MPC2 variants disrupt mitochondrial pyruvate metabolism and cause an early-onset mitochondriopathy

Abstract

Pyruvate is an essential metabolite produced by glycolysis in the cytosol and must be transported across the inner mitochondrial membrane into the mitochondrial matrix, where it is oxidized to fuel mitochondrial respiration. Pyruvate import is performed by the mitochondrial pyruvate carrier (MPC), a hetero-oligomeric complex composed by interdependent subunits MPC1 and MPC2. Pathogenic variants in the MPC1 gene disrupt mitochondrial pyruvate uptake and oxidation and cause autosomal-recessive early-onset neurological dysfunction in humans. The present work describes the first pathogenic variants in MPC2 associated with human disease in four patients from two unrelated families. In the first family, patients presented with antenatal developmental abnormalities and harboured a homozygous c.148T>C (p.Trp50Arg) variant. In the second family, patients that presented with infantile encephalopathy carried a missense c.2T>G (p.Met1?) variant disrupting the initiation codon. Patient-derived skin fibroblasts exhibit decreased pyruvate-driven oxygen consumption rates with normal activities of the pyruvate dehydrogenase complex and mitochondrial respiratory chain and no defects in mitochondrial content or morphology. Re-expression of wild-type MPC2 restored pyruvate-dependent respiration rates in patient-derived fibroblasts. The discovery of pathogenic variants in MPC2 therefore broadens the clinical and genetic landscape associated with inborn errors in pyruvate metabolism.

Keywords: metabolism; mitochondria; pyruvate carrier.

Figure 1

Pedigree and imaging details of MPC2-affected familial individuals carrying deleterious variants. (A) Pedigree of Family 1 (F1) carrying the c.148T>C, p.(Trp50Arg) variant. (B) Cerebral ultrasonographic examination of 24 weeks foetus (Patient F1-II.3) showing important radial vascularization from peri callosal artery (PCA), also seen in Fowler’s disease. (C) Normal brain vascularization in a control foetus at the same term, with basilary artery (BA); cerebral anterior artery (CAA); peri callosal artery (PCA); radial vascularization (RV); cerebral intern vein (CIV); vein of Galen (GV). (D) Pedigree of Family 2 (F2) carrying the c.2T>G, p.(Met1?) variant. (E) Locations and conservation analysis of the detected variants in MPC2 (in red), conservation of the mutated amino acids is indicated by the alignment of eight species. (F–I) MRI features associated with MPC2 variants in Patient F2-II.2 showing hypoplasia and partial agenesis of the corpus callosum with axial T₂-weighted images (F and H), axial FLAIR (fluid-attenuated inversion recovery) weighted image (G) and sagittal weighted image (I) showing bilateral T₂ and FLAIR hyperintensities of the thalamus as well as cortical, subcortical and corpus callosum atrophy (arrow).

Figure 2

Analysis of MPC1 and MPC2 expression levels. (A) Representative immunoblot of equal amounts of lysates from control (Control 1 and 2), MPC2-deficient fibroblasts before (MPC2^Trp50Arg or MPC2^Met1?) and after functional complementation with human MPC2-HA (MPC2^Met1?+MPC2), and MPC1-deficient (MPC1^Arg97Trp or MPC1^Lys79His) patient-derived fibroblasts, separated by SDS-PAGE and immunoblotted with the indicated antibodies. An number sign denotes MPC2 native form, triangle denotes truncated MPC2; asterisk denotes MPC2-HA form. (B) Densitometric quantification of MPC2 and MPC1 protein levels in A relative to VINCULIN in patient-derived fibroblasts (n = 2–4). (C) Relative MPC2 and MPC1 mRNA levels in patient-derived fibroblasts as compared with controls (n = 4). (D and E) Pyruvate-dependent respiration of control (Control 1 and 2), MPC2-deficient fibroblasts (MPC2^Trp50Arg or MPC2^Met1?) before and after functional complementation with human MPC2-HA (MPC2^{Trp50Arg + MPC2} or MPC2^Met1?+MPC2), and MPC1-deficient (MPC1^Arg97Trp or MPC2^Lys79His) patient-derived fibroblasts measured by seahorse flux analyser. Basal oxygen consumption was measured in the incubation media without glucose and with pyruvate and maximal respiration was measured after injection of CCCP (carbonyl cyanide m-chlorophenyl hydrazone). All data are presented as mean ± SEM and analysed by two-tailed unpaired Student’s t-test with (**P ≤ 0.01, *P ≤ 0.05, ns = not significant).