and38.2%in the LN patients and19.3%,48.5%and32.2%inthe SLE patients without nephritis, and significant differences were exhibited in theallele and genotype frequencies between LN patients and SLE patients without nephritis(C vs. G: χ~2=7.453, P=0.006, OR=1.240,95%CI:1.063-1.448; CC vs. GG: χ~2=7.860,P=0.005, OR=1.621,95%CI:1.156-2.272; GC+CC vs. GG: χ~2=4.958, P=0.026, OR=1.416,95%CI:1.043-1.924); To SNP rs2292832of hsa-miRNA-149, genotypefrequencies for CC, CT and TT were9.7%,40.0%and50.3%in the LN patients and12.0%,45.9%and42.2%in the SLE patients without nephritis, and significantdifferences were existed both in allele frequencies and in genotype frequencies betweenLN patients and SLE patients without nephritis (T vs. C: χ~2=8.186, P=0.004, OR=1.269,95%CI:1.078-1.494; TT vs. CC: χ~2=4.560, P=0.033, OR=1.517,95%CI:1.035-2.225);To SNP rs11614913of hsa-miRNA-196a2, genotype frequencies for CC, CT and TTwere22.1%,49.6%and28.4%in the LN patients and20.0%,48.1%and31.9%in theSLE patients without nephritis, and there were no significant differences existed inallele and genotype frequencies between LN patients and SLE patients without nephritis(P>0.05).(4) Association analysis of pre-miRNA gene SNPs and SLE patients with an initialsymptom There were1382SLE patients (93.4%) with information of initial symptomsof1479SLE patients. Analyze of the associations between nine main initial symptoms(malar rash, discoid rash, photosensitivity, oral ulcers, arthritis, pleuritis, pericarditis,renal involvement and nervous system involvement) and three SNPs, we found thatrs2910164of hsa-miRNA-146a was associated with the initial symptoms of arthritis(GC vs. GG: χ~2=4.809, P=0.028, OR=1.423,95%CI:1.038-1.950; GC+CC vs. GG:χ~2=4.583, P=0.032, OR=1.383,95%CI:1.028-1.860）and renal involvement (C vs. G:χ~2=7.973, P=0.005, OR=1.548,95%CI:1.141-2.101; GC vs. GG: χ~2=5.167, P=0.023,OR=2.596,95%CI:1.140-5.910; CC vs. GG: χ~2=7.915, P=0.005, OR=3.295,95%CI:1.436-7.561; GC+CC vs. GG: χ~2=6.809, P=0.009, OR=2.887,95%CI:1.302-6.402),rs2292832of hsa-miRNA-149was associated with the initial symptoms of arthritis (T vs.C: χ~2=4.580, P=0.032, OR=0.849,95%CI:0.731-0.986; TT vs. CC: χ~2=4.273, P=0.039,OR=0.715,95%CI:0.521-0.983) and rs11614913of hsa-miRNA-196a2was notassociated with the initial symptoms.(5) Pre-miRNA haplotype analysis We analyzed the haplotypes constructed of rs2910164-rs2292832-rs11614913and determined eight haplotypes: CCC, CCT, CTC,CTT, GCC, GCT, GTC and GTT, and found that eight haplotypes frequencies were notsignificantly different between SLE patients and those in controls (P>0.05).(6) Pre-miRNA gene-gene interaction analysis We analyzed gene-gene interaction ofthree SNPs (rs2910164, rs2292832and rs11614913) in pre-miRNA, and found nogene-gene interaction exist in pre-miRNA in SLE and LN.Conclusions(1) No significant differences existed between the SLE patients and controls in SNPs(rs2910164, rs2292832and rs11614913) in the precursor region of hsa-miRNA-146,hsa-miRNA-149and hsa-miRNA-196a2, their haplotypes and gene-gene interaction,which suggests that the polymorphisms of pre-miRNA genes might not contribute toSLE susceptibility in the Chinese population.(2) The SNPs rs2910164and rs2292832in hsa-miRNA-146a and hsa-miRNA-149maybe associated with susceptibility to LN, and there was no interaction between genes.(3) The SNPs rs2910164in hsa-miRNA-146a may be associated with the initialsymptom of arthritis and renal involvement in SLE patients and the SNPs rs11614913inhsa-miRNA-196a2may be associated with the initial symptom of arthritis in SLEpatients.