Hove, Johan L.K. Van

Abstract Genome sequencing of the heterogeneous primary mitochondrial disorders (PMD) frequently reveals variants of uncertain significance that require functional tests for diagnosis, and does not identify variants in all patients. We analyzed mitochondrial enzyme assays, blue native polyacrylamide gel electrophoresis (BN-PAGE) with in-gel activity staining, complex I assembly blot, and select protein abundances in fibroblasts of a case series of 204 PMD patients divided into functional classes, in comparison to 51 controls and 53 differential diagnostic conditions. Overall, sensitivity and specificity for respiratory chain enzyme assays were 46% and 93% respectively, for BN-PAGE 40% and 98%, for complex I assembly assay 49% and 99%. The overall sensitivity of all tests was 76%, specificity 93%, with positive predictive value 96% and negative predictive value 67%. Categories with high sensitivity were isolated complex deficiencies, nuclear DNA-encoded mitochondrial protein synthesis defects, co-factor defects, and mitochondrial amino-acyl-tRNA synthetase conditions when aided by protein abundance. Mitochondrial DNA mutations and maintenance disorders showed poor sensitivities. Secondary dysfunctions were rare. A complete battery of functional tests showed strong diagnostic clinical utility in fibroblasts. One sentence summary A combination of four mitochondrial functional tests to identify or confirm suspected primary mitochondrial disease in fibroblasts had good sensitivity and excellent specificity, well beyond what was perceived using enzyme assays only.

AbstractClinical recognition of primary mitochondrial disorders (PMD) is difficult due to the clinical and genetic heterogeneity. Whereas lactate has low sensitivity and specificity, in structured clinical studies growth differentiation factor 15 (GDF15) has shown promise with elevations in mitochondrial DNA (mtDNA)‐related PMD, but its specificity has been questioned. In a tertiary care hospital‐wide study, medical records were retrospectively reviewed from 418 cases where GDF15 levels were obtained by clinicians. Patients were classified into patients with PMD due to mtDNA‐related defects (mtDNA maintenance, mtDNA deletions, and mtDNA‐encoded tRNA variants), PMD due to structural defects or other nuclear causes, and in non‐mitochondrial disease. Patients with liver disease or systemic critical illness were excluded. GDF15 was assayed in a clinical laboratory with a cutoff of 750 ng/L. There were 38 mtDNA‐related PMD (GDF15 >750 pg/mL in 76%), 35 other nuclear DNA‐encoded PMD or structural subunits (31% elevated GDF15), 309 non‐mitochondrial disorders (13% elevated GDF15). Based on the highest Youden J‐index, the optimal cut‐off value to identify these target mtDNA‐related disorders was 815 pg/mL, with sensitivity 76%, specificity 88%, positive predictive value of 41% and negative predictive value of 97%. At this optimized cutoff level, mtDNA‐encoded PMD patients had elevated GDF15 in 76%, nuclear DNA‐encoded PMD in 26%, and non‐mitochondrial disorders in 11% of patients. Thus, in a real‐life clinical setting, after excluding abnormal liver function and critical illness, GDF15 had good clinical utility increasing the odds at predicting mtDNA‐related primary mitochondrial disorders 14‐fold, but not for structural or other nuclear‐encoded primary mitochondrial disorders.

Congenital disorders of glycosylation (CDG) and mitochondrial disorders are multisystem disorders with overlapping symptomatology. Pathogenic variants in the PMM2 gene lead to abnormal N-linked glycosylation. This disruption in glycosylation can induce endoplasmic reticulum stress, contributing to the disease pathology. Although impaired mitochondrial dysfunction has been reported in some CDG, cellular bioenergetics has never been evaluated in detail in PMM2-CDG. This prompted us to evaluate mitochondrial function and autophagy/mitophagy in vitro in PMM2 patient-derived fibroblast lines of differing genotypes from our natural history study. We found secondary mitochondrial dysfunction in PMM2-CDG. This dysfunction was evidenced by decreased mitochondrial maximal and ATP-linked respiration, as well as decreased complex I function of the mitochondrial electron transport chain. Our study also revealed altered autophagy in PMM2-CDG patient-derived fibroblast lines. This was marked by an increased abundance of the autophagosome marker LC3-II. Additionally, changes in the abundance and glycosylation of proteins in the autophagy and mitophagy pathways further indicated dysregulation of these cellular processes. Interestingly, serum sorbitol levels (a biomarker of disease severity) and the CDG severity score showed an inverse correlation with the abundance of the autophagosome marker LC3-II. This suggests that autophagy may act as a modulator of biochemical and clinical markers of disease severity in PMM2-CDG. Overall, our research sheds light on the complex interplay between glycosylation, mitochondrial function, and autophagy/mitophagy in PMM2-CDG. Manipulating mitochondrial dysfunction and alterations in autophagy/mitophagy pathways could offer therapeutic benefits when combined with existing treatments for PMM2-CDG.