Hereditary optic neuropathies (HON) are genetic diseases which lead to the loss of central vision and it has been estimated that in Europe they affect more than 50.000 people. HON are characterized by selective loss of retinal ganglion cells (RGCs), leading to optic nerve atrophy and different degrees of visual impairment and blindness. Thanks to next generation sequencing, three heterozygous mutations in SDHA gene were identified in patients, that could be associated with these pathologies. SDHA encodes for one of the four subunits of the succinate dehydrogenase complex, involved in both the Krebs cycle and the mitochondrial electron transport chain. To evaluate whether these mutations are the specific cause of the pathology, it was necessary to validate them. The yeast Saccharomyces cerevisiae is extensively used to prove with high confidence the link between novel mutations and mitochondrial diseases, since yeast can survive without mitochondrial DNA or with large deletions of it, and can grow on fermentable carbon sources in absence of an oxidative metabolism. Besides, several human genes encoding for mitochondrial proteins are present and often conserved in yeast, giving the chance to introduce the mutation in the yeast orthologous gene or to introduce the human pathological allele in a strain disrupted in its orthologue. SDHA and SDH1, its yeast orthologue, encodes for proteins which share more than 60% of identity, and the three variants found in patients affect amino acids which are conserved in most organisms, including fungi and mammals. The three mutations were introduced in SDH1, and these mutant alleles were inserted in a SDH1-disrupted strain. Our results showed that the presence of mutant alleles leads to a decrease of the oxidative growth and of the oxygen consumption rate compared to the strain harboring SDH1 wild type allele. Furthermore, a strong depletion in the succinate dehydrogenase activity was also observed. Western blots showed that the steady state levels of the mutant proteins were similar or slightly reduced compared to the levels of wild type Sdh1, whereas Sdh2, the second subunit of the complex which interacts directly with Sdh1, is absent or strongly decreased. These results suggest that the substitutions in Sdh1/SDHA are pathological and that affects the stability of the complex. Besides, through the construction of heterozygous diploid strains, we demonstrated that two of three mutations are dominant negative, thus explaining the observation that the patients are heterozygous for each of these mutations.

Saccharomyces cerevisiae as a model to study the role of mutations in SDHA gene associated to hereditary optic neuropathies / Degiorgi, Andrea; Tagliavini, Francesca; Palombo, Flavia; La Morgia, Chiara; Carelli, Valerio; Caporali, Leonardo; Baruffini, Enrico. - STAMPA. - (2019), pp. P8-P8. ((Intervento presentato al convegno Mitochondrial Medicine tenutosi a Wellcome Genome _Campus, Hinxton, Cambridge nel 11-13/12/2019.

Saccharomyces cerevisiae as a model to study the role of mutations in SDHA gene associated to hereditary optic neuropathies

Andrea Degiorgi;Enrico Baruffini
2019-01-01

Abstract

Hereditary optic neuropathies (HON) are genetic diseases which lead to the loss of central vision and it has been estimated that in Europe they affect more than 50.000 people. HON are characterized by selective loss of retinal ganglion cells (RGCs), leading to optic nerve atrophy and different degrees of visual impairment and blindness. Thanks to next generation sequencing, three heterozygous mutations in SDHA gene were identified in patients, that could be associated with these pathologies. SDHA encodes for one of the four subunits of the succinate dehydrogenase complex, involved in both the Krebs cycle and the mitochondrial electron transport chain. To evaluate whether these mutations are the specific cause of the pathology, it was necessary to validate them. The yeast Saccharomyces cerevisiae is extensively used to prove with high confidence the link between novel mutations and mitochondrial diseases, since yeast can survive without mitochondrial DNA or with large deletions of it, and can grow on fermentable carbon sources in absence of an oxidative metabolism. Besides, several human genes encoding for mitochondrial proteins are present and often conserved in yeast, giving the chance to introduce the mutation in the yeast orthologous gene or to introduce the human pathological allele in a strain disrupted in its orthologue. SDHA and SDH1, its yeast orthologue, encodes for proteins which share more than 60% of identity, and the three variants found in patients affect amino acids which are conserved in most organisms, including fungi and mammals. The three mutations were introduced in SDH1, and these mutant alleles were inserted in a SDH1-disrupted strain. Our results showed that the presence of mutant alleles leads to a decrease of the oxidative growth and of the oxygen consumption rate compared to the strain harboring SDH1 wild type allele. Furthermore, a strong depletion in the succinate dehydrogenase activity was also observed. Western blots showed that the steady state levels of the mutant proteins were similar or slightly reduced compared to the levels of wild type Sdh1, whereas Sdh2, the second subunit of the complex which interacts directly with Sdh1, is absent or strongly decreased. These results suggest that the substitutions in Sdh1/SDHA are pathological and that affects the stability of the complex. Besides, through the construction of heterozygous diploid strains, we demonstrated that two of three mutations are dominant negative, thus explaining the observation that the patients are heterozygous for each of these mutations.
Saccharomyces cerevisiae as a model to study the role of mutations in SDHA gene associated to hereditary optic neuropathies / Degiorgi, Andrea; Tagliavini, Francesca; Palombo, Flavia; La Morgia, Chiara; Carelli, Valerio; Caporali, Leonardo; Baruffini, Enrico. - STAMPA. - (2019), pp. P8-P8. ((Intervento presentato al convegno Mitochondrial Medicine tenutosi a Wellcome Genome _Campus, Hinxton, Cambridge nel 11-13/12/2019.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2869037
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