The thymidylate synthase enhancer region (TSER) polymorphism increases the risk of thymic lymphoid hyperplasia in patients with Myasthenia Gravis

Abstract

Background

Myasthenia Gravis (MG) is caused, in approximately 80% of the patients, by autoantibodies against the nicotinic acetylcholine receptor (AChR). The disease is often associated with pathological changes of the thymus: thymic epithelial tumours are present in about 10–20% of the patients, while up to 80% of the patients with early disease onset have thymic hyperplasia. Folate metabolism is required for the production of DNA precursors and for proper DNA methylation reactions, and impaired folate metabolism has been often associated with cellular growth and cancer.

Methods

We investigated if major polymorphisms of folate-related genes, namely MTHFR c.677C > T, MTR c.2756A > G, MTRR c.66A > G and TYMS TSER (a 28-bp tandem repeat in the 5′ promoter enhancer region of TYMS) increase the risk of pathological changes of the thymus in AChR + MG patients. A total of 526 AChR + MG patients, including 132 patients with normal (involuted) thymus, 146 patients with thymic hyperplasia, and 248 patients with a thymoma were included in the study. Allele and genotype comparisons were performed among the three study groups, after correcting for multiple testing.

Results

The frequency of the TYMS TSER 3R allele was significantly higher in MG patients with thymic hyperplasia (P = 0.004), and the TYMS TSER 3R3R genotype was significantly associated with increased risk of thymic hyperplasia [OR 2.71 (95% CI: 1.34–5.47)].

Conclusions

The 3R allele in the thymidylate synthase promoter enhancer region results in increased protein production, required for the synthesis of DNA precursors. The present study suggests that the TYMS TSER 3R allele increases the risk of thymic lymphoid hyperplasia in AChR + MG patients.

Introduction

Myasthenia Gravis (MG) is a prototypic antibody-mediated autoimmune disease characterized by muscle weakness and abnormal fatigability. In approximately 80% of the patients autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction are found. The remaining cases result from autoantibodies directed to other targets at the neuromuscular junction, including muscle specific kinase (MuSK) and lipoprotein-related protein 4 (LRP4). The origin of the autoimmune dysfunction in MG patients is largely unknown, but thymic abnormalities, defects in immune regulation, sex hormones, and genetic predisposition are among major players in AChR positive (AChR +) patients (Berrih-Aknin and Le Panse, 2014, Gilhus, 2016).

The thymus is essential for T-cell differentiation and for the establishment of central tolerance, and MG is often associated with pathological changes of the thymus. Indeed, thymic epithelial tumours are present in about 10–20% of AChR + cases, while up to 80% of the patients with early disease onset (EOMG, age of onset < 50 years) have follicular hyperplasia, i.e. ectopic germinal centers (GCs) developing in the thymus and containing a large number of B cells producing anti-AChR antibodies (Eymard and Berrih-Aknin, 1995, Marx et al., 2015). Interestingly, follicular hyperplasia is very common in EOMG, particularly in women, suggesting that estrogens promote the proliferation of B cells. On the contrary, thymomas are common in late-onset MG (LOMG, age of onset ≥ 50 years) (Berrih-Aknin and Le Panse, 2014).

Folate metabolism is required for the production of DNA precursors and for a proper DNA methylation (Fig. 1). Impaired folate metabolism has been often linked to cell proliferation and cancer, and polymorphisms of folate-related genes have been associated with risk and severity of certain autoimmune disorders and of a number of malignancies, including those of the lymphatic system (Skibola et al., 2004, Mao et al., 2010, Arakawa et al., 2012, Coppedè et al., 2014, Goričar et al., 2015). However, to the best of our knowledge, no previous study has investigated if common polymorphisms in folate-related genes might account for an increased risk of pathological changes of the thymus among AChR + MG patients. In order to address this issue we compared allele and genotype frequencies of the major polymorphisms of folate-related genes among AChR + MG patients with normal (involuted) thymus, with thymic hyperplasia and with thymomas. Particularly, we investigated a common polymorphism in the methylenetetrahydrofolate reductase gene (MTHFR c.677C > T, rs1801133) linked to reduced enzyme activity and increased plasma homocysteine levels (Schwahn and Rozen, 2001), a G variant at nucleotide 2756 of the methionine synthase gene (MTR c.2756A > G, rs1805087) and a G variant at nucleotide 66 of the methionine synthase reductase gene (MTRR c.66A > G, rs1801394), both leading to impaired methionine synthesis and aberrant DNA methylation (Coppedè et al., 2014), and a 28-bp short tandem repeat polymorphism in the promoter enhancer region of the thymidylate synthase gene (TYMS), known as TSER (thymidylate synthase enhancer region) polymorphism or VNTR (variable number of tandem repeats) polymorphism (rs45445694). Two major alleles are known in the TSER region, the first containing two 28-bp repeats (2R) and the latter containing three repeats (3R) and resulting in increased TYMS mRNA expression; some rare alleles containing four, five or more repeats have been also reported (Skibola et al., 2004).