Systemic sclerosis (SSc), a multifaceted connective tissue disease, is distinguished by widespread fibrosis and vasculopathy. With a complex clinical presentation, SSc can lead to significant morbidity and mortality due to its impact on the skin, blood vessels, and internal organs . The epidemiology of SSc reveals a challenging global health burden, particularly affecting women and exhibiting a heterogeneous geographic distribution . Studies have shown that ILD is one of the most common visceral complications in SSc patients. In addition, interstitial lung disease (ILD) is the most common cause of death in patients with SSc, with a prevalence of up to 30% and a 10-year mortality rate of up to 40%.  And SSc-associated ILD most commonly presents with dyspnoea, cough, and a non-specific interstitial pneumonia pattern on CT scan, with a minority of cases fulfilling the criteria for usual interstitial pneumonia. The intricate interplay of genetic and environmental factors in the etiology of SSc underscores the need for a deeper understanding of its risk factors to inform prevention and therapeutic strategies.

Mitochondrial DNA copy number (mtDNA-CN), often measured in peripheral blood leukocytes as an indicator of mitochondrial content and cellular bioenergetic state, has gained attention for its potential role in various diseases. Observational studies have increasingly suggested a possible association between altered mtDNA-CN and the risk or progression of autoimmune rheumatic diseases, including SSc . For instance, Bogatyreva AI et al. reported an inverse association between blood mtDNA-CN and SSc disease duration ,  while Movassaghi S et al. found lower mean mtDNA-CN in SSc patients compared to healthy controls . However, these observational findings are susceptible to confounding factors (e.g., age, disease severity, treatment effects) and reverse causation (i.e., SSc pathology leading to mtDNA-CN changes), leaving the causal nature of the association uncertain.

Mendelian randomization (MR) offers a valuable approach to infer causality by using genetic variants as instrumental variables (IVs) for an exposure . Leveraging the random allocation of alleles at meiosis, MR can mitigate conventional confounding and minimize bias from reverse causation, providing more robust causal estimates than traditional observational studies. In this study, we applied a two-sample MR framework to investigate the potential causal relationship between genetically predicted mtDNA-CN and the risk of SSc. Furthermore, to explore the specificity of this potential association concerning lung involvement, we included distinct ILD-related endpoints: overall ILD, idiopathic pulmonary fibrosis (IPF), autoimmune-related ILD, and specifically ILD within the context of systemic autoimmune diseases. This allows differentiation between potential effects on general lung pathology versus SSc-specific lung manifestations.

 In a two-sample MR study using large-scale GWAS data from European-ancestry individuals, we assessed the effect of genetically predicted mtDNA-CN on ILD, ILD related to systemic autoimmune diseases, SSc, and strict SSc. We employed the inverse-variance weighted (IVW) method for primary analysis, which was validated with multiple complementary MR methods and robust sensitivity analyses.

After removing two outlier SNPs (rs73349121, rs7705526) identified by MR-PRESSO in the ILD analysis, the IVW method revealed no significant causal effect of genetically predicted mtDNA-CN on SSc (OR=0.67, 95% CI 0.35-1.30, P=0.24), strict SSc (OR=0.45, 95% CI 0.14-1.47, P=0.19), ILD (OR=0.76, 95% CI 0.53-1.07, P=0.12), or ILD related to systemic autoimmune diseases (OR=1.02, 95% CI 0.22-4.65, P=0.98). Sensitivity analyses consistently supported these null findings.

Our study does not support a causal relationship between genetically predicted mtDNA-CN and the risk of SSc or associated ILD. These findings suggest that earlier associations may result from confounding or reverse causation, indicating that mtDNA-CN may not be a primary driver of SSc pathogenesis. Further research is needed to explore tissue-specific mtDNA dynamics in SSc.