Congenital Hypermetabolism and Uncoupled Oxidative Phosphorylation

Abstract

We describe the case of identical twin boys who presented with low body weight despite excessive caloric intake. An evaluation of their fibroblasts showed elevated oxygen consumption and decreased mitochondrial membrane potential. Exome analysis revealed a de novo heterozygous variant in ATP5F1B, which encodes the β subunit of mitochondrial ATP synthase (also called complex V). In yeast, mutations affecting the same region loosen coupling between the proton motive force and ATP synthesis, resulting in high rates of mitochondrial respiration. Expression of the mutant allele in human cell lines recapitulates this phenotype. These data support an autosomal dominant mitochondrial uncoupling syndrome with hypermetabolism. (Funded by the National Institutes of Health.).

Figure 1.. Oxidative phosphorylation in normal and variant cells.

The oxidative phosphorylation (OXPHOS) pathway is a means of generating chemical energy, in the form of ATP, by oxidizing nutrients that are normally obtained from food. Electrons from nutrients are transferred by the respiratory chain complexes (CI-CIV) to oxygen, and in the process, energy is conserved in the form of a proton gradient, or proton motive force, across the mitochondrial inner membrane that consists primarily of a membrane potential. This proton gradient then drives efficient formation of ATP by complex V (CV). In healthy cells (top panel) the dissipation of the proton gradient is tightly coupled to the formation of ATP by complex V. The Leu335Pro variant in the ATP5B protein in twins A and B is predicted to loosen the coupling between the proton motive force and ATP synthesis by Complex V (bottom panel). As a consequence, these mitochondria are predicted to exhibit higher rates of respiration to defend the proton gradient for ATP production.

Figure 2.. Newborn twins with evidence of hypermetabolism and uncoupled respiration.

A. Caloric intake of Twin A (blue) and Twin B (red) at representative time points and the calculated caloric need for catch-up growth (shaded area). B. Weight of Twin A and Twin B calculated as Z-score based on the WHO weight for age standards. C. Pedigree of the reported family including the affected monozygotic twins. D. Multiple sequence alignment of the ATP5B protein indicating evolutionarily conserved residues (shaded), as well as the Leu335Pro mutation. Yeast mgi mutation sites are also denoted. E. Overview of the ovine CV structure (PDB: 6TT7) with a zoom into the patient mutation site (red sphere) in the beta subunit and the yeast mgi mutations (yellow spheres). F. Basal oxygen consumption in intact fibroblasts. G. Oxygen consymption in permeabilized fibroblasts in the presence of complex I substrates glutamate/malate (G/M) prior to addition of ADP and in the presence of saturating ADP. H. Change in polarization of the mitochondrial membrane in response to glutamate/malate (G/M), followed by ADP as represented by (1-TMRM intensity). Individual points represent n=6 (Fig. 1F), n=5 (Fig. 1G), n=5 (Fig. 1H) biological replicates. Means ± 95 % CI are shown.

Figure 3.. Bioenergetic characterization of engineered human cell lines.

A. CRISPR was used to engineer the observed heterozygous mutation in isogenic A375 cell lines. The left panel shows Seahorse intact cell oxygen consumption rate measurements with sequential additions of oligomycin (CV inhibitor), CCCP (protonophore), and piericidin+antimycin (CI/CIII inhibitors). The right panel shows basal mitochondrial respiration and oligomycin-insensitive mitochondrial respiration estimated by subtracting the non-mitochondrial OCR (after piericidin+antimycin addition) from OCR at the baseline and OCR upon oligomycin addition, respectively. B. Seahorse permeabilized cell oxygen consumption rate measurements in the CRISPR engineered isogenic cell lines with sequential additions of ADP, oligomycin, CCCP, and piericidin+antimycin. C. TMRM intact cell membrane potential measurements in the CRISPR engineered isogenic cell lines under baseline, oligomycin, and CCCP conditions. D. HeLa cells were used for heterologous expression of the WT and Leu335Pro variant. Western blot analysis of GFP control, FLAG-tagged wild type ATP5B, or the FLAG-tagged Leu335Pro variant. E. Seahorse intact cell oxygen consumption measurements in the heterologous expression cell lines under basal condition. F. TMRM intact cell membrane potential measurements in the heterologous expression cell lines under baseline condition. OCR: oxygen consumption rate; A.U.: arbitrary units. Means ± standard deviations are shown. Individual points represent n=36 (Fig 3A), n=6 (Fig 3B), n=8 (Fig 3C), and n=18 (Fig 3E) biological replicates. Means ± 95 % CI of >300 single-cell measurements are shown for Fig 3F. In Panels A, C, and E, mean values for biologic replicates are shown; I bars indicate standard deviations. In Panel F, mean values for more than 300 single-cell measurements are shown; I bars indicate 95% confidence intervals. AU denotes arbitrary units.