Proteomics into pathological mechanisms of Chronic Progressive External Ophthalmoplegia (CPEO): implications of the pathogenic variability

Chronic Progressive External Ophthalmoplegia (CPEO) is a primary mitochondrial myopathy (PMM) characterized by defects in oxidative phosphorylation (OXPHOS) and marked by genetic and clinical heterogeneity [1]. Defective OXPHOS leads to a lack of ATP production, compromising tissues with high-energy demand. This condition primarily affects muscles controlling eye and eyelid movement, resulting in ptosis, ophthalmoplegia, and myopathy. The clinical presentation of CPEO can also include impairment of multiple organs and varying degrees of systemic involvement. CPEO can arise from direct mitochondrial DNA (mtDNA) variants, such as single large-scale mtDNA deletions, or nuclear DNA (nDNA) mutations secondarily affecting mtDNA function [2]. Single large-scale deletions can vary in size and location, and remove multiple mtDNA genes essential for mitochondrial function. Secondary mtDNA defects are due to mutations in nDNA genes with a critical role in mtDNA replication and maintenance, such as POLG and TWNK. POLG encodes the catalytic subunit of mitochondrial DNA polymerase, and its mutations impair mtDNA replication and repair. TWNK mutations, which encodes a helicase essential for mtDNA replication, lead to mtDNA instability and multiple deletions. Understanding the pathogenic mechanisms of CPEO remains a challenge due to factors such as its genetic basis and the spectrum of symptoms, which result in inter-individual phenotypic variability. The aim of the study was to explore the urinary proteome of 10 CPEO patients with pathogenic variants in POLG and TWNK or single large-scale mtDNA deletions, sex and age matched with 10 healthy controls to individuate alteration in the proteomic profile related to pathological and mutation-dependent metabolic mechanisms. A bottom-up proteomic approach was performed with an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific) followed by a label-free quantification. The exploratory phase, conducted with a Principal Component Analysis (PCA), showed a clear separation between CPEO patient group and the control group, and a mutation-guided sub-grouping within CPEO. Differential expression and pathways analyses confirmed this evidence, revealing how the clustering was driven by proteins and pathways differing according to mutation. The proteomic data obtained documented significant metabolic differences attributable to the pathogenic mechanisms in the distinct mutation CPEO sub-groups and how the proteomic approach could represent a valid contribution in unveiling the mechanisms of this specific mitochondrial disease.

References:
[1] Mancuso, M.; et al. International Workshop : Outcome Measures and Clinical Trial Readiness in Primary Mitochondrial Myopathies in Children and Adults. Consensus Recommendations. 2017, 27, 1126–1137.
[2] Alston, C. L. et al. The Genetics and Pathology of Mitochondrial Disease. Journal of Pathology. 2017, 241, 236–250.