Osteoarthritis (OA) is a common joint disease with degenerative changes in articular cartilage and remains major impact on public health worldwide (Nelson, 2018). According to the estimation of the Global Burden of Disease Study (GBD), nearly 300 million individuals throughout the world are suffering from OA, and the number is still increasing. OA is one of the leading causes of pain, disability, and great socioeconomic burden in developed countries (Glyn-Jones et al., 2015). However, the mechanism of OA remains unclear. Identifying risk factors such as a disorder-related biomarker is essential to understand OA pathogenesis, decrease the incidence of the disease and develop efficiency prevention strategies.

Mitochondria are complex multifunctional organelles involved in various cell functions, e.g., heat regulation, calcium homeostasis, reactive oxygen species (ROS) production, and proinflammatory cytokines production, the damage of which may affect chondrocyte health at an extent (Blanco et al., 2011; Fernandez-Moreno et al., 2017; Blanco et al., 2018). They have their own genome (mitochondrial DNA, mtDNA) which contain 37 genes and encode 13 proteins, 22 transfer RNAs (tRNAs) and two ribosomal RNAs (rRNAs) (Anderson et al., 1981). As multicopy genome, sequence mutation and copy number variation of mtDNA are prevalent in population (McArdle et al., 2013; McGuire, 2019). As one of the mtDNA quantity characteristics, mtDNA abundance is recognized as a rough estimation for the number of mitochondria (Castellani et al., 2020). Previous studies provided supportive evidence for extensive pathogenicity of mtDNA abundance (Frahm et al., 2005; Hu et al., 2016). As one of the mtDNA quality characteristics, mitochondrial herteroplasmy (MtHz), where mtDNA with distinct sequences coexist, has been found in a large spectrum of human disease, including classical mitochondrial diseases and complex disorders (Ye et al., 2014). MtDNA abundance and MtHz can integrate many aspects of mitochondrial function and serve as promising biomarkers in probing interactions between mitochondria and disorders (Castellani et al., 2020; Tian et al., 2021).

In a recent review, OA was recognized as a potential mitochondrial disease considering the impact of mitochondrial dysfunction on cartilage degradation (Fernandez-Moreno et al., 2022). However, the causality between mitochondria and OA has rarely been investigated. Therefore, the goal of our study was to probe causal effects of characterization of mtDNA quantity and quality on OA development which is crucial to understand the role of mitochondria in OA etiology. A two-sample Mendelian randomization (MR) analysis was performed to investigate potential causal associations between mtDNA abundance and MtHz with OA using summary statistics from large-scale genome-wide association study (GWAS). MR-Steiger test was applied to ascertain whether variation of mtDNA characterization is a cause or a consequence of OA development.

The causal roles of MtHz and mtDNA abundance in OA pathogenesis were poorly studied in previous works. To our best knowledge, this is the first MR study to evaluate the causal association between mitochondrial genome traits and OA. Four MR methods were employed to estimate the causal association between mtDNA characterization and OA. The results of MR steiger test verified the causal direction of our research (MtHz and mtDNA abundance were exposure and OA were outcome). No effect of mtDNA abundance and MtHz on overall OA was detected. In subgroup analysis, a suggestively protective role of MtHz on knee OA was observed, but not on other sites. And we did not find that mtDNA abundance was causally associated with any site-specific OA.

Although mitochondria is widely recognized as an important factor of OA development (Blanco et al., 2011), the role of MtHz has not been thoroughly investigated (Suliman and Piantadosi, 2016). Indeed, heteroplasmic mutations in mtDNA are often pathogenetic (Ye et al., 2014). An animal study has indicated that the state of heteroplasmy itself was deleterious when the two mtDNA sequences contain no pathogenic variants (Sharpley et al., 2012). However, there is potentially a particular level threshold for MtHz (McCormick et al., 2020). For instance, when the A3243G mutation in mitochondrial DNA is present in more than 10%, patients can manifest Type 2 diabetes (Wallace, 2005). And low-frequency mtDNA variants (0.2%–2% heteroplasmy) are extensively presented in healthy subjects (Payne et al., 2013). Furthermore, MtHz can be beneficial in health promotion as an intermediate state in emergence of novel mtDNA haplogroups (Wallace, 2016). In previous study, MtHz was found to be significantly relevant to several haplogroups (haplogroup H, J, K, T, U and X) with different characterizations among haplogroups (Nandakumar et al., 2021). Mitochondrial haplogroup J has been extensively found to mediate the development of OA (5). A Spanish cohort-study ( n c a s e = 457; n c o n t r o l = 262) had reported that haplogroup J was associated with a decreased risk of knee OA (OR = 0.460, 95% CI: 0.282–0.748, p= 0.002) (Rego-Perez et al., 2008). Furthermore, a meta-analysis in European cohorts also suggested that haplogroup J was associated with a lower risk of knee OA (HR = 0.702, 95% CI: 0.541–0.912, p= 0.008) (Fernandez-Moreno et al., 2017). However, the association between mitochondrial DNA variants and OA has not been verified in a large sample observational study (Hudson et al., 2013). The study has only explored the causal relationship in terms of single mutation, but we take MtHz (variation at a whole mtDNA level) as an exposure which contribute to understand the causal role of mitochondria in OA development. In terms of biological mechanism, MtHz has been reported a regulation role of metabolic and epigenomic changes. Kopinski PK et al. had found the levels of mitochondrially drove acetyl-CoA decreased at high heteroplasmy (Kopinski et al., 2019). And the reduction of acetyl-CoA levels might influence histone acetylation and activate AMP-activated protein kinase (AMPK) to protect from OA development and progression (Chen et al., 2018). Besides, MtHz could play a role in other processes involved in OA pathogenesis to reduce the risk of OA, including regulating energy production, maintaining mitochondrial proteostasis, suppressing matrix metalloproteinase expression, reducing ROS generation, and promoting mitophagy (Blanco et al., 2018). In conjunction with prior observational findings and mechanism studies, our epidemiologic and genetic findings provided supportive evidence that MtHz may have therapeutic value in knee OA and can be regarded as a candidate biomarker after precisely identifying the relationship between MtHz and each haplogroup (Blanco et al., 2018).

However, the reason that MtHz showed diverse effects on different sites of OA remains unclear. So far, we did not find relevant research that investigated the relationship between MtHz and hand, finger, and thumb OA. A case-control study suggested that mitochondrial haplogroups was associated with hip OA (Rego et al., 2010) (OR = 0.661, 95% CI: 0.440–0.993, p= 0.045), however, with no evidence on the causality. In addition, the data sources and sample size used in MR analysis varied in site-specific OA, which mainly included more knee OA cases and less other skeletal joints cases. More MR studies and additional GWAS covering more other site-specific OAs are needed to determine a role of MtHz in the risk of different OA sites.

MtDNA abundance has been regarded as a potential biomarker of mitochondrial function and plays a role in several human diseases (Castellani et al., 2020; Clyde, 2022). Existing literature has suggested that mtDNA abundance may involve in production of inflammatory mediators and regulation of immune function that can influence OA development (Blanco et al., 2018; Zhan et al., 2020). In addition, mtDNA abundance is also associated with sex, advanced age, and elevated BMI, which are also risk factors for OA (16). However, there are few studies to estimate the effects of mtDNA abundance on OA. Only two case-control studies in Asian population were found that reported an association between mtDNA copy number and OA, but their findings were inconsistent (Fang et al., 2014; Zhan et al., 2020). One in Thailand ( n c a s e = 204; n c o n t r o l = 169) had found mtDNA abundance in the OA group was significantly lower than that in the control group (p < 0.0001), whereas the results were not adjusted by sex and age (Zhan et al., 2020). Another case-control study carried out in southern Chinese ( n c a s e = 187; n c o n t r o l = 420) observed a general increase of mtDNA abundance in OA patients (p= 0.019), but obesity was not adjusted in the analysis (Fang et al., 2014). These findings from case-control studies could not determine causal relationships between mtDNA abundance and OA, and the potential confounding variables were not comprehensively considered. Our MR analysis overcame these shortcomings and suggested that genetically determined mtDNA abundance was unrelated to OA. Furthermore, previous study has implicated that the relative contribution of mtDNA abundance might differ between different ethnic groups (Ruiz-Pesini et al., 2004). Considering that our studies have been carried out in European population, the comparisons with results from other ethnic groups such as Asian ancestry require careful considerations.

Our research applies MR methods to investigate causal relationships between mitochondrial genome characterization and OA. However, there are some limitations in our analysis. Firstly, we did not estimate causal effects of mitochondrial genome traits on OA stratified by gender. Mitochondrial genome are maternally inherited, and females may have lower MtHz than males from the perspective of mitochondrial inheritance (Nandakumar et al., 2021). Therefore, the effects of MtHz and mtDNA abundance on OA may differ in gender. Secondly, only participants of European descent are included in the study, but the impact of specific mtDNA variants on diseases could vary in different ethnic groups (Ruiz-Pesini et al., 2004). Additional MR studies on other ethnic groups are needed to probe a causal association between mtDNA characterization and OA. Thirdly, the genetic instruments for mtDNA abundance explain a relatively small amount of phenotypic variance (8.3%), IVs that can account for more variance of mtDNA abundance are warranted to draw robust conclusions. Besides, considering the Bonferroni correction of multiple independent tests, our findings about the association between MtHz and knee OA are deemed suggestive evidence of possible associations (0.01 < p< 0.05). Furthermore, the variation of mtDNA abundance and the levels of MtHz varied in different tissues and cell types. And in original GWASs, mtDNA abundance was measured from leukocyte in blood and MtHz from saliva samples rather than cartilage or synovial tissues. Considering that OA is mainly characterized by progressive loss of cartilage and synovial hyperplasia, the application of data from blood samples and saliva samples would limit the explanation ability of our study to a certain extent (Sellam and Berenbaum, 2010). Observational epidemiology studies exploring an association between concrete levels of MtHz and a risk of OA are needed to improve causal inference.

In conclusion, our MR analyses elucidated that MtHz is a suggestively protective factor of knee OA, implying that MtHz could be a genetically prediction factor and a therapeutic target in the development of knee OA. No causal association was found between mtDNA abundance and OA. Additional MR analyses are warranted to probe causal relationships between mitochondrial genome traits and OA stratified by gender. Moreover, GWAS covering more than one ethnic population are needed to detect the effect of characterization of mtDNA quantity and quality in different ethnic groups.