Elevated circulating cell-free mitochondrial DNA in fabry disease: insights into inflammatory activation

Objective This study aimed to investigate mitochondrial dysfunction and its role in the pathogenesis of Fabry disease (FD) by analyzing circulating cell-free DNA (ccf-DNA) in patients with FD. Methods Sixty-six FD patients and 21 healthy controls (ctrls) were enrolled. Levels of plasma mitochondrial- (ccf-mtDNA) and nuclear-derived ccf-DNA (ccf-nDNA) were quantified by quantitative reverse-transcription PCR (RT-qPCR), and 14 inflammatory cytokines were measured in treatment-naïve patients. Associations among ccf-DNA levels, cytokine profiles, disease biomarkers, and clinical markers were analyzed, with subgroup analyses stratified by sex, genotype, clinical subtype, and disease severity. Results Treatment-naïve patients exhibited significantly higher ccf-mtDNA ( z =–4.530, P -adj<0.001) and mtDNA/nDNA ratio ( z =–2.613, P -adj=0.014) compared with ctrls. In the long-term enzyme replacement therapy (ERT) group (> 12 months), ccf-mtDNA copy number remained elevated ( z =–3.141, P -adj=0.006), whereas the mtDNA/nDNA ratio did not differ significantly ( z =–1.013, P -adj=0.311). No differences in ccf-nDNA were observed between treatment-naïve patients or the long-term ERT group compared with ctrls. Receiver operating characteristic analysis demonstrated the strong diagnostic performance of ccf-mtDNA (area under the curve=0.860), with 70% sensitivity and 91% specificity at an optimal cut-off value of 1,793,188.04 copies. Both ccf-mtDNA and mtDNA/nDNA ratio correlated positively with inflammatory cytokines including interleukin-17F and tumor necrosis factor-β, with stronger associations observed in male patients with classic FD. No correlations were observed with disease duration, α-galactosidase A activity, plasma globotriaosylsphingosine or clinical markers after adjustment for age and sex. Similarly, ccf-DNA measures did not differ significantly by sex, GLA mutation type (truncated vs . non-truncated), FD subtype (classic vs . non-classic), or across subgroups defined by disease severity or organ involvement (high vs . low MSSI, with or without hypertrophic cardiomyopathy, with or without chronic kidney disease, mild vs . severe white matter lesions, with or without neuralgia, or mild vs . severe pain). Conclusions Mitochondrial dysfunction, reflected by elevated ccf-mtDNA, is implicated in FD pathogenesis and may be linked to inflammatory activation. ccf-mtDNA represents a promising diagnostic biomarker for FD, potentially offering an additional therapeutic target when combined with ERT.