<i>CACNA2D2</i> mRNA and protein expression.

<p>Left panel: the mRNA content of the proband was evaluated by Real Time-PCR and normalized on that of control individuals (n = 4). <i>CACNA2D2</i> mRNA resulted strongly reduced in the proband (II:1, 18%), father (I:1, 28%) and mother (I:2, 54%) compared to controls (Anova test, p<0.001). Data are shown as mean ± standard error of three independent experiments. Right panel: the protein content was evaluated by Western blot and normalized on that of control individuals (n = 3), using GAPDH as reference protein and loading control. Graph shows data obtained by densitometric analysis. The proband (II:1) showed a 3% of protein expression compared to controls (Anova on Ranks test, p = 0.034), whereas both parents (I:1 and I:2) had ∼50% of expression. Data are expressed as mean ± standard error of three independent experiments. Representative western blot is shown.</p

Instrumental findings in the proband.

<p>Left panel: EEG recording during wakefulness showing generalized spike-wave complex at 4 Hz lasting thirteen seconds with absence, eye up deviation and eyelid myoclonia. Right panel: Sagittal T1 MRI showing prominent cerebellar atrophy.</p

Features of the CACNA2D2 and CELSR3 mutations.

<p>−12.3 to 6.17, with 6.17 being the most conserved <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082154#pone.0082154-Cooper1" target="_blank">[30]</a>. GERP (Genomic Evolutionary Rate Profiling) is a measure of the nucleotide evolutionary conservation. It ranges from </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

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

List of complete mtDNA sequences included in Figure 1.

a<p>Family ID numbers correspond to the numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042242#pone-0042242-g001" target="_blank">Figure 1</a>.</p>b<p>This family is originally from Lebanon.</p>c<p>This family is originally from Turkey.</p>d<p>This family is originally from Benin.</p>e<p>Haplogroup classification based on the most updated human mitochondrial phylogeny <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042242#pone.0042242-vanOven1" target="_blank">[35]</a>.</p>f<p>Nucleotide positions and changes refer to rCRS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042242#pone.0042242-Andrews1" target="_blank">[57]</a>. The non-synonymous nucleotide changes at nps 8860 (8860G) and 15326 (15326G), present in all LHON samples, are not included because they are private mutations of the reference sequence. LHON mutations are in bold, while those with a possible synergistic effect are underlined.</p>g<p>Only the entire mitochondrial sequence #6 was already published (Olivieri et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042242#pone.0042242-Olivieri1" target="_blank">[44]</a>).</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

Phylogenetic tree of 16 complete mtDNA sequences from LHON patients.

<p>Rare LHON mutations are shown in bold. The position of rCRS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042242#pone.0042242-Andrews1" target="_blank">[57]</a> is indicated for reading off sequence motifs. Mutations are shown on the branches; they are transitions unless a base is explicitly indicated. The prefix @ designates reversions, while suffixes indicate: transversions (to A, G, C, or T), indels (+, d), gene locus (∼t, tRNA; ∼r, rRNA), synonymous or non-synonymous changes (s or ns) and heteroplasmies (h). Recurrent mutations are underlined. One mtDNA sequence (I; black circle) from a control subject (GenBank accession number FJ190383) was also included to illustrate that sequences 11 and 12 acquired independently the LHON mutation m.14568C>T. The haplogroup affiliation of each haplotype is based on mutational motifs and follows the most updated human phylogeny <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042242#pone.0042242-vanOven1" target="_blank">[35]</a>.</p