Hereditary Optic Neuropathy Mutation Research Articles

Mitochondrial DNA m.3243A>G mutation in ethambutol-induced optic neuropathy: A case report

A 35-year-old man with maternally inherited diabetes presented with blurring of vision in both eyes for 6 months. He previously received antituberculous therapy including ethambutol (EMB) for 2 years. Temporal disc pallor and central scotomas were noticed in both eyes. P100 latency was delayed in visual evoked potential bilaterally. In addition, an elevated blood lactate concentration was observed. Interestingly, although EMB's potential optic neuropathy toxicity was our first consideration, a m.3243A>G mutation of mitochondrial DNA was found in the blood. Herein, we reported a young patient carrying a novel Leber's hereditary optic neuropathy mutation who developed EMB-induced optic neuropathy.

The top 10 most frequently involved genes in hereditary optic neuropathies in 2186 probands.

Hereditary optic neuropathies are caused by the degeneration of retinal ganglion cells whose axons form the optic nerves, with a consistent genetic heterogeneity. As part of our diagnostic activity, we retrospectively evaluated the combination of Leber hereditary optic neuropathy mutations testing with the exon sequencing of 87 nuclear genes on 2186 patients referred for suspected hereditary optic neuropathies. The positive diagnosis rate in individuals referred for Leber hereditary optic neuropathy testing was 18% (199/1126 index cases), with 92% (184/199) carrying one of the three main pathogenic variants of mitochondrial DNA (m.11778G>A, 66.5%; m.3460G>A, 15% and m.14484T>C, 11%). The positive diagnosis rate in individuals referred for autosomal dominant or recessive optic neuropathies was 27% (451/1680 index cases), with 10 genes accounting together for 96% of this cohort. This represents an overall positive diagnostic rate of 30%. The identified top 10 nuclear genes included OPA1, WFS1, ACO2, SPG7, MFN2, AFG3L2, RTN4IP1, TMEM126A, NR2F1 and FDXR. Eleven additional genes, each accounting for less than 1% of cases, were identified in 17 individuals. Our results show that 10 major genes account for more than 96% of the cases diagnosed with our nuclear gene panel.

A broad comparative genomics approach to understanding the pathogenicity of Complex I mutations

Disease caused by mutations of mitochondrial DNA (mtDNA) are highly variable in both presentation and penetrance. Over the last 30 years, clinical recognition of this group of diseases has increased. It has been suggested that haplogroup background could influence the penetrance and presentation of disease-causing mutations; however, to date there is only one well-established example of such an effect: the increased penetrance of two Complex I Leber's hereditary optic neuropathy mutations on a haplogroup J background. This paper conducts the most extensive investigation to date into the importance of haplogroup context in the pathogenicity of mtDNA mutations in Complex I. We searched for proven human point mutations across more than 900 metazoans finding human disease-causing mutations and potential masking variants. We found more than a half of human pathogenic variants as compensated pathogenic deviations (CPD) in at least in one animal species from our multiple sequence alignments. Some variants were found in many species, and some were even the most prevalent amino acids across our dataset. Variants were also found in other primates, and in such cases, we looked for non-human amino acids in sites with high probability to interact with the CPD in folded protein. Using this “local interactions” approach allowed us to find potential masking substitutions in other amino acid sites. We suggest that the masking variants might arise in humans, resulting in variability of mutation effect in our species.

Mitochondrial Mutations in Multiple Sclerosis Patients with Atypical Optic Neuropathy

Background: Multiple sclerosis-related optic neuritis is mostly associated with good recovery. The aim of this study was to investigate the causes of progressive visual worsening in multiple sclerosis patients despite treatment. Methods: We retrospectively reviewed the medical records of multiple sclerosis patients with optic neuritis admitted to the ward of our Neurology Department between 2001 and 2020. The patients with unilateral/bilateral progressive visual loss or non-substantial recovery of visual acuity were screened for genetic testing for Leber's hereditary optic neuropathy. Results: Of 1014 multiple sclerosis patients, 411 (39%) reported having optic neuritis. During follow-up, 11 patients manifested atypical characteristics of multiple sclerosis-related optic neuritis (presence of one of the following clinical findings: bilateral simultaneous or sequential eye involvement, progressive visual loss, or no response to corticosteroids during hospitalization), while others presented with typical multiple sclerosis-related optic neuritis. Those multiple sclerosis patients with atypical characteristics of optic neuritis were screened for other possible etiologies of optic neuropathy. We found pathogenic mitochondrial mutations in 5 patients with multiple sclerosis in our study group. Conclusion: In our study group, the prevalence of mitochondrial mutations among all multiple sclerosis patients with optic neuritis was 0.12%. We strongly recommend investigating Leber's hereditary optic neuropathy mutations in MS patients if they suffer from severe or bilateral visual loss without recovery during follow-up. Because Leber's hereditary optic neuropathy mitochondrial mutations indicate relatively poor visual prognosis and have important implications for genetic counseling.

Smoking and alcohol, health-related quality of life and psychiatric comorbidities in Leber\u2019s Hereditary Optic Neuropathy mutation carriers: a prospective cohort study

BackgroundLeber’s hereditary optic neuropathy (LHON) is a rare mitochondrial disorder, characterized by acute or subacute bilateral vision loss, frequently leading to significant chronic disability, mainly in young people. The causal LHON mutations of the mitochondrial DNA have incomplete penetrance, with the highest risk of disease manifestation for male mutation carriers in the second and third decades of life. Here we evaluated smoking, alcohol drinking habits, health-related quality of life (QOL) and psychiatric comorbidities in a cohort of LHON patients and asymptomatic mutation carriers from a tertiary referral centre.MethodsCross-sectional analysis of the ongoing Munich LHON prospective cohort study. Participants included all LHON patients and asymptomatic LHON mutation carriers older than 16 years at baseline, who were recruited between February 2014 and June 2015 and consented to participate. General, neurological and ophthalmological investigations were performed, including validated questionnaires on smoking, alcohol drinking habits, depressive symptoms and health-related QOL.ResultsSeventy-one participants were included, 34 LHON patients (82% male) and 37 asymptomatic mutation carriers (19% male). Median age at baseline was 36 years (range 18–75 years). For LHON patients, median age at visual loss onset was 27 years (9 to 72 years). Smoking is more frequent in LHON patients than asymptomatic LHON mutation carriers, and significantly more frequent in both groups than in the general population. Sixty percent of LHON patients, who smoked at disease onset, stopped or significantly reduced smoking after visual loss onset, yet 40% of LHON patients continued to smoke at study baseline. Excessive alcohol consumption is more frequent in male LHON patients than in LHON asymptomatic and more frequent than in the male general population. Further, female asymptomatic LHON mutation carriers are at risk for depression and worse mental QOL scores.ConclusionsGiven the high prevalence of smoking and excessive drinking in LHON mutation carriers, implementing effective measures to reduce these risk factors may have a significant impact in reducing LHON disease conversion risk. The underrecognized prevalence of mental health issues in this population of LHON mutation carriers highlights the need for awareness and more timely diagnosis, which may lead to improved outcomes.

Mitochondrial DNA Variation of Leber's Hereditary Optic Neuropathy in Western Siberia.

Our data first represent the variety of Leber’s hereditary optic neuropathy (LHON) mutations in Western Siberia. LHON is a disorder caused by pathogenic mutations in the mitochondrial DNA (mtDNA), inherited maternally and presents mainly in young adults, predominantly males. Clinically, LHON manifests itself as painless central vision loss, resulting in early onset of disability. The epidemiology of LHON has not been fully investigated yet. In this study, we report 44 genetically unrelated families with LHON manifestation. We performed whole mtDNA genome sequencing and provided genealogical and molecular genetic data on mutations and haplogroup background of LHON patients. Known “primary” pathogenic mtDNA mutations (MITOMAP) were found in 32 families: m.11778G>A represents 53.10% (17/32), m.3460G>A—21.90% (7/32), m.14484T>C–18.75% (6/32), and rare m.10663T>C and m.3635G>A represent 6.25% (2/32). We describe potentially pathogenic m.4659G>A in one subject without known pathogenic mutations, and potentially pathogenic m.6261G>A, m.8412T>C, m.8551T>C, m.9444C>T, m.9921G>A, and m.15077G>A in families with known pathogenic mutations confirmed. We suppose these mutations could contribute to the pathogenesis of optic neuropathy development. Our results indicate that haplogroup affiliation and mutational spectrum of the Western Siberian LHON cohort substantially deviate from those of European populations.

Demographics of a Large International Population of Patients Affected by Leber’s Hereditary Optic Neuropathy

To study the demographics of Leber's hereditary optic neuropathy (LHON) using a large international database of people affected by LHON. Cross-sectional study. One thousand five hundred seventeen people affected by LHON with a known pathogenic genetic mutation. Self-reported genetic and demographic data were collected. The data were de-identified and then analyzed. Leber's hereditary optic neuropathy mutation, gender, age at vision loss onset, and geographical region. The data showed that both females and males can experience symptom onset at any age. We found a 3:1 male-to-female ratio. Interestingly, at younger than 5 years and older than 45 years, the male-to-female ratio of those becoming affected was approximately 1:1. A dramatic peak in age at onset of vision loss was found among males between 14 and 26 years of age. Disease onset in females occurred across all age groups, without any comparable dramatic peak of onset age. This study found that 10% of individuals become affected with LHON after 50 years of age. According to the literature, we found that the m.11778, m.14484, and m.3460 mutations were the most common LHON point mutations in both males and females, with a similar age at onset distribution. This was the largest study of LHON demographics to date. It showed that women carrying an LHON mutation are at higher risk of losing vision than is generally expected. Unlike the traditional 5:1 male-to-female ratio commonly reported in the literature, we found a 3:1 male-to-female ratio. Earlier studies may have harbored an ascertainment bias of overemphasizing the confirmation of this being a disease of young men. However, our data suggest that LHON is a disease that affects both females and males of all ages. This should prompt physicians to conduct genetic testing for LHON in all patients who meet the clinical criteria, regardless of whether they fit the demographics traditionally associated with the disease. Counseling about LHON should be offered to all maternal bloodline relatives, females and males of all ages, because they are at risk of sudden-onset legal blindness.

What can a comparative genomics approach tell us about the pathogenicity of mtDNA mutations in human populations?

Mitochondrial disorders are heterogeneous, showing variable presentation and penetrance. Over the last three decades, our ability to recognize mitochondrial patients and diagnose these mutations, linking genotype to phenotype, has greatly improved. However, it has become increasingly clear that these strides in diagnostics have not benefited all population groups. Recent studies have demonstrated that patients from genetically understudied populations, in particular those of black African heritage, are less likely to receive a diagnosis of mtDNA disease. It has been suggested that haplogroup context might influence the presentation and penetrance of mtDNA disease; thus, the spectrum of mutations that are associated with disease in different populations. However, to date there is only one well‐established example of such an effect: the increased penetrance of two Leber's hereditary optic neuropathy mutations on a haplogroup J background. This paper conducted the most extensive investigation to date into the importance of haplogroup context on the pathogenicity of mtDNA mutations. We searched for proven human point mutations across 726 multiple sequence alignments derived from 33 non‐human species absent of disease. A total of 58 pathogenic point mutations arise in the sequences of these species. We assessed the sequence context and found evidence of population variants that could modulate the phenotypic expression of these point mutations masking the pathogenic effects seen in humans. This supports the theory that sequence context is influential in the presentation of mtDNA disease and has implications for diagnostic practices. We have shown that our current understanding of the pathogenicity of mtDNA point mutations, primarily built on studies of individuals with haplogroups HVUKTJ, will not present a complete picture. This will have the effect of creating a diagnostic inequality, whereby individuals who do not belong to these lineages are less likely to receive a genetic diagnosis.

Food derived respiratory complex I inhibitors modify the effect of Leber hereditary optic neuropathy mutations

Mitochondrial DNA mutations in genes encoding respiratory complex I polypeptides can cause Leber hereditary optic neuropathy. Toxics affecting oxidative phosphorylation system can also cause mitochondrial optic neuropathy. Some complex I inhibitors found in edible plants might differentially interact with these pathologic mutations and modify their penetrance. To analyze this interaction, we have compared the effect of rotenone, capsaicin and rolliniastatin-1 on cybrids harboring the most frequent Leber hereditary optic neuropathy mutations and found that m.3460G > A mutation increases rotenone resistance but capsaicin and rolliniastatin-1 susceptibility. Thus, to explain the pathogenicity of mitochondrial diseases due to mitochondrial DNA mutations, their potential interactions with environment factors will have to be considered.

LHON: A look into nuclear and environmental factors; What is “sufficient”?

PurposeIn Leber's Hereditary Optic Neuropathy (LHON) mutations of the mtDNA, are necessary but not sufficient for visual loss. We studied nuclear/environmental interactions in patients and in the Wallace LHON mouse model, to consider what is “sufficient”. The LHON mutation causes a modest decrease in ATP production, but a large increase in ROS production. Nuclear genetic and environmental factors may play upon these factorsMethodsWe noted in the data of Kirkman et al (Brain 2009) that heavy smoking mitigated visual loss by delaying disease onset. We reproduced these peculiar results (Carelli et al, Brain 2013). We also studied the ND6 mutant Wallace mouse model of LHON. 30 mice of 12 groups were sacrificed at 23 months including ND6 mutant vs ND6 wildtype and nicotinamide nucleotide transhydrogenase‐NNT +/+ vs ‐/‐ (NNT deletion), under different environments of air and smoke. 10 experiments compared the axonal nerve fiber spectrums.ResultsIn our patient pedigrees, heavy smoking also mitigated visual loss, but we saw in this, the existence of two subtypes of LHON. Type I, with an abrupt and severe loss of vision in the teens, was characterized by severe losses of both structure and function. Type II occured decades later with a more insidious onset, with less structural than functional loss. Type II patients were more likely to smoke. In the mice, we found the predominant loss of small fibers in NNT −/− mice carrying the ND6 mutation which were exposed to smoke.ConclusionsBoth nuclear modifiers and environmental factors in combination with LHON mutations can lead to vision loss. NNT is a transhydrogenase that couples hydride transfer from NAD(H) to NADP(+) and proton translocation across the inner mitochondrial membrane. NNT is potentially an important nuclear modifying gene for LHON.

Identification of small molecules that improve ATP synthesis defects conferred by Leber's hereditary optic neuropathy mutations

Inherited mitochondrial complex I mutations cause blinding Leber's hereditary optic neuropathy (LHON), for which no curative therapy exists. A specific biochemical consequence of LHON mutations in the presence of trace rotenone was observed: deficient complex I-dependent ATP synthesis (CIDAS) and mitochondrial O2 consumption, proportional to the clinical severity of the three primary LHON mutations. We optimized a high-throughput assay of CIDAS to screen 1600 drugs to 2, papaverine and zolpidem, which protected CIDAS in LHON cells concentration-dependently. TSPO and cAMP were investigated as protective mechanisms, but a conclusive mechanism remains to be elucidated; next steps include testing in animal models.

Enhanced tumorigenicity by mitochondrial DNA mild mutations.

To understand how mitochondria are involved in malignant transformation we have generated a collection of transmitochondrial cybrid cell lines on the same nuclear background (143B) but with mutant mitochondrial DNA (mtDNA) variants with different degrees of pathogenicity. These include the severe mutation in the tRNALys gene, m.8363G>A, and the three milder yet prevalent Leber's hereditary optic neuropathy (LHON) mutations in the MT-ND1 (m.3460G>A), MT-ND4 (m.11778G>A) and MT-ND6 (m.14484T>C) mitochondrial genes. We found that 143B ρ0 cells devoid of mtDNA and cybrids harboring wild type mtDNA or that causing severe mitochondrial dysfunction do not produce tumors when injected in nude mice. By contrast cybrids containing mild mutant mtDNAs exhibit different tumorigenic capacities, depending on OXPHOS dysfunction.The differences in tumorigenicity correlate with an enhanced resistance to apoptosis and high levels of NOX expression. However, the final capacity of the different cybrid cell lines to generate tumors is most likely a consequence of a complex array of pro-oncogenic and anti-oncogenic factors associated with mitochondrial dysfunction.Our results demonstrate the essential role of mtDNA in tumorigenesis and explain the numerous and varied mtDNA mutations found in human tumors, most of which give rise to mild mitochondrial dysfunction.

Tobacco–alcohol amblyopia: A diagnostic dilemma

IntroductionTobacco alcohol amblyopia is an outdated term for a rare condition characterized by visual impairment due to tobacco and alcohol abuse usually associated with nutritional deficiencies. The more accurate term used now is Nutritional Optic Neuropathy. The visual impairment generally presents as a centrocecal scotoma. Its pathophysiology is poorly understood but it is generally attributed to toxic effects of cyanide and B12 deficiency. Design/subjects61year old male presented with one month history of altered mental status and progressive, painless, bilateral vision loss in the setting of severe alcohol and tobacco abuse and poor nutrition. Patient developed loss of central vision in the left-eye and blurring of vision in the right-eye after 1week, which was reduced to only perception of hand movements by the time of presentation. His BMI was 19.3 with an emaciated appearance. On ophthalmological exam, visual-acuity was 20/200 (right) and 20/300 (left), with a left central scotoma and normal fundoscopic exam. Neurological examination was significant for confabulation and gait instability consistent with Wernickes Korsakoff. Workup included negative imaging and normal B12, folate and thiamine levels, undetectable cyanide levels, and negative Leber's Hereditary Optic Neuropathy mutation. He was discharged after 30days, after a steady reversal of his symptoms in the setting of abstinence and nutritional supplementation. A diagnosis of tobacco alcohol amblyopia was made by exclusion and by history. ResultsThis case of tobacco alcohol amblyopia has an atypical presentation because of normal fundoscopic exam, undetectable cyanide levels and normal B12 levels. It reinforces the need for biomarkers for this disease. Nutritional fortification has made these cases rare, but a neurological perspective may be essential for accurate diagnosis, treatment and positive outcome for these patients. ConclusionTobacco alcohol amblyopia can be a difficult diagnosis due to lack of biomarkers and rare occurrence. A neurologist can facilitate diagnosis in atypical cases.

Leber hereditary optic neuropathy mutations and toxic‐genetic optic neuropathy — Authors' response

Leber hereditary optic neuropathy mutations and toxic‐genetic optic neuropathy — Authors' response

Effects of Topiramate on afferent visual pathways: controversies and uncertainties

We read the report by Rinalduzzi et al. [1] on a case carrying Leber hereditary optic neuropathy mutation (LHON) where Topiramate (TPM) use was presumed as a possible trigger of visual loss. In a systematic review on 74 available studies, we have established a classification for the wide range of TPM visual side effects [2]. Herein, we wish to provide a concise outline of the controversies regarding the effects of TPM on afferent visual pathways and further comments on the above-mentioned case. In our review, we could not identify any experimental or clinical evidence for any positive or negative effect of TPM on optic nerves. However, there are corresponding findings on retinal function; and the histopathology of retinal cells (RCs) and retinal ganglion cells (RGCs). A number of case studies described the presentation of maculopathy, scotoma; pigment abnormality and visual field defect in TPM consumers. Moreover, there are some clinical or experimental studies in this regard [2]. Ozturk et al. in their longitudinal clinical study on TPM consumers, investigated alterations of retina and retinal nerve fiber layer thickness. During the follow-up period (3 months) no statistically significant difference was observed in retinal thickness, though, retinal nerve fiber layer became significantly thickened [3]. Kjellstrom et al. in their rabbit experiments, evaluated the effects of TPM on full-field electroretinography (ERG) and retinal morphology. Full-field ERG revealed normal rod function in both case and control groups; though, in the case group, a significant reduction was observed in 30 Hz flicker b-wave amplitude. The cause of such a finding was disclosed in retinal histopathological investigations, where the inner plexiform layer and amacrine cells of twothird of treated rabbits were extensively accumulated with c-aminobutyric acid (GABA) substance [4]. Inconsistently, in the rat experiments of Sills et al. [5], TPM did not affect any of the measured GABA-related neurochemical parameters and did not concentrate appreciably in the retina. Yoneda et al. in their rat study investigated the effects of TPM via two experiments. In the in vitro experiment, they induced neurotoxicity in the RGCs or RCs and showed that TPM reduces neurotoxin-induced death of either of cell groups. In this experiment, they also found that TPM prevented glutamate induced RGC death at certain concentrations. In the in vivo experiment, through which retinal ischemia was induced; evaluation of the retinal histopathology (thickness of the inner plexiform layer) and S.-H. Abtahi M.-A. Abtahi H. Attarzadeh Isfahan Eye Research Center (IERC), Feiz Hospital, Isfahan University of Medical Sciences, Isfahan, Iran

Topiramate and visual loss in a patient carrying a Leber hereditary optic neuropathy mutation

We describe a 43-year-old patient who experienced visual loss 4 years after beginning antiepileptic therapy with topiramate. Ophthalmological and neurological examinations led to a preliminary diagnosis of bilateral toxic optic neuritis. Mitochondrial genome sequence analysis detected a Leber hereditary optic neuropathy 11778G>A mutation. The possibility that topiramate might favor a conversion disease, alerts physicians to seek a history of blindness in patients undergoing chronic antiepileptic therapy.

Mitochondrial DNA haplogroups and mutations in children with acquired central demyelination

We investigated mitochondrial DNA (mtDNA) variants in children with a first episode of acquired demyelinating syndromes (PD-ADS) of the CNS and their relationship to disease phenotype, including subsequent diagnosis of multiple sclerosis (MS). This exploratory analysis included the initial 213 children with PD-ADS in the prospective Canadian Pediatric Demyelinating Study and 166 matched healthy sibling controls from the Canadian Autism Genome Project. A total of 31 single nucleotide polymorphisms (SNPs) were analyzed, including haplogroup-defining SNPs and mtDNA variants previously reported to be associated with MS. Primary Leber hereditary optic neuropathy (LHON) mutations and other known pathogenic mtDNA mutations were absent in both patients with pediatric acquired demyelinating syndromes and controls. The 13708A haplogroup J-associated variant, previously linked to adult MS, was more frequent among subjects with PD-ADS (13.0%) compared to controls (6.2%; odds ratio [OR] 2.27; 95% confidence interval [CI] 1.06 to 4.83) and haplogroup M was associated with an earlier age at onset of PD-ADS (-1.74 years; 95% CI -3.33 to -0.07). In contrast, the haplogroup cluster UKJT, as well as 3 other SNPs, were each associated with a lower risk of PD-ADS. A total of 33 subjects with PD-ADS were diagnosed with MS during a mean follow-up period of 3.11 ± 1.14 (SD) years. No single SNP was associated with the risk of subsequent diagnosis of MS. However, haplogroup H was associated with an increased risk of MS (OR 2.60; 95% CI 1.21 to 5.55). These data suggest an association between mtDNA variants and the risk of PD-ADS and of a subsequent MS diagnosis. Replication of these findings in an independent population of subjects with PD-ADS is required.

Assessing mitochondrial DNA nucleotide changes in spontaneous optic neuropathies

Purpose: The high mutation rate in the mitochondrial genome makes it difficult to be certain about mtDNA pathology, and yet we now recognize several primary and provisional Leber hereditary optic neuropathy (LHON) mutations (which are commonly pathologic) and a larger number of secondary LHON mutations (which are often associated with certain primary LHON mutations and may contribute to pathogenicity), haplogroup-specific mitochondrial DNA (mtDNA) sequence variants, and simple polymorphisms (which are not commonly pathologic).Conclusions: An enormous amount of information is now known about mitochondria, the apparent dependence of the optic nerve on mitochondria, various metabolic effects of primary LHON mutations, and certain ways in which these nucleotide changes might harm the optic nerve are discussed.

Genetic Screening for OPA1 and OPA3 Mutations in Patients with Suspected Inherited Optic Neuropathies

Autosomal-dominant optic atrophy (DOA) is one of the most common inherited optic neuropathies, and it is genetically heterogeneous, with mutations in both OPA1 and OPA3 known to cause disease. Approximately 60% of cases harbor OPA1 mutations, whereas OPA3 mutations have been reported in only 2 pedigrees with DOA and premature cataracts. The aim of this study was to determine the yield of OPA1 and OPA3 screening in a cohort of presumed DOA cases referred to a tertiary diagnostic laboratory. Retrospective case series. One hundred eighty-eight probands with bilateral optic atrophy referred for molecular genetic investigations at a tertiary diagnostic facility: 38 patients with an autosomal-dominant pattern of inheritance and 150 sporadic cases. OPA1 and OPA3 genetic testing was initially performed using polymerase chain reaction-based sequencing methods. The presence of large-scale OPA1 and OPA3 genomic rearrangements was assessed further with a targeted comparative genomic hybridization microarray platform. The 3 primary Leber hereditary optic neuropathy (LHON) mutations, m.3460G→>A, m.11778G→A, and m.14484T→C, also were screened in all patients. The proportion of patients with OPA1 and OPA3 pathogenic mutations. The clinical profile observed in molecularly confirmed DOA cases. Twenty-one different OPA1 mutations were found in 27 (14.4%) of the 188 probands screened. The mutations included 6 novel pathogenic variants and the first reported OPA1 initiation codon mutation at c.1A→T. An OPA1 missense mutation, c.239A→G (p.Y80C), was identified in an 11-year-old black girl with optic atrophy and peripheral sensorimotor neuropathy in her lower limbs. The OPA1 detection rate was significantly higher among individuals with a positive family history of visual failure (50.0%) compared with sporadic cases (5.3%). The primary LHON screen was negative in the patient cohort, and additional molecular investigations did not reveal any large-scale OPA1 rearrangements or OPA3 genetic defects. The mean baseline visual acuity for the OPA1-positive group was 0.48 logarithm of the minimum angle of resolution (units mean Snellen equivalent, 20/61; range, 20/20-20/400; 95% confidence interval, 20/52-20/71), and visual deterioration occurred in 54.2% of patients during follow-up. OPA1 mutations are the most common genetic defects identified in patients with suspected DOA, whereas OPA3 mutations are very rare in isolated optic atrophy cases.

Multiplex analysis of mitochondrial DNA pathogenic and polymorphic sequence variants

The mitochondrial DNA (mtDNA) encompasses two classes of functionally important sequence variants: recent pathogenic mutations and ancient adaptive polymorphisms. To rapidly and cheaply evaluate both classes of single nucleotide variants (SNVs), we have developed an integrated system in which mtDNA SNVs are analyzed by multiplex primer extension using the SNaPshot system. A multiplex PCR amplification strategy was used to amplify the entire mtDNA, a computer program identifies optimal extension primers, and a complete global haplotyping system is also proposed. This system genotypes SNVs on multiplexed mtDNA PCR products or directly from enriched mtDNA samples and can quantify heteroplasmic variants down to 0.8% using a standard curve. With this system, we have developed assays for testing the common pathogenic mutations in four multiplex panels: two genotype the 13 most common pathogenic mtDNA mutations and two genotype the 10 most common Leber Hereditary Optic Neuropathy mutations along with haplogroups J and T. We use a hierarchal system of 140 SNVs to delineate the major global mtDNA haplogroups based on a global phylogenetic tree of coding region polymorphisms. This system should permit rapid and inexpensive genotyping of pathogenic and lineage-specific mtDNA SNVs by clinical and research laboratories.