Abstract
Objectives
The pathogenesis of Graves’ orbitopathy (GO) remains to be fully elucidated. Here, we reviewed the role of genetics and epigenetics.
Design
We conducted a PubMed search with the following keywords: GO, thyroid eye disease; or Graves’ ophthalmopathy; or thyroid-associated ophthalmopathy; and: genetic, or epigenetic, or gene expression, or gene mutation, or gene variant, or gene polymorphism, or DNA methylation, or DNA acetylation. Articles in which whole DNA and/or RNA sequencing, proteome, and methylome analyses were performed were chosen.
Results
The different prevalence of GO in the two sexes, as well as racial differences, suggest that genetics play a role in GO pathogenesis. In addition, the long-lasting phenotype of GO and patient-derived orbital fibroblasts suggests a genetic or epigenetic mechanism. Although no genes have been found to confer a specific risk for GO, differential gene expression has been reported in orbital fibroblasts from GO patients vs control fibroblasts, suggesting that an epigenetic mechanism may be involved. In this regard, a different degree of DNA methylation, which affects gene expression, has been found between GO and control fibroblasts, which was confirmed by whole methylome analysis. Histone acetylation and deacetylation, which also affect gene expression, remain to be investigated.
Conclusions
Although no pathogenic gene variants have been reported, epigenetic mechanisms elicited by an initial autoimmune insult seem to be needed for differential gene expression to occur and, thus, for GO to develop and persist over time.
Introduction
Graves’ orbitopathy
Graves’ orbitopathy (GO), also known as thyroid eye disease (TED), is a relatively common extrathyroidal manifestation of Graves’ disease, affecting approximately 30% of patients with Graves’ hyperthyroidism (1, 2). GO can also be observed in a minority of patients with hypothyroid autoimmune thyroiditis or in the absence of overt thyroid dysfunction (2). Overall, GO is quite rare (0.54–0.9 cases/100,000/year in men, 2.67–3.3 cases/100,000/year in women), and the majority of patients experience only mild forms, whereas moderate-to-severe and severe forms account for 5–6% of GO patients (3, 4). Nevertheless, GO has a strong impact on patients’ quality of life, even in mild forms (5, 6, 7), because of its functional and disfiguring consequences. The clinical manifestations of GO reflect an increased volume within the orbital space, due to augmented fibroadipose tissue and the release of large amounts of glycosaminoglycans (GAGs), especially hyaluronic acid (HA), by orbital fibroblasts (8). Features of GO include exophthalmos, eyelid edema or erythema, conjunctival redness and swelling, increased palpebral aperture, eye muscle enlargement with consequent diplopia, and in the most severe cases, corneal breakdown or optic neuropathy, the latter mainly due to compression of the optic nerves at the orbital apex (9).
As far as treatment is concerned, whereas mild GO is usually managed with local ointments (artificial tears, ophthalmic gels) and selenium, the treatment of moderate-to-severe and active GO is generally performed with high-dose systemic glucocorticoids (9), based on activity defined by a clinical score reflecting inflammatory signs and symptoms. More recently, thanks to advancements in the knowledge of GO pathogenesis, new treatment modalities have been to some extent introduced in clinical practice. In particular, a monoclonal anti-insulin-like growth factor-1 receptor (IGF-1R) antibody, known as Teprotumumab, which is now the first-line treatment in certain countries (10). Other promising medications, some of which are under investigation, are Azathioprine (11), Linsitinib (IGF-1R inhibitor) (12), Batoclimab (13) (anti-FcRn monoclonal antibody), Efgartigimod alfa (FcRn antagonist) (14), Tocilizumab (15) and Satralizumab (16) (both anti IL6-receptor monoclonal antibodies), Rituximab (anti-CD20 monoclonal antibody) (17), Sirolimus (molecular target of rapamycin (mTOR) inhibitor) (18), and the use of Atorvastatin as an add-on therapy to glucocorticoids (19). Radiotherapy and surgery (orbital decompression, squint, and palpebral surgery) are also used in selected patients (9).
GO pathogenesis
GO is believed to reflect an autoimmune pathogenesis (8). In this regard, the thyrotropic hormone receptor (TSHR), autoimmunity against which is responsible for Graves’ hyperthyroidism, is commonly recognised as the most important autoantigen (8). According to the current view of the molecular mechanisms underlying GO, the TSHR expressed by orbital fibroblasts is recognised by autoantibodies and/or autoreactive T-cells to then interact with IGF-1R (20). Following this interaction, both TSHR and IGF-1R are activated, resulting in augmented fibroblast growth and secretion of GAGs (8, 20). To some extent, these events are also elicited by cytokines secreted by immune-competent cells and by orbital fibroblasts themselves, as well as by the consequent oxidative stress in the orbital microenvironment (8). Whether IGF-1R also functions as an autoantigen is debated (21, 22).
As it occurs in many diseases of autoimmune origin, in addition to thyroid autoimmune disease, GO can coexist with a number of organ-specific and nonorganspecific autoimmune conditions (9). These associations can be ascribed to genetic predisposition, but also to the environment patients are exposed to. In this regard, in recent years, emphasis has been posed to the gut microbiota, which was found to be involved in the pathogenesis of several autoimmune diseases (23, 24), in some of which it can elicit epigenetic changes that concur to the pathogenesis (25). Very interestingly, specific patterns of the gut microbiota have been associated with GO (26).
Known risk factors for the development of GO in patients with Graves’ hyperthyroidism are age, sex, uncontrolled hyper- or hypothyroidism, radioiodine treatment, smoking, and hypercholesterolemia (4, 27). Whether genetics and epigenetics play a role has been the subject of several studies and is the main topic of the present review.
Genetics
General concepts
The different prevalence of GO in the two sexes (28), as well as racial differences (29), suggests that genetics may play a role in its pathogenesis. Thus, the female-to-male ratio of GO is approximately 2:1, and males are at a higher risk of developing severe forms of the eye disease (27). In addition, Asians have a lower likelihood of developing GO compared to Caucasians (29).
A large number of studies have allowed the identification of gene variants associated with the occurrence of Graves’ hyperthyroidism, including immune regulatory genes, human leukocyte antigen (HLA)-DRβ1-Arg74, cytotoxic T lymphocyte antigen (CTLA)-4, PTPN22, CD40, thyroglobulin, and TSHR (30). However, the contribution of specific genetic variants to disease pathogenesis appears to be low, and none of them seems to be necessary or sufficient for the development of Graves’ hyperthyroidism. Furthermore, no gene variants have been shown to confer a specific risk for GO (30). In this regard, it is certainly possible that the genetic basis for the development of Graves’ disease is quite complex, probably involving several susceptibility genes (31, 32), none of which have variants sufficient for the disease to occur. In particular, concerning GO, as discussed in detail below, epigenetic factors are likely needed for genetic modifications (differential gene expression) to occur and, thus, for the eye disease to develop and persist over time.
Microarray studies
After the development of the microarray method, a number of studies investigated the genomic profile of GO, with several genes found to be either under- or overexpressed.
Lantz et al. (33) performed a case–control study on orbital adipose tissues from five GO patients and five control subjects. They found overexpression of several genes, including stearoyl-coenzyme A desaturase, a marker of adipose tissue. In addition, certain immediate early genes were overexpressed, namely CYR61 (cysteine-rich, angiogenic inducer 61), cyclooxygenase-2, dual-specificity phosphatase 1, B cell translocation gene 2, and early growth response 1. CYR61-responsive genes known to participate in inflammation, such as IL-1β, matrix metalloproteinase-3, and vascular endothelial growth factor, were also overexpressed. Furthermore, the expression of CYR61 was greater in the active phase than in the chronic phase of GO.
A similar study was conducted by Ezra et al. (34), who found that the most differentially expressed genes were related to the IGF-1 pathway, namely IGF-1, IGF-1R binding/signalling genes (SOCS3 and IRS2), and downstream signalling and transcriptional regulators, including SGK and c-JUN. In addition, they found dysregulation of wingless-type MMTV (Wnt) signalling, including Wnt5a, sFRPs, and DKK.
In 2022, Verma et al. (35) published a microarray pathway analysis of orbital tissues from 83 patients with orbital inflammatory disease, including GO (25 patients), with the latter being the most prevalent cause of orbital inflammation (36). In confirmation of previous findings (35), they demonstrated a perturbation of the downstream gene expressions of IGF-1R (MAPK/RAS/RAF/MEK/ERK and PI3K/Akt/mTOR pathways), peroxisome proliferator-activated receptor-γ (PPARγ), adipocytokine, and AMPK signalling pathways.
DNA and RNA sequencing
In 2017, Tao et al. (37) first performed RNA sequencing analysis on orbital adipose-derived stem cells isolated from orbital fat of patients with GO compared to controls. They found a substantially different gene expression profile between patients and controls, encompassing 54 genes. Genes related to developmental morphogenesis and lineage commitment were predominantly dysregulated in GO, but no significant differences were detected concerning genes related to inflammation. In this regard, the authors correctly pointed out that extrinsic factors, such as inflammatory cytokines, are less likely to be detected by RNA sequencing due to cell passages in the culture system. Thus, the genetic differences between the stem cell populations could be related to their developmental origins, which may, however, affect how these cells are implicated in the tissue remodelling of orbital fat in GO.
In 2021, Rotondo Dottore et al. (38) analysed primary cultures of orbital fibroblasts established from tissue samples taken during surgery from six patients with GO vs six controls patients. Next-generation sequencing of the whole genome was performed on individual samples. Gene variants were distinguished into insertions/deletions (INDELs) and single nucleotide polymorphisms. The impact of these variants was assessed using the scale-invariant feature transform damage score (39) or the Polymorphism Phenotyping (PolyPhen) version 2.0 score based on genecards (40). However, none of the many INDELs and SNPs found proved to be detrimental.
In the same study (38), gene expression was evaluated in the whole genome, and 58 genes were found to be differentially expressed to a significant extent in GO compared with control fibroblasts. Of the 33 underexpressed genes, 24 were protein-coding genes, five long ncRNA (LncRNA) genes, three pseudogenes, and one RNA gene. Six of the underexpressed genes encode proteins involved in downregulation of cell growth, apoptosis, or cell cycle, (RASSF2, CYP19A1, ASPN, NOD1, AIFM2, and EIF1AY), three encode proteins with antioxidant activity (AIFM2, LINC01423, and UTY), one encodes a protein involved in T-lymphocyte establishment in non-lymphoid organs (PRDM1), notably a key plasma cell transcription factor, one a protein with stimulatory action in the interferon α and β pathways (DDX3Y), one a protein that reduces fat breakdown (IMPA2), and one a protein involved in the stabilisation of the extracellular matrix (ITIH5) (35). It is to be noted that 12 (36.3%) underexpressed genes found are Y-linked. Twenty-five genes were found to be overexpressed in GO fibroblasts, of which 18 are protein-coding genes, five LncRNA genes, and one a pseudogene. Again, and interestingly, three overexpressed genes encode proteins favouring cell growth, proliferation, and differentiation downregulation (PAX9, FGF14, and EGF), one a protein involved in lipid and fibronectin binding (PLEKHA2), one a protein involved in signal transduction (TGFBR3L), and one a protein involved in the inhibition of apoptosis (RNF144B) (38).
In another study (41), bulk RNA sequencing of intraconal orbital fat from controls and patients with GO was performed. In addition, cultured OFs obtained from GO patients were analysed by single nucleus RNA sequencing, following exposure to adipogenic media. Gene expression profiles were found to be different between control and GO orbital fat, in particular regarding the following signalling pathways: PI3K-Akt, cAMP, AGE-RAGE, lipolysis, and thyroid hormone. Orbital fibroblasts undergoing adipogenesis showed differential expression of the adipocyte-specific genes FABP4/5, APOE, PPARG, and ADIPOQ during adipogenic differentiation. In addition, IGF-1R and Wnt signalling pathways were enriched in the early phase of adipogenesis. Interestingly, genes overexpressed in GO orbital fat were also upregulated in orbital adipocytes upon differentiation.
In 2022, Bai et al. (42) performed RNA sequencing of orbital adipose tissue from GO patients vs controls, and found 468 differentially expressed genes, of which 245 were downregulated and 223 were overexpressed. The main enriched biological processes were related to extracellular matrix organisation and extracellular structure organisation; the main enriched cellular components were related to extracellular matrix and collagen; and the main enriched molecular functions were sulfur compound binding and receptor regulator activity.
Still in 2022 (43), Wu et al. performed single-cell RNA sequencing of orbital connective tissue and found that IL-11Rα was dominantly expressed in orbital fibroblasts. RNA sequencing of paired unstimulated and transforming growth factor (TGF)-β1-stimulated samples demonstrated that the upregulation of IL-11 expression defined the dominant transcriptional response.
In another study (44) single-cell RNA sequencing was performed on orbital connective tissue from GO patients, allowing the identification of three subsets of orbital fibroblasts based on the expression of THY1, RASD1, and other functional genes, including matrix remodelling-related genes (COL1A1, DCN, MMP2, COL12A1, CTHRC1, and POSTN), one inflammatory gene (CXCL14), and muscle contraction-related genes (TAGLN and ACTA2). Gene Ontology analysis showed enriched pathways related to adipogenesis and inflammation in fibroblasts with adipogenic features, with a transition between the fibroblast subsets.
Proteomics
In one study (45), the proteomic profile of GO orbital tissues was investigated, showing a number of proteins, especially proinflammatory ones, to be differentially expressed compared with control tissues, which seemed to be reversible following immunosuppressive treatment in non-smokers, but not in smokers.
Considerations on genetic and proteomic studies
The various studies conducted over the last ten years indicate a different gene or protein expression profile in adipose tissue, orbital-derived stem cells, or orbital fibroblasts from patients with GO vs controls. The genes and proteins found to be differentially expressed differ from one study to another, which may reflect the type of tissues or cell analysed and the techniques employed. Thus, studies in cultured cells may be hampered by the modifications that can occur with cell passages; but, at the same time, have the advantage of comparing cells grown in the same microenvironment, namely without autoantibodies, immune cells, cytokines, and oxidative stress, as it occurs in vivo, which, along with treatments that patients undergo, may affect gene or protein expression. Another reason for the apparent discrepancies may be due to technical issues, in particular to the different methods employed. In this regard, RNA sequencing is generally considered superior to microarray technology in differentiating biologically critical isoforms and in allowing the identification of genetic variants that may not be detected by microarrays (46), which lacks sufficient specificity. In other words, those genes that are found to be differentially expressed by microarray may not be detected by RNA sequencing, whereas genes detected by RNA sequencing, especially low-abundance transcripts, may not be detected by microarrays. In addition, the results obtained by proteomics of orbital tissues cannot be compared with those obtained by RNA sequencing.
A major limitation of studies conducted in search of gene variants or altered gene expression is that virtually all of them are underpowered, which is due to the fact that only a minority of GO patients undergo surgery, namely orbital decompression. Thus, the availability of tissue samples is quite low. In this regard, multicentre studies may overcome this limitation, shedding light on a more precise definition of the genes involved.
Given these considerations, overall, gene variants do not seem to be responsible for GO development, whereas an altered gene expression in orbital fibroblasts, involving several genes, seems to be more likely to be causative. The observed differential gene expression in the absence of significant alterations in the DNA sequence can be ascribed to an epigenetic mechanism, as discussed below.
Epigenetics
Basis for an epigenetic mechanism in GO
In the vast majority of patients, GO does not disappear after treatment, sometimes regardless of whether signs of autoimmunity (i.e. circulating autoantibodies against the TSHR) are still present (47). A similar behaviour is observed in thyroid dermopathy, the second most frequent extrathyroidal manifestation of Graves’ disease, which is believed to reflect a pathogenesis similar to that of GO (48). An in vitro counterpart of this phenomenon is the observation of a different phenotype of cultured orbital fibroblasts depending on their origin (GO vs non-GO patients) (49, 50, 51, 52, 53), namely different degrees of cell proliferation and HA secretion. Because fibroblasts are usually grown in media in which the in vivo microenvironment of orbital tissues is not present, namely immune cells, autoantibodies, cytokines, and reactive oxygen species, none of these factors can be responsible for the different phenotype of GO vs control fibroblasts. Thus, the different degrees of cell proliferation and HA secretion can be ascribed only to intrinsic (genetic or epigenetic) differences between GO and control fibroblasts, which may also explain the persistence of signs and symptoms of GO over time. Given the absence of causative gene variants in the presence of an altered gene expression profile, a possible explanation for the latter may be explained by epigenetic mechanisms.
DNA methylation, acetylation, and deacetylation
Epigenetics are generally defined as differential gene expression reflecting DNA methylation or acetylation. DNA methylation is a process by which methyl groups are added to the DNA molecule (54). As a consequence, the activity of a DNA segment can change without changing the sequence. In gene promoters, methylation typically represses gene transcription (55).
Histone acetylation and deacetylation are the processes by which the lysine residues within the N-terminal tail protruding from the histone core of the nucleosome are acetylated and deacetylated as part of gene regulation. Acetylation and deacetylation are also involved in the regulation of gene expression (56). In particular, in acetylation, an acetyl functional group is transferred from one DNA molecule to another, whereas deacetylation is the reverse reaction. To our knowledge, no studies on DNA acetylation and deacetylation have been performed in GO.
Role of DNA methylation
In the same study in which differential gene expression was reported, Rotondo Dottore et al. found that global DNA methylation was greater in GO fibroblasts compared with control cells (38). In a separate study (54), the same authors found that global DNA methylation increased significantly both in GO and control fibroblasts following incubation with the TSHR-stimulating antibody M22, suggesting that an autoimmune insult elicits DNA methylation. To understand the extent to which the increased DNA methylation was associated with gene expression, they studied by RT-PCR nine of the genes previously found to be under- or overexpressed (57). The expression of one of the underexpressed genes, namely CYP19A1, was significantly reduced by M22 in both control and GO fibroblasts, whereas the expression of one overexpressed gene, namely AIFM2, was increased by IL-6 in control fibroblasts and by M22 in GO fibroblasts, suggesting a parallel behaviour between DNA methylation and gene expression under autoimmune insult. In addition, cell proliferation, which was increased by M22, correlated with global DNA methylation, indicating that the latter has functional consequences in GO fibroblasts. Whole DNA methylation analysis resulted in a total number of 19,869 DNA regions differently methylated (8781 hypermethylated and 11,088 hypomethylated) in GO vs control fibroblasts (54). The differently methylated regions encompass 3957 genes, of which 2092 contained hypermethylated and 1865 contained hypomethylated regions in GO vs control fibroblasts. In this regard, differential methylation of the so-called CpG islands (CGIs) (regions rich in G and C bases) is known to affect gene expression (58). CGIs include the CpG island, the CpG shores (2 Kb upstream/downstream of the two ends of the CpG island), the CpG shelves (2 Kb upstream/downstream of the limits of CpG shores), and the so-called open-sea regions, namely those between CGIs (Fig. 1). Interestingly, a remarkable number of differentially methylated sites were in CpG islands (3558, 17.9%), shores (2954, 14.8%), or shelves (1101, 5.5%), namely in positions where methylation is likely to affect gene expression. A functional analysis showed that the differentially methylated genes involved 119 families and subfamilies of genes, including extracellular matrix components. Eighty-nine genes encoding proteins with similar molecular functions were significantly hyper- or hypomethylated, of which those of particular interest were groups encoding G protein-coupled receptor activity. A large number of biological processes were found to be involved (no. 402), among which the following were considered of relevance: regulation of G protein-coupled receptor signalling; connective tissue development; cell differentiation; positive regulation of cell proliferation; and regulation of secretion. Seven pathways were involved, namely cadherin signalling, metabotropic glutamate receptor group III, muscarinic acetylcholine receptor 1 and 3 signalling, Wnt signalling, heterotrimeric G-protein signalling, angiogenesis, and gonadotropin-releasing hormone receptor. Of the 33 genes underexpressed in GO fibroblasts (57), two were hypermethylated, and of the 25 genes previously found to be overexpressed in GO fibroblasts, one was hypomethylated. Among the differently methylated genes, genes encoding TSHR, mTOR, B-cell activating factor (BAFF), CD44, fibronectin, IL1A and IL1B, IL2, TNFα, IFNγ, and their receptors, as well as proteins involved in the adipogenicity cascade, were not differentially methylated. Despite the lack of differential gene expression, IGF-1R, fibroblast growth factor-12 (FGF12), fibroblast growth factor-16 (FGF16), fibroblast growth factor-17 (FGF17), fibroblast growth factor-22 (FGF22), fibroblast growth factor receptor-1 (FGFR1), fibroblast growth factor receptor-3 (FGFR3), fibroblast growth factor receptor-4 (FGFR4), and IL6 were differentially methylated. Interestingly, except for FGF17 and FGF3, the remaining above-mentioned genes were hypomethylated and, yet not significantly, more expressed in GO vs control fibroblasts. In general, there was a good concordance between DNA methylation and gene expression.
In another recent investigation, Virakul et al. (59) studied the proteome and methylome in primary cultures of orbital fibroblasts from five patients with active GO vs four patients with inactive GO. Proteins involved in inflammation, cellular proliferation, hyaluronan synthesis, and adipogenesis were overexpressed in orbital fibroblasts from active GO patients, whereas extracellular matrix and fibrotic components were overexpressed in fibroblasts from inactive GO patients. Orbital fibroblasts from active GO patients were found to display hypermethylation of genes involved in inflammation and hypomethylation of genes involved in adipogenesis and autoimmunity. Additional analysis revealed networks containing molecules to which both hypermethylated and hypomethylated genes were linked, including NF-κB, ERK1/2, Alp, RNA polymerase II, Akt, and IFNα. A relatively poor correlation between protein expression, DNA methylation, and mRNA expression was observed. Overall, findings were different from those reported by Rotondo Dottore et al. (57), who, in contrast, had compared GO vs normal fibroblasts rather than active vs inactive GO fibroblasts.
The available knowledge on DNA methylation seems to indicate that, following exposure to an autoimmune environment, orbital fibroblasts undergo hyper- or hypomethylation of certain genes, which results in differential gene expression and finally in maintenance of functional features, particularly cell proliferation, which are then responsible for the long-lasting phenotype of the eye disease. Interestingly, among the differentially expressed genes found, several were LncRNA, which are known to be involved in the regulation of gene transcription (60).
In addition to DNA methylation, acetylation and deacetylation may also play a role that should be investigated. In this context, the identification of key genes that are either differentially methylated or acetylated, resulting in sustained, altered gene expression, associated with the demonstration that these hypothetical genes play a role in GO pathogenesis, may establish the basis for ‘epigenetic treatments,’ which should be investigated in clinical trials. Thus, the variability of the epigenome has raised the possibility of its reversion through pharmacological treatments, especially in the field of cancer (61).
Conclusions
The pathogenesis of GO remains to be fully elucidated. The long-lasting phenotype of GO patients, regardless of the treatments they underwent, finds a counterpart in cultured orbital fibroblasts, suggesting that either genetic or epigenetic mechanisms persisting when immunosuppression is achieved are responsible. Although pathogenetic gene variants have not been reported, the gene expression profile of orbital fibroblasts is different from that of normal fibroblasts, which, in the absence of gene variants, suggests an epigenetic mechanism. The differential DNA methylation of GO fibroblasts suggests an important role in this process, whereas DNA acetylation or deacetylation remains to be investigated. A putative, simplified view of how genetic and especially epigenetic mechanisms are involved in the pathogenesis of GO is depicted in Fig. 2.
A putative view of the pathogenesis of Graves’ Orbitopathy (GO), with the role of genetic and epigenetic mechanisms. TSHR, TSH receptor; IgF-1R, insulin-like growth factor-1 receptor.
Citation: European Thyroid Journal 13, 6; 10.1530/ETJ-24-0179
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
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