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Case Reports
. 2016 Oct 6;99(4):860-876.
doi: 10.1016/j.ajhg.2016.08.014. Epub 2016 Sep 29.

Recurrent De Novo Dominant Mutations in SLC25A4 Cause Severe Early-Onset Mitochondrial Disease and Loss of Mitochondrial DNA Copy Number

Kyle Thompson  1 , Homa Majd  2 , Cristina Dallabona  3 , Karit Reinson  4 , Martin S King  2 , Charlotte L Alston  1 , Langping He  1 , Tiziana Lodi  3 , Simon A Jones  5 , Aviva Fattal-Valevski  6 , Nitay D Fraenkel  7 , Ann Saada  8 , Alon Haham  9 , Pirjo Isohanni  10 , Roshni Vara  11 , Inês A Barbosa  12 , Michael A Simpson  12 , Charu Deshpande  13 , Sanna Puusepp  4 , Penelope E Bonnen  14 , Richard J Rodenburg  15 , Anu Suomalainen  16 , Katrin Õunap  4 , Orly Elpeleg  17 , Ileana Ferrero  3 , Robert McFarland  1 , Edmund R S Kunji  2 , Robert W Taylor  18
Affiliations
Case Reports

Recurrent De Novo Dominant Mutations in SLC25A4 Cause Severe Early-Onset Mitochondrial Disease and Loss of Mitochondrial DNA Copy Number

Kyle Thompson et al. Am J Hum Genet. .

Erratum in

Abstract

Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.

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Figures

Figure 1
Figure 1
The c.239G>A (p.Arg80His) and c.703C>G (p.Arg235Gly) SLC25A4 Mutations Are De Novo Affecting Evolutionary Conserved Residues (A) Pedigrees of six families showing each clinically affected subject and confirmatory sequencing chromatograms to show that the c.239G>A (p.Arg80His) or c.703C>G (p.Arg235Gly) heterozygous variants in each affected individual are not present in respective parental samples, indicating de novo occurrence. (B) Multiple sequence alignment of this region of the AAC1 amino acid sequence (GenBank: NP_001142.2) was performed using ClustalOmega and confirms that the p.Arg80His (left) and p.Arg235Gly (right) alterations affect evolutionarily conserved residues (shaded blue).
Figure 2
Figure 2
The p.Arg80His and p.Arg235Gly Alterations Cause Decreased AAC1 Levels and Loss of OXPHOS Subunits Specifically in Skeletal Muscle (A) Western blot analysis of OXPHOS complex subunits and AAC (Abcam cat# ab110322, RRID: AB_10862212) performed on skeletal muscle lysates from subject 1 (p.Arg80His). (B) Western blot analysis of OXPHOS complex subunits and AAC (all isoforms) performed on skeletal muscle samples from subject 5 (p.Arg235Gly). (C) Western blot analysis of OXPHOS complex subunits performed on fibroblast lysates from subject 1.
Figure 3
Figure 3
The Functional Elements of the Human Mitochondrial ADP/ATP Carrier (A) Transport cycle of the ADP/ATP carrier. Disruption and formation of the cytoplasmic and matrix salt bridge networks (top and bottom, respectively) change the accessibility of the central substrate binding site (hexagon) to either side of the membrane., (B) Lateral view of the human ADP/ATP carrier from the membrane, showing the residues of the matrix and cytoplasmic networks (blue and red sticks) and substrate binding site (green sticks, hexagon). ADP (light blue ball and stick) and the glutamine brace (light green stick) are also shown. The residues that are mutated are shown in yellow. (C) Cytoplasmic view of the carrier showing only the residues of the proposed substrate-binding site. The residues of the p.Arg80His (R80H) mutation are shown in yellow and magenta, respectively. The contact points of the substrate binding site are indicated by roman numerals. (D) Cytoplasmic view of the carrier showing only the residues of the matrix salt bridge network. The residues of the p.Arg235Gly (R235G) mutation are shown in yellow and magenta, respectively. The comparative model of human AAC1 is based on the structure of the bovine carrier and was extended at the C terminus. The ionic interactions are indicated with plus and minus signs.
Figure 4
Figure 4
The p.Arg80His and p.Arg235Gly Alterations Impair ADP/ATP Transport (A) ADP uptake curves of whole cells of L. lactis-expressing AAC1 (open circle), AAC1 p.Arg80His (open square), and AAC1 p.Arg235Gly (open triangle). To determine the non-specific binding, the specific inhibitor carboxyatractyloside was added (closed symbols). The error bars represent the standard deviation of eight assays. (B) Residual transport activity relative to wild-type AAC1, corrected for background binding and differences in expression levels, as determined by the uptake rate of radiolabeled ADP into L. lactis-expressing human AAC1 with de novo mutations described here (black bars), adPEO mutations (white bars), or the recessive mitochondrial myopathy and cardiomyopathy mutations (gray bars). The data are represented by the average and standard deviation of eight transport assays.
Figure 5
Figure 5
Phenotypic Analysis of the WB12 Strain Transformed with the Empty Vector, Wild-Type AAC2, aac2Arg96His, or aac2Arg252Gly Mutant Allele (A) Assessment of an oxidative growth phenotype. Equal amounts of serial dilutions of cells from exponentially grown cultures (105, 104, 103, and 102 cells) were spotted onto SC plates without uracil, supplemented with either 2% glucose, 2% ethanol, or 2% glycerol. The growth was scored after 3 days of incubation at 28°C. (B) Cytochrome profiles of cells grown in SC without uracil supplemented with 0.6% glucose at 37°C. The peaks at 550, 560, and 602 nm (vertical bars) correspond to cytochromes c, b, and aa3, respectively. All the experiments were performed in triplicate. (C) Respiratory activity (normalized to wild-type, performed in triplicate, error bars show standard deviation) and corresponding western blot analysis to show Aac2 expression. (D) Transport activity as determined by the uptake rate of [14C]-labeled ADP measured with fused mitochondrial membranes isolated from yeast strains and corresponding western blot analysis to illustrate appropriate Aac2 expression. Error bars describe the standard deviations of four assays.
Figure 6
Figure 6
De Novo SLC25A4 Mutations Do Not Decrease Mitochondrial Protein Import (A) Western blot analysis of inner mitochondrial membrane proteins performed on skeletal muscle lysates from subject 2 (p.Arg80His). (B) Western blot analysis of inner mitochondrial membrane proteins performed on skeletal muscle samples from subject 5 (p.Arg235Gly).
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