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SSBP1 mutations cause mtDNA depletion underlying a complex optic atrophy disorder
Valentina Del Dotto, … , Tommaso Pippucci, Valerio Carelli
Valentina Del Dotto, … , Tommaso Pippucci, Valerio Carelli
Published September 24, 2019
Citation Information: J Clin Invest. 2020;130(1):108-125. https://doi.org/10.1172/JCI128514.
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Research Article Genetics Ophthalmology

SSBP1 mutations cause mtDNA depletion underlying a complex optic atrophy disorder

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Abstract

Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single-strand binding protein (SSBP1) in 4 families with dominant and 1 with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect the amount of SSBP1 protein and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids, and 7S-DNA amounts as well as mtDNA replication, affecting replisome machinery. The variable mtDNA depletion in cells was reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits, and complex amount and assembly. mtDNA depletion and cytochrome c oxidase–negative cells were found ex vivo in biopsies of affected tissues, such as kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by WT mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as a cause of human pathology.

Authors

Valentina Del Dotto, Farid Ullah, Ivano Di Meo, Pamela Magini, Mirjana Gusic, Alessandra Maresca, Leonardo Caporali, Flavia Palombo, Francesca Tagliavini, Evan Harris Baugh, Bertil Macao, Zsolt Szilagyi, Camille Peron, Margaret A. Gustafson, Kamal Khan, Chiara La Morgia, Piero Barboni, Michele Carbonelli, Maria Lucia Valentino, Rocco Liguori, Vandana Shashi, Jennifer Sullivan, Shashi Nagaraj, Mays El-Dairi, Alessandro Iannaccone, Ioana Cutcutache, Enrico Bertini, Rosalba Carrozzo, Francesco Emma, Francesca Diomedi-Camassei, Claudia Zanna, Martin Armstrong, Matthew Page, Nicholas Stong, Sylvia Boesch, Robert Kopajtich, Saskia Wortmann, Wolfgang Sperl, Erica E. Davis, William C. Copeland, Marco Seri, Maria Falkenberg, Holger Prokisch, Nicholas Katsanis, Valeria Tiranti, Tommaso Pippucci, Valerio Carelli

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Figure 8

Energetic profile of SSBP1 mutated fibroblasts.

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Energetic profile of SSBP1 mutated fibroblasts.
(A) OCR of fibroblasts, ...
(A) OCR of fibroblasts, expressed as pmoles O2/min normalized for protein content, under basal conditions and after injection of oligomycin (O), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; F), rotenone (R) and antimycin A (AA). Data are shown as mean ± SEM of control (n = 7) and mutant cells (n = 3). (B) Basal, ATP-linked, maximal respiration, and spare respiratory capacity were calculated from OCR traces and are reported in the graph as mean ± SEM. OCR experiments show a severe reduction of respiratory capacity in p.R107Q and a partial defect in p.G40V mutants. (C) Western blot of OXPHOS subunit expression levels; ACTIN was used as a loading control. One representative experiment out of 3 is shown. (D) Densitometric analysis of data shown in C shows a variable reduction of OXPHOS subunits in p.R107Q and p.G40V cells. Data, normalized to the control cells, are shown as mean ± SEM of 3 independent experiments. (E) Analysis of complexes assembly was carried out in digitonin-treated mitoplasts resolved by CN and BN-PAGE, as described in Methods. SDHA (CII) was used as a loading control. One representative experiment out of 3 is shown. (F) Densitometric analysis of CI and CIII complexes. Data are shown as mean ± SEM of 3 independent experiments. (G) Densitometric analysis of CV complex, showing an increase of F1 subunit not assembled in R107Q and G40V fibroblasts. (H) Western blot of CS, TIM23, and TOM20; ACTIN was used as a loading control. One representative experiment out of 3 is shown. (I) Densitometric analysis of the mitochondrial mass proteins. Data, normalized to the control cells, are shown as mean ± SEM of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way (G) or 2-way ANOVA (B, D, F, and I) with Dunnett’s test.
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