Integrated Genotype, Phenotype and Neuro-Immune Biomarker Profiling in Primary Mitochondrial Disease

Background and Objectives: Primary mitochondrial diseases (PMDs) represent a genetically and clinically heterogeneous group of disorders caused by dysfunction of the mitochondrial respiratory chain with limited treatment options. The absence of disease specific biomarkers is a key factor impeding the development of new treatments. Methods: We performed an unbiased, exploratory approach integrating standardised phenotyping using Human Phenotype Ontology (HPO) with genetic information, mtDNA heteroplasmy levels, and biomarkers of neurodegeneration (NfL, GFAP, total Tau, UCHL1) and inflammation (FGF21, IL-6, IL-10, IFNγ, CCL3, GDF15, TNFα). We applied correlation-based clustering to identify recurrent phenotypic patterns across the cohort and within specific genetic subgroups. Results: Among the neurodegenerative biomarkers, NfL and total Tau were significantly elevated in PMDs compared to controls with good group-level discriminatory performance, and exploratory evidence of increased NfL levels in a subset of asymptomatic mtDNA carriers. PMD patients also showed alterations in selected cytokines, including increased GDF15 and reduced IL-10, while other markers were not consistently significant after adjustment. Integrated analysis of biomarker profiles and HPO terms highlighted heterogeneity across genetic sub-groups, but could not identify consistent genotype-specific patterns. Apart from NfL levels, which showed a significant positive correlation with HPO term burden and, in a limited subset, with age-adjusted mtDNA heteroplasmy, most biomarkers could not distinguish asymptomatic from symptomatic individuals, and did not correlate with overall disease burden. Discussion: Together, these exploratory results highlight the heterogeneity of PMDs and support the feasibility of integrated biomarker and HPO-based approaches to complement diagnosis and screening in PMDs. Further studies in larger longitudinal cohorts will determine the value of this approach for patient stratification and pharmacodynamic biomarker development. Classification of Evidence: This study provides Class III evidence that serum NfL and total Tau levels can distinguish patients with primary mitochondrial disorders from healthy controls