Recessive pathogenic variants in MCAT cause combined oxidative phosphorylation deficiency
- Department of Pediatrics and Center for Human Genomics and Precision Medicine, University of Wisconsin School of Medicine and Public Health, United States
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, United States
- Department of Pediatrics, Icahn School of Medicine at Mount Sinai, United States
- Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, United States
- Department of Metabolism and Nutritional Programming, Van Andel Institute, United States
- Children’s Medical Center Research Institute, University of Texas Southwestern Medical Center, United States
- Department of Pediatrics, Nijmegen Center for Mitochondrial Disorders, Radboud University Medical Center, Netherlands
- Department of Radiology, Icahn School of Medicine at Mount Sinai, United States
- Howard Hughes Medical Institute, United States
- Department of Biochemistry, University of Utah, United States
Figures
Figure 1
Axial T2-weighted MRI sections of the brain displayed from inferior to superior.
(A–B) Sections through the brainstem and cerebellum. Sections through the medulla (A) and mid pons (B) demonstrate well-defined, symmetrical signal increase in the central tegmental tracts (1) and …
Figure 2 with 4 supplements
Western blot analysis in patient lymphoblasts (A–C) and fibroblasts (D).
(A) Western blot analysis to assess the expression level of one subunit of each of the five different oxidative phosphorylation complexes in the patient lymphoblast sample (P) compared to three …
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Figure 2—source data 1
Uncropped immunoblots for Figure 2.
- https://cdn.elifesciences.org/articles/68047/elife-68047-fig2-data1-v1.zip
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Figure 2—source data 2
Unlabeled immunoblots for Figure 2.
- https://cdn.elifesciences.org/articles/68047/elife-68047-fig2-data2-v1.zip
Figure 2—figure supplement 1
Quantification of western blot analysis in patient lymphoblasts (A–C) and fibroblasts (D).
The intensity of the protein of interest was normalized to GAPDH. The average of the controls was set to 100% and the individual values are expressed as a percentage.
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Figure 2—figure supplement 1—source data 1
Excel spreadsheet containing quantitative data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/68047/elife-68047-fig2-figsupp1-data1-v1.zip
Figure 2—figure supplement 2
Measurement of 2-oxoglutaric acid dehydrogenase complex (OGDHc) activity.
Oxidative decarboxylation of 2-oxoglutaric acid by OGDHc is measured in cell lysates from lymphoblasts from the patient (P) and three healthy controls (C1–3). Control 1 (C1) was a single observation …
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Figure 2—figure supplement 2—source data 1
Excel spreadsheet containing quantitative data for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/68047/elife-68047-fig2-figsupp2-data1-v1.zip
Figure 2—figure supplement 3
Relative abundance of lactate, 2-oxoglutarate, and 2-oxoadipate from quantitative metabolomics.
Three cell pellets were collected for the patient fibroblast cell line (P) and each of five control fibroblast lines (C1–5). Lactate was elevated in the patient line, while 2-oxoglutarate and …
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Figure 2—figure supplement 3—source data 1
Excel spreadsheet containing quantitative data for Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/68047/elife-68047-fig2-figsupp3-data1-v1.zip
Figure 2—figure supplement 4
Protein modeling of malonyl-CoA-acyl carrier protein transacylase (MCAT) p.T271I mutation.
The T235 residue in the crystal structure corresponds to the residue at the site of the T271I mutation. This mutation occurs one residue away from the active site histidine (H234) and is located in …
Figure 3
Mitochondrial respiratory supercomplexes visualized by blue-native PAGE.
Mitochondrial lysates generated from four control fibroblast lines (C1–4), the patient fibroblast line (P), the patient line expressing the control plasmid mtGFP (P+mtGFP), and the patient line with …
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Figure 3—source data 1
Uncropped immunoblots for Figure 3.
- https://cdn.elifesciences.org/articles/68047/elife-68047-fig3-data1-v1.zip
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Figure 3—source data 2
Unlabeled immunoblots for Figure 3.
- https://cdn.elifesciences.org/articles/68047/elife-68047-fig3-data2-v1.zip
Figure 4
Rescue of hypomorphic malonyl-CoA-acyl carrier protein transacylase (MCAT) mutant C2C12 with wild-type (WT) and p.T271I mutant MCAT constructs.
(A) Mitochondrial lysates generated from MCAT hypomorphic CRISPR mutant C2C12 mouse skeletal myoblasts (MCAT) and isogenic controls (GFP), stably infected with either WT human MCAT or p.T271I mutant …
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Figure 4—source data 1
Uncropped immunoblots for Figure 4.
- https://cdn.elifesciences.org/articles/68047/elife-68047-fig4-data1-v1.zip
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Figure 4—source data 2
Unlabeled immunoblots for Figure 4.
- https://cdn.elifesciences.org/articles/68047/elife-68047-fig4-data2-v1.zip
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Figure 4—source data 3
Excel spreadsheet containing quantitative data for Figure 4 Panel B.
- https://cdn.elifesciences.org/articles/68047/elife-68047-fig4-data3-v1.xlsx
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Figure 4—source data 4
Excel spreadsheet containing quantitative data for Figure 4 Panel C.
- https://cdn.elifesciences.org/articles/68047/elife-68047-fig4-data4-v1.xlsx
Tables
Table 1
Clinical biochemical testing of respiratory chain enzyme activities in the proband’s fibroblasts.
| Proband | Reference range | |
|---|---|---|
| Complex I | 119 mU/U CS | 163–599 mU/U CS |
| Complex II | 286 mU/U CS | 335–888 mU/U CS |
| Complex III | 632 mU/U CS | 570–1383 mU/U CS |
| Complex IV | 103 mU/U CS | 288–954 mU/U CS |
| Complex V | 630 mU/U CS | 193–819 mU/U CS |
| CS | 412 mU/mg | 151–449 mU/mg |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Mus musculus) | C2C12 | ATCC | #CRL-1772, RRID:CVCL_0188 | |
| Cell line (Homo sapiens) | HEK293T | ATCC | #CRL-11268, RRID:CVCL_1926 | |
| Antibody | Anti-human OXPHOS cocktail (mouse monoclonal) | MitoSciences | ab110411 | WB: (1:1000) |
| Antibody | Anti-GAPDH (rabbit monoclonal) | Abnova | H00002597-K | WB: (1:2500) |
| Antibody | Anti-MCAT (mouse monoclonal) | Sigma-Aldrich | HPA035471, RRID: AB_10670590 | WB: (1:1000) |
| Antibody | Anti-MCAT (mouse monoclonal) | Santa Cruz | sc-390858, RRID:AB_2827536 | WB: (1:100) |
| Antibody | Anti-DLAT (rabbit monoclonal) | Abcam | ab172617, RRID:AB_2827534 | WB: (1:1000) |
| Antibody | Anti-DLST (rabbit polyclonal) | Cell Signaling | 5556, RRID:AB_106951 | WB: (1:1000) |
| Antibody | Anti-GRIM19 (mouse monoclonal) | Abcam | ab110240, RRID:AB_10863178 | WB: (1:1000) |
| Antibody | Anti-SDHA (mouse monoclonal) | Abcam | ab14715, RRID:AB_301433 | WB: (1:10,000) |
| Antibody | Anti-UQCRQ (mouse monoclonal) | Abcam | ab110255 | WB (1:1000) |
| Antibody | Anti-MTCO1 (mouse monoclonal) | Abcam | ab14705, RRID:AB_2084810 | WB (1:1000) |
| Antibody | Anti-ATP5A (mouse monoclonal) | Abcam | ab14748, RRID:AB_301447 | WB (1:1000) |
| Antibody | Anti-Lipoic Acid (rabbit polyclonal) | Abcam | ab58724, RRID:AB_880635 | WB (1:1000) |
| Antibody | Anti-NDUFA9 (mouse monoclonal) | Abcam | ab14713, RRID:AB_301431 | WB (1:1000) |
| Antibody | Anti-HA (rabbit polyclonal) | BioLegend | PRB-101C | WB (1:1000) |
