Identification and biochemical characterization of the novel mutation m.8839G>C in the mitochondrial ATP6 gene associated with NARP syndrome

Mutations in the ATP6 gene are reported to be associated with Leber hereditary optic neuropathy, bilateral striatal necrosis, coronary atherosclerosis risk and neuropathy, ataxia and retinitis pigmentosa (NARP)/maternally inherited Leigh syndromes. Here, we present a patient with NARP syndrome, in whom a previously undescribed mutation was detected in the ATP6 gene: m.8839G>C. Several observations support the concept that m.8839G>C is pathogenically involved in the clinical phenotype of this patient: (1) the mutation was heteroplasmic in muscle; (2) mutation load was higher in the symptomatic patient than in the asymptomatic carriers; (3) cybrids carrying this mutation presented lower cell proliferation, increased mitochondrial DNA (mtDNA) copy number, increased steady‐state OxPhos protein levels and decreased mitochondrial membrane potential with respect to isogenic wild‐type cybrids; (4) this change was not observed in 2959 human mtDNAs from different mitochondrial haplogroups; (5) the affected amino acid was conserved in all the ATP6 sequences analyzed; and (6) using in silico prediction, the mutation was classified as ‘probably damaging’. However, measurement of ATP synthesis showed no differences between wild‐type and mutated cybrids. Thus, we suggest that m.8839G>C may lower the efficiency between proton translocation within F₀ and F₁ rotation, required for ATP synthesis. Further experiments are needed to fully characterize the molecular mechanisms involved in m.8839G>C pathogenicity .     

Mitochondrial haplotypes may modulate the phenotypic manifestation of the LHON-associated m.14484T>C (MT-ND6) mutation in Chinese families

Mitochondrial m.14484T>C (MT-ND6) mutation has been associated with Leber's hereditary optic neuropathy. Previous investigations revealed that the m.14484T>C mutation is a primary factor underlying the development of optic neuropathy but is not sufficient to produce a clinical phenotype. However, mitochondrial haplogroups have been proposed to modulate the phenotypic manifestation of the m.14484T>C mutation. Here, we performed the clinical, genetic evaluation and complete mitochondrial genome sequence analysis of 41 Han Chinese pedigrees carrying the m.14484T>C mutation. These families exhibited a wide range of penetrances and expressivities of optic neuropathy. The average ratio between affected male/female matrilineal relatives from 41 families was 2:1. The penetrance of optic neuropathy in these Chinese pedigrees ranged from 5.6% to 100%, with the average of 23.8%. Furthermore, the age-of-onset for optic neuropathy varied from 4 to 44 years, with the average of 19.3 years. Sequence analysis of their mitochondrial genomes identified distinct sets of polymorphisms belonging to ten Eastern Asian haplogroups, indicating that the m.14484T>C mutation occurred through recurrent origins and founder events. We showed that mitochondrial haplogroups M9, M10 and N9 increased the penetrance of optic neuropathy in these Chinese families. In particular, these mitochondrial haplogroup specific variants: m.3394T>C (MT-ND1), m.14502T>C (MT-ND4) and m.14693A>G (MT-TE) enhanced the penetrance of visual loss in these Chinese families. These data provided the direct evidence that mitochondrial modifiers modulate the variable penetrance and expressivity of optic neuropathy among Chinese pedigrees carrying the m.14484T>C mutation.     

Mechanistic insights into mitochondrial tRNAAla 3’-end metabolism deficiency

Mitochondrial tRNA 3’-end metabolism is critical for the formation of functional tRNAs. Deficient mitochondrial tRNA 3’-end metabolism is linked to an array of human diseases, including optic neuropathy, but their pathophysiology remains poorly understood. In this report, we investigated the molecular mechanism underlying the Leber’s hereditary optic neuropathy (LHON)-associated tRNA^Ala 5587A>G mutation, which changes a highly conserved adenosine at position 73 (A73) to guanine (G73) on the 3’-end of the tRNA acceptor stem. The m.5587A>G mutation was identified in three Han Chinese families with suggested maternal inheritance of LHON. We hypothesized that the m.5587A>G mutation altered tRNA^Ala 3’-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3’-end processing of tRNA^Ala precursors by RNase Z and inhibited the addition of CCA by tRNA nucleotidyltransferase (TRNT1). Northern blot analysis revealed that the m.5587A>G mutation perturbed tRNA^Ala aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNA^Ala in these cells. The impact of m.5587A>G mutation on tRNA^Ala function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNA^Ala metabolism impaired mitochondrial translation, perturbed assembly and activity of oxidative phosphorylation complexes, diminished ATP production and membrane potential, and increased production of reactive oxygen species. These pleiotropic defects elevated apoptotic cell death and promoted mitophagy in cells carrying the m.5587A>G mutation, thereby contributing to visual impairment. Our findings may provide new insights into the pathophysiology of LHON arising from mitochondrial tRNA 3’-end metabolism deficiency.     

Mitochondrial DNA Haplogroups M7b1′2 and M8a Affect Clinical Expression of Leber Hereditary Optic Neuropathy in Chinese Families with the m.11778G→A Mutation

Leber hereditary optic neuropathy (LHON) is the most extensively studied mitochondrial disease, with the majority of the cases being caused by one of three primary mitochondrial DNA (mtDNA) mutations. Incomplete disease penetrance and gender bias are two features of LHON and indicate involvement of additional genetic or environmental factors in the pathogenesis of the disorder. Haplogroups J, K, and H have been shown to influence the clinical expression of LHON in subjects harboring primary mutations in European families. However, whether mtDNA haplogroups would affect the penetrance of LHON in East Asian families has not been evaluated yet. By studying the penetrance of LHON in 1859 individuals from 182 Chinese families (including one from Cambodia) with the m.11778G→A mutation, we found that haplogroup M7b1′2 significantly increases the risk of visual loss, whereas M8a has a protective effect. Analyses of the complete mtDNA sequences from LHON families with m.11778G→A narrow the association of disease expression to m.12811T→C (Y159H) in the NADH dehydrogenase 5 gene (MT-ND5) in haplogroup M7b1′2 and suggest that the specific combination of amino acid changes (A20T-T53I) in the ATP synthase 6 protein (MT-ATP6) caused by m.8584G→A and m.8684C→T might account for the beneficial background effect of M8a. Protein secondary-structure prediction for the MT-ATP6 with the two M8a-specific amino acid changes further supported our inferences. These findings will assist in further understanding the pathogenesis of LHON and guide future genetic counseling in East Asian patients with m.11778G→A.     

Biochemical phenotypes associated with the mitochondrial ATP6 gene mutations at nt8993

Two point mutations (T > G and T > C) at the same 8993 nucleotide of mitochondrial DNA (at comparable mutant load), affecting the ATPase 6 subunit of the F₁F₀-ATPase, result in neurological phenotypes of variable severity in humans. We have investigated mitochondrial function in lymphocytes from individuals carrying the 8993T > C mutation: the results were compared with data from five 8993T > G NARP (Neuropathy, Ataxia and Retinitis Pigmentosa) patients. Both 8993T > G and 8993T > C mutations led to energy deprivation and ROS overproduction. However, the relative contribution of the two pathogenic components is different depending on the mutation considered. The 8993T > G change mainly induces an energy deficiency, whereas the 8993T > C favours an increased ROS production. These results possibly highlight the different pathogenic mechanism generated by the two mutations at position 8993 and provide useful information to better characterize the biochemical role of the highly conserved Leu-156 in ATPase 6 subunit of the mitochondrial ATP synthase complex.     

A yeast model of the neurogenic ataxia retinitis pigmentosa (NARP) T8993G mutation in the mitochondrial ATP synthase-6 gene

NARP (neuropathy, ataxia, and retinitis pigmentosa) and MILS (maternally inherited Leigh syndrome) are mitochondrial disorders associated with point mutations of the mitochondrial DNA (mtDNA) in the gene encoding the Atp6p subunit of the ATP synthase. The most common and studied of these mutations is T8993G converting the highly conserved leucine 156 into arginine. We have introduced this mutation at the corresponding position (183) of yeast Saccharomyces cerevisiae mitochondrially encoded Atp6p. The "yeast NARP mutant" grew very slowly on respiratory substrates, possibly because mitochondrial ATP synthesis was only 10% of the wild type level. The mutated ATP synthase was found to be correctly assembled and present at nearly normal levels (80% of the wild type). Contrary to what has been reported for human NARP cells, the reverse functioning of the ATP synthase, i.e. ATP hydrolysis in the F(1) coupled to F(0)-mediated proton translocation out of the mitochondrial matrix, was significantly compromised in the yeast NARP mutant. Interestingly, the oxygen consumption rate in the yeast NARP mutant was decreased by about 80% compared with the wild type, due to a selective lowering in cytochrome c oxidase (complex IV) content. This finding suggests a possible regulatory mechanism between ATP synthase activity and complex IV expression in yeast mitochondria. The availability of a yeast NARP model could ease the search for rescuing mechanisms against this mitochondrial disease.     

Juvenile parkinsonism,hypogonadism and Leigh-like MRI changes in a patient with m.4296G>A mutation in mitochondrial DNA

Leigh syndrome is a mitochondrial disease with considerable clinical and genetic variation. We present a 16-year-old boy with Leigh-like syndrome and broad developmental retardation, parkinsonism and hypogonadism. Sequencing of the entire mitochondrial DNA from blood revealed the m.4296G>A mutation in the MT-TI gene. The mutation was heteroplasmic with a 95% proportion of the mutant genome, while the proportion was 58% in the blood of the patient's clinically healthy mother. Our results suggest that m.4296G>A is pathogenic in humans, and that the phenotype related to this change includes Leigh-like syndrome in adolescence with parkinsonism and hypogonadism, in addition to the previously reported early infantile Leigh syndrome.     

Presence of mutation m.14484T>C in a Chinese family with maternally inherited essential hypertension but no expression of LHON

Essential hypertension (EH, MIM 145500) is the most common cardiovascular disease and affects one-quarter of the world's adult population. Families with EH in a mode of maternal transmission have been occasionally observed in clinical settings and suggested an involvement of mitochondrial DNA (mtDNA) mutation. We aimed to characterize the role of mtDNA mutation in EH. We reported a large Han Chinese family with a maternally inherited EH and an extraordinarily high percentage of sudden death mainly in affected females. Analysis of the entire mtDNA genome of the proband identified a homoplasmic primary mutation m.14484T>C for Leber's hereditary optic neuropathy (LHON), along with several variants indicating haplogroup F1 status. Intriguingly, no maternal member in this family had LHON though they all harbored m.14484T>C. The arterial stiffness of the members carrying mutation m.14484T>C was significantly increased than that of non-maternal members without this mutation. No environmental factor (including age, sex, smoking, diabetes, hyperlipidemia) was correlated with the decreased aortic elastic properties observed in affected members. Mitochondrial respiration rate and membrane potential (ΔΨ_m) were significantly reduced in lymphoblastoid cell lines established from affected members carrying m.14484T>C when compared to control cell lines (P < 0.05). There was an elevation of reactive oxygen species and a compensatory increase of mitochondrial mass in mutant cell lines. Our results suggest that m.14484T>C causes EH under certain circumstance. This study provides a paradigm for diverse phenotypes of the primary LHON mutation and suggests for the necessity of routine cardiac evaluation in patients with the primary LHON mutation.     

Mitochondrial DNA mutation m.3635G>A may be associated with Leber hereditary optic neuropathy in Chinese

Leber hereditary optic neuropathy (LHON) was the first disease to be linked to the presence of a mitochondrial DNA (mtDNA) mutation. Nowadays over 95% of LHON cases are known to be caused by one of three primary mutations (m.11778G>A, m.14484T>C, and m.3460G>A). Reports for other (rare) primary mutations in LHON patients are not infrequent. Among those is the mutation m.3635G>A in the MT-ND1 gene which was reported to be pathogenic in a Russian LHON family. In this study, we report on a Chinese family with clinical features of LHON but without any of the three well-known primary mutations. Analysis of the complete mitochondrial genome in the proband revealed the presence of m.3635G>A and m.6228C>T, along with a full array of other variants that suggest the haplogroup M7b1. Evolutionary analysis indicates that site 3635, but not 6228, is highly conserved in vertebrates. Protein secondary-structure modeling for the MT-ND1 protein harboring amino acid change S110N indicates that mutant m.3635G>A decreases the protein hydrophobicity. Our current observations provide further support for a pathogenic role of m.3635G>A in patients with LHON.     

Heterozygous p.Y955C mutation in DNA polymerase γ leads to alterations in bioenergetics,complex I subunit expression,and mtDNA replication

In human cells, ATP is generated using oxidative phosphorylation machinery, which is inoperable without proteins encoded by mitochondrial DNA (mtDNA). The DNA polymerase gamma (Polγ) repairs and replicates the multicopy mtDNA genome in concert with additional factors. The Polγ catalytic subunit is encoded by the POLG gene, and mutations in this gene cause mtDNA genome instability and disease. Barriers to studying the molecular effects of disease mutations include scarcity of patient samples and a lack of available mutant models; therefore, we developed a human SJCRH30 myoblast cell line model with the most common autosomal dominant POLG mutation, c.2864A>G/p.Y955C, as individuals with this mutation can present with progressive skeletal muscle weakness. Using on-target sequencing, we detected a 50% conversion frequency of the mutation, confirming heterozygous Y955C substitution. We found mutated cells grew slowly in a glucose-containing medium and had reduced mitochondrial bioenergetics compared with the parental cell line. Furthermore, growing Y955C cells in a galactose-containing medium to obligate mitochondrial function enhanced these bioenergetic deficits. Also, we show complex I NDUFB8 and ND3 protein levels were decreased in the mutant cell line, and the maintenance of mtDNA was severely impaired (i.e., lower copy number, fewer nucleoids, and an accumulation of Y955C-specific replication intermediates). Finally, we show the mutant cells have increased sensitivity to the mitochondrial toxicant 2′-3′-dideoxycytidine. We expect this POLG Y955C cell line to be a robust system to identify new mitochondrial toxicants and therapeutics to treat mitochondrial dysfunction.     

Antioxidant defence systems and generation of reactive oxygen species in osteosarcoma cells with defective mitochondria: Effect of selenium

Mitochondrial diseases originate from mutations in mitochondrial or nuclear genes encoding for mitochondrial proteome. Neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) syndrome is associated with the T8993G transversion in ATP6 gene which results in substitution at the very conservative site in the subunit 6 of mitochondrial ATP synthase. Defects in the mitochondrial respiratory chain and the ATPase are considered to be accompanied by changes in the generation of reactive oxygen species (ROS). This study aimed to elucidate effects of selenium on ROS and antioxidant system of NARP cybrid cells with 98% of T8993G mutation load. We found that selenium decreased ROS generation and increased the level and activity of antioxidant enzymes such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). Therefore, we propose selenium to be a promising therapeutic agent not only in the case of NARP syndrome but also other diseases associated with mitochondrial dysfunctions and oxidative stress.     

Impaired ATP synthase assembly associated with a mutation in the human ATP synthase subunit 6 gene

Mutations in human mitochondrial DNA are a well recognized cause of disease. A mutation at nucleotide position 8993 of human mitochondrial DNA, located within the gene for ATP synthase subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome. To enable analysis of this mutation in control nuclear backgrounds, two different cell lines were transformed with mitochondria carrying NARP mutant mitochondrial DNA. Transformant cell lines had decreased ATP synthesis capacity, and many also had abnormally high levels of two ATP synthase sub-complexes, one of which was F(1)-ATPase. A combination of metabolic labeling and immunoblotting experiments indicated that assembly of ATP synthase was slowed and that the assembled holoenzyme was unstable in cells carrying NARP mutant mitochondrial DNA compared with control cells. These findings indicate that altered assembly and stability of ATP synthase are underlying molecular defects associated with the NARP mutation in subunit 6 of ATP synthase, yet intrinsic enzyme activity is also compromised.     

Illness-induced exacerbation of Leigh syndrome in a patient with the MTATP6 mutation,m. 9185 T>C

The most common mitochondrial DNA (mtDNA) mutations giving rise to Leigh syndrome reside in the MTATP6 gene. We report a rare mutation, m. 9185 T>C that gives rise to a progressive, but episodic pattern of neurological impairment with partial recovery. Disease progression corresponded to febrile viral illness and nuclear magnetic resonance imaging (MRI) changes. The patient displayed nearly 100% homoplasmy, while his asymptomatic mother was 30%. Phenotypically, exacerbations of muscle weakness with endurance intolerance, dysarthric speech, ataxia, and eyelid ptosis accompanied febrile viral illness. This case demonstrates an episodic pattern of febrile illness-induced disease exacerbation with corresponding MRI changes.     

Cybrid studies establish the causal link between the mtDNA m.3890G>A/MT-ND1 mutation and optic atrophy with bilateral brainstem lesions

Complex I (CI) deficiency is a frequent cause of mitochondrial disorders and, in most cases, is due to mutations in CI subunit genes encoded by mitochondrial DNA (mtDNA). In this study, we establish the pathogenic role of the heteroplasmic mtDNA m.3890G>A/MT-ND1 (p.R195Q) mutation, which affects an extremely conserved amino acid position in ND1 subunit of CI. This mutation was found in a young-adult male with optic atrophy resembling Leber's hereditary optic neuropathy (LHON) and bilateral brainstem lesions. The only previously reported case with this mutation was a girl with fatal infantile Leigh syndrome with bilateral brainstem lesions. Transfer of the mutant mtDNA in the cybrid cell system resulted in a marked reduction of CI activity and CI-dependent ATP synthesis in the presence of a normally assembled enzyme.These findings establish the pathogenicity of the m.3890G>A/MT-ND1 mutation and remark the link between CI mutations affecting the mtDNA-encoded ND subunits and LHON-like optic atrophy, which may be complicated by bilateral and symmetric lesions affecting the central nervous system. Peculiar to this mutation is the distribution of the brainstem lesions, with sparing of the striatum in both patients.     

Retinal ganglion cell neurodegeneration in mitochondrial inherited disorders

Since the early days of mitochondrial medicine, it has been clear that optic atrophy is a very common and sometimes the singular pathological feature in mitochondrial disorders. The first point mutation of mitochondrial DNA (mtDNA) associated with the maternally inherited blinding disorder, Leber's hereditary optic neuropathy (LHON), was recognized in 1988. In 2000, the other blinding disorder, dominant optic atrophy (DOA) Kjer type, was found associated with mutations in the nuclear gene OPA1 that encodes a mitochondrial protein. Besides these two non-syndromic optic neuropathies, optic atrophy is a prominent feature in many other neurodegenerative diseases that are now recognized as due to primary mitochondrial dysfunction.We will consider mtDNA based syndromes such as LHON/dystonia/Mitochondrial Encephalomyopahty Lactic Acidosis Stroke-like (MELAS)/Leigh overlapping syndrome, or nuclear based diseases such as Friedreich ataxia (mutations in FXN gene), deafness-dystonia-optic atrophy (Mohr-Tranebjerg) syndrome (mutations in TIMM8A), complicated hereditary spastic paraplegia (mutations in SPG7), DOA “plus” syndromes (mutations in OPA1), Charcot-Marie-Tooth type 2A (CMT2A) with optic atrophy or hereditary motor and sensory neuropathy type VI (HMSN VI) (mutations in MFN2), and Costeff syndrome and DOA with cataract (mutations in OPA3). Thus, genetic errors in both nuclear and mitochondrial genomes often lead to retinal ganglion cell death, a specific target for mitochondrial mediated neurodegeneration. Many mechanisms have been studied and proposed as the bases for the pathogenesis of mitochondrial optic neuropathies including bioenergetic failure, oxidative stress, glutamate toxicity, abnormal mitochondrial dynamics and axonal transport, and susceptibility to apoptosis.     

Bioenergetics of mitochondrial diseases associated with mtDNA mutations

This mini-review summarizes our present view of the biochemical alterations associated with mitochondrial DNA (mtDNA) point mutations. Mitochondrial cytopathies caused by mutations of mtDNA are well-known genetic and clinical entities, but the biochemical pathogenic mechanisms are often obscure. Leber's hereditary optic neuropathy (LHON) is due to three main mutations in genes for complex I subunits. Even if the catalytic activity of complex I is maintained except in cells carrying the 3460/ND1 mutation, in all cases there is a change in sensitivity to complex I inhibitors and an impairment of mitochondrial respiration, eliciting the possibility of generation of reactive oxygen species (ROS) by the complex. Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa (NARP), is due to a mutation in the ATPase-6 gene. In NARP patients ATP synthesis is strongly depressed to an extent proportional to the mutation load; nevertheless, ATP hydrolysis and ATP-driven proton translocation are not affected. It is suggested that the NARP mutation affects the ability of the enzyme to couple proton transport to ATP synthesis. A point mutation in subunit III of cytochrome c oxidase is accompanied by a syndrome resembling MELAS: however, no major biochemical defect is found, if we except an enhanced production of ROS. The mechanism of such enhancement is at present unknown. In this review, we draw attention to a few examples in which the overproduction of ROS might represent a common step in the induction of clinical phenotypes and/or in the progression of several human pathologies associated with mtDNA point mutations.     

Hereditary spastic paraplegia-like disorder due to a mitochondrial ATP6 gene point mutation

Hereditary spastic paraplegia refers to a genetically heterogeneous syndrome. We identified five members of a family suffering from a late-onset spastic paraplegia-like disorder, carrying the homoplasmic m.9176 T > C mutation in the mitochondrial ATP6 gene. The clinical severity of the disease observed in the family was correlated with the biochemical and assembly defects of the ATP synthase. The m.9176 T > C mutation has been previously associated to Leigh syndrome or familial bilateral striatal necrosis. Other factors such as modifying genes may be involved in the phenotypic expression of the disease. The family belongs to the mitochondrial haplogroup J, previously shown to play a role in modulating the phenotype of mitochondrial diseases and be associated with longevity. Moreover other nuclear modifying genes or environmental factors may contribute to the disease phenotype. This finding extends the genetic heterogeneity of the hereditary spastic paraplegia together with the clinical spectrum of mutations of the ATP6 gene.     

Influence of a mitochondrial genetic defect on capacitative calcium entry and mitochondrial organization in the osteosarcoma cells

Effects of T8993G mutation in mitochondrial DNA (mtDNA), associated with neurogenical muscle weakness, ataxia and retinitis pigmentosa (NARP), on the cytoskeleton, mitochondrial network and calcium homeostasis in human osteosarcoma cells were investigated. In 98% NARP and rho(0) (lacking mtDNA) cells, the organization of the mitochondrial network and actin cytoskeleton was disturbed. Capacitative calcium entry (CCE) was practically independent of mitochondrial energy status in osteosarcoma cell lines. The significantly slower Ca(2+) influx rates observed in 98% NARP and rho(0), in comparison to parental cells, indicates that proper actin cytoskeletal organization is important for CCE in these cells.     

Resistance of Dynamin-related Protein 1 Oligomers to Disassembly Impairs Mitophagy,Resulting in Myocardial Inflammation and Heart Failure

We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure.     

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。