OPA1-associated disorders: Phenotypes and pathophysiology

The OPA1 gene, encoding a dynamin-like mitochondrial GTPase, is involved in autosomal dominant optic atrophy (ADOA, OMIM #165500). ADOA, also known as Kjer's optic atrophy, affects retinal ganglion cells and the axons forming the optic nerve, leading to progressive visual loss. OPA1 gene sequencing in patients with hereditary optic neuropathies indicates that the clinical spectrum of ADOA is larger than previously thought. Specific OPA1 mutations are responsible for several distinct clinical presentations, such as ADOA with deafness (ADOAD), and severe multi-systemic syndromes, the so-called “ADOA plus” disorders, which involve neurological and neuromuscular symptoms similar to those due to mitochondrial oxidative phosphorylation defects or mitochondrial DNA instability. The study of the various clinical presentations of ADOA in conjunction with the investigation of OPA1 mutations in fibroblasts from patients with optic atrophy provides new insights into the pathophysiological mechanisms of the disease while underscoring the multiple physiological roles played by OPA1 in energetic metabolism, mitochondrial structure and maintenance, and cell death. Finally, OPA1 represents an important new paradigm for emerging neurodegenerative diseases affecting mitochondrial structure, plasticity and functions.     

Mitochondrial oxidative phosphorylation compensation may preserve vision in patients with OPA1-linked autosomal dominant optic atrophy

Autosomal Dominant Optic Atrophy (ADOA) is the most common inherited optic atrophy where vision impairment results from specific loss of retinal ganglion cells of the optic nerve. Around 60% of ADOA cases are linked to mutations in the OPA1 gene. OPA1 is a fission-fusion protein involved in mitochondrial inner membrane remodelling. ADOA presents with marked variation in clinical phenotype and varying degrees of vision loss, even among siblings carrying identical mutations in OPA1. To determine whether the degree of vision loss is associated with the level of mitochondrial impairment, we examined mitochondrial function in lymphoblast cell lines obtained from six large Australian OPA1-linked ADOA pedigrees. Comparing patients with severe vision loss (visual acuity [VA]<6/36) and patients with relatively preserved vision (VA>6/9) a clear defect in mitochondrial ATP synthesis and reduced respiration rates were observed in patients with poor vision. In addition, oxidative phosphorylation (OXPHOS) enzymology in ADOA patients with normal vision revealed increased complex II+III activity and levels of complex IV protein. These data suggest that OPA1 deficiency impairs OXPHOS efficiency, but compensation through increases in the distal complexes of the respiratory chain may preserve mitochondrial ATP production in patients who maintain normal vision. Identification of genetic variants that enable this response may provide novel therapeutic insights into OXPHOS compensation for preventing vision loss in optic neuropathies.     

Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity,leading to cytochrome c release and apoptosis

OPA1 encodes a large GTPase related to dynamins, anchored to the mitochondrial cristae inner membrane, facing the intermembrane space. OPA1 haplo-insufficiency is responsible for the most common form of autosomal dominant optic atrophy (ADOA, MIM165500), a neuropathy resulting from degeneration of the retinal ganglion cells and optic nerve atrophy. Here we show that down-regulation of OPA1 in HeLa cells using specific small interfering RNA (siRNA) leads to fragmentation of the mitochondrial network concomitantly to the dissipation of the mitochondrial membrane potential and to a drastic disorganization of the cristae. These events are followed by cytochrome c release and caspase-dependent apoptotic nuclear events. Similarly, in NIH-OVCAR-3 cells, the OPA1 siRNA induces mitochondrial fragmentation and apoptosis, the latter being inhibited by Bcl2 overexpression. These results suggest that OPA1 is a major organizer of the mitochondrial inner membrane from which the maintenance of the cristae integrity depends. As loss of OPA1 commits cells to apoptosis without any other stimulus, we propose that OPA1 is involved in the cytochrome c sequestration and might be a target for mitochondrial apoptotic effectors. Our results also suggest that abnormal apoptosis is a possible pathophysiological process leading to the retinal ganglion cells degeneration in ADOA patients.     

The <Emphasis Type="Italic">OPA1</Emphasis> Gene Mutations Are Frequent in Han Chinese Patients with Suspected Optic Neuropathy

While many patients with hereditary optic neuropathies are caused by mitochondrial DNA (mtDNA) mutations of Leber’s hereditary optic neuropathy (LHON), a significant proportion of them does not have mtDNA mutation and is caused by mutations in genes of the nuclear genome. In this study, we investigated whether the OPA1 gene, which is a pathogenic gene for autosomal dominant optic atrophy (ADOA), is frequently mutated in these patients. We sequenced all 29 exons of the OPA1 gene in 105 Han Chinese patients with suspected LHON. mtDNA copy number was quantified in blood samples from patients with and without OPA1 mutation and compared to healthy controls. In silico program-affiliated prediction, evolutionary conservation analysis, and in vitro cellular assays were performed to show the potential pathogenicity of the mutations. We identified nine OPA1 mutations in eight patients; six of them are located in exons and three are located in splicing sites. Mutation c.1172T?>?G has not been reported before. When we combined our data with 193 reported Han Chinese patients with optic neuropathy and compared to the available data of 4327 East Asians by the Exome Aggregation Consortium (ExAC), we found a significant enrichment of potentially pathogenic OPA1 mutations in Chinese patients. Cellular assays for OPA1 mutants c.869G?>?A and c.2708_2711del showed abnormalities in OPA1 isoforms, mitochondrial morphology, and cellular reactive oxygen species (ROS) level. Our results indicated that screening OPA1 mutation is needed for clinical diagnosis of patients with suspected optic neuropathy.     

Effects of OPA1 mutations on mitochondrial morphology and apoptosis: relevance to ADOA pathogenesis

To characterize the molecular links between type-1 autosomal dominant optic atrophy (ADOA) and OPA1 dysfunctions, the effects of pathogenic alleles of this dynamin on mitochondrial morphology and apoptosis were analyzed, either in fibroblasts from affected individuals, or in HeLa cells transfected with similar mutants. The alleles were missense substitutions in the GTPase domain (OPA1(G300E) and OPA1(R290Q)) or deletion of the GTPase effector domain (OPA1(Delta58)). Fragmentation of mitochondria and apoptosis increased in OPA1(R290Q) fibroblasts and in OPA1(G300E) transfected HeLa cells. OPA1(Delta58) did not influence mitochondrial morphology, but increased the sensitivity to staurosporine of fibroblasts. In these cells, the amount of OPA1 protein was half of that in control fibroblasts. We conclude that GTPase mutants exert a dominant negative effect by competing with wild-type alleles to integrate into fusion-competent complexes, whereas C-terminal truncated alleles act by haplo-insufficiency. We present a model where antagonistic fusion and fission forces maintain the mitochondrial network, within morphological limits that are compatible with cellular functions. In the retinal ganglion cells (RGCs) of patients suffering from type-1 ADOA, OPA1-driven fusion cannot adequately oppose fission, thereby rendering them more sensitive to apoptotic stimuli and eventually leading to optic nerve degeneration.     

Dominant optic atrophy: exclusion and fine genetic mapping of the candidate gene, HRY

Autosomal dominant optic atrophy (OPA1) maps to Chromosome (Chr) 3q28, and the disease interval has been refined to within 1.4 cM, flanked by the markers D3S3669 and D3S3562. HRY, the human homolog of the Drosophila segmentation gene, hairy, maps by in situ hybridization to the chromosomal region 3q28-q29. We screened for mutations in HRY in 36 patients from 18 pedigrees with dominant optic atrophy and a group of normal control individuals. Heteroduplex mutation analysis and direct sequencing of all four coding exons and one upstream putative untranslated exon were performed. No disease-associated sequence alterations were identified. A polymorphism in the untranslated region of exon 2 was found, with four alleles. PCR amplification of this part of exon 2 in four of the pedigrees affected by autosomal dominant optic atrophy mapping to chromosome 3q, followed by haplotype analysis, showed recombination between HRY and OPA1 in one pedigree. This allows us to genetically position HRY in relation to known microsatellite markers in the region, placing HRY telomeric to marker D3S3562 and centromeric to D3S1305. This is outside the published critical disease interval for dominant optic atrophy. We have, therefore, excluded HRY as the gene for dominant optic atrophy by sequence analysis, mapped it genetically, and identified a polymorphism in our population. Received: 27 February 1998 / Accepted: 8 June 1998     

线粒体融合、分裂与神经变性疾病

线粒体是一种处于高度运动状态的频繁地进行融合与分裂的细胞器.在生理状态下,线粒体的融合与分裂处于一种平衡的状态,这种平衡受线粒体融合蛋白1/2（Mfn1/2）、视神经萎缩蛋白1（OPA1）和动力相关蛋白1（Drp1）的调节. Mfn1/2介导线粒体外膜的融合,而OPA1则参与线粒体内膜的融合,这些蛋白受泛素化和蛋白水解的调控. Drp1参与线粒体的分裂过程,受多种翻译后修饰的调节,如磷酸化、泛素化、SUMO化和S 硝基化.对于神经元来说,线粒体融合分裂的动态平衡对保证神经元末梢长距离运输和能量平均分布是非常重要的.因此,线粒体融合分裂异常可能是许多神经变性疾病的致病因素之一.对线粒体融合而言,Mfn2错义突变将导致遗传性运动感觉神经病2型（CMT2A）;OPA1错义突变将引起显性遗传性视神经萎缩(ADOA),而就线粒体分裂而言,Drp1突变与多系统功能障碍的新生儿致死性相关.     

Unexpected heterogeneity due to recessive and de novo dominant mutations of GJB2 in an Iranian family with nonsyndromic hearing loss: implication for genetic counseling

Mutations in the GJB2 gene are the most common cause of nonsyndromic autosomal recessive sensorineural hearing loss (HL). A few mutations in GJB2 have also been reported to cause dominant nonsyndromic HL. Here we report a large inbred family including two individuals with nonsyndromic sensorineural hearing loss. A dominant GJB2 mutation, c.551G>A (p.R184Q), was detected in the proband, yet his parents were negative for the mutation. The second affected person had heterozygous c.35delG mutation, which was inherited from his father. Large deletions of the GJB6 gene were not detected in this family. This study highlights the importance of mutation analysis in all affected cases within a pedigree.     

Ethambutol-induced optic neuropathy linked to OPA1 mutation and mitochondrial toxicity

Ethambutol (EMB), widely used in the treatment of tuberculosis, has been reported to cause Leber’s hereditary optic neuropathy in patients carrying mitochondrial DNA mutations. We study the effect of EMB on mitochondrial metabolism in fibroblasts from controls and from a man carrying an OPA1 mutation, in whom the drug induced the development of autosomal dominant optic atrophy (ADOA). EMB produced a mitochondrial coupling defect together with a 25% reduction in complex IV activity. EMB induced the formation of vacuoles associated with decreased mitochondrial membrane potential and increased fragmentation of the mitochondrial network. Mitochondrial genetic variations may therefore be predisposing factors in EMB-induced ocular injury.     

Mutation spectrum and splicing variants in the OPA1 gene

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy that features low visual acuity leading in many cases to legal blindness. We have recently shown, with others, that mutations in the OPA1 gene encoding a dynamin-related mitochondrial protein, underlie the dominant form of optic atrophy. Here we report that OPA1 has eight mRNA isoforms as a result of the alternative splicing of exon 4 and two novel exons named 4b and 5b. In addition, we screened a cohort of 19 unrelated patients with dominant optic atrophy by direct sequencing of the 30 OPA1 exons (including exons 4b and 5b) and found mutations in 17 (89%) of them of which 8 were novel. A majority of these mutations were truncative (65%) and located in exons 8 to 28, but a number of them were amino acid changes predominantly found in the GTPase domain (exons 8 to 15). We hypothesize that at least two modifications of OPA1 may lead to dominant optic atrophy, that is alteration in GTPase activity and loss of the last seven C-terminal amino acids that putatively interact with other proteins.     

Cosegregation of the ND4 G11696A mutation with the LHON-associated ND4 G11778A mutation in a four generation Chinese family

We report here the characterization of a four-generation Han Chinese family with Leber's hereditary optic neuropathy (LHON). This Chinese family exhibited a variable severity and age-at-onset of visual loss. Notably, the average age-at-onset of vision impairment changed from 26 years (generation III) to 14 years (generation IV), with the average of 18 years in this family. In addition, 30% and 50% of matrilineal relatives in generation III and IV of this family developed visual loss with a variability of severity, ranging from blindness to normal vision. Sequence analysis of the complete mitochondrial DNA in this pedigree revealed the presence of the homoplasmic ND4 G11778A mutation and 33 other variants, belonging to the Asian haplogroup D4. Of other variants, the homoplasmic G11696A mutation in the ND4 gene is of special interest as it was implicated to be associated with LHON in a large Dutch family and five Chinese pedigrees with extremely penetrance of visual loss. In fact, the G11696A mutation caused the substitution of an isoleucine for valine at amino acid position 313, located in a predicted transmembrane region of ND4. These imply that the G11696A mutation may act in synergy with the primary LHON-associated G11778A mutation in this Chinese pedigree.     

A frameshift mutation in exon 28 of the OPA1 gene explains the high prevalence of dominant optic atrophy in the Danish population: evidence for a founder effect

Dominant optic atrophy (DOA) is a hereditary optic neuropathy characterised by decreased visual acuity, colour vision deficits, centro-coecal scotoma and optic nerve pallor. The gene OPA1, encoding a dynamin-related GTPase, has recently been identified within the genetic linkage interval for the major locus for DOA on chromosome 3q28 and shown to harbour genetic aberrations segregating with disease in DOA families. The prevalence of the disorder in Denmark is reported to be the highest of any geographical location, suggestive of a founder effect. In order to establish the genetic basis of disease in a sample of 33 apparently unrelated Danish families, we screened DNA from affected members for OPA1 gene mutations by heteroduplex analysis and direct sequencing. A novel identical mutation in exon 28 (2826delT) was associated with DOA in 14 pedigrees and led to a frameshift and abnormal OPA1 protein -COOH terminus. Haplotype analysis of a region of approximately 1 Mb flanking the OPA1 gene using eight polymorphic markers revealed a common haplotype shared by all 14 patients; this haplotype was markedly over-represented compared with ethnically matched controls. Statistical analysis confirmed significant linkage disequilibrium with DOA over approximately 600 kb encompassing the disease mutation. We have therefore demonstrated that the relatively high frequency of DOA in Denmark is attributable to a founder mutation responsible for approximately 42% of the examined families and suggest that presymptomatic screening for the (2826delT) mutation may facilitate diagnosis and genetic counselling in a significant proportion of DOA patients of Danish ancestry.     

A new vicious cycle involving glutamate excitotoxicity, oxidative stress and mitochondrial dynamics

Glutamate excitotoxicity leads to fragmented mitochondria in neurodegenerative diseases, mediated by nitric oxide and S-nitrosylation of dynamin-related protein 1, a mitochondrial outer membrane fission protein. Optic atrophy gene 1 (OPA1) is an inner membrane protein important for mitochondrial fusion. Autosomal dominant optic atrophy (ADOA), caused by mutations in OPA1, is a neurodegenerative disease affecting mainly retinal ganglion cells (RGCs). Here, we showed that OPA1 deficiency in an ADOA model influences N-methyl-D-aspartate (NMDA) receptor expression, which is involved in glutamate excitotoxicity and oxidative stress. Opa1^enu/+ mice show a slow progressive loss of RGCs, activation of astroglia and microglia, and pronounced mitochondrial fission in optic nerve heads as found by electron tomography. Expression of NMDA receptors (NR1, 2A, and 2B) in the retina of Opa1^enu/+ mice was significantly increased as determined by western blot and immunohistochemistry. Superoxide dismutase 2 (SOD2) expression was significantly decreased, the apoptotic pathway was activated as Bax was increased, and phosphorylated Bad and BcL-xL were decreased. Our results conclusively demonstrate that not only glutamate excitotoxicity and/or oxidative stress alters mitochondrial fission/fusion, but that an imbalance in mitochondrial fission/fusion in turn leads to NMDA receptor upregulation and oxidative stress. Therefore, we propose a new vicious cycle involved in neurodegeneration that includes glutamate excitotoxicity, oxidative stress, and mitochondrial dynamics.     

Heterozygous Mutation of Drosophila Opa1 Causes the Development of Multiple Organ Abnormalities in an Age-Dependent and Organ-Specific Manner

Optic Atrophy 1 (OPA1) is a ubiquitously expressed dynamin-like GTPase in the inner mitochondrial membrane. It plays important roles in mitochondrial fusion, apoptosis, reactive oxygen species (ROS) and ATP production. Mutations of OPA1 result in autosomal dominant optic atrophy (DOA). The molecular mechanisms by which link OPA1 mutations and DOA are not fully understood. Recently, we created a Drosophila model to study the pathogenesis of optic atrophy. Heterozygous mutation of Drosophila OPA1 (dOpa1) by P-element insertion results in no obvious morphological abnormalities, whereas homozygous mutation is embryonic lethal. In eye-specific somatic clones, homozygous mutation of dOpa1 causes rough (mispatterning) and glossy (decreased lens deposition) eye phenotypes in adult Drosophila. In humans, heterozygous mutations in OPA1 have been associated with mitochondrial dysfunction, which is predicted to affect multiple organs. In this study, we demonstrated that heterozygous dOpa1 mutation perturbs the visual function and an ERG profile of the Drosophila compound eye. We independently showed that antioxidants delayed the onset of mutant phenotypes in ERG and improved larval vision function in phototaxis assay. Furthermore, heterozygous dOpa1 mutation also caused decreased heart rate, increased heart arrhythmia, and poor tolerance to stress induced by electrical pacing. However, antioxidants had no effects on the dysfunctional heart of heterozygous dOpa1 mutants. Under stress, heterozygous dOpa1 mutations caused reduced escape response, suggesting abnormal function of the skeletal muscles. Our results suggest that heterozygous mutation of dOpa1 shows organ-specific pathogenesis and is associated with multiple organ abnormalities in an age-dependent and organ-specific manner.     

The novel heterozygous Thr377Arg MYOC mutation causes severe Juvenile Open Angle Glaucoma in a large Pakistani family

Glaucoma is one of the major causes of blindness worldwide with characteristic optic disc changes and elevated intraocular pressure. It is subcategorized into Primary Open Angle Glaucoma (POAG) and Juvenile Open Angle Glaucoma (JOAG) depending upon age of the disease onset. Myocilin (MYOC) is the frequently mutated gene in familial cases of glaucoma. MYOC mutations show variable phenotype and penetrance. This study was aimed to identify disease causing mutation in 8 affected of a consanguineous family diagnosed with severe form of Juvenile Open Angle Glaucoma. Homozygosity mapping with four microsatellite markers and subsequent direct sequencing of MYOC revealed a novel heterozygous transition c.1130 C>G, substituting Threonine in to Arginine at codon 377 (p.Thr377Arg) of MYOC. This mutation was segregating with phenotype in all affected and was not found in control subjects. Ophthalmological findings revealed JOAG with severe and rapidly progressive phenotype. The age of onset was in the first decade of life and maximum Intra Ocular Pressure (IOP) recorded was 25 mm Hg. Bioinformatic tools predicted C to G transition at c.1130 as pathogenic and no structural changes were predicted in protein. This is the first report of novel MYOC mutation from Pakistan; segregating as autosomal dominant trait in large family diagnosed with JOAG. Identification of novel disease causing allele in MYOC indicates genetic heterogeneity of the population. This finding will help to provide genetic counseling to the affected family and carriers of this mutation may be advised for early therapeutic intervention to avoid irreversible visual loss.     

The ND4 G11696A mutation may influence the phenotypic manifestation of the deafness-associated 12S rRNA A1555G mutation in a four-generation Chinese family

We report here the clinical, genetic and molecular characterization of a large Han Chinese family with aminoglycoside-induced and nonsyndromic hearing loss. The penetrance of hearing loss (affected matrilineal relatives/total matrilineal relatives) in this pedigree was 53%, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrance of hearing loss in this pedigree was 42%. These matrilineal relatives exhibited a wide range of severity of hearing loss, varying from profound to normal hearing. Furthermore, these affected matrilineal relatives shared some common features: bilateral hearing loss of high frequencies and symmetries. Sequence analysis of mitochondrial DNA (mtDNA) in the pedigree identified the homoplasmic 12S rRNA A1555G mutation and other 35 variants belonging to Eastern Asian haplogroup D4. Of these, the V313I (G11696A) mutation in ND4 was associated with vision loss. However, the extremely low penetrance of visual loss, and the mild biochemical defect and the presence of one/167 Chinese controls indicted that the G11696A mutation is itself not sufficient to produce a clinical phenotype. Thus, the G11696A mutation may act in synergy with the primary deafness-associated 12S rRNA A1555G mutation in this Chinese family, thereby increasing the penetrance and expressivity of hearing loss in this Chinese pedigree.     

Linkage analysis in dominant optic atrophy

A kindred of German descent was studied for dominant optic atrophy, type Kjer (McKusick catalog no. 16540). One hundred twenty-three family members were examined clinically, and 36 affected, 81 normal, and six uncertain members were ascertained. Twenty-seven markers were analyzed for 121 members. The maximum lod score obtained was 2.0 at theta = .18 for linkage between the Kidd locus and dominant optic atrophy. Twenty-eight offspring were informative with 2-generation data. There was insufficient information for the acid phosphatase locus to aid gene localization. These data suggest that the locus for dominant optic atrophy is on chromosome 2.     

Use of Targeted Exome Sequencing in Genetic Diagnosis of Chinese Familial Hypercholesterolemia

Familial hypercholesterolemia is an autosomal dominant inherited disease characterized by elevated plasma low-density lipoprotein cholesterol (LDL-C). It is mainly caused by mutations of the low-density lipoprotein receptor (LDLR) gene. Currently, the methods of whole genome sequencing or whole exome sequencing for screening mutations in familial hypercholesterolemia are not applicable in China due to high cost. We performed targeted exome sequencing of 167 genes implicated in the homozygous phenotype of a proband pedigree to identify candidate mutations, validated them in the family of the proband, studied the functions of the mutant protein, and followed up serum lipid levels after treatment. We discovered that exon 9 c.1268 T>C and exon 8 c.1129 T>G compound heterozygous mutations in the LDLR gene in the proband derived from the mother and father, respectively, in which the mutation of c.1129 T>G has not been reported previously. The mutant LDL-R protein had 57% and 52% binding and internalization functions, respectively, compared with that of the wild type. After 6 months of therapy, the LDL-C level of the proband decreased by more than 50% and the LDL-C of the other family members with heterozygous mutation also reduced to normal. Targeted exome sequencing is an effective method for screening mutation genes in familial hypercholesterolemia. The exon 8 and 9 mutations of the LDLR gene were pedigree mutations. The functions of the mutant LDL-R protein were decreased significantly compared with that of the wild type. Simvastatin plus ezetimibe was proven safe and effective in this preschool-age child.