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.

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.

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;

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).

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).

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 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.

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).

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)

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.

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

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