Genetic association between autoimmune thyroiditis and microscopic polyangiitis: A two-sample Mendelian randomization study

Abstract

Anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) is a group of life-threatening autoimmune small vessel vasculitis and the prognosis depends heavily on whether a prompt diagnosis is achieved. Autoimmune thyroiditis is the most common autoimmune endocrine disease and could overlap with other autoimmune diseases. It remains elusive whether autoimmune thyroiditis affects the risk of AAV development. We performed a 2-sample Mendelian randomization analysis to explore the true association between autoimmune thyroiditis and microscopic polyangiitis (MPA), a subtype of AAV. Independent single-nucleotide polymorphisms associated with Hashimoto thyroiditis or Grave disease with genome-wide significance were selected as instrumental variables from large genome-wide association study. MPA genome-wide association study summary statistics were obtained from FinnGen consortium. The inverse-variance weighted method was conducted as the primary analysis for estimating the effect of the exposure on the outcome. Mendelian randomization-Egger and the weighted median method were used to confirm the results. We found a causal association between Hashimoto thyroiditis and MPA while no causal effect of Grave disease on MPA. This study contributed a genetic viewpoint to the understanding of the link between autoimmune thyroiditis and AAV.

1. Introduction

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a disease entity for a group of autoimmune small vessel vasculitis accompanied by the presence of serum ANCAs, including microscopic polyangiitis (MPA), granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis.^[1] AAV diseases can have multisystem manifestations with specific features of organ involvement and consequently lead to serious morbidity and mortality.^[2] Premature mortality and societal economic costs caused by AAV are considerable all over the world.^[3] Delayed or missed diagnosis will lead to life-threatening prognosis worse than most cancers.^[2] Prompt recognition is vital for AAV patients.

Genetic susceptibility and environmental triggers contribute to the pathogenesis of AAV. Some variants in genes (such as PTPN22, SERPINA1, PRTN3) have been confirmed to link with AAV.^[3] Experimental data implicated that a plasmid-encoded 6-phosphogluconate dehydrogenase sequence from Staphylococcus aureus induced anti-myeloperoxidase (MPO) nephritogenic autoimmunity in MPA.^[4] Disorders of autoimmune are instilled into each step of AAV from the initiation to end-organ damage, which comprise excessive activation of neutrophils, disturbed humoral and cell immune homeostasis, and abnormal complement system.^[5]

Autoimmune thyroiditis is the most common autoimmune endocrine disease, with an estimated prevalence of about 3% to 5% of the general population.^[6] The spectrum of autoimmune thyroiditis mainly includes Hashimoto thyroiditis and Graves disease, characterized by the production of thyroid autoantibodies and by thyroidal lymphocytic infiltration. Similar to AAV, autoimmune thyroiditis is well known to result from susceptibility genes associated with immune system and environmental factors.^[7] Some case reports revealed the connection between autoimmune thyroiditis and AAV.^[8,9] However, there is a lack of evidence indicating the genetic relationship between autoimmune thyroiditis and AAV.

Mendelian randomization (MR) utilizes genetic variants as instrumental variables (IVs) to effectively estimate the causal relationship between risk factors and a disease outcome, which imitates the design of a randomized controlled trial. In the present study, we aim to perform a 2-sample MR analysis to explore whether autoimmune thyroiditis affects the risk of MPA and clarify the true association between autoimmune thyroiditis and MPA.

2. Methods

2.1. Study design

In our study, single-nucleotide polymorphisms (SNPs) were defined as IVs. The performance of MR analysis required 3 assumptions to be satisfied: all selected IVs must be strongly correlated with Hashimoto thyroiditis or Grave disease, all selected IVs are not associated with confounders, and none of the selected IVs affected MPA by pathways other than Hashimoto thyroiditis or Graves disease (Fig. 1).

Figure 1.

An overview of the study design. IV = instrumental variable, SNP = single-nucleotide polymorphism.

2.2. Data sources

Genetic associations for autoimmune thyroiditis including Hashimoto thyroiditis and Graves disease were derived from a large-scale genome-wide association study (GWAS), which respectively included 395,640 and 458,620 participants of European descent. Similarly, GWAS data for MPA were obtained from a European ancestry population through FinnGen consortium release data R9, which included 375,364 controls and 143 MPA. Summary data for all 3 phenotypes are listed in Table 1.

Table 1.

The detailed data information of the Mendelian randomization study.

Phenotype	Sample size	Sample case	Sample control	Ancestry	Number of SNPs
Hashimoto thyroiditis	395,640	15,654	379,986	European	24,146,037
Grave disease	458,620	1678	456,942	European	24,189,816
Microscopic polyangiitis	375,507	143	375,364	European	20,170,184

SNP = single-nucleotide polymorphism.

2.3. Selection of genetic variants

To obtain exposure group data, the MR Base database (http://www.mrbase.org/) was searched and the SNPs closely associated with Hashimoto thyroiditis or Graves disease (P < 5 × 10⁸) at a genome-wide significance level were selected. Independence of IVs was validated based on the linkage disequilibrium clumping algorithm with a cutoff of r² = 0.001. The power of each variant was quantified with F-statistics and the weak ones (F-statistics <10) should be excluded.

2.4. Ethical approval

Our analyses were conducted using publicly available GWAS summary data. Each GWAS included in this study had already obtained ethical approval.

2.5. Statistical analysis for MR

Inverse-variance weighted (IVW) analyses were performed as the main method to estimate the causal effect of autoimmune thyroiditis on MPA risk, which analyzed each Wald ratio and provided a consistent estimate of the causal effect when all IVs were valid.^[10] MR-Egger and weighted median analyses were further conducted to explore and adjust for pleiotropy in the IVW analyses. The slope from MR-Egger regression represented the causal effect estimate and the intercept was interpreted as an estimate of the average horizontal pleiotropic effect across the genetic variants.^[11] The weighted median method gave unbiased estimates provided at least 50% of the information comes from non-pleiotropic SNPs.^[12] The results were considered statistically significant at P < .05. All statistical analysis was realized with R software (www.r-project.org). We performed MR analysis by “Two Sample MR” and “plyr” packages.

2.6. Pleiotropy and heterogeneity analysis

Pleiotropy was assessed based on the intercept from MR-Egger regression and the MR-PRESSO global test. A “leave-one-out” sensitivity analysis was performed to evaluate whether the analysis was biased by a single SNP that had a particularly large horizontal pleiotropic effect. Heterogeneities between SNPs were assessed based on Cochran Q-statistics and funnel plots.

3. Results

3.1. Causal effects of Hashimoto thyroiditis on MPA

A total of 8 independent SNPs were screened from a GWAS of Hashimoto thyroiditis as IVs. The F-statistics ranged from 32.52 to 194.22, with an average F value of 81.61, which indicated that the strength of the variables satisfied the relevance assumption of MR. The MR results are illustrated in Figures 2 and 3. According to the IVW method, we found a statistically significant positive causal effect of Hashimoto thyroiditis on MPA (odds ratio [OR] = 2.234, 95% confidence interval [CI] = 1.248–3.999, P = .007). The positive causal association between Hashimoto thyroiditis and MPA was also confirmed by the weighted median approach (OR = 2.196, 95% CI = 1.028–4.695, P = .042). The MR-Egger analysis showed no causal association between Hashimoto thyroiditis and MPA (OR = 1.355, 95% CI = 0.279–6.574, P = .719). The scatter plot revealed a positive correlation between Hashimoto thyroiditis and the risk of MPA. Cochran Q test showed no evidence of heterogeneity of IVW estimates (P = .256). The MR-Egger regression demonstrated that directional pleiotropy is unlikely to introduce bias to the results (intercept = 0.095; P = .526). The MR-PRESSO global test results indicated the absence of overall pleiotropy across all IVs (R_ssobs = 12.28, P = .300). The “leave-one-out” analysis demonstrated that no single SNP was driving the IVW point estimate.

Figure 2.

Mendelian randomization estimates of autoimmune thyroiditis and the risk of microscopic polyangiitis. MR = Mendelian randomization.

Figure 3.

Effects of Hashimoto thyroiditis on microscopic polyangiitis. (A) Forrest plot of SNPs associated with Hashimoto thyroiditis and microscopic polyangiitis. (B) Leave-one-out analysis of SNPs associated with Hashimoto thyroiditis and microscopic polyangiitis. (C) Scatter plot of SNPs associated with Hashimoto thyroiditis and microscopic polyangiitis. The slopes of each line represent the causal association for each method. MR = Mendelian randomization, SNP = single-nucleotide polymorphism.

3.2. Causal effects of Grave disease on MPA

A total of 22 independent SNPs were screened from a GWAS of Grave disease as IVs. These 22 SNPs had F-statistics ranged from 30.42 to 306.74, thus indicating that our instruments were strongly associated with Grave disease. The MR results are illustrated in Figures 2 and 4. The results of IVW showed that there was no causal association between Grave disease and MPA (OR = 1.151, 95% CI = 0.872, 1.520, P = .321). The MR-Egger method (OR = 1.509, 95% CI = 0.697–3.270, P = .309) and weighted median approach (OR = 1.191, 95%CI = 0.797–1.779, P = .393) also failed to support a causal association between Grave disease and MPA. Cochran Q test indicated no evidence of heterogeneity between IV estimates based on the individual variants (P = .455). The MR-Egger regression and the MR-PRESSO global test results indicated the absence of directional pleiotropy (intercept = −0.061; P = .469) and overall pleiotropy (R_ssobs = 23.77, P = .458) across all IVs respectively. No single SNP strongly drove the overall effect of Grave disease on MPA in the leave-one-out analysis. The funnel plot suggested that there were no violations of the IV assumptions.

Figure 4.

Effects of Grave disease on microscopic polyangiitis. (A) Forrest plot of SNPs associated with Grave disease and microscopic polyangiitis. (B) Leave-one-out analysis of SNPs associated with Grave disease and microscopic polyangiitis. (C) Scatter plot of SNPs associated with Grave disease and microscopic polyangiitis. The slopes of each line represent the causal association for each method. MR = Mendelian randomization, SNP = single-nucleotide polymorphism.

4. Discussion

It has been reported that autoimmune thyroiditis could overlap with other autoimmune diseases such as AAV,^[13] systemic lupus erythematosus,^[14] rheumatoid arthritis,^[15] and type 1 diabetes.^[16] A case-control study, conducted in a southeastern US population, found that 20% of the cases with AAV reported a history of thyroid disease, compared to only 7% of the control group, indicating a significantly higher odds ratio for developing AAV in individuals with a history of thyroid disease. This association was even more pronounced in women, with 38% of female cases having a history of thyroid disease, compared to 9% of female controls. Within the cases, those with a history of thyroid disease were more likely to have MPO-ANCA, rather than proteinase 3-ANCA.^[13] We performed a 2-sample MR analysis to investigate the causal relationship between autoimmune thyroiditis including Hashimoto thyroiditis and Grave disease and MPA. This study suggested that there was causal association between Hashimoto thyroiditis and MPA while no causal association between Grave disease and MPA. The underlying mechanisms for the association between autoimmune thyroiditis and other autoimmune diseases may be the common genetic predispositions to autoimmunity and similar environmental triggers to induce immune disorder.

PTPN22 is a known susceptibility gene associated with increased risks of multiple autoimmune diseases. Gong et al^[17] identified a rare variant of PTPN22 (NM_015967.5: c.A77G; p.N26S) to Hashimoto thyroiditis, providing further evidence of the disease-causing or susceptibility role of PTPN22 in autoimmune thyroid disease. Kyrgios et al^[18] found that DNA methylation in the PTPN22 gene promoter was significantly higher in HT patients in comparison with controls. In addition, PTPN22 gene promoter DNA methylation was associated with thyroid autoimmunity, proposing a possible etiological association between thyroiditis and abnormalities of PTPN22 function. The Vasculitis Clinical Research Consortium provided the first evidence for genetic variants, for example, in PTPN22, in both MPA and granulomatosis with polyangiitis, suggesting that there is also a shared genetic component to different subtypes of AAV.^[19,20]

T regulator cells (Treg) are a subset of T lymphocytes, which have been considered able to inhibit harmful immune responses. It has been speculated that there is an alteration in the number and function of Treg cells in Hashimoto thyroiditis.^[21,22] Helios is an important mediator for Treg cells through upregulating Foxp3 expression. Hu et al^[23] revealed that lower percentage of Tregs and activated Tregs in Hashimoto thyroiditis patients and decreased expression of Helios in activated Tregs, in comparison with those in healthy controls. Several studies showed that patients with AAV had low numbers and functional impairment of Treg cells. Chavele et al^[24] further indicated that MPO-specific T-cell frequencies were regulated during MPA disease remission, which was in association with tryptophan degradation. In a mouse model of crescentic glomerulonephritis, blocking IL-6 activity increased the migration of Tregs into the kidney and the regional lymph nodes and ameliorated the disease.^[25] Alteration of Treg cells could be the intersection of the pathogenesis of Hashimoto thyroiditis and MPA.

Neutrophil extracellular traps (NETs) are well known to play a pivotal role in AAV development. Patients with AAV exhibit low NET degradation activity, leading to disruption of tolerance to specific self-antigens, including MPO and proteinase 3 and subsequently production of ANCAs.^[5] Xiao et al^[26] found that neutrophils from patients with Hashimoto thyroiditis formed NETs more markedly than from controls after PMA stimulation in vitro and levels of circulating NET-associated components were increased and positively associated with TGAb/TPOAb titers in the patients, indicating the involvement of NETs in Hashimoto thyroiditis.

Cytokine production is disturbed both in Hashimoto thyroiditis and ANCA-AAV. Serum level of IL-12 cytokine has been shown to increase in patients with Hashimoto thyroiditis.^[27] IL-12 was also elevated in MPA patients and correlated positively with disease activity.^[28]

Recent studies have provided evidence for the common molecular mechanisms between Hashimoto thyroiditis and MPA. NLRP3 inflammasome and its downstream cytokines were significantly increased in thyroid tissues from patients with autoimmune thyroiditis.^[29] Interestingly, the expression of NLRP3 was significantly higher in kidneys from AAV patients than those from normal controls, minimal change disease or class IV lupus nephritis.^[30]

The 3-dimensional model of thyroid peroxidase presented in a study shared significant sequence homology with MPO, suggesting a potential for cross-reactivity between the autoantibodies directed against these 2 peroxidases.^[31] This insight could be pivotal for understanding autoimmune thyroid diseases where antibodies to thyroid peroxidase might also recognize MPO, indicating a shared immunological mechanism.

There are some limitations in our study. The datasets used in this study are from participants of European descent and the results need to be further investigated in other populations because causality may depend on ethnicity. In the analyses of causal effects of Hashimoto thyroiditis on MPA, several MR methods demonstrated inconsistencies, which may be related to the small case size. This limited number of MPA cases may potentially introduce biases and reduce the statistical power of our study, which might affect the reliability and generalizability of our findings. The prevalence of autoimmune thyroiditis is higher in women than men, and the causal relationships between autoimmune thyroiditis and MPA stratified by gender could not be directly estimated. The MR analyses reflected an individual’s cumulative lifetime exposure to risk factors rather than assessing specific periods in the life course. The results cannot reflect the effect of current treatment on prognosis and population characteristics should be taken into account in the interpretation.

In conclusion, our MR study offers valuable insights into the potential genetic linkage between Hashimoto thyroiditis and MPA. The findings suggest a causal association between Hashimoto thyroiditis and MPA, while no such link was identified for Grave disease. This study enriches the existing body of evidence by providing a genetic perspective on the relationship between autoimmune thyroiditis and AAV, specifically MPA. These findings highlight the need for further research to explore the broader clinical implications, including the potential benefits and limitations of ANCA screening in patients with Hashimoto thyroiditis. Future studies with diverse populations and larger cohorts are essential to validate these results and to determine the precise role of genetic factors in the pathogenesis of MPA within the context of autoimmune thyroid diseases.

Author contributions

Conceptualization: Yujia Wang, Zhimin Chen, Yanfang Xu.

Data curation: Yujia Wang, Zhimin Chen.

Formal analysis: Yujia Wang.

Funding acquisition: Yujia Wang, Yanfang Xu.

Methodology: Yujia Wang, Zhimin Chen, Yanfang Xu.

Project administration: Yujia Wang, Yanfang Xu.

Software: Yujia Wang, Zhimin Chen.

Supervision: Yujia Wang, Zhimin Chen, Yanfang Xu.

Validation: Yujia Wang, Yanfang Xu.

Visualization: Yujia Wang, Zhimin Chen.

Writing—original draft: Yujia Wang.

Writing—review & editing: Yujia Wang, Zhimin Chen.