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Case Reports
. 2018 Feb 1;27(3):499-504.
doi: 10.1093/hmg/ddx419.

SLC25A10 Biallelic Mutations in Intractable Epileptic Encephalopathy With Complex I Deficiency

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SLC25A10 Biallelic Mutations in Intractable Epileptic Encephalopathy With Complex I Deficiency

Giuseppe Punzi et al. Hum Mol Genet. .
Free PMC article

Abstract

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability.

Figures

Figure 1.
Figure 1.
SLC25A10 genetic and RNA analyses. (A) Pedigree of the affected family (filled symbol: patient; open symbols: unaffected relatives). (B) Compound heterozygous mutations identified by trio-WES, reported as the “Integrative genomic viewer” (26) output. (C) Structure of the SLC25A10 gene and location of mutations. The nucleotide and amino acid changes are indicated with respect to the reference sequences NM_001270888 and NP_001257817, respectively. (D) qRT-PCR analysis of fibroblast cDNA revealed a decrease in endogenous SLC25A10 RNA quantity in the patient (II: 2) compared with control (Ctrl). (E) Gel electrophoresis of cDNA PCR products revealed the presence of a fragment spanning exons 3-8 in patient fibroblasts (II: 2) and control fibroblasts (Ctrl), while (F) patient fibroblasts lack the fragment spanning exons 3-11. (G) Structure of the minigene construct spannig exons 9-11 (E: exon). (H) Ratio between the quantity of the minigene-derived RNA splicing fragments including and excluding intron 10 (I10), exon 10 (E10) or both of them (E10-I10) in minigene E9-E11. (I) Inclusion/Exclusion ratios in minigene E10-E11. Data are presented as mean+SEM of at least three experiments; ***: non parametric Wilcoxon test P-value<0.05.
Figure 2.
Figure 2.
SLC25A10 protein and functional analyses. (A) Western-blot of primary fibroblast mitochondrial extracts (30 μg of protein) in patient (II: 2) and two control cell lines (Ctrl1 and Ctrl2). (B) Transport activity of SLC25A10 in fibroblast mitochondrial extracts of patient (II: 2) and control (Ctrl). Liposomes were reconstituted with mitochondrial extracts, preloaded with excess of Pi or malate (20 mM) and incubated with radio-labeled [14C]malate or [33P]phosphate (1 mM), respectively, for 60 min. Pi: phosphate. (C) Mass spectrometry quantification of NADPH and GSH (reduced forms) and NADP+ and GSSG (oxidized forms), reported as reduced/oxidized form ratios, in the fibroblasts of patient (II: 2) and control (Ctrl). (D) Lack of transport of GSH and GSSG by DIC (Slc25a10). Liposomes were reconstituted with the recombinant purified rat DIC, preloaded with excess of malate, GSH or GSSG or NaCl (10 mM) and incubated with radio-labeled [14C]malate (0.5 mM) for 30 min. (E) Percentage of “petite” respiratory-deficient yeast colonies generated from S. cerevisiae knockout strain (ΔDIC1) and isogenic wild-type strain (WT) after 2 or 4 days of growth in glycerol at 30 °C or 37 °C. ∼1000 colonies were screened per strain per assay. °°°: binomial test P-value<0.05, n.s.: not significant. (F) Yeast quantitative PCR analysis of relative mtDNA copy number after 4 days of growth in glycerol compared with 2 days. (G) Yeast growth curves in glycerol for WT and ΔDIC1 in absence (open symbols) or in presence (filled symbols) of H2O2 (2.5 mM). Data are presented as mean+SEM of at least three experiments except for Figure 2E, ***: non parametric wilcoxon test P-value<0.05, n.s.: not significant.

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