Severe mitochondrial encephalomyopathy caused by de novo variants in OPA1 gene

Background Mitochondria adjust their shape in response to the different energetic and metabolic requirements of the cell, through extremely dynamic fusion and fission events. Several highly conserved dynamin-like GTPases are involved in these processes and, among those, the OPA1 protein is a key player in the fusion of inner mitochondrial membranes. Hundreds of monoallelic or biallelic pathogenic gene variants have been described in OPA1, all associated with a plethora of clinical phenotypes without a straightforward genotype-phenotype correlation. Methods Here we report two patients harboring novel de novo variants in OPA1. DNA of two patients was analyzed using NGS technology and the pathogenicity has been evaluated through biochemical and morphological studies in patient’s derived fibroblasts and in yeast model. Results The two patients here reported manifest with neurological signs resembling Leigh syndrome, thus further expanding the clinical spectrum associated with variants in OPA1. In cultured skin fibroblasts we observed a reduced amount of mitochondrial DNA (mtDNA) and altered mitochondrial network characterized by more fragmented mitochondria. Modeling in yeast allowed to define the deleterious mechanism and the pathogenicity of the identified gene mutations. Conclusion We have described two novel-single OPA1 mutations in two patients characterized by early-onset neurological signs, never documented, thus expanding the clinical spectrum of this complex syndrome. Moreover, both yeast model and patients derived fibroblasts showed mitochondrial defects, including decreased mtDNA maintenance, correlating with patients’ clinical phenotypes.


Functional studies in yeast
Synthetic complete medium (SC) contained 6.9 g/l yeast nitrogen base without amino acids (Formedium, UK), 1 g/l Kaiser drop-out mix, without the appropriate amino acid or base.YP medium contained 0.5% yeast extract (Formedium, UK) and 1% peptone (Formedium, UK); YPA medium was YP 2× supplemented with 40 mg/ml adenine base (Formedium, UK).Carbon sources were added as indicated in the text.Solid medium was obtained by adding 2% agar (Formedium, UK).The plasmids used in this work were pFL39TEToffCHIM3 (TRP1 marker), pFL36TEToffCHIM3 (LEU2 marker), pFL38MGM1 (URA3 marker) (2,3).chim3 mutant alleles cloned in pFL39TEToff were obtained by site-directed mutagenesis, using the QuikChange TM site-directed mutagenesis technique using KOD HotStart DNA Polymerase (Merck Millipore, US) and oligos reported in Supplementary Table 2. Mutant and wt CHIM3 alleles were introduced in the W3031-1B mgm1Δ strain already transformed with pFL38MGM1, whose loss was then induced through the plasmid shuffling technique as previously reported (4).
The petite status, i.e. the presence of whole mtDNA, of the mutant haploid strains was evaluated by crossing the haploid strains transformed with the mutant chim3 alleles or the empty vector with the BY4741 mip1 rho 0 strain as tester strain.Briefly, cells were grown on solid YP medium supplemented with 2% glucose for 48 hours and crossed on the same medium for 72 hours.Then, cells were replicated on solid SC medium supplemented with 2% glucose without methionine and tryptophan to confirm the formation of diploid cells and the maintenance of the vector and on solid YP medium supplemented with 2% ethanol and 2% glycerol to evaluate the oxidative growth.It was expected that if the chim3 tested strains were rho + , i.e. containing whole mtDNA, they would be able to complement the rho 0 status, i.e. the lack of mtDNA, of the tester strain devoid of Mip1, the mitochondrial DNA polymerase fundamental for the replication of the mtDNA, and thus that derived diploid strains are respiratory proficient.On the contrary, if the tested strains are rho 0 , the derived diploid strains are still respiratory deficient.
To test the dominance/recessivity of the mutations, diploid strains were obtained by crossing W303-1B mgm1Δ harboring MGM1 cloned pFL38 and the chim3 wild type or mutant allele cloned in pFL39TEToff with W303-1A mgm1Δ harboring CHIM3 wild type allele cloned in pFL36TEToff, thus obtaining DW1.1 strains.Diploid DW1.1 strains were spotted as previously reported (5).For each diploid strain, a small patch of cells was at first inoculated in 200 μl of SC medium supplemented with 2% glucose without leucine, tryptophan and uracil (SCD-L,W,U) in order to maintain the three plasmids inside the cells (pFL38MGM1, pFL36TEToffCHIM3 and pFL39TEToff harboring wt or mutant chim3 alleles) and incubated for 28 hours without shaking into a 96-well microtiter plate.Then, 2 μl of this culture were inoculated in 200 μl of SCD-L,W medium, which contain uracil in order to allow the loss of the pFL38MGM1 plasmid, and grown until the early stationary phase (30 hours).For all strains, cell concentration was between 7.5 and 8.5 OD600.5 μl of undiluted culture and 1:10 dilutions were then spotted on several media, including SCD-L,W, SCD-L,W,U, SCD-L,W supplemented with 1 mg/ml 5-fluoroorotic acid (5-FOA) and SC-l,W supplemented with 2% galactose and 5-FOA (SCGal-L,W+5-FOA).Plates were incubated for 5 days.On the first medium, all cells will grow, including those that have lost MGM1 cloned in pFL38: it is expected that the number of colonies grown at each dilution is similar.On the second medium, cells that have lost MGM1 cloned in pFL38 will not grow: if the plasmid lost is similar among the different diploid strains, it is expected that the number of colonies grown at each dilution is similar.On the third medium, only cells that have lost pFL38MGM1 will grow: it is expected that if the plasmid loss does not affect the fermentative growth, the growth of the serial spots will be similar.On the fourth medium, only cells that have lost pFL38MGM1 and which can use galactose through oxidation will grow: it is expected that if the plasmid loss affects the oxidative growth, the growth of the serial spots will be decreased.
To measure the respiratory activity, DW1.1 diploid strains were pre-grown as in the spot assay technique, but after growth in SCD-L,W medium, 20 l of this culture were inoculated in 2ml of SCD-L,W+5-FOA in order to counter-select the cells which maintained the pFL38MGM1 plasmid.After a 36-hour growth under shaking, cells were inoculated at a final concentration of 0.08 OD/ml in SC-L,W supplemented with 0.6% glucose and grown until glucose was exhausted for approximately 20 h.The oxygen consumption rate (OCR) was measured on whole cells using a Clark-type oxygen electrode (Oxygraph System Hansatech Instruments England) with 1 ml of air-saturated respiration buffer (0.1M phthalate-KOH, pH 5.0), 10mM glucose at 30 °C.The reaction started by adding 20 mg of wet-weight cells, as described previously (3).The oxygen consumption of each clone was at first normalized for the dry weight of the cells and then for the absolute OCR of the CHIM3/CHIM3 homoallelic strain.
The frequency of respiratory proficient cells, i.e. rho + cells, was measured by growing the original diploid strains on SCD-L,W solid medium in order to allow the loss of pFL38MGM1.After a 48-hour growth, cells were replica-plated on SCGal-L,W+5-FOA solid medium to counter-select both cells containing pFL38MGM1 and respiratory deficient cells, i.e. petite cells, since they are not able to oxidize galactose, then on SCD-L,W+5-FOA solid medium in order to allow the formation and the growth of petite cells.After 48 hours, a small patch of cells was diluted in water and, after dilution, plated on SC-L,W medium supplemented with 2% ethanol, 2% glycerol and 0.4% glucose in order to determine the frequency of petite colonies as previously reported (6).The frequency of respiratory proficient cells is reported as the ratio between cells forming grande colonies, which were able to use ethanol and glycerol as carbon sources after glucose exhaustion, and the sum of grande colonies and petite colonies, the latter being unable to oxidize ethanol and glycerol.