Cytochrome b mutations in Leber hereditary optic neuropathy.

New mutations were discovered in the apocytochrome b gene in Leber hereditary optic neuropathy probands who did not harbor either of the two known Complex I mutations (positions 3,460 and 11,778). A mutation at position 15,257 was found in eight independent probands which changed a highly conserved aspartate to asparagine, was not found in controls, and appears to be pathogenetically significant. The 15,257 mutation occurred in association with a known synergistic mutation at position 13,708 in 7/8 probands and in association with a new apocytochrome b mutation at position 15,812 in 4/8 probands. Mutations in Complex III genes may be involved in Leber hereditary optic neuropathy and multiple, simultaneous mutations occur frequently.     

Phylogenetic Analysis of the Mitochondrial Genomes from Leber Hereditary Optic Neuropathy Pedigrees

The nucleotide sequences of the mitochondrial genomes from patients with Leber hereditary optic neuropathy (LHON) were used for phylogenetic analysis to study the origin and population history of pathogenic mitochondrial mutations. Sequences of both the coding region (8300 bp) and the more rapidly evolving noncoding control region (1300 bp) were analyzed. Patients with the primary LHON mutations at nucleotides 3460, 11,778, and 14,484 were included in this study, as were LHON patients and non-LHON controls that lacked these primary mutations; some of the subjects also carried secondary LHON mutations. The phylogenetic analyses demonstrate that primary LHON mutations arose and were fixed multiple times within the population, even for the small set of LHON patients that was analyzed in these initial studies. In contrast, the secondary LHON mutations at nucleotides 4216, 4917, and 13,708 arose once: the mitochondrial genomes that carried these secondary mutations formed a well supported phylogenetic cluster that apparently arose 60,000 to 100,000 years ago. Previous studies found secondary LHON mutations at a higher frequency among LHON patients than among control subjects. However, this finding does not prove a pathogenetic role of these mutations in LHON. Instead, the increased frequency is more likely to reflect the population genetic history of secondary mutations relative to that of primary LHON mutations.     

Sequence analysis of the complete mitochondrial genome in patients with Leber's hereditary optic neuropathy lacking the three most common pathogenic DNA mutations

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder characterized by central vision loss in young adults. The majority of LHON cases around the world are associated with mutations in the mitochondrial genome at nucleotide positions (np) 3460, 11,778, and 14,484. Usually, these three mutations are screened in suspected LHON patients. The result is important not only in respect to the diagnosis but also as different LHON mutations lead to variations in expression, severity, and recovery of the disease. There are, however, a significant number of patients without any of these primary mutations. In these situations, genetic counselling of a patient and his family can be difficult. We sequenced the complete mitochondrial DNA (mtDNA) in 14 LHON patients with the typical clinical features but without a primary mtDNA mutation to evaluate the potential of extensive mutation screening for clinical purposes. Our results suggest to include the mutation at np 15,257 in a routine screening as well as the ND6 gene, a hot spot for LHON mutations. Screening for the secondary LHON mutations at np 4216 and np 13,708 may also help in making the diagnosis of LHON as these seem to modify the expression of LHON mutations. Although they do not allow to prove the clinical diagnosis, their presence increases the probability of LHON. Sequencing the complete mitochondrial genome can reveal novel and known rare disease causing mutations. However, considering the effort it adds little value for routine screening.     

Genetic and biochemical impairment of mitochondrial complex I activity in a family with Leber hereditary optic neuropathy and hereditary spastic dystonia.

A rare form of Leber hereditary optic neuropathy (LHON) that is associated with hereditary spastic dystonia has been studied in a large Dutch family. Neuropathy and ophthalmological lesions were present together in some family members, whereas only one type of abnormality was found in others. mtDNA mutations previously reported in LHON were not present. Sequence analysis of the protein-coding mitochondrial genes revealed two previously unreported mtDNA mutations. A heteroplasmic A-->G transition at nucleotide position 11696 in the ND4 gene resulted in the substitution of an isoleucine for valine at amino acid position 312. A second mutation, a homoplasmic T-->A transition at nucleotide position 14596 in the ND6 gene, resulted in the substitution of a methionine for the isoleucine at amino acid residue 26. Biochemical analysis of a muscle biopsy revealed a severe complex I deficiency, providing a link between these unique mtDNA mutations and this rare, complex phenotype including Leber optic neuropathy.     

Leber hereditary optic neuropathy: involvement of the mitochondrial ND1 gene and evidence for an intragenic suppressor mutation.

A large Queensland family has an extreme form of Leber hereditary optic neuropathy (LHON) in which several neurological abnormalities and an infantile encephalopathy are present in addition to the characteristic ophthalmological changes. Sequence analysis of the seven mitochondrial genes encoding subunits of respiratory chain complex I (NADH-ubiquinone oxidoreductase) reveals two novel features of the etiology of this mitochondrial genetic disease. The first conclusion from these studies is that the ophthalmological and neurological deficits in this family are produced by a mutation at nucleotide 4160 of the ND1 gene. This nucleotide alteration results in the substitution of proline for the highly conserved leucine residue at position 285 of the ND1 protein. Secondary-structure analysis predicts that the proline replacement disrupts a small alpha helix in a hydrophilic loop. All nine family members analyzed were homoplasmic for this mutation. The second major result from these studies is that the members of one branch of this family carry, at nucleotide 4136 of the same gene, a second mutation, also homoplasmic, which produces a cysteine-for-tyrosine replacement at position 277. The clinical and biochemical phenotypes of the family members indicate that this second nucleotide substitution may function as an intragenic suppressor mutation which ameliorates the neurological abnormalities and complex I deficiency.     

线粒体tRNAGlu A14693G可能是与Leber遗传性视神经病变相关的基因突变

收集了3个具有典型临床特征的中国汉族Leber遗传性视神经病变(Leber's hereditary optic neuropathy, LHON)家系。通过对先证者和家系其他成员进行眼科临床(如视力损害程度和发病年龄)检查, 发现这些家系成员中视力损害的外显率很低, 经mtDNA测序分析, 在tRNA^Glu 上发现了A14693G同质性突变位点, 多态性位点分别属于东亚单体型Y1b、Y1和Y1, 没有发现其他高度保守和有功能意义的突变位点。A14693G突变位于线粒体tRNAGlu高度保守区(通用位点为54位), 可能导致tRNA空间结构和稳定性发生改变, 继而影响tRNA的代谢, 导致线粒体蛋白合成功能受损和ATP障碍, 最终导致视力损害。所以, tRNAGlu A14693G突变可能是与视神经病变相关的致病性线粒体突变位点。     

A novel mtDNA C11777A mutation in Leigh syndrome

A novel mitochondrial DNA point mutation, a C-to-A mutation at nucleotide position (np) 11,777, was identified in two unrelated patients out of 100 with Leigh syndrome. This mutation converted a highly evolutionary conserved arginine to a serine at codon 340 in ND4 gene. This codon was also converted by a G-to-A mutation at np 11,778, the most common mutation associated with Leber's hereditary optic neuropathy (LHON), but the amino acid replacement was different (R340S vs. R340H). Cybrid study revealed that the percentage of heteroplasmy was correlated with complex I function and that the novel mutation caused a much more deleterious effect than the np 11,778 LHON mutation in complex I activity.     

Leber hereditary optic neuropathy: identification of the same mitochondrial ND1 mutation in six pedigrees.

Biochemical and molecular genetic evidence is presented that in six independent pedigrees the development of Leber hereditary optic neuropathy (LHON) is due to the same primary mutation in the mitochondrial ND1 gene. A LHON family from the Newcastle area of Great Britain was analyzed in depth to determine the mitochondrial genetic etiology of their disease. Biochemical assays of mitochondrial electron transport in organelles isolated from the platelet/white-blood-cell fraction have established that the members of this family have a substantial and specific lowering of flux through complex I (NADH-ubiquinone oxidoreductase). To determine the site of the primary mitochondrial gene mutation in this pedigree, all seven mitochondrial complex I genes were sequenced, in their entirety, from two family members. The primary mutation was identified as a homoplasmic transition at nucleotide 3460, which results in the substitution of threonine for alanine at position 52 of the ND1 protein. This residue occurs within a very highly conserved hydrophilic loop, is invariantly alanine or glycine in all ND1 proteins, and is adjacent to an invariant aspartic acid residue. This is only the second instance in which both a biochemical abnormality and a mitochondrial gene mutation have been identified in an LHON pedigree. The sequence analysis of the ND81 gene was extended to a further 11, unrelated LHON pedigrees that had been screened previously and found not to carry the mitochondrial ND4/R340H mutation. The ND1/A52T mutation at nucleotide 3460 was found in five of these 11 pedigrees. In contrast, this sequence change was not found in any of the 47 non-LHON controls. The possible role of secondary complex I mutations in the etiology of LHON is also addressed in these studies.     

Mitochondrial DNA mutation in an Italian family with Leber hereditary optic neuropathy

Summary Mitochondrial (mt) DNA from a Southern Italian family with Leber hereditary optic neuropathy was analyzed for the presence of the reported mutation at position 11778 of the ND4 subunit gene. The point mutation was found in mt DNA extracted from peripheral blood in all members of the family with the exclusion of the father, and was present in a homoplasmic fashion, despite the phenotypic heterogeneity of disease presentation among family members.     

Heteroplasmy analysis in the Polish patients with 11778A mutation responsible for Leber hereditary optic neuropathy

We have analysed the heteroplasmy level in 11 individuals from 3 families harbouring the mitochondrial 11778A mutation responsible for Leber hereditary optic neuropathy using last cycle hot PCR. The mutation level exceeded 90% both in affected and in unaffected individuals. We also checked whether any of the families belonged to the J haplogroup of mitochondrial DNA and obtained a negative result.     

A variant of Leber hereditary optic neuropathy characterized by recovery of vision and by an unusual mitochondrial genetic etiology.

The Tas2 and Vic2 Australian families are affected with a variant of Leber hereditary optic neuropathy (LHON). The risk of developing the optic neuropathy shows strict maternal inheritance, and the ophthalmological changes in affected family members are characteristic of LHON. However, in contrast to the common form of the disease, members of these two families show a high frequency of vision recovery. To ascertain the mitochondrial genetic etiology of the LHON in these families, both (a) the the nucleotide sequences of the seven mitochondrial genes encoding subunits of respiratory-chain complex I and (b) the mitochondrial cytochrome b gene were determined for representatives of both families. Neither family carries any of the previously identified primary mitochondrial LHON mutations: ND4/11778, ND1/3460, or ND1/4160. Instead, both LHON families carry multiple nucleotide changes in the mitochondrial complex I genes, which produce conservative amino acid changes. From the available sequence data, it is inferred that the Vic2 and Tas2 LHON families are phylogenetically related to each other and to a cluster of LHON families in which mutations in the mitochondrial cytochrome b gene have been hypothesized to play a primary etiological role. However, sequencing analysis establishes that the Vic2 and Tas2 LHON families do not carry these cytochrome b mutations. There are two hypotheses to account for the unusual mitochondrial genetic etiology of the LHON in the Tas2 and Vic2 LHON families. One possibility is that there is a primary LHON mutation within the mitochondrial genome but that it is at a site that was not included in the sequencing analyses. Alternatively, the disease in these families may result from the cumulative effects of multiple secondary LHON mutations that have less severe phenotypic consequences.     

Use of transmitochondrial cybrids to assign a complex I defect to the mitochondrial DNA-encoded NADH dehydrogenase subunit 6 gene mutation at nucleotide pair 14459 that causes Leber hereditary optic neuropathy and dystonia.

A heteroplasmic G-to-A transition at nucleotide pair (np) 14459 within the mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) gene has been identified as the cause of Leber hereditary optic neuropathy (LHON) and/or pediatric-onset dystonia in three unrelated families. This ND6 np 14459 mutation changes a moderately conserved alanine to a valine at amino acid position 72 of the ND6 protein. Enzymologic analysis of mitochondrial NADH dehydrogenase (complex I) with submitochondrial particles isolated from Epstein-Barr virus-transformed lymphoblasts revealed a 60% reduction (P < 0.005) of complex I-specific activity in patient cell lines compared with controls, with no differences in enzymatic activity for complexes II plus III, III and IV. This biochemical defect was assigned to the ND6 np 14459 mutation by using transmitochondrial cybrids in which patient Epstein-Barr virus-transformed lymphoblast cell lines were enucleated and the cytoplasts were fused to a mtDNA-deficient (p 0) lymphoblastoid recipient cell line. Cybrids harboring the np 14459 mutation exhibited a 39% reduction (p < 0.02) in complex I-specific activity relative to wild-type cybrid lines but normal activity for the other complexes. Kinetic analysis of the np 14459 mutant complex I revealed that the Vmax of the enzyme was reduced while the Km remained the same as that of wild type. Furthermore, specific activity was inhibited by increasing concentrations of the reduced coenzyme Q analog decylubiquinol. These observations suggest that the np 14459 mutation may alter the coenzyme Q-binding site of complex I.     

High frequency of mutations at position 11778 in mitochondrial ND4 gene in Japanese families with Leber's hereditary optic neuropathy

We have investigated the presence of a point mutation at position 11778 in the ND4 gene of mitochondrial DNA in 17 Japanese families with Leber's hereditary optic neuropathy (LHON), and have identified the mutation in 14 (82.4%) of the 17 families. The prevalence of this mutation appears to be much higher in Japanese patients with LHON than in patients of other ethnic origins, such as Finnish, Dutch, German, and English families.     

Rapid shift in genotype of human mitochondrial DNA in a family with Leber's hereditary optic neuropathy

Mitochondrial DNA isolated from white blood cells was investigated in families suffering from Leber's hereditary optic neuropathy. A recently described mutation at nucleotide position 11778 was present in 5 out of 12 families and heteroplasmic mitochondrial DNA was observed in 2 of these 5 families. A rapid shift in genotype was found in one of the families with heteroplasmy: the grandmother had 60 percent mitochondrial DNA mutated at nucleotide position 11778, the mother 55 percent, and the two sons at least 95 percent. These data indicate that the number of mitochondrial DNA molecules transmitted to the progeny passes a developmental bottleneck, as previously proposed to occur in bovine oogenesis.     

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.     

7例携带线粒体tRNAAlaC5601T突变的Leber遗传性视神经病变家系的相关研究

文章收集了7例携带线粒体tRNAAl。C5601T突变的中国Leber遗传性视神经病变(Leber’s hereditary opticneuropathy,LHON)的家系,通过眼科检查和遗传学分析,发现7个家系的外显率很低,分别为9.5%、14.3%、4.5%、8.3%、10.0%、22.2%和25.0%。用24对有部分重叠的引物对7个先证者线粒体DNA(Mitochondrial DNA,mtDNA)全序列进行扩增,并进行相关的分子生物学分析,结果发现这些家系均未携带G11778A、G3460A和T14484C这3个常见的原发突变位点,而在tRNAAla上发现了C5601T同质性突变,多态性位点分析分别属于东亚线粒体单体型G2、G2a1、G2a1、G2、G2b、G2a1、G2。C5601T突变位于线粒体tRNAAla的高度保守区(通用位点为59位),可能引起tRNA空间结构和稳定性发生改变,继而影响tRNA的代谢,导致线粒体蛋白和ATP合成障碍,最终导致视力损害。因此,tRNAAlaC5601T突变可能是与LHON相关的线粒体突变位点。同时低外显率提示其他因素(包括核修饰基因、环境因素)可能影响这7个中国C5601T突变家系的表型表达。     

线粒体T12338C突变可能是与Leber遗传性视神经病变相关的突变位点

Leber遗传性视神经病变变(Leber’s hereditary optic neuropathy,LHON)是一种与线粒体DNA(Mito-chondrial DNA,mtDNA)突变相关的母系遗传性眼科疾病。文章报道了两例具有典型LHON临床、分子遗传特征的中国汉族家系。首先通过对家系先证者和其他成员进行眼科相关检查,发现两个家系成员中视力都仅有先证者一人损害严重,即外显率很低。经常规的方法对母系成员进行mtDNA测序及相关软件分析,结果发现携带ND4 G11696A和ND5 T12338C同质性突变位点,多态性变异位点均属于东亚单体型F2。线粒体DNA ND4 G11696A是一个已知的与LHON相关的突变位点,而T12338C位于线粒体氧化磷酸化复合体I亚基ND5的第2个碱基,该突变使起始密码子由蛋氨酸转变成苏氨酸,并且紧连tRNALeu(CUN)的3′末端。这可能影响tRNA Leu(CUN)空间结构和稳定性发生改变,以及起始密码子改变导致线粒体ND5蛋白合成功能受损和ATP障碍,最终导致需求能量高的视神经受损和视力损害。因此,线粒体ND4 G11696A和ND5 T12338C突变可能协同作用Leber遗传性视神经病变的发生,是与LHON相关的mtDNA突变位点,但外显率很低说明突变本身不足以造成LHON的表型表达,提示其他修饰因子(核修饰基因、环境等)可能对这两个家系发病起协同作用。     

Hereditäre Optikusatrophien

Hereditary optic neuropathies comprise a group of clinically and genetically heterogeneous disorders, which can be divided into 2 subgroups: isolated hereditary optic atrophies and optic neuropathies as part of complex disorders. In the first group of isolated hereditary optic neuropathies, optic nerve dysfunction is typically the only manifestation of the disease. This group comprises autosomal dominant, autosomal recessive and X-linked recessive optic atrophy, and the mitochondrial inherited Leber’s hereditary optic neuropathy (LHON). In the second group of complex disorders, various neurologic and other systemic abnormalities are regularly observed. The most frequent cause in this group are mitochondrial DNA (mtDNA) mutations, inherited peripheral neuropathies, Charcot–Marie–Tooth disorders (CMT2A2, CMTX5), hereditary sensory neuropathy type 3 (HSAN3), Friedreich ataxia, leukodystrophies, sphingolipidoses, ceroid-lipofuscinoses, and neurodegeneration with brain iron accumulation (NBIA). In the present article, the clinical phenotypes and underlying genetic predispositions are described.     

Leber Hereditary Optic Neuropathy: How Do Mitochondrial DNA Mutations Cause Degeneration of the Optic Nerve?

Leber hereditary optic neuropathy (LHON) is an inherited form of bilateral optic atrophy in which the primary etiological event is a mutation in the mitochondrial genome. The optic neuropathy involves a loss of central vision due to degeneration of the retinal ganglion cells and optic nerve axons that subserve central vision. The primary mitochondrial mutation is necessary—but not sufficient—for development of the optic neuropathy, and secondary genetic and/or epigenetic risk factors must also be present although they are poorly defined at the present time. There is broad agreement that mutations at nucleotides 3460, 11778, and 14484 are primary LHON mutations, but there may also be other rare primary mutations. It appears that the three primary LHON mutations are associated with respiratory chain dysfunction, but the derangements may be relatively subtle. There is also debate on whether there are mitochondrial mutations that have a secondary etiological or pathogenic role in LHON. The specific pattern of the optic neuropathy may arise from a chokepoint in the optic nerve in the region of the nerve head and lamina cribosa, and which may be more severe in those LHON family members who become visually affected. It is hypothesized that the respiratory chain dysfunction leads to axoplasmic stasis and swelling, thereby blocking ganglion cell function and causing loss of vision. In some LHON patients, this loss of function is reversible in a substantial number of ganglion cells, but in others, a cell death pathway (probably apoptotic) is activated with subsequent extensive degeneration of the retinal ganglion cell layer and optic nerve.     

Leber hereditary optic neuropathy--a disease with a known molecular basis but a mysterious mechanism of pathology

Leber hereditary optic neuropathy is a maternally inherited type of blindness caused by degeneration of the optic nerve. It is caused by point mutations in mitochondrial DNA. Like in other mitochondrial diseases, its penetrance and inheritance is complicated by heteroplasmy, tissue distribution, and the bottleneck phenomenon in oocyte maturation. On the cellular level, the mechanism of the disease development is still mysterious. Currently three theories of pathomechanism of LHON are considered: biochemical, ROS (reactive oxygen species) and apoptotic.