In most cases, DOA presents as a non syndromic, bilateral optic neuropathy. Although DOA is usually diagnosed in school-aged children complaining of reading problems, the condition can manifest later, during adult life 15 16 17 . DOA patients typically experience a slowly progressive, insidious decrease of vision, which can rarely be asymmetric, although rapid decline has also been reported in adults 18 19 . The visual impairment is irreversible, usually moderate (visual acuity: 6/10 to 2/10) and highly variable between and within families. However, extreme severity (legal blindness) or very mild presentation (subclinical decrease in visual acuity) can be encountered 20 21 .

On fundus examination, the optic disk typically presents a bilateral and symmetrical pallor of its temporal side, witnessing the loss of RGC fibers entering the optic nerve (Figure 1A). The optic nerve rim is atrophic and a temporal grey crescent is often present. Optic disc excavation is not unusual, but its clinical features vary in most of the cases from that of glaucoma. Optical Coherence Tomography (OCT) discloses the reduction of the thickness of the peripapillary retinal nerve fiber layer in all four quadrants, but does not disclose alteration of other retinal layers 22 23 (Figure 1B). The visual field typically shows a caecocentral scotoma, and less frequently a central or paracentral scotoma, while peripheral visual field remains normal (Figure 1C). Importantly, there is a specific tritanopia, i.e. a blue-yellow axis of color confusion, which, when found, is strongly indicative of Kjer disease 24 25 (Figure 1D). However, in severe cases or in patients with congenital dyschromatopsia (daltonism), interpretation of the color vision defect may be more difficult. The pupillary reflex and circadian rhythms are not affected, suggesting that the melanopsin RGC are spared during the course of the disease 26 27 .

Some patients harboring the pathogenic OPA1 mutation can be asymptomatic; at the opposite end of the clinical variability spectrum, mutations of the OPA1 gene have been reported to enhance multisystemic deficits while sparing totally the optic nerve.

Anterior and/or posterior blue-dot cerulean cataract occurs in the rare DOA patients with an OPA3 mutation 28 .

Although typical DOA is associated with a progressive and irreversible loss of vision, we reported the case of a young man (23 years) who developed an isolated, progressive, painless bilateral optic neuropathy as a result of central scotomas that spontaneously recovered partial vision six months later. The patient harbored a novel heterozygous mutation in OPA1 exon 5b (c.740G > A) which was the first mutation to be described in one of the three alternative OPA1 exons 29 . In addition, we identified another original case presenting a late onset (62 years) sequencial and acute loss of vision, associated to a novel dominant mutation (c.2794C > T) in OPA1 30 , suggesting that atypical natural histories of DOA can be related to OPA1 mutations.

Syndromic DOAD and DOAplus patients experience full penetrance and usually more severe visual deficits 9 31 32 .

DOAD and DOAplus with extra-ophthalmological abnormalities represent up to 20% of DOA patients with an OPA1 mutation 6 . The most common extra-ocular sign in DOA is sensori-neural hearing loss, but other associated findings may occur later during life (myopathy and peripheral neuropathy), suggesting that there is a continuum of clinical presentations ranging from a mild “pure DOA” affecting only the optic nerve, to a severe and multisystemic presentations. Sensori-neural hearing loss associated to DOA may range from severe and congenital to subclinical 31 32 33 34 35 36 with intra- and inter- familial variations, and mostly segregate with the OPA1 R445H (c.1334G>A) mutation. In general, auditory brain stem responses, which reflect the integrity of the auditory pathway from the auditory nerve to the inferior colliculus, are absent, but both ears show normal evoked oto-acoustic emissions, reflecting the functionality of presynaptic elements and in particular that of the outer hair cells 37 .

Peripheral axonal sensory and/or motor neuropathy and proximal myopathy may be diagnosed in some DOA patients from their third decade of life onwards, as well as a combination of cerebellar and sensory ataxia in adulthood, multiple sclerosis-like illness and spastic paraplegia 9 16 38 39 . Progressive external ophthalmoplegia is also frequently diagnosed in syndromic DOAplus patients 9 . One report of a Behr syndrome associating DOA to pyramidal signs, ataxia and mental retardation was linked to an OPA1 mutation 40 and another report describing a severe neuromuscular phenotype associated to optic atrophy was described in two OPA1 compound heterozygote siblings 41 . Muscle biopsy from DOAplus patient revealed features typical of mitochondrial myopathy, as approximately 5% of all fibers were deficient in histochemical COX activity and several fibers showed evidence of subsarcolemmal accumulation of abnormal mitochondria, a phenotype known as ragged red fibers 9 31 32 .

DOA is not genetically highly heterogeneous, in comparison with many other ophthalmologic or neurodegenerative disorders (Table 1). The first DOA locus, OPA1, localised on 3q28 was initially considered as unique 42 43 . But since the discovery of the OPA1 gene in 2000 44 45 , two other loci, OPA4 and OPA5, were further identified in few families (1 for OPA4 and 2 for OPA5) presenting pure DOA 46 47 . Additional loci and genes were identified as responsible for Optic Atrophy, but either with a X-linked mode of inheritance (OPA2) 48 49 , a recessive mode of inheritance (OPA6 and OPA7) 50 51 or as syndromic recessive or dominant forms (OPA3 and OPA8) 28 52 53 . Thus to date, OPA1 is the major gene responsible for DOA, accounting for at least 75% of all the patients, whereas all the other genes or loci only contribute each for less than 1% of the patient cohort 7 .

legend: dom: dominant; ress: recessive.

Three genes have been identified to date, OPA1, OPA3 and TMEM126A (OPA7) (Table 1); all encode mitochondrial proteins ubiquitously expressed and associated to the inner mitochondrial membrane, due to the presence of at least one transmembrane domain in their sequence 51 54 55 . In OPA1, 27% of the mutations are missense, 27% are splice variant, 23.5% lead to frame shift, 16.5% are nonsense and 6% are deletion or duplication 7 . Most of them are leading to a haplo-insufficiency situation where the mutated transcript is degraded by mRNA decay, thus leading to a reduction of 50% in the amount of OPA1 protein. As a direct consequence, the different mutations in OPA1 are not related to the severity of the disease, and genotype/phenotype correlations are difficult to infer 25 . In this respect, secondary nuclear genes, but not the mitochondrial genome, are suspected to control the severity of the disease in non-syndromic patients 56 . Conversely, few missense mutations in the GTPase domain of OPA1 are responsible for syndromic cases with severe dominant negative effects 9 , because the mutated protein might interfere with and inhibit the wild-type protein. Importantly, sporadic cases, cases with de novo mutation and cases with unknown familial history, account together for 50% of all patients. Concerning OPA3, indirect evidences suggest that the 2 mutations so far reported in DOAC affect the trans-membrane domain, are fully penetrant and act in a dominant negative manner, as heterozygous carriers of a recessive mutation leading to the inhibition of OPA3 expression are asymptomatic 52 . In the case of TMEM126a, the consanguineous recessive disease is associated to a mutation introducing a stop codon at position 55, thus deleting 140 out of the 195 amino-acids composing the protein 51 .

Analysis of OPA1 functions in common cell lines (HeLa, COS) and dysfunctions in patient fibroblasts revealed a systematic susceptibility to apoptosis and mild to severe alteration of the mitochondrial respiration activity, essentially associated to a reduced energetic coupling 28 51 57 58 59 60 . In addition, the 8 OPA1 isoforms that result from alternate splicing of 3 exons (4, 4b and 5b) have discrete functions in structuring the cristae, in mitochondrial membrane dynamics, maintenance of the membrane potential, calcium clearance, interaction with the respiratory chain complexes and maintenance of mitochondrial genome integrity 61 62 63 64 65 . As a consequence, and as revealed by numerous patient fibroblast studies, mutations in OPA1 can have a direct although variable impact on these functions, 31 33 57 58 59 66 , and possibly the genetic background and aging might contribute to the mitochondrial phenotype, either in a compensatory or in an accentuating manner.

Importantly, the OPA1 gene is the fifth identified nuclear gene responsible for generating multiple deletions in the mitochondrial DNA, together with POLG1 (DNA polymerase γ), PEO1 (twinkle), SLC25A4 (ANT1) and TP (thymidine phosphorylase). The presence of multiple deletions in the mtDNA has been found in the skeletal muscle of the majority of patients harbouring OPA1 mutations, even in those with isolated optic atrophy 67 . This OPA1 related genomic instability is likely to play a crucial role in the pathophysiology of DOA, taken into account its direct functional consequence on respiratory chain capacities and may explain the convergence of clinical expressions between DOAplus syndromes and other disorders related to mutations in mtDNA.

The major concern in studying DOA pathophysiology concerns the question why RGCs are most specifically affected by this disease, while the OPA genes are expressed in all cells of the body. Histochemical studies revealed a peculiar distribution of mitochondria in retinal ganglion cells. Indeed, they are accumulated in the cell bodies and in the intra-retinal unmyelinated axons, where they form varicosities, and are conversely scarce in the myelinated part of axons after the lamina cribosa 68 69 70 71 . These observations emphasize the importance of mitochondrial network dynamic in order to maintain the appropriate intracellular distribution of the mitochondria that is critical for axonal and synaptic functions, and point to a possible pathophysiological mechanism associated to OPA1 that could jeopardize RGC survival. Alternatively, RGCs are the only neurons of the body that are exposed to the day long stress of light, which generates oxidative species favoring the apoptotic process 72 . Therefore the mitochondrial fragility conferred by OPA1 mutations, together with the photo-oxidative stress could precipitate RGCs in premature cell death. A third pathophysiological hypothesis involves the tremendous energetic requirements of RGCs, as these neurons permanently fire action potentials, in addition through axons that are not myelinated in the eye globe. As the energetic fuelling of RGCs soma is restricted in the central part of the retina, due to the physical constraints imposed by the macula blood vessel organization, one can hypothesize that due to the uncoupling of mitochondrial respiration in OPA1 cells, the ATP synthesis in patient RGCs is limited and can not fulfill the physiological energetic requirements for long term cell survival. Which of these hypotheses represents the princeps mechanism responsible for the RGC degeneration remains unknown. Nevertheless, in the last years, two mouse models with an Opa1 mutation have been generated and deeply analyzed in terms of vision; both summarize DOA in that loss of RGCs is preeminent 73 74 . Reduction of the scotopic, but not the photopic evoked potential response was found in one mouse model 75 , whereas light-adapted ERG and VEP responses revealed a significant reduction in their amplitudes in another mouse model 76 . Histological examinations revealed a decrease of the dentritic length of the RGC-On subpopulation in the retina 77 , and abnormal myelin structures, increase in micro-glia and autophagy were noticed in the optic nerve 78 . In addition, some mild neuromuscular symptoms were found, as locomotor activity was reduced and tremor observed in old animals, but no alteration of the audition was detected 79 , thus these Opa1 animals show some features of the syndromic DOA forms.

Interviewing patients about the natural history of the disease, at best in the presence of the family, is mandatory to define the timing of visual loss over time. Suspicion of DOA prompts also the search of similar visual signs among relatives. Special attention should be paid on sensorial or peripheral neuropathy symptoms that would support the hypothesis of a pathophysiology related to a mitochondrial deficit. Finding at least one affected member in two consecutive generations is indicative of a dominant trait, or eventually of a mitochondrial maternal transmission, that will further orientate the genetic investigations.

DOA is characterized by a bilateral symmetric vision loss. On funduscopic examination, the cardinal sign consists in an optic nerve pallor usually bilateral and symmetric on the temporal side in about 50% of patients and global in the other 50% 80 , especially in old or severely affected patients. In moderate cases, the optic nerve atrophy may not be visible. The neuroretinal rim is often pale and sometimes associated with a temporal pigmentary grey crescent. OCT examination discloses and quantifies the thinning of the fiber layer in the 4 cardinal directions at the optic nerve rim 23 81 . Profound papillary excavation is reported in 21% of eyes from OPA1 patients 82 . Visual fields examination typically reveals a central, centrocecal or paracentral scotoma, which may be large in severely affected individuals, and the sparing of the peripheral visual field 20 . Color vision, evaluated by the desaturated 15-Hue test discloses often a blue-yellow loss dyschromatopsy, or tritanopia 25 .

Visual evoked potentials (VEPs) are typically absent or delayed, but are not characteristic of the disease. In subclinical or mildly affected patients, no alteration of the VEPs can be found. Pattern electroretinogram (PERG) shows an abnormal N95:P50 ratio, with reduction in the amplitude of the N95 waveform suggesting alterations of the ganglion cells layer 83 .

The clinical diagnosis of an optic atrophy will orientate the genetic investigations. After collecting 5ml of blood of the patient and its relatives and preparing total DNA, the analysis of OPA1 gene will be performed on the DNA sample of the index patient by amplifying and sequencing all the 31 coding exons and their flanking intronic regions. If a mutation is identified, its segregation in the family must be analyzed and its identity has to be compared to the database hosted by the CHU of Angers, France ( http://lbbma.univ-angers.fr/lbbma.php?id=9) to find out if the mutation is already recognized as pathogenic. If not, the possible consequence of the mutation on OPA1 transcript and protein integrity should be analyzed in silico, and by assessing the expression of the mutated allele by RT-PCR amplification and sequencing. If no significant mutation is found in OPA1, then the presence of a deletion in the gene can be tested with the Multiplex Ligation Probe Amplification methodology 84 85 86 . Otherwise careful reconsideration of the anamnesis might orientate to test either OPA3 gene or the full length mitochondrial genome. If results are still negative, then when the family is large and many members are affected, genetic analysis of chromosome markers can be performed to identify the causative locus and eventually a novel pathogenic gene. Nevertheless, the identification of a morbid mutation greatly helps the genetic counseling.

Patients with extra-ophthalmological symptoms should be referred to diagnostic centers specialized in mitochondrial disorders, in order to obtain additional examinations by a multidisciplinary team including geneticists, neuro-ophthalmologists, neurologists, otorhynolaryngologists.

The diagnosis of such multisystemic mitochondrial disorders often requires the study of the functionality of the respiratory chain in order to evaluate the severity of the energetic deficiency. A skeletal muscle biopsy is usually performed to measure the enzymatic activity of the 5 respiratory complexes and the mitochondrial oxygraphy. In addition, it allows anatomo-pathological examinations to check for the presence of mtDNA deletions, cytochrome c deficient fibers and ragged red fibers. Alternatively, skin fibroblasts are also useful to evaluate the severity of respiratory chain dysfunction.

Athough OPA loci are all primarly associated to optic atrophy, in some cases they can be differentiated by the presence of secondary symptoms (Table 2) that may orient toward a particular gene or locus. OPA2: Two families mapping on the OPA2 locus Xp11.4-p11.21 were identified 48 , both presented optic atrophy from early childhood 49 , and one associated in some instances optic atrophy to mental retardation and neurological symptoms as jerks, dysarthria, dysdiadochokinesia, tremor and gait 88 89 . In both families, only male are affected and female carriers showed no abnormalities.

legend: (+): systematic; (+/-): possible; (-): never reported.

OPA3: Patients presenting dominant mutation in OPA3 gene display an early optic atrophy followed by a later anterior and/or posterior cortical cataract and dyschromatopsy without systematic axis. In some cases, patients present tremor, extrapyramidal rigidity, pes cavus and absence of deep tendon reflex 28 . Patients with OPA3 recessive mutations present the syndromic Costeff syndrome (see next paragraph).

OPA4 and OPA5: three families linked to the OPA4 or OPA5 loci present an optic atrophy that can not be differentiated from the phenotype observed in OPA1 patients: i.e. optic nerve pallor, decreased visual acuity, color vision defects, impaired VEP, and normal ERG and no extra-ocular findings 46 47 .

OPA6 and OPA7: Recessive forms of optic atrophy were described linked to the OPA6 and OPA7 loci. OPA6 patients present an early onset optic atrophy slowly progressing with a red-green dyschromatopsia 50 . Concerning OPA7, a severe juvenile-onset optic atrophy with central scotoma was found in a large multiplex inbred Algerian family and subsequently in three other Maghreb families with the same mutation in the TMEM126A gene, suggesting a founder effect. In these family, some patients presented mild auditory alterations and hypertrophic cardiopathy 51 .

OPA8: One large family with a optic atrophy undistinguishable from that related to OPA1 was recently described. In this family late-onset sensorineural hearing loss, increases of central conduction times at somato-sensory evoked potentials, and various cardiac abnormalities were also described in some patients 53 .

Leber Hereditary Optic Neuropathy (LHON) is the major differential diagnosis for optic atrophy type 1 (OPA1). LHON typically presents in young adults as painless acute or subacute visual failure, occurring sequentially in both eyes, within six months. The acute phase begins with blurring of central vision and color desaturation. The central visual acuity deteriorates to the level of counting fingers in up to 80% of cases, associated with a large centrocecal scotoma. In few cases, in particular in patients with the m.14484 G > A mutation, visual acuity may partially improve over time. Males are more commonly affected than females and women tend to develop the disorder slightly later in life and may be more severely affected, sometimes with associated multiple sclerosis like symptoms. Other neurologic abnormalities, such as a postural tremor or the loss of ankle reflexes are also found. LHON is maternally inherited by mutation in the mitochondrial genome, in most patients (95% of cases) by one of the three mutations m.11778G > A, m.14484T > C, and m.3460G > A 90 .