Yaron Tomer

A CD40 Kozak sequence polymorphism and susceptibility to antibody-mediated autoimmune conditions: the role of CD40 tissue-specific expression.

Previously, we and others have demonstrated the association of a C/T single nucleotide polymorphism (SNP), in the Kozak sequence of CD40, with Graves disease (GD). Here, using an expanded data set of patients, we confirm the association of the CD40 SNP with GD (n=210, P=0.002, odds ratio (OR)=1.8). Subset analysis of patients with persistently elevated thyroid peroxidase (TPO) and/or thyroglobulin (Tg) antibodies (Abs), (TPO/Tg Abs), after treatment (n=126), revealed a significantly stronger association of the SNP with disease (P=5.2 × 10−5, OR=2.5) than in GD patients who were thyroid antibody-negative. However, the CD40 SNP was not associated with TPO/Tg Abs in healthy individuals. Next, we tested the CD40 SNP for association with Myasthenia Gravis (MG), which, like GD is an antibody-mediated autoimmune condition. Analysis of 81 MG patients found no association of the SNP with disease. Functional studies revealed significant expression of CD40 mRNA and protein in the thyroid (target tissue in GD) but not in skeletal muscle (target tissue in MG). Combined, our genetic and tissue expression data suggest that the CD40 Kozak SNP is specific for thyroid antibody production involved in the etiology of GD. Increased thyroidal expression of CD40 driven by the SNP may contribute to this disease specificity.

Thyroid epigenetics: X chromosome inactivation in patients with autoimmune thyroid disease.

Abstract:u2002 The autoimmune thyroid diseases (AITDs) are female‐predominant diseases with a ratio of approximately seven females to each male. X chromosome inactivation (XCI), an epigenetic phenomenon, has been suggested to be skewed in many such female patients with AITD. We analyzed female genomic DNA from 87 patients with Graves disease (GD), 47 patients with Hashimotos thyroiditis (HT), and 69 healthy controls. Using an XCI assay based on Hpa II digestion and PCR and DNA sequencing, we found skewed heterozygous XCI (≥80%) in 20 of 70 GD patients (28.6%) and 11 of 43 HT patients (25.6%), giving a total of 31 of 113 AITD patients (27.4%) with skewed XCI. In contrast, only 5 of 58 healthy controls had skewed XCI (8.6%). Statistical analysis confirmed that XCI skewing was significantly associated with AITD (P= 0.004, OR = 4.0), demonstrating that the degree of XCI is an important contributor to the increased risk of females in developing AITD.

Influence of the TSH Receptor Gene on Susceptibility to Graves' Disease and Graves' Ophthalmopathy

BACKGROUNDnA large gene region, called GD-1, was first described by this laboratory as linked to Graves disease (GD) and included the gene for the thyroid-stimulating hormone receptor (TSHR). Recent studies have now suggested an association of TSHR intronic polymorphisms with GD. We have taken the opportunity to examine a population of well-characterized patients with autoimmune thyroid disease (AITD) typed for an additional thyroid susceptibility gene, the immunoregulatory gene for cytotoxic T-lymphocyte antigen 4 (CTLA-4), to examine its relationship with the susceptibility to GD endowed by TSHR gene polymorphisms.nnnMETHODSnWe used TSHR-SNP-rs2268458, located in intron 1 of the TSHR gene, measured using standard PCR-RFLP procedures, as our marker for the TSHR gene association. We genotyped 200 patients with GD, 83 patients with Hashimotos thyroiditis (HT), and 118 healthy controls (all female Caucasians).nnnRESULTSnThe allele and genotype frequencies from GD patients, but not HT patients, were significantly different from controls. The frequency of the combined genotype (allele) CC + TC was significantly higher in GD patients versus controls, suggesting that the C-containing genotype increased the risk for GD in a dominant manner (p = 0.018, odds ratio [OR] = 1.8). When compared with CTLA-4 (A/G)(49) single-nucleotide polymorphism (SNP), we were unable to demonstrate additive risk in patients with established AITD. Further, subsetting the patients (n = 120) into those with clinically significant Graves ophthalmopathy (GO) showed no association with the TSHR SNP.nnnCONCLUSIONSnThese results demonstrated that the intronic TSHR-SNP-rs2268458 was associated with GD, but not with HT, thus indicating that the TSHR gene has the potential to increase susceptibility to GD. However, we were not able to demonstrate any additive risk with the CTLA-4 (A/G)(49) SNP, which is, therefore, an independent risk factor for AITD. This suggested that, within the limits of the study population, each of these two genes provided a small contribution to GD susceptibility and that neither was essential. In addition, there was no evidence for the TSHR gene association adding to the risk of developing GO. Direct functional analyses are now needed to help explain the mechanisms of this TSHR gene susceptibility to GD.

A Multilocus Model of the Genetic Architecture of Autoimmune Thyroid Disorder, with Clinical Implications

We report here a preliminary model of the genetic architecture of Autoimmune Thyroid Disorder (AITD). Using a flexible class of mathematical modeling techniques, applied to an established set of data and supplemented with information both from candidate-gene and genome-wide-association studies and from basic bioinformatics, we find strong statistical support for a model in which AITD is the result of hits along three distinct genetic pathways: affected individuals have (1) a genetic susceptibility to clinical AITD, along with (2) a separate predisposition to develop the autoantibodies characteristic of AITD, and they also have (3) a predisposition to develop high levels of autoantibodies once they occur. Genes underlying each of these factors then appear to interact with one another to cause clinical AITD. We also find that a genetic variant in CTLA4 that increases risk for AITD in some people might actually protect against AITD in others, depending on which additional risk variants an individual carries. Our data show that the use of statistical methods for the incorporation of information from multiple sources, combined with careful modeling of distinct intermediate phenotypes, can provide insights into the genetic architecture of complex diseases. This model has several clinical implications, which we believe will prove relevant to other complex diseases as well.

Employing a Recombinant HLA-DR3 Expression System to Dissect Major Histocompatibility Complex II-Thyroglobulin Peptide Dynamism A GENETIC, BIOCHEMICAL, AND REVERSE IMMUNOLOGICAL PERSPECTIVE

Previously, we have shown that statistical synergism between amino acid variants in thyroglobulin (Tg) and specific HLA-DR3 pocket sequence signatures conferred a high risk for autoimmune thyroid disease (AITD). Therefore, we hypothesized that this statistical synergism mirrors a biochemical interaction between Tg peptides and HLA-DR3, which is key to the pathoetiology of AITD. To test this hypothesis, we designed a recombinant HLA-DR3 expression system that was used to express HLA-DR molecules harboring either AITD susceptibility or resistance DR pocket sequences. Next, we biochemically generated the potential Tg peptidic repertoire available to HLA-DR3 by separately treating 20 purified human thyroglobulin samples with cathepsins B, D, or L, lysosomal proteases that are involved in antigen processing and thyroid biology. Sequences of the cathepsin-generated peptides were then determined by matrix-assisted laser desorption ionization time-of-flight-mass spectroscopy, and algorithmic means were employed to identify putative AITD-susceptible HLA-DR3 binders. From four predicted peptides, we identified two novel peptides that bound strongly and specifically to both recombinant AITD-susceptible HLA-DR3 protein and HLA-DR3 molecules expressed on stably transfected cells. Intriguingly, the HLA-DR3-binding peptides we identified had a marked preference for the AITD-susceptibility DR signatures and not to those signatures that were AITD-protective. Structural analyses demonstrated the profound influence that the pocket signatures have on the interaction of HLA-DR molecules with Tg peptides. Our study suggests that interactions between Tg and discrete HLA-DR pocket signatures contribute to the initiation of AITD.

TYPE 1 DIABETES AND AUTOIMMUNE THYROIDITIS: THE GENETIC CONNECTION

Genetic studies have transformed our understanding of autoimmune diseases. As new genes and loci are being identified, novel mechanisms underlying autoimmunity are being unravelled. For example, with the mapping of NOD2 as a major Crohn’s disease gene, it became clear that the innate immune response is critical for the development of Crohn’s disease, and the entire field moved in this direction (1). Similarly, the identification of HLA-DR (2) and 5 additional non-MHC genes (3) as major susceptibility genes for autoimmune thyroid diseases (AITD) highlighted the central role of the immunological synapse (i.e. the interface between antigen presenting cells and T-cells during antigen presentation (4;5)) in the etiology of thyroid autoimmunity (6).

Chapter 6 The Genetics of Autoimmune Thyroid Diseases

Abstract Autoimmune thyroid diseases (AITD), including Graves’ disease (GD) and Hashimotos thyroiditis (HT) are complex diseases that arise due to interplay between environmental and genetic factors. In the past decade, several AITD susceptibility genes have been identified and characterized, including both immune-regulatory genes and thyroid-specific genes. Some of these susceptibility genes are specific to either GD or HT, while others confer susceptibility to thyroid autoimmunity in general. Recent studies began dissecting the mechanisms by which these new genes predispose to thyroid autoimmunity, and the emerging mechanisms focus on abnormalities of the immunological synapse. In this chapter, we will summarize the recent data on the genes predisposing to AITD and the emerging mechanisms by which they confer susceptibility to disease.