Nb 60 M – c.768C>G p.S256R + + + + Spasticity c.854A>G p.Q285R Ob 64 F – c.768C>G p.S256R + + + + Spasticity c.854A>G p.Q285R

Molecular screening of 980 cases of suspected hereditary optic neuropathy with a report on 77 novel OPA1 mutations

AU-135F++++++c.1635C>Gp.S545RDynamin

FR-943M+++++++c.1635C>Gp.S545RDynamin

UK-1170aF++++++c.1635C>Gp.S545RDynaminUK-1164F+++++++Diabetesc.1635C>Gp.S545RDynaminUK-1142M++++c.1635C>Gp.S545RDynaminUK-1138F+++++++Migrainec.1635C>Gp.S545RDynaminUK-1130M++++++c.1635C>Gp.S545RDynamin

NO-1 60 M + + + + + Spasticity c.768C>G p.S256R CC c.854A>G p.Q285R GTPase

FR-130M++++++c.1635C>Gp.S545RDynamin

Carelli et al. [28] 1 y M c.1311A>G/p.lIle437Met c.1705+1G>T LP Yes Yes Yes No Delayed Normal

Yu-Wai-Man et al. [6] unknown M c.768C>G/p.Ser256Arg c.854A>G/p.Gln285Arg Unk Yes Yes Yes Not reported unknown unknown

Indeed, in contrast to fibroblasts bearing the I382M mutation, which displayed mild mitochondrial network fragmentation, fibroblasts with the p.R445H and p.S545R mutations were marked by the disappearance of filamentous mitochondria in favour of fragmented and punctuated mitochondria (patients 1–3) (Fig. ​(Fig.2B2B and Fig. S1). Quantification of the ratio of mitochondrial to cellular volume showed a 20% decrease of mitochondrial content in fibroblasts with the dominant‐negative p.R445H and p.S545R mutations (patients 1–3), compared to control fibroblasts, whereas no significant loss of mitochondrial volume was observed in the haploinsufficient OPA1 mutated fibroblasts (patients 4–7) (Fig. ​(Fig.2C).2C).

OPA1 OPA1 mitochondrial dynamin like GTPase 1601T>G

We here add our observation of a similar bi-allelic OPA1 case with Behr syndrome. This proband is a 20-year-old Italian boy, born from unrelated parents (Fig. 1A, IV-4 in the pedigree), who presented a congenital nystagmus at birth and bilateral optic atrophy was recognized in the first year of life. He acquired autonomous walking at 14 months of age, but clumsy gait and frequent falls were reported. Motor difficulties and visual acuity worsened during childhood whereas cognitive development was normal. Brain MRI performed at the age of 4 and 7 years were normal. At 6 years of age, neurological examination showed ataxic-spastic gait with positive Romberg sign. Fundus exam confirmed bilateral optic atrophy (Fig. 1B). Tendon reflexes were brisk at the lower limbs and there was bilateral Babinski sign. The patient also had bilateral talipes equinovarus. Nerve conduction studies showed delayed sensory-motor velocities with reduced amplitudes of motor potentials and loss or severe reduction of sensory action potentials. Somatosensory evoked potentials could not be recorded and motor evoked potentials showed absent potentials at lower limbs and delayed latency at upper limbs. Brainstem evoked potentials were delayed bilaterally. Repeated urine organic acid gas chromatography/mass spectrometry analysis revealed mild elevation of 3-methylglutaconic acid (8–20 μmol/mmol creatinine). At muscle biopsy histology was normal and spectrophotometric assessment of the OXPHOS enzymatic activities was normal in muscle homogenate. Sural nerve biopsy demonstrated a marked reduction of the large-diameter myelinated axons (Fig. 1E–G). Teased fibre analysis disclosed regions of demyelination confined to the paranodal region, clustered in some axons, suggesting secondary demyelination. Ultrastructural examination confirmed the reduction of myelinated axons (not shown). A clinical revaluation at 15 years of age demonstrated elevated serum lactic acid after standardized exercise (3.9 mM, normal range 0.6–2.4mM) and optical coherence tomography revealed a generalized reduction of the retinal fibre layer thickness (Fig. 1D). At neurological examination, the patient was wheelchair bound due to severe sensory ataxia and spastic paraparesis and signs of cerebellar dysfunction, such as dysmetria, dysdiadochokinesia and positive Holmes rebound phenomenon, were evident. His vision was light perception with severe nistagmus in any direction of gaze. This clinical picture remained stable in the subsequent years. Figure 1. Open in a new tab (A) Reconstruction of the paternal and maternal genealogies of the proband with Behr syndrome (IV-4). The maternal side is remarkable for segregating isolated optic neuropathy in a dominant fashion associated with the heterozygous c.1705+1G>T splicing mutation. (B–D) Ophthalmological investigations. The fundus picture of the proband (IV-4) reveals severe optic atrophy with bilateral pale optic disc (B). The fundus picture of the mother (III-4) shows only a mild optic atrophy with temporal pallor of the optic discs bilaterally (C). Optical coherence tomography examination of retinal nerve fibre layer (RNFL) thickness in the proband shows a diffuse reduction of thickness compatible with the severe optic atrophy (D). (E–G) Semithin cross-section of the sural nerve biopsy. A marked depletion of large myelinated axons affects the sural nerve (E and F). At higher magnification some axons appear to have a thin myelin sheath related to the axonal diameter (G). (H–L) Brain MRI and 1HMR spectroscopy studies. On the left (H) the sagittal FSPGR (Fast SPoiled GRadient echo) T1 shows a mild cerebellar atrophy in the proband (IV-4), and on the right (I) a mild pathological accumulation of lactate (rectangle) was detected in the CSF of lateral ventriculi. On the axial FLAIR T2 sequence no signal changes were detected in the proband (IV-4) (J), whereas few focal hyperintensities, related to not specific areas of gliosis, in the cerebral white matter of the proband’s father (III-3) (K) and mother (III-4) (L) were present. TEMP = temporal; SUP = superior; NAS = nasal; INF = inferior; MRI = magnetic resonance imaging; HMR = proton magnetic resonance; CSF = cerebrospinal fluid; FLAIR = Fluid attenuated inversion recovery.The 43-year-old proband’s mother (Fig. 1A, III-4 in the pedigree) had isolated optic atrophy, most evident on the temporal side (Fig. 1C), which segregated as a dominant trait in her family (Fig. 1A). The remaining neurological examination was normal. Nerve conduction velocities and somatosensory evoked potentials were normal. The other affected individuals in this family all suffered a non-syndromic optic atrophy, which started in childhood and progressively worsened, but remained as an isolated symptom. The proband’s father and the elder sister (Fig. 1A, III-3 and IV-3, respectively) are healthy. In particular, neither signs of optic neuropathy nor abnormal nerve conduction velocities were recorded upon neuro-ophthalmological exam. Both parents and the proband have been investigated by brain MRI and proton magnetic resonance spectroscopy (1H-MRS) in a 1.5 T scanner. There were no MRI structural or metabolic abnormalities in the father (III-3) and the mother (III-4) of the proband, except for a few focal hyperintensities in the cerebral white matter (Fig. 1K–L), whereas in the proband mild cerebellar atrophy (Fig. 1H,J) and pathological accumulation of lactate in the CSF of lateral ventriculi (Fig. 1I) were evident. Direct sequencing of the entire coding regions of the OPA1 gene and the flanking intronic boundaries disclosed two heterozygous mutations. The first c.1705+1G>T mutation occurs in intron 17 and involves the highly conserved donor splice site consensus sequence. Analysis of cDNA revealed aberrant transcript splicing with the activation of a cryptic splice donor site in exon 17, which predicts a prematurely truncated gene product (data not shown). This haploinsufficiency mutation was also identified in the affected mother and in nine maternal relatives, three of which (III-10, III-14 and III-18) are clinically unaffected (Fig. 1A). The second mutation identified in the proband is missense, c.1146A>G (p.I382M), which lies in exon 12, being also present in the clinically unaffected father and sister. Interestingly this is the same missense mutation already reported in other bi-allelic cases (Schaaf et al., 2011; Bonifert et al., 2014). This mutation was found only in one other patient in Italy, out of 132 OPA1 complete sequences. This patient is currently under investigation, being another case of Behr syndrome with optic atrophy complicated by ataxia, but with only the heterozygous c.1146A>G (p.I382M) mutation at the moment (Leonardo Caporali and Valerio Carelli, unpublished data). In the Tübingen cohort of patients with bilateral optic atrophy (in most cases isolated optic atrophy) no further case with the c.1146A>G mutation was found among ∼500 patients that underwent sequencing of all coding exons of the OPA1 gene. This mutation has also been found at low frequency (about 1/1000) in large public data sets (1000 Genome Project, NHLBI GO Exome Sequencing Project). Sequence analysis of the OPA3 gene excluded the presence of pathogenic mutations. Fibroblasts were grown from skin biopsies obtained from the proband and both parents, after approval of the Internal Review Board and signed informed consent. In fibroblasts we first assessed mitochondrial network morphology, after loading mitochondria with Mitotracker Red and examination by fluorescence microscopy as previously reported (Zanna et al., 2008). The mitochondrial network of fibroblasts grown in standard Dulbecco’s modified Eagle’s medium-glucose medium bearing the heterozygous c.1705+1G>T (III:4) and c.1146A>G (III:3) mutations, respectively was similar to controls (Fig. 2A–C), whereas the fibroblasts from the proband were scored into three categories, those with a typical normal filamentous network (Fig. 2D), those with a hyperfragmented pattern (Fig. 2E) and those with filamentous mitochondria containing balloon-like structures (Fig. 2F), each class being present at approximately the same percentage. To investigate whether these OPA1 mutations could affect the bioenergetic efficiency, we measured the rate of mitochondrial ATP synthesis in digitonin-permeabilized fibroblasts. Compared to controls, mitochondrial ATP synthesis driven by complex I substrates (pyruvate and malate) was increased in the proband’s father fibroblasts (III:3) bearing the c.1146A>G mutation, and incrementally reduced in the mother’s fibroblasts bearing the c.1705+1G>T (III:4) mutation and in the proband’s fibroblasts (IV:4) carrying both mutations. The latter was significantly lower compared to his father’s fibroblasts. A similar but less severe situation was observed for the ATP synthesis driven by the complex II substrate (succinate) (Fig. 2G). Overall, these results indicate that the biallelic mutation in the proband led to abnormal network dynamics and impaired ATP synthesis through complex I. Both parent’s mutations were not severe enough to impair mitochondrial network dynamics under the experimental conditions used, but the mother’s mutation impaired partially complex I-dependent ATP synthesis. Figure 2. Open in a new tab (A–F) Mitochondrial network morphology. Control (A) and patient (B–F) fibroblasts were grown in Dulbecco’s modified Eagle’s medium-glucose and loaded with Mitotracker Red as described in Zanna et al. (2008). Representative out of eight similar images are shown for each individual. (G) ATP synthesis. Fibroblasts were treated with 50 µg/ml digitonin, the rate of ATP synthesis was measured as described in Zanna et al. (2008) and was normalized for citrate synthase (CS) activity. Data (mean ± SEM) were obtained from five controls and the three OPA1 mutated fibroblasts. The experiment was performed at least in triplicate. Asterisk denotes values significantly different (P < 0.05) by Kruskal-Wallis one-way ANOVA on Ranks. GPD = Glyceraldehyde 3-phosphate dehydrogenase.

c.1705+1G>T p.(V556Qfs*40) Intron 17 CS061312

a 60-year-old man and his 64-year-old sister (p.[S256R];[Q285R]), who both show ataxia, myopathy, peripheral neuropathy, and spasticity in addition to optic atrophy [42],

While the Q785R mutation presented normal mtDNA and partially affected mitochondrial morphology, similar to the D603H mutation, the S545R mutation had an intermediate behavior, borderline across phenotypes. Indeed, the complete fragmentation of the mitochondrial network was associated with only a slight, non-significant reduction of mtDNA amount, supporting a borderline nature of this variant, causing either DOA or DOA “plus” possibly depending on modulation of other genetic and/or environmental modifiers. MEFs analysis suggests that a complete fragmentation of the mitochondrial network, in association with mtDNA depletion, hallmarks the DOA “plus” phenotype, whereas if these two parameters are mildly affected the final outcome is expected to be pure DOA. This hypothesis is represented in the scheme of Fig. 10.

Instead, in P3 we found a homozygous p.Ala394Thr change; this is the second case of homozygous missense mutation in OPA1 reported to date, after the description of a patient with a severe fatal cardiomyopathy associated with the p.Leu589Arg mutation [8].

This observation was consistent with the reduced OPA1 protein levels observed in another patient (NO-1) with a complicated syndromic DOA phenotype characterised by optic atrophy and marked generalised neurodegenerative features, who has been confirmed to harbour compound heterozygous OPA1 missense mutations in exon 5b (c.768C>G, p.Ser256Arg) and exon 8 (c.854A>G, p.Gln285Arg; figure 1C).

The subjects of this study are two sisters of Arab Muslim origin and their parents are entirely healthy first-degree cousins with no visual or neurological complaints. Patient I-1 was born prematurely at 35 weeks gestation and failure to thrive was a problem since birth. At 2 months of age, she was also noted to have truncal hypotonia that was replaced by multiple episodes of hypertonia and opisthotonic posturing. Furthermore, she had a weak cry and abnormal eye pursuits. An initial echocardiography and ophthalmological examinations performed at that time were unremarkable. Metabolic investigations revealed elevated serum alanine and lactate levels, but a normal cerebrospinal fluid (CSF) lactate concentration. Disease progression was marked by a persisting failure to thrive with severe neurodevelopmental delay, but brain MRI was normal. The patient subsequently developed a progressive, non-obstructive, generalised hypertrophic cardiomyopathy, in the absence of overt heart failure. The patient died at 10 months of age following an apnoeic episode.

The clinical application of a new, widely applicable method known as Karyomapping to carry out a total of 55 clinical cases of preimplantation genetic diagnosis (PGD) for single gene disorders is reported.

II:5. A 66-year-old woman had had visual impairment since childhood and had attended a special school for the blind. She presented with a further slowly progressive deterioration in her visual acuity, a progressive gait disturbance of 20 years duration, and a 5-year history of deteriorating hearing. Shortly after presentation she developed acute paranoid delusions, low mood and visual hallucinations which resolved spontaneously. On examination, she was just able to perceive light and had bilateral optic atrophy. There was a complete external ophthalmoplegia, bilateral ptosis and bifacial weakness. Her gait was ataxic, tendon reflexes depressed and plantar responses flexor. Clinical investigations revealed an elevated serum creatine kinase (417 U/l, normal <190 U/l) with a normal fasting blood glucose and lactate. An electrocardiogram showed left axis deviation but an echocardiogram was normal. A chest X-ray and pulmonary function tests were normal. An EEG revealed evidence of a mild cortical disturbance. Brain MRI revealed mild cortical and cerebellar atrophy, and prominent small-vessel ischaemic lesions in the cerebral white matter bilaterally.

We prospectively recruited 40 patients with confirmed pathogenic OPA1 mutations from our neuro-ophthalmology clinic: 26 patients with isolated optic atrophy and 14 patients manifesting DOA+ features (Table 1). The clinical and molecular descriptions of these patients have been detailed previously.5,9 Best-corrected visual acuity was measured using the Snellen chart and converted to logarithm of the minimum angle of resolution (LogMAR) decimal values for the purpose of statistical analysis.20 The peripapillary RNFL profile in our DOA cohort was compared with age-matched normal controls recruited among members of staff of our eye unit (N=15), and the normative database (N=328) included in the Stratus OCT operating software (Carl Zeiss Meditec, Dublin, CA, USA;

As Bonneau et al. (2014) correctly point out, we did describe two siblings from a non-consanguineous Norwegian family in our original Brain paper, who developed a particularly aggressive disease course characterized by ataxia, spasticity, peripheral neuropathy and myopathy (Yu-Wai-Man et al., 2010). OPA1 sequencing identified two pathogenic variants in both the affected brother and sister: the c.768C>G (p.Ser256Arg) missense mutation in exon 5b and the c.854A>G (p.Gln285Arg) missense mutation in exon 8. Bonneau et al. (2014) rightly queried whether we had actually proven compound heterozygosity in these two affected Norwegian siblings. Although DNA was not available from their deceased parents, we did have access to DNA samples from the brother’s two unaffected daughters and both harboured only the c.768C>G (p.Ser256Arg) substitution in exon 5b. Furthermore, haplotype analysis provided additional evidence that the proband and his affected sister were indeed compound heterozygous for the c.768C>G (p.Ser256Arg) and the c.854A>G (p.Gln285Arg) OPA1 mutations (Yu-Wai-Man et al., 2010).

Download : Download high-res image (1MB)Download : Download full-size imageFig. 3. Mutated forms of fusion genes modify the shape of the mitochondrial network. Skin fibroblasts from patients with dominant mutations: MFN2:p.R94Q, OPA1:p.S545R and OPA1:p.R733X, show altered organization of the mitochondrial networks. (A) Representative images of controls and fibroblasts with OPA1:p.S545R and MFN2:p.R94Q mutations. Individual mitochondrial tubules are shown distinctively coloured. (B) Histograms indicate the number of individual mitochondrial tubules per total mitochondrial length; (C) the number of network branch points per total mitochondrial length; (D) the mitochondrial volume per cellular volume (convex hull algorithm); (E) and the bar diagram shows the distribution of mitochondrial lengths. Values of mutated skin fibroblasts are the mean ± s.d. of at least 40 measurements from two independent acquisitions. Control values are the mean ± s.d. of at least 160 measurements (X, XL or XXL cell size) from four healthy control cell lines. *P < 0.01, **P < 0.001, Mann–Whitney U-test. Scale bar: 10 μm.Dominant OPA1 mutations are associated with fragmented mitochondrial networks in fibroblasts from ADOA patients (Amati-Bonneau et al., 2005), and in HeLa cells transfected with OPA1 mutants (Olichon et al., 2007) but no reduction of mitochondrial volume has been described. In our study, the OPA1:p.S545R and OPA1:p.R733X mutations were associated with a higher ratio of individual mitochondrial tubules to total mitochondrial length (Fig. 3B). Unconnected mitochondria consisted of small tubular structures (Fig. 3E) with few dichotomous branches and a small number of connection points (Fig. 3C). Significantly, mitochondrial fragmentation in the OPA1:p.S545R cells was associated with a 20.5% reduction of mitochondrial volume compared to controls (P < 0.001; Mann–Whitney U-test) (Fig. 3D). The mitochondrial fragmentation observed in OPA1:p.R733X cells did not affect the total mitochondrial volume since the length of each individual tubule in these cells is longer than in OPA1:p.S545R cells.This fragmentation was associated with a disorganization of the mitochondrial network that did not affect cell size or shape. Interestingly, in OPA1:p.S545R cells, most mitochondrial tubular structures were found close to the MTOC (Fig. 3, Fig. 4) whereas isolated mitochondria were mainly found in the three extension arms of the triangular cell and around the nucleus. It would have been interesting to perform a statistical analysis of the variation of mitochondrial volume according to the various cell regions but the precise definition of these subregions for automatic anaylsis presented considerable difficulties. However, our visual analysis indicated that each region of the cell – central, perinuclear, and periplasmic – was equally affected by the reduction of mitochondrial volume in OPA1:p.S545R cells.Download : Download high-res image (848KB)Download : Download full-size imageFig. 4. Mitochondrial network of OPA1:p.S545R fibroblasts. (A) Mitochondrial network (in black) in control fibroblast (MitoTracker Green staining). (B) Mitochondrial network fragmentation induced by CCCP 10 μM 24 h. (C1–4) In OPA1:p.S545R fibroblasts, the major portion of the mitochondrial network was fragmented except for mitochondria emerging from the MTOC (white spots are supposed to indicate the position the MTOC). Representative images of at least 160 cells (X, XL or XXL cell size) from three control cell lines, acquired in two independent times. Scale bars: 10 μm.We noted that no tubular structures were preserved when mitochondrial fragmentation was induced by the mitochondrial uncoupler CCCP (Fig. 4B) or by siRNA OPA1 (supplementary material Fig. S4B).The expression of the GTPase-domain mutant of MFN2 (MFN2:p.K109T) in HeLa cells was shown to induce mitochondrial fragmentation (Detmer and Chan, 2007). However, standard fluorescent imaging revealed no mitochondrial network defects in heterozygous MFN2 CMT2A fibroblasts (Loiseau et al., 2007, Amiott et al., 2008). In contrast, our standardized technique of mitochondrial imaging showed that the missense mutation MFN2:p.R94Q induced mitochondrial biogenesis and the over-fusion of mitochondria. Reticulated mitochondria were distributed all over the cell (Fig. 3A) with a 24% higher number of mitochondrial network branch points compared to controls (P < 0.01; Mann–Whitney U-test) (Fig. 3C) and a 14.8% increase of mitochondrial content per cell compared to controls (P < 0.01; Mann–Whitney U-test) (Fig. 3D).In contrast to the mitochondrial fragmentation observed after neuroblastoma cell lines were transfected with GDAP1 mutants (Niemann et al., 2009), no alterations in mitochondrial network morphology were found in fibroblasts with the GDAP1:p.R120W mutation (Fig. 3).

Family 5/Case 1/M/43 + +/+ + +/+ Diffuse myalgia + RRFs/COX - + NA c.1635 C > G (p.S545R)

Here, we report the identification and cDNA analysis of four intronic and three exonic OPA1 gene mutations that cause a variety of splicing defects including activation of cryptic splice sites in either flanking exon or intron sequences, and a leaky splicing mutation. Our results show that cDNA analysis is of prime importance for the full evaluation of the effect of putative splicing mutations in the OPA1 gene.

OA-16c.1635C>Gp.S545R333.71.0-5.020/117020/200 - HM