Pooled genome wide association detects association upstream of FCRL3 with Graves' disease.

BACKGROUND: Graves' disease is an autoimmune thyroid disease of complex inheritance. Multiple genetic susceptibility loci are thought to be involved in Graves' disease and it is therefore likely that these can be identified by genome wide association studies. This study aimed to determine if a genome wide association study, using a pooling methodology, could detect genomic loci associated with Graves' disease. RESULTS: Nineteen of the top ranking single nucleotide polymorphisms including HLA-DQA1 and C6orf10, were clustered within the Major Histo-compatibility Complex region on chromosome 6p21, with rs1613056 reaching genome wide significance (p = 5 × 10-8). Technical validation of top ranking non-Major Histo-compatablity complex single nucleotide polymorphisms with individual genotyping in the discovery cohort revealed four single nucleotide polymorphisms with p ≤ 10-4. Rs17676303 on chromosome 1q23.1, located upstream of FCRL3, showed evidence of association with Graves' disease across the discovery, replication and combined cohorts. A second single nucleotide polymorphism rs9644119 downstream of DPYSL2 showed some evidence of association supported by finding in the replication cohort that warrants further study. CONCLUSIONS: Pooled genome wide association study identified a genetic variant upstream of FCRL3 as a susceptibility locus for Graves' disease in addition to those identified in the Major Histo-compatibility Complex. A second locus downstream of DPYSL2 is potentially a novel genetic variant in Graves' disease that requires further confirmation.

Association of Polymorphisms in MACRO Domain Containing 2 With Thyroid-Associated Orbitopathy.

PURPOSE: Thyroid-associated orbitopathy (TO) is an autoimmune-mediated orbital inflammation that can lead to disfigurement and blindness. Multiple genetic loci have been associated with Graves' disease, but the genetic basis for TO is largely unknown. This study aimed to identify loci associated with TO in individuals with Graves' disease, using a genome-wide association scan (GWAS) for the first time to our knowledge in TO. METHODS: Genome-wide association scan was performed on pooled DNA from an Australian Caucasian discovery cohort of 265 participants with Graves' disease and TO (cases) and 147 patients with Graves' disease without TO (controls). Top-ranked single nucleotide polymorphisms (SNPs) then were genotyped in individual DNA samples from the discovery cohort, and two replication cohorts totaling 584 cases and 367 controls. RESULTS: In the GWAS of pooled DNA samples, several SNPs showed suggestive association with TO at genome-wide P ≤ 10-6; rs953128 located on chr10q21.1, rs2867161 on chr7q11.22, rs13360861 on chr5q12.3, rs7636326 on chr3q26.2, rs10266576 on chr 7q11.22, rs60457622 on chr3q23, and rs6110809 on chr20p12.1. However, the only SNP consistently associated with TO on individual genotyping in the discovery and replication cohorts was rs6110809, located within MACROD2 on chromosome 20p12.1. On combined analysis of discovery and replication cohorts, the minor A allele of rs6110809 was more frequent in TO than in Graves' disease controls without TO (P = 4.35 × 10-5; odds ratio [OR] = 1.77; 95% confidence interval [CI], 1.35-2.32) after adjusting for age, sex, duration of Graves' disease, and smoking. CONCLUSIONS: In patients with Graves' disease, a common genetic variant in MACROD2 may increase susceptibility for thyroid-associated orbitopathy. This association now requires confirmation in additional independent cohorts.

Uroplakin Ib gene transcription in urothelial tumor cells is regulated by CpG methylation.

Uroplakin Ib is a structural protein on the surface of urothelial cells. Levels of uroplakin Ib mRNA are dramatically reduced or absent in many transitional cell carcinomas, but the molecular mechanisms responsible remain undetermined. Previously, we showed that loss of uroplakin Ib expression correlated with CpG methylation of Sp1/NFkappaB-binding motifs within the proximal promoter. In this study, we show that reporter activity was completely blocked by the methylation of three CpG pairs in this promoter region. Gel shift analysis using purified proteins or nuclear extracts showed that Sp1 and NFkappaB bound to motifs encompassing two of the three CpG pairs. Interestingly, the methylation of these two CpG sites did not prevent the binding of proteins to the promoter in gel shift analyses. Additionally, mutation of these two CpGs did not affect reporter activity, but mutation of 6-bp fragment spanning each CpG partially inhibited reporter activity, suggesting that these sites were functional. A requirement for both Sp1 and NFkappaB in regulating reporter activity was confirmed in transfection experiments using plasmids expressing individual proteins. Our data suggest that the methylation of specific CpG sites can silence the uroplakin Ib promoter, at least in part, by blocking the binding of Sp1 and NFkappaB, although other factors may be involved.

Methylation of a CpG island within the uroplakin Ib promoter: a possible mechanism for loss of uroplakin Ib expression in bladder carcinoma.

Uroplakin Ib is a structural protein on the surface of urothelial cells. Expression of uroplakin Ib mRNA is reduced or absent in many transitional cell carcinomas (TCCs) but molecular mechanisms underlying loss of expression remain to be determined. Analysis of the uroplakin Ib promoter identified a weak CpG island spanning the proximal promoter, exon 1, and the beginning of intron 1. This study examined the hypothesis that methylation of this CpG island regulates uroplakin Ib expression. Uroplakin Ib mRNA levels were determined by reverse transcription polymerase chain reaction and CpG methylation was assessed by bisulfite modification of DNA, PCR, and sequencing. A correlation was demonstrated in 15 TCC lines between uroplakin Ib mRNA expression and lack of CpG methylation. In support of a regulatory role for methylation, incubating uroplakin Ib-negative lines with 5-aza-2'-deoxycytidine reactivated uroplakin Ib mRNA expression. A trend between uroplakin Ib mRNA expression and CpG methylation was also observed in normal urothelium and bladder carcinomas. In particular, loss of uroplakin Ib expression correlated with methylation of a putative Sp1/NFkappaB binding motif. The data are consistent with the hypothesis that methylation of specific sites within the uroplakin Ib promoter may be an important factor in the loss of uroplakin Ib expression in TCCs.