Differential Gene Expression Profiling of Orbital Adipose Tissue in Thyroid Orbitopathy

Published under a Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/legalcode)PURPOSE. We aimed to determine differentially expressed genes relevant to orbital inflammation and orbital fat expansion in thyroid orbitopathy (TO) using microarray gene profiling in a case-control study. METHODS. Human orbital adipose samples were obtained from individuals with active TO (n ¼ 12), inactive TO (n ¼ 21), and normal controls (n ¼ 21). Gene expression profiles were examined using microarray analysis and were compared between active and inactive TO, and between active TO and normal controls. Top ranked differentially expressed genes were validated by real-time RT-PCR in an additional eight active TO, 13 inactive TO, and 11 normal controls and correlated with gene set enrichment analysis (GSEA) and molecular pathways analysis. RESULTS. Seven hundred twenty-one probes (683 genes) and 806 probes (735 genes) were significantly differentially expressed in comparing active to inactive TO and in comparing active TO to healthy controls, respectively. All selected genes were confirmed to be differentially expressed by real-time RT-PCR. Multiple top ranked genes in active versus inactive TO comparison are overrepresented by immune and inflammatory response genes. They include defensins (DEFA1, DEFA1B, DEFA3), which were overexpressed by 3.05- to 4.14-fold and TIMD4 by 4.20-fold. Markers for adipogenesis were overexpressed including SCD, FADS1, and SCDP1. Gene set enrichment analysis revealed dysregulation of epigenetic signatures, T-cell activation, Th1 differentiation, defensin pathway, cell adhesion, cytoskeleton organization, apoptosis, cell cycling, and lipid metabolism in active TO. CONCLUSIONS. Active TO is characterized by upregulation of genes involved in cell-mediated immune, innate immune, and inflammatory response and enhanced orbital adipogenesis. TIMD4, DEFA1, DEFA1B, and DEFA3 genes may be involved in the innate immune-mediated orbital inflammation in TO. Epigenetic mechanisms may play a role in the pathogenesis of TO

Genetic study of thyroid eye disease

© 2016 Dr. Jwu Jin KhongThyroid-associated orbitopathy (TO) is an autoimmune-mediated orbital inflammation that affects 25% of patients with Graves’ disease. The close temporal relationship between onset of Graves’ disease and TO suggests they share a common aetiology. While the complex inheritance of Graves’ disease is better characterized, little is known of the genetic susceptibility in TO. Multiple environmental factors such as smoking, male, older age are known risk factors for development of TO, however the extent of gene-environmental interaction remains largely unknown. The molecular mechanisms driving the development of TO is incompletely understood, hence targeted treatment options for TO remained limited. This thesis is undertaken to test the hypothesis that there is genetic susceptibility that predispose to development of TO. The research project initially examined exogenous risk factors associated with TO in a large Australian cohort with Graves’ disease, in order to identify environmental factors important for subsequent covariates adjustment when analyzing genetic findings. The risk factors association study found smoking, older age and longer duration of Graves’ disease correlated positively with TO, and secondarily there was relative selenium deficiency in TO cases compared to Graves’ disease patients without eye involvement. A genome-wide association study using deoxyribonucleic acid pooling approach and high-throughout array platform were used to discover gene variants associated with thyroid-associated orbitopathy in a case-control study design. The genetic findings were followed by a second stage individual genotyping targeting fewer markers to validate the genetics variants identified through genome-wide association study in the discovery cohort and independent replication study cohort. MACROD2, a novel gene that encodes an eraser of mono-ADP-ribosylation, possibly has a role in nuclear factor κβ signaling, showed evidence of association with TO in genome-wide association study and also in validation genotyping. A secondary aim of the thesis is to determine differentially expressed genes a priori in active TO orbital adipose tissue using microarray to explore molecular mechanisms of TO and to correlate gene expression findings with the genetic study. The study found TIMD4, DEFA1, DEFA1B, and DEFA 3 were over-expressed in active TO compared with inactive TO suggesting a pathogenic role of the innate immune response in TO. Active TO was marked by up-regulation of multiple genes involved in cell-mediated, innate and inflammatory responses with concurrent enhancement of orbital adipogenesis. For the first time, epigenetic factors was implicated in the pathogenesis of TO. However MACROD2 were not differentially expressed in active TO when compared with either inactive TO or normal control. Overall the findings from this thesis further our understanding on the genetic and environmental risk factors involved in thyroid-associated orbitopathy and give new insights into the underlying complex molecular mechanisms. The novel insights into candidate molecules and pathways can be explored to develop alternative treatment strategies for TO

Serum selenium status in Graves' disease with and without orbitopathy: A case-control study

Objective Selenium is effective in improving quality of life and reducing the progression of active Graves’ orbitopathy. The effect of correcting relative selenium deficiency on improving Graves’ orbitopathy is unknown, as baseline selenium levels have not previously been measured. The study aims to determine whether serum selenium levels are reduced in patients with Graves’ disease with orbitopathy (GO) compared with without orbitopathy (GD). Design A prospective, case–control study performed between 2009 and 2012 at endocrine and ophthalmology clinics in Australia. Patients A total of 198 patients with Graves’ disease participated in the study: 101 with Graves’ orbitopathy and 97 without Graves’ orbitopathy. Measurements Serum selenium levels in both groups. Results Mean serum selenium levels were significantly lower in GO (1·10 ± 0·18 μm) than in GD (1·19 ± 0·20 μm) (P = 0·001). Mean selenium levels appeared to decrease in parallel with increasing severity of GO; selenium level was 1·19 ± 0·20 μm in GD, 1·10 ± 0·19 μm in moderate-to-severe GO and 1·09 ± 0·17 μm in sight-threatening GO (P = 0·003). Serum selenium levels remained significantly lower in GO after adjusting for age, smoking status, thyroidectomy, radioactive iodine treatment and residential location. Conclusion Serum selenium levels are lower in patients with GO compared with GD in an Australian study population with marginal selenium status. Relative selenium deficiency may be an independent risk factor for orbitopathy in patients with Graves’ disease

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.9 page(s

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.Jwu Jin Khong, Kathryn P. Burdon, Yi Lu, Kate Laurie, Lefta Leonardos, Paul N. Baird, Srujana Sahebjada, John P. Walsh, Adam Gajdatsy, Peter R. Ebeling, Peter Shane Hamblin, Rosemary Wong, Simon P. Forehan, Spiros Fourlanos, Anthony P. Roberts, Matthew Doogue, Dinesh Selva, Grant W. Montgomery, Stuart Macgregor and Jamie E. Crai