A plasma metabolomic signature of Leber hereditary optic neuropathy showing taurine and nicotinamide deficiencies.

Leber's hereditary optic neuropathy (LHON) is the most common disorder due to mitochondrial DNA mutations and complex I deficiency. It is characterized by an acute vision loss, generally in young adults, with a higher penetrance in males. How complex I dysfunction induces the peculiar LHON clinical presentation remains an unanswered question. To gain an insight into this question, we carried out a non-targeted metabolomic investigation using the plasma of 18 LHON patients, during the chronic phase of the disease, comparing them to 18 healthy controls. A total of 500 metabolites were screened of which 156 were accurately detected. A supervised Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) highlighted a robust model for disease prediction with a Q2 (cum) of 55.5%, with a reliable performance during the permutation test (cross-validation analysis of variance, P-value = 5.02284e-05) and a good prediction of a test set (P = 0.05). This model highlighted 10 metabolites with variable importance in the projection (VIP) > 0.8. Univariate analyses revealed nine discriminating metabolites, six of which were the same as those found in the Orthogonal Projections to Latent Structures Discriminant Analysis model. In total, the 13 discriminating metabolites identified underlining dietary metabolites (nicotinamide, taurine, choline, 1-methylhistidine and hippurate), mitochondrial energetic substrates (acetoacetate, glutamate and fumarate) and purine metabolism (inosine). The decreased concentration of taurine and nicotinamide (vitamin B3) suggest interesting therapeutic targets, given their neuroprotective roles that have already been demonstrated for retinal ganglion cells. Our results show a reliable predictive metabolomic signature in the plasma of LHON patients and highlighted taurine and nicotinamide deficiencies.

Mitochondrial tRNAAla 5601C>T variant may affect the clinical expression of the LHON­related ND4 11778G>A mutation in a family.

Certain mutations in mitochondrial DNA (mtDNA) are associated with Leber's hereditary optic neuropathy (LHON). In particular, the well­known NADH dehydrogenase 4 (ND4) m.11778G>A mutation is one of the most common LHON­associated primary mutations worldwide. However, how specific mtDNA mutations, or variants, affect LHON penetrance is not fully understood. The aim of the current study was to explore the relationship between mtDNA mutations and LHON, and to provide useful information for early detection and prevention of this disease. Following the molecular characterization of a Han Chinese family with maternally inherited LHON, four out of eight matrilineal relatives demonstrated varying degrees of both visual impairment and age of onset. Through PCR amplification of mitochondrial genomes and direct Sanger sequencing analysis, a homoplasmic mitochondrial­encoded ND4 m.11778G>A mutation, alongside a set of genetic variations belonging to human mtDNA haplogroup B5b1 were identified. Among these sequence variants, alanine transfer RNA (tRNA)Ala m.5601C>T was of particular interest. This variant occurred at position 59 in the TψC loop and altered the base pairing, which led to mitochondrial RNA (mt­RNA) metabolism failure and defects in mitochondrial protein synthesis. Bioinformatics analysis suggested that the m.5601C>T variant altered tRNAAla structure. Therefore, impaired mitochondrial functions caused by the ND4 m.11778G>A mutation may be enhanced by the mt­tRNAAla m.5601C>T variant. These findings suggested that the tRNAAla m.5601C>T variant might modulate the clinical manifestation of the LHON­associated primary mutation.

Performance of the MLPA technique for detecting common mutations in Leber hereditary optic neuropathy.

Leber hereditary optic neuropathy (LHON) causes painless vision loss resulting from mitochondrial DNA (mtDNA) mutation. Over 95% of LHON cases result from one of three mtDNA point mutations (m.3460G>A, m.11778G>A, and m.14484T>C). There is no established cure for LHON; early and accurate diagnosis would enable patients to be given appropriate treatments leading to a reduction of the disease progression. To increase the accessibility to molecular genetic testing for LHON, an accurate and cost-effective technique is required. The purpose of this study was to evaluate the accuracy of multiplex ligation-dependent probe amplification (MLPA) for detecting the three common mutations in 18 LHON blood specimens. Validation of the results using direct DNA sequencing technology proved that the MLPA technique had 100% accuracy, with no false-positive results. This study demonstrates that MLPA could provide a highly accurate, economical, and widely accessible technique for routine molecular genetic testing for mitochondrial disorders.

Neuromyelitis optica antibody in Leber hereditary optic neuropathy: case report.

Neuromyelitis optica antibody (or aquaporin-4 antibody) is a well established serum marker associated to high-risk neuromyelitis optica syndrome that presents as an inflammatory demyelinating disease characterized by the occurrence of bilateral and simultaneous optic neuritis without complete visual recovery or it occurs as an isolated episode of transverse myelitis accompanied by longitudinally extensive spinal cord lesions. On the other hand, Leber hereditary optic neuropathy is a primarily hereditary disorder that affects all tissues of the body and its clinical presentation is tissue-specific for the optic nerve and, eventually, it might reach the spinal cord. Overlapping clinical features of neuromyelitis optica and Leber hereditary optic neuropathy may suggest common target organ diseases. The case report described herein emphasizes the coexistence of serum markers of both diseases, and suggests that further investigation of this challenging clinical presentation is warranted to confirm or rule out this association.

Neuromyelitis optica antibody in Leber hereditary optic neuropathy: case report / Anticorpo da neuromielite óptica em neuropatia óptica hereditária de Leber: relato de caso

Neuromyelitis optica antibody (or aquaporin-4 antibody) is a well stablished serum marker associated to high-risk neuromyelitis optica syndrome that presents as an inflammatory demyelinating disease characterized by the occurrence of bilateral and simultaneous optic neuritis without complete visual recovery or it occurs as an isolated episode of transverse myelitis accompanied by longitudinally extensive spinal cord lesions. On the other hand, Leber hereditary optic neuropathy is a primarily hereditary disorder that affects all tissues of the body and its clinical presentation is tissue-specific for the optic nerve and, eventually, it might reach the spinal cord. Overlapping clinical features of neuromyelitis optica and Leber hereditary optic neuropathy may suggest common target organ diseases. The case report described herein emphasizes the coexistence of serum markers of both diseases, and suggests that further investigation of this challenging clinical presentation is warranted to confirm or rule out this association.

Anticorpo da neuromielite óptica (ou anticorpo aquaporina-4) é um marcador sorológico bem estabelecido associado à síndrome de alto risco para neuromielite óptica, doença inflamatória desmielinizante, caracterizada por ocorrência bilateral, simultânea de neurite óptica ou por episódio isolado de mielite transversa com achado de lesões espinais longitudinais extensas. Por outro lado, a neuropatia óptica hereditária de Leber é uma doença primariamente hereditária que afeta todos os tecidos do corpo e sua apresentação clínica envolve o nervo óptico e, eventualmente, a medula espinal. Aspectos clínicos comuns sugerem que neuromielite óptica e neuropatia óptica hereditária de Leber possam atingir os mesmos órgãos. O caso descrito enfatiza a coexistência de marcadores sorológicos das duas doenças e sugere a necessidade de investigação futura desta apresentação clínica atípica para confirmar ou não esta associação.

Oxidative phosphorylation differences between mitochondrial DNA haplogroups modify the risk of Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy is a maternally inherited optic atrophy caused by mitochondrial DNA point mutations. Previous epidemiological studies have shown that individuals from mitochondrial genetic backgrounds (haplogroups) J/Uk and H have a higher and a lower risk, respectively, of suffering this disorder. To analyze the bases of these associations at cellular and molecular levels, functional studies with cybrids provide high quality evidence. Cybrids from haplogroup J contain less mitochondrial deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and synthesize a smaller amount of mitochondrial DNA-encoded polypeptides than those from haplogroup H. Haplogroup J cybrids also display lower oxygen consumption, mitochondrial inner membrane potential and total adenosine-5'-triphosphate (ATP) levels. Moreover, mitochondrial DNA levels correlate with many parameters of the oxidative phosphorylation system. These results suggest that the mitochondrial DNA amount determines oxidative phosphorylation capacity and, along with other recently published observations, support the possibility that mitochondrial DNA levels may be responsible for the bias of the disorder toward males, for the incomplete penetrance of mutations causing Leber's hereditary optic neuropathy and for the association of the disease with particular mitochondrial DNA haplogroups.

[Evaluation of serum levels of SOD and MDA in patients with Leber's hereditary optic neuropathy carrying the mitochondrial DNA G11778A mutation].

OBJECTIVE: To determine the serum levels of total superoxide dismutase (SOD) activity and malondialdehyde (MDA), and to evaluate the oxidant-antioxidant status in patients with Leber's hereditary optic neuropathy (LHON) carrying the mitochondrial G11778A mutation. METHODS: Nineteen patients and 12 carriers from three Chinese G11778A LHON families were enrolled in this study, and 30 age-matched healthy volunteers were recruited as normal controls. The serum levels of total SOD activity and MDA in all subjects were measured by xanthine oxidase test and thiobarbituric acid technique, respectively. RESULTS: The serum level of total SOD activity in LHON patients was significantly less than that in carriers and normal controls (q = 7.085 and 8.351, respectively, both P < 0.05), however, there was no significant difference between the carriers and normal controls (q = 0.269, P > 0.05). The serum level of MDA in patients and carriers was significantly higher than that in normal controls (q = 9.069 and 4.748, respectively, both P < 0.05), and it was also significantly higher in patients than that in carriers (q = 3.618, P < 0.05). CONCLUSIONS: Antioxidant capacity decreased significantly in patients with LHON, which indicates that the onset of LHON was related with the oxidation-antioxidation imbalance.

MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR.

OBJECTIVE: Leber's hereditary optic neuropathy (LHON) is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA (mtDNA). Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing. This study aims to develop a minor groove binder (MGB) probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction (PCR). METHODS: Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation, with 20 normal individuals as a control group at the same time. A real-time PCR involving two MGB probes was used to detect the mtDNA11778 mutation and heteroplasmy. A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones. RESULTS: All 48 LHON patients and their maternal relatives were positive for mtDNA11778 mutation in our assay, 27 heteroplasmic and 21 homoplasmic. Eighteen cases did not show an occurrence of the disease, while 9 developed the disease among the 27 heteroplasmic mutation cases. Eleven did not show an occurrence of the disease, while 10 cases developed the disease among 21 homoplasmic mutation cases. There was a significant difference in the incidence between the heteroplasmic and the homoplasmic mutation types. The time needed for running a real-time PCR assay was only 80 min. CONCLUSION: This real-time PCR assay is a rapid, reliable method for mtDNA mutation detection as well as heteroplasmy quantification. Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.

Oxidative stress in Chinese patients with Leber's hereditary optic neuropathy.

The cellular mechanisms of Leber's hereditary optic neuropathy (LHON) are poorly understood and there is little information on the onset of blindness and neurological degeneration. Here we define the relationship between oxidative stress and LHON pathogenicity at the cellular level. Venous blood was obtained from 14 patients with LHON, 21 asymptomatic maternal relatives and 30 normal individuals (controls). The level of free radicals in blood was assessed as luminol luminescence immediately and at 10 min after addition of phytohaemagglutinin. In LHON patients and their asymptomatic relatives, free radicals increased significantly immediately after adding phytohaemagglutinin compared with baseline and normal controls. After 10 min, however, there were no significant differences between and within the groups. These results suggest that the antioxidant capacity is reduced in the blood of patients with LHON and in asymptomatic relatives, and that oxidative stress plays a significant role in the pathogenesis of LHON.

Leber's hereditary optic neuropathy and vitamin B12 deficiency.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a maternally inherited optic neuropathy caused by mutations in mitochondrial DNA (mtDNA). It is also believed that several epigenetic factors have an influence on the development of LHON. METHODS: A case series was observed. RESULTS: Three patients who developed bilateral optic neuropathy are presented. All patients had a primary LHON mutation in their mtDNA, but also a subnormal vitamin B12 serum level at the time of presentation. CONCLUSIONS: The clinical picture of optic neuropathy associated with vitamin B12 deficiency shows similarity to that of LHON. Both involve the nerve fibres of the papillomacular bundle. The present case reports suggest that optic neuropathy in patients carrying a primary LHON mtDNA mutation may be precipitated by vitamin B12 deficiency. Therefore, known carriers should take care to have an adequate dietary intake of vitamin B12 and malabsorption syndromes like those occurring in familial pernicious anaemia or after gastric surgery should be excluded.

[Rapid genetic screening of Leber's hereditary optic neuropathy with mtDNA G11778A mutation by AS-PCR with whole blood].

OBJECTIVE: Rapid Genetic Screening of Leber's hereditary optic neuropathy (LHON) with mtDNA G11778A mutation by allele-specific polymerase chain reaction (AS-PCR) with whole blood. METHODS: Whole blood with anticoagulant was used as a template of AS-PCR for the analysis of LHON with mtDNA G11778A point mutation. The amplified DNA fragment was directly observed by electrophoretogram with ethidium bromide stained. RESULTS: The accuracy was 100% by using this method in 24 blood samples tested, and the specific of PCR of which used whole blood as template was better than one of the purified mtDNA. The reliability of the method for screening of LHON with mtDNA G11778A mutation was checked by double-blind test in 22 blood samples. CONCLUSION: This method does not need purified DNA from blood and only required one step of PCR. Thus, it is very simple, rapid and accurate for the clinical genetic screening of LHON with mtDNA G11778A point mutation.

Segregation pattern and biochemical effect of the G3460A mtDNA mutation in 27 members of LHON family.

Inheritance and expression of mitochondrial DNA (mtDNA) mutations are crucial for the pathogenesis of Leber hereditary optic neuropathy (LHON). We have investigated the segregation and functional consequences of G3460A mtDNA mutation in 27 members of a three-generation family with LHON syndrome. Specific activity of respiratory chain complex I in platelets was reduced in average to 56%, but no direct correlation between the mutation load and its biochemical expression was found. Heteroplasmy in blood, platelets and hair follicles varied from 7% to 100%. Segregation pattern exhibited tissue specificity and influence of different nuclear backgrounds in four branches of the pedigree. Longitudinal analysis revealed a significant (p=0.02) decrease in blood mutation load. Although enzyme assay showed reduction of complex I activity, our results give additional support to the hypothesis that expression of LHON mutation depends on complex nuclear-mitochondrial interaction.

mtDNA/nDNA ratio in 14484 LHON mitochondrial mutation carriers.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disease caused by mitochondrial DNA (mtDNA) mutations. In this study, the mtDNA/nuclear DNA ratio was evaluated in 11 LHON patients with the 14484 mutation, 13 asymptomatic carriers and 18 non-carrier relatives as controls, to reveal possible relationships between the disease and mtDNA content. DNAs from peripheral blood lymphocytes were subjected to quantitative PCR. Gender differences and age-dependent changes in the mtDNA content were not observed. Significant increase in the mtDNA content was observed only in the asymptomatic carriers (P<0.05). This indicated that individuals whose mtDNA content had increased and been maintained at certain levels were free from LHON development, whereas those whose levels had not, had developed LHON. Since the asymptomatic carriers are the stock of the future LHON patients, monitoring the mtDNA content in patients and their relatives may help to predict the prognosis of the disease.

Segregation of the ND4/11778 and the ND1/3460 mutations in four heteroplasmic LHON families.

Leber hereditary optic neuropathy (LHON) is an ocular disease associated with mutations in the mitochondrial DNA (mtDNA). The level of heteroplasmy in the mtDNA mutations ND4/11778 and ND1/3460 was followed over a period of 4-12 years in blood samples taken from nine members of four heteroplasmic LHON families. In addition, hair follicle and urinary tract epithelium samples of one individual were studied. The quantification of heteroplasmy was performed using the solid-phase minisequencing method. Only minor and random shifts in the heteroplasmy levels were observed over time, but there were no systematic changes towards an increasing or decreasing proportion of either LHON mutant in the individuals. This indicates that there is no selection for either mtDNA genotype but the segregation of the wild-type mtDNAs and those carrying LHON mutations is a stochastic process governed by random genetic drift. In this respect, LHON mutations seem to behave like neutral polymorphisms.

Increase of mitochondrial DNA in blood cells of patients with Leber's hereditary optic neuropathy with 11778 mutation.

AIMS: To investigate the change of mitochondrial DNA (mtDNA) content in Leber's hereditary optic neuropathy (LHON) with 11778 mutation. METHODS: Mitochondrial DNA content in 27 LHON patients with 11778 mutation, 26 asymptomatic maternal relatives, and 23 normal controls was measured using a competitive polymerase chain reaction (PCR) method. RESULTS: The mean relative content of mtDNA (with respect to the beta actin gene) in LHON patients, asymptomatic maternal relatives, and normal controls was 245.5 (162.3), 238.2 (118.4), and 156.5 (61.6), respectively. There was a statistically significant difference between patients and controls and between relatives and controls. However, no statistically significant difference between patients and unaffected relatives was found. There was no statistically significant difference in the relative content of mtDNA between all males and females carrying 11778 mtDNA mutation CONCLUSION: The results suggest that the increase in mtDNA content in LHON patients with 11778 mtDNA mutation may be due to a compensatory effect for respiratory chain defects of mitochondria. However, the increase of mtDNA content is the result rather than the cause of defective mtDNA. It still cannot explain the pathogenesis of LHON.