The Positive Phase Of Two-color Fluorescence Hybridization Assay Snp Dynamics Of Complex Disease Susceptibility Genes Association Analysis And Meta-analysis

Microarray-based dual-color fluorescence hybridization can realize screening a large number of genomic DNA samples in parallel for a given SNP locus. The technique is especially suitable for genotyping some known candidate SNP loci in large-scale association study for complex disease. However, the hybridization dynamics about this technique hasn't been reported until now. In this study, by observing signal value (Cy5, Cy3) and ratio value (Cy5/Cy3), we discuss how spotting concentration of PCR products, PCR products length and fluorescence labeled probe length affect SNP genotyping. Results indicate that as the concentration of spotted PCR product increases, the signal values of Cy5, Cy3 increase and ratio value for three genotypes appeared to change regularly. By dye swap experiment, we tended to dominate the systematic bias. The results indicated that ratio values of three genotypes tended to be stable as concentration of spotted PCR product increases; Signal value declined obviously as the PCR products length increased and ability of ratio value for genotyping also declined. When using DMSO spotting technique to make PCR products maintain single-strand, signal value didn't change as the length. According to the results, we suggest that dye-swap for the same samples should be tried to get balanced results. If spotting double-strand DNA, the length of PCR products shouldn't be too long. Or else, we can use DMSO spotting to raise the signal intensity for good genotyping. Appropriate spotting concentration of PCR products, PCR product length and fluorescence labeled probe length as well as dye-swap labeling may provide stable and correct genotyping results.Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a key negative regulator of T-cell activation and proliferation during the immune response, and thereby has been an attractive candidate susceptible gene for T cell-mediated autoimmune diseases such as Graves" disease (GD). However, most association studies about GD susceptibility restricted their analysis to the exon 1 +49 A/G polymorphism and few studies investigated the role of polymorphisms in the promoter region and 3'UTR in GD susceptibility. There is strong evidence to support the Thyroglobulin gene (TG) as a thyroid-specific AITD susceptibility gene. However, the association study results were confusing rather than conclusive Additional studies are needed to confirm this association. In this study, we performed a more detailed association study about the role of five CTLA-4 SNPs (-1722A/G, -1661A/G, -318C/T +49G/A, CT60) and two TG SNPs (rs180223 and rs11535853) in GD susceptibility in the Chinese populationusing dual-color fluorescence hybridization technique for genotyping. Our results demonstrated that -1661A/G, +49G/A, CT60 and rs11535853 were significantly associated with GD susceptibility. Moreover, we found that the +49G and rs11535853C risk allele have a trend to cause the disease susceptibility in a recessive genetic manner implying a dose effect of these alleles. Our results also showed that the -1661A/G and CT60 polymorphisms were associated with GD in a gender specific manner. Haplotype analysis revealed a most related haplotype -1722G~-1661G~ -318C~+49G~CT60G The elevated risk of haplotype than single SNP on GD susceptibility possibly implies an interaction between different SNP sites. Haplotype analysis of two SNPs of TG showed that only the haplotype including the rs11535853C allele demonstrated an increased risk of diseaseIn our study, we also investigated the role of 5 SNPs of CTLA-4 gene 2SNPs of TG gene in the susceptibility of clinically evident GO in Chinese GD patients. Our results demonstrated that the -318T allele was negatively associated with GO under both additive and dominant genetic models in a gender specific manner. As far as the other polymorphisms were concerned (-1722A/G, -1661A/G, +49G/A, CT60, rsl80223 and rsll535853), we did not detect any association between them and GO individually. Haplotype analyses showed that only the haplotype containing the -318T allele played a protective role in GO. These findings suggest that -318C/T polymorphism may contribute to the etiology of GO or in linkage disequilibrium with a nearby real causal variant especially for male GD patients.As genetic association study always produce conflicting results for the same variant and disease between different studies, meta-analysis of genetic epidemiology studies has been considered as a good way to resolve the discrepancies between studies. On one hand, meta-analysis can effectively improve the statistical power for detecting the small effect genes and give a more conclusive conclusion, on the other hand, meta-analysis is a powerful tool to explore the sources of heterogeneity between studies and can guide the design and interpret of genetic association studies. We have focued on three conflicting topics of genetic association study and made some useful conclusions by meta-analysis: 1) meta-analysis of CTLA-4-exonl +49A/G polrmorphism with ophthalmopathy in Graves" disease patients. Our meta-analysis results demonstrated no significant association between the G allele and GO risk in Asian GD patients. However, a significant association between this polymorphism and GO risk might exist in European GD patients. We suggest that more or largerstudies should be performed to further clarify its role in Asian GD patients. 2) meta-analysis of the association of ctla-4 exonl +49A/G polrmorphism with rheumatoid arthritis. Our meta-analysis, along with other studies, suggests that the CTLA-4 exon-1 +49 G allele might be a risk factor for RA in Asians but not in Europeans. More studies or large case-control studies should be performed to clarify its possible role in Asian populations. 3) DNA repair gene XRCC3 polymorphisms and cancer risk: a meta-analysis of 40 case-control Studies. Our meta-analysis supports that the XRCC3 could not be a major increased risk factor for cancer but it might represent a low-penetrance susceptible gene especially for cancer of breast, bladder, head and neck, and non-melanoma skin cancer.