Association Studies In The Genetic Susceptibility Of Systemic Lupus Erythematosus

Part 1 Candidate gene approach-Association of PDCD1 polymorphisms with systemic lupus erythematosusObjective: To investigate the genetic associations of PDCD1 polymorphisms,SNP-7809(rs11568821),SNP-7872 and SNP-8162(rs6705653)with systemic lupus erythematosus(SLE)in Chinese Han population from Fujian province;to test the utility of DNA-pooling technique when applied to association studies.Methods: Polymerase Chain Reaction-Restriction Fragment Length Polymorphism(PCR-RFLP)and Primer-Introduced Restriction Analysis PCR(PIRA-PCR)were applied to genotype SNP-7872 and SNP-8162 in 240 patients of SLE and 210 healthy controls in Fujian population. The allelic ratio of two alleles in the DNA pools was determined with the method of standard curve. By comparing the results,the validity of applying DNA-pooling technique to association study of SNPs was determined.Results: In Fujian Han population,SNP-7809 was not polymorphic. There were no significant differences in the genotypic frequencies of PDCD1 SNP-7872 and SNP-8162 between SLE and control group(sP=0.81,0.86; OR=1.02,0.99). Multiple logistic regression analyses revealed that PDCD1 SNP-7872 and SNP-8162 were not associated with SLE after adjusting for both potential confounding factors of gender and age(P=0.75,0.81). This study also demonstrated that assaying odd ratios with DNA-pooling technique can produce similar results as that obtained by genotyping individual samples.Conclusion: In Fujian Han population,SNP-7809 of PDCD1 was not polymorphic and SNP-7872 and SNP-8162 were not associated with SLE. Our experiments also demonstrated that'genotyping'SNPs with DNA pools can be used as a fast and labor-saving method in the association studies of a portion of SNPs. Part 2 Genome-wide screening approach-Application of DNA pooling and SNP microarray technology in the association studies of SLEObjective: To identify genetic loci associated with human SLE via genome-wide screening. To develop a high throughput method (combining DNA-pooling and microarray, SNP-MaP) in assaying the difference of allelic ratio of SNP in patients and controls with only a minimum number of DNA chips; To evaluate the utility of this method in our studies of susceptible loci of SLE.Methods: Affymetric 250K StyI microarray containing ~250K SNP and covering the whole genome, was employed to determine the allelic ratios of SNPs in DNA-pools of control and patient samples; By proper adjustments of both confidence and the odd ratio levels, major genetic loci related to the occurrence of SLE with high confidence were identified. Using real-time PCR (Taqman) to individually genotype the samples and determine the allelic frequencies at the SNPs resulted from the first round of screening using SNP-MaP. Confirmation and evaluation of the effectiveness of this method were achieved by comparison of odd ratios obtained from two rounds of screening.Results: In the first of screening by microarray, the allelic ratios of 238,304 SNPs were determined in both DNA-pools derived from 240 SLE patients and 210 controls. 108 SNPs were found to be statistically associated with the occurrence of SLE with odd ratios higher than 1.5. Among them, 6 were further validated by the heterozygosity-assaying method derived from DNA-sequencing. So far one SNP, located at 7q31, rs4731117, has been ultimately confirmed to be highly significantly associated with the occurrence of SLE in our cohort with an odd ratio of 2.2.Conclusion: A new method combining SNP microarray and DNA-pooling (SNP-MaP) has been developed to perform the high throughout genome-wide association studies. This method has been demonstrated to be a potential highly effective and inexpensive in our case of genetic study of SLE. A novel SNP, rs4731117 was discovered to be strongly associated with the occurrence SLE in Fujian population with high risk ratio. The candidate genes includes interferon regulatory factor 5, a gene which plays an important role in immune regulation.