Comparison of optical coherence tomography parameters (different macular thickness layers) between all patients with and without macular microcysts and healthy controls.

<p>Data are mean (SD). LHON = Leber hereditary optic neuropathy. DOA = Dominant optic atrophy. MM = macular microcysts. mRNFL = macular retinal nerve fiber layer. GCL-IPL = ganglion cell and inner plexiform layers. INL = inner nuclear layer including outer plexiform layers. ONL = outer nuclear layer including inner and outer photoreceptor segments.</p><p>Comparison of optical coherence tomography parameters (different macular thickness layers) between all patients with and without macular microcysts and healthy controls.</p

Segmentation of ROIs including prefrontal white matter (A), cerebellar white matter (B) and optic radiation (C) on axial T2 images.

<p>D: 3D reconstruction of optic radiation ROIs on a registered T1 volumetric image. E: Box-plot of MD values of optic radiations in controls, LHON healthy carriers and LHON patients. (Each box shows the median, quartiles, extreme values; * = P<0.01).</p

MD values of optic radiation, prefrontal white matter and cerebellar white matter in LHON patients, LHON healthy carriers and controls with group comparison results (first two sections).

<p>The bottom of the table shows the results of GLM analysis used to evaluate the effect of genetic, clinical and demographic data on optic radiation MD values in LHON patients.</p>*<p> = mean of left and right MD values.</p>#<p> = corrected for multiple comparisons.</p><p>Values are reported as mean and standard deviation.</p><p>MD: mean diffusivity; WM: white matter; n.s.: not significant; GLM: general linear model.</p

Comparison of optical coherence tomography parameters (different macular thickness layers) between LHON and DOA patients with and without macular microcysts and healthy controls.

<p>Data are mean (SD). MM = macular microcysts. INL = inner nuclear layer including outer plexiform layers. ONL = outer nuclear layer including inner and outer photoreceptor segments.</p><p>Comparison of optical coherence tomography parameters (different macular thickness layers) between LHON and DOA patients with and without macular microcysts and healthy controls.</p

Top: neuron soma size by layer type for LHON and control LGN.

<p>The ratio of the magnocellular to parvocellular layers for the two LGN is similar suggesting that the atrophy seen in the LHON case was consistent across all layers. Middle: average cell density of the magnocellular and parvocellular layers for both the LHON and control LGNs. LHON LGN exhibits a decrease in neuron density consistent across both cell layer types. Bottom: axonal counts for LHON and control left optic nerve.</p

Top panel: optic nerves in cross-section and stained by p-Phenylenediamine for control and LHON patient (25x magnification).

<p>In the LHON patient only a small patch of fibers remains (arrow) in the super-nasal quadrant. Bottom panel: lateral geniculate nuclei (LGN) of control and LHON patients with all magnocellular (1 and 2) and parvocellular (3–6) layers identified (25x magnification). Insets represent samples of each zone at 200x magnification.</p

Patients OCT foveal scans.

<p>Horizontal OCT scans revealed macular microcysts (MM) of the inner nuclear layer in the four LHON patients and three DOA patients.</p

Peculiar combinations of individually non-pathogenic missense mitochondrial DNA variants cause low penetrance Leber’s hereditary optic neuropathy

<div><p>We here report on the existence of Leber’s hereditary optic neuropathy (LHON) associated with peculiar combinations of individually non-pathogenic missense mitochondrial DNA (mtDNA) variants, affecting the <i>MT-ND4</i>, <i>MT-ND4L</i> and <i>MT-ND6</i> subunit genes of Complex I. The pathogenic potential of these mtDNA haplotypes is supported by multiple evidences: first, the LHON phenotype is strictly inherited along the maternal line in one very large family; second, the combinations of mtDNA variants are unique to the two maternal lineages that are characterized by recurrence of LHON; third, the Complex I-dependent respiratory and oxidative phosphorylation defect is co-transferred from the proband’s fibroblasts into the cybrid cell model. Finally, all but one of these missense mtDNA variants cluster along the same predicted fourth E-channel deputed to proton translocation within the transmembrane domain of Complex I, involving the ND1, ND4L and ND6 subunits. Hence, the definition of the pathogenic role of a specific mtDNA mutation becomes blurrier than ever and only an accurate evaluation of mitogenome sequence variation data from the general population, combined with functional analyses using the cybrid cell model, may lead to final validation. Our study conclusively shows that even in the absence of a clearly established LHON primary mutation, unprecedented combinations of missense mtDNA variants, individually known as polymorphisms, may lead to reduced OXPHOS efficiency sufficient to trigger LHON. In this context, we introduce a new diagnostic perspective that implies the complete sequence analysis of mitogenomes in LHON as mandatory gold standard diagnostic approach.</p></div

Biochemical characterization of cybrid clones.

<p><b>A.</b> Cell viability after different time of incubation in galactose medium (0, 24, 48, 72h). Data are expressed as percentage of T0 (<i>n</i> = 12; mean ± SEM). <b>B.</b> Rotenone sensitive redox activity of respiratory complex I normalized for CS activity (<i>n</i> = 9; mean ± SD). <b>C.</b> OCR traces as pmol O₂/min, after the injection of 1μM oligomycin (O), 0.2μM FCCP (F), 1μM rotenone (R) and 1μM antimycin A (AA) (mean ± SEM). Asterisks indicate statistical significance (<i>n></i> = 3; * p<0.05). <b>D.</b> XFe Metabolic Phenogram. Basal OCR (pmol/min) and ECAR (mpH/min) rates were plotted in controls vs LHON cybrids, showing a metabolic shift in LHON cybrids towards glycolysis. <b>E.</b> ATP synthesis rates normalized for CS activity driven by complex I substrates (malate/glutamate) and complex II substrate succinate (mean ± SD). Asterisks indicate statistical significance (<i>n</i> = 16; * p<0.05).</p

Complex I model.

<p>Localization of polymorphic variants on the cryo-EM structure of the ovine complex I, [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007210#pgen.1007210.ref034" target="_blank">34</a>] using the UCSF Chimera software. The ovine amino acids Ala140 (corresponding to human p.P139L, m.14258G>A/<i>MT-ND6</i>), Gly32 (corresponding to human p.V31A, m.14582A>G/<i>MT-ND6</i>) and Ala71 (corresponding to human p.A71T, m.10680G>A/<i>MT-ND4L</i>) are shown as red-labelled spheres; whereas residues Glu143/ND1, Glu192/ND1, Glu34/ND4L, Tyr60/ND6, the key residues for the E-channel (near Q site), are shown as blue-labelled spheres. The structures of ND1, ND4L, ND6 and ND3 subunits are shown as ribbons, in green, blue, yellow and red, respectively. The combination of variants in Family 1 (<b>A-B</b>) and Family 2 (<b>C-D</b>) are displayed as front (<b>A-C</b>) and upper (<b>B-D</b>) views. Light blue arrows indicate the proposed proton translocation pathway [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007210#pgen.1007210.ref034" target="_blank">34</a>].</p