Background: Primary mitochondrial diseases (MTDs) form a clinically and genetically heterogeneous group of inherited disorders that arise due to a defect in mitochondrial energetic metabolism. PMPCA gene encodes the alpha subunit of mitochondrial processing peptidase (aMPP), a heterodimeric enzyme responsible for the cleavage nuclear-encoded mitochondrial precursor proteins after import into the mitochondria. The patient fibroblasts showed decreased α-MPP levels and reduction and fragmentation of the mitochondrial network, although the lactate level was increased only once during illness.

Patient and methods: The proband is an 8-year-old boy with progressive spastic quadriparesis, delayed psychomotor development, and cortical atrophy. MRI showed cerebellar atrophy, leukoencephalopathy, and reduced volume of basal ganglia. Laboratory tests revealed elevated levels of lactate in plasma. The whole-exome sequencing was performed and suspected variants were confirmed by Sanger sequencing. Western blot analysis was performed using anti-PMPCA antibody (NBP1-89126, 1:250 dilutions, Novus biologicals) and Immunofluorescence staining using anti-PMPCA antibody (NBP1-89126, 1:200 dilutions, Novus biologicals) and OXPHOS Rodent antibody cocktail from Abcam (ab110413, 1:500) for staining of mitochondria were used as a primary antibody for double-staining of PMPCA and mitochondria, respectively. In ImageJ, we analyzed 200 cells immunostained with the OXPHOS antibody for each patient and a control sample to confirm the mitochondrial morphology.

Results: Using whole exome sequencing, we have identified two novel compound heterozygous variants (p.Tyr241Ser and p.Met251Val) in the PMPCA gene that encodes the alpha subunit of mitochondrial processing peptidase (α-MPP). The In silico missense variant prediction methods, such as PolyPhen-2, MutationTaster, and SIFT, identified these missense variants as likely to be deleterious and according to the ACMG guidelines, both are variants of unknown significance. To evaluate the functional effects of PMPCA mutations, Patient fibroblasts contained a lower amount of α-MPP than control fibroblasts in western blot. Immunofluorescence staining results suggest abnormal mitochondrial α-MPP distribution as compared to the control. Mitochondrial morphology is statistically different, confirming increased fragmentation of mitochondria in the patient cells.

Conclusion: We have identified two novel variants in the PMPCA gene, that is responsible for the clinical picture of Leigh-like syndrome in the patient. The functional studies we have performed suggest now that the quantity and distribution of the α-MPP and mitochondrial morphology are impaired in the patient fibroblasts.