Haplotype and phylogenetic analyses suggest that one European-specific mtDNA background plays a role in the expression of Leber hereditary optic neuropathy by increasing the penetrance of the primary mutations 11778 and 14484.

mtDNAs from 37 Italian subjects affected by Leber hereditary optic neuropathy (LHON) (28 were 11778 positive, 7 were 3460 positive, and 2 were 14484 positive) and from 99 Italian controls were screened for most of the mutations that currently are associated with LHON. High-resolution restriction-endonuclease analysis also was performed on all subjects, in order to define the phylogenetic relationships between the mtDNA haplotypes and the LHON mutations observed in patients and in controls. This analysis shows that the putative secondary/intermediate LHON mutations 4216, 4917, 13708, 15257, and 15812 are ancient polymorphisms, are associated in specific combinations, and define two common Caucasoid-specific haplotype groupings (haplogroups J and T). On the contrary, the same analysis shows that the primary mutations 11778, 3460, and 14484 are recent and are due to multiple mutational events. However, phylogenetic analysis also reveals a different evolutionary pattern for the three primary mutations. The 3460 mutations are distributed randomly along the phylogenetic trees, without any preferential association with the nine haplogroups (H, I, J, K, T, U, V, W, and X) that characterize European populations, whereas the 11778 and 14484 mutations show a strong preferential association with haplogroup J. This finding suggests that one ancient combination of haplogroup J-specific mutations increases both the penetrance of the two primary mutations 11778 and 14484 and the risk of disease expression.     

Genetic heterogeneity in Leber hereditary optic neuroretinopathy revealed by mitochondrial DNA polymorphism.

The presence or absence of a recently observed mitochondrial DNA (mtDNA) mutation associated with Leber hereditary optic neuroretinopathy (LHON) was tested in 19 Finnish families with cases of LHON. Leukocyte and muscle DNA from individuals with optic atrophy, microangiopathy, or normal fundi from maternal lineages were studied by Southern blot analysis, using mouse mtDNA as a hybridization probe. The mtDNA mutation, detected as SfaNI site polymorphism, was seen in 10 of the 19 families. In one family, the mutation was seen only in the two affected individuals, indicating recent origin for the mutation. Nine families and 28 maternally unrelated controls did not show the mutation. The results imply that alternative mtDNA mutations are associated with LHON and that this genetic heterogeneity may be the cause of the interfamilial variation in the clinical expression of LHON. In the families showing the SfaNI site mutation, the mutation was homoplasmic in all individuals irrespective of their disease status, suggesting that the intrafamilial variation in the clinical expression is not due to different ratios of mutant versus normal mtDNA.     

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.     

The MT-ND1 and MT-ND5 genes are mutational hotspots for Chinese families with clinical features of LHON but lacking the three primary mutations

LHON is one of the most common and primary causes of acute blindness in young male adults. Over 95% of LHON cases are caused by one of the three primary mutations (m.11778G>A, m.14484T>C, and m.3460G>A). In contrast to these genetically diagnosed LHON patients, there are many patients with clinical features of LHON but without the three primary mutations, and these patients have been insufficiently analyzed. We reported 10 suspected Chinese LHON families without the three primary mutations. The overall penetrance (53.4%) in these families is significantly higher than in those families with m.11778G>A (33.3%) or m.3460G>A (25.6%). Complete mtDNA genome sequencing of the 10 families showed that they belonged to different haplogroups and all identified variants (excluding m.12332A>G in mt-tRNA^Leu) were previously reported. Eight of 12 private non-synonymous variants in the probands are located in the MT-ND1 and MT-ND5 genes, which is substantially higher than that of individuals from general Chinese populations. Comparison of the private variants in the 10 families and in 10 randomly selected mtDNAs from general Chinese populations using resampling simulation strategy further confirmed this pattern. Our results suggest that the MT-ND1 and MT-ND5 genes are mutational hotspots for Chinese families with suspected LHON lacking the common primary mutations. Variants m.3736G>A (p.V144I) in family Le1235 and m.10680G>A (p.A71T) in Le1107 can be the pathogenic mutations for LHON.     

Novel A14841G mutation is associated with high penetrance of LHON/C4171A family

We report the clinical and genetic characterization of a Chinese LHON family carrying an ND1/C4171A mutation. This family has high penetrance of visual impairment and extremely low frequency of vision recovery, which is in marked contrast to previously reported results for Korean LHON families with the same mutation. Sequence analysis of the complete mtDNA in the partially defined East Asian haplogroup N9a1 revealed the presence of 29 other variants. A novel heteroplasmic A14841G mutation, one of the variants with a serine substituted for a highly conserved asparagine at amino acid 32 of Cytochrome b (Cytb), may play a synergistic role with the C4171A mutation, leading to significantly different clinical manifestations of LHON among these families. The study further confirmed that C4171A was a rare primary LHON mutation, and the mtDNA background could also contribute to the clinical manifestation of the LHON/C4171A mutation.     

The epidemiology of Leber hereditary optic neuropathy in the North East of England

We performed the first population-based clinical and molecular genetic study of Leber hereditary optic neuropathy (LHON) in a population of 2,173,800 individuals in the North East of England. We identified 16 genealogically unrelated families who harbor one of the three primary mitochondrial DNA (mtDNA) mutations that cause LHON. Two of these families were found to be linked genetically to a common maternal founder. A de novo mtDNA mutation (G3460A) was identified in one family. The minimum point prevalence of visual failure due to LHON within this population was 3.22 per 100,000 (95% CI 2.47-3.97 per 100,000), and the minimum point prevalence for mtDNA LHON mutations was 11.82 per 100,000 (95% CI 10.38-13.27 per 100,000). These results indicate that LHON is not rare but has a population prevalence similar to autosomally inherited neurological disorders. The majority of individuals harbored only mutant mtDNA (homoplasmy), but heteroplasmy was detected in approximately 12% of individuals. Overall, however, approximately 33% of families with LHON had at least one heteroplasmic individual. The high incidence of heteroplasmy in pedigrees with LHON raises the possibility that a closely related maternal relative of an index case may not harbor the mtDNA mutation, highlighting the importance of molecular genetic testing for each maternal family member seeking advice about their risks of visual failure.     

Evidence against an X-linked locus close to DXS7 determining visual loss susceptibility in British and Italian families with Leber hereditary optic neuropathy.

Leber hereditary optic neuropathy (LHON) is associated with mutations of mtDNA, but two features of LHON pedigrees are not explicable solely on the basis of mitochondrial inheritance. There is a large excess of affected males, and not all males at risk develop the disease. These observations could be explained by the existence of an X-linked visual loss susceptibility gene. This hypothesis was supported by linkage studies in Finland, placing the susceptibility locus at DXS7, with a maximum lod score of 2.48 at a recombination fraction of 0. Linkage studies in 1 Italian and 12 British families with LHON, analyzed either together or separately depending on the associated mtDNA mutation, have excluded the presence of such a locus from an interval of about 30 cM around DXS7 in these kindreds, with a total lod score of -26.51 at a recombination fraction of 0.     

Low penetrance of Leber's hereditary optic neuropathy in ten Han Chinese families carrying the ND6 T11484C mutation

Background

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited disorder. The purpose of this investigation is to understand the role of mitochondrial haplotypes in the development of LHON associated with ND6 T14484C mutation in Chinese families.

Methods

One hundred fourteen subjects from ten Han Chinese families with LHON were studied by the clinical and genetic evaluation as well as molecular and biochemical analyses of mitochondrial DNA (mtDNA).

Results

Clinical evaluation revealed that ten families exhibited extremely low penetrance of visual impairment, with an average of 10%. In particular, ten (8 males/2 females) of 114 matrilineal relatives in these families exhibited the variable severity and age-at-onset in visual dysfunction. The average age-of-onset of vision loss was 19 years old. Molecular analysis of mitochondrial DNA (mtDNA) identified the homoplasmic T14484C mutation and distinct sets of variants, belonging to the Asian haplogroups B5b, D4, D4g1b, G3a2, R11, R11a and Z3, respectively. However, there was the absence of secondary LHON-associated mtDNA mutations in these ten Chinese families.

Conclusion

The low penetrance of vision loss in these Chinese pedigrees strongly indicated that the T14484C mutation was itself insufficient to produce a clinical phenotype. The absence of secondary LHON mtDNA mutations suggests that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the T14484C mutation in those Chinese families with low penentrace of vision loss. However, nuclear modifier genes and environmental factors appear to be modifier factors for the phenotypic manifestation of the T14484C mutation in these Chinese families.     

Sequence analysis of the mitochondrial genomes from Dutch pedigrees with Leber hereditary optic neuropathy

The complete mitochondrial DNA (mtDNA) sequences for 63 Dutch pedigrees with Leber hereditary optic neuropathy (LHON) were determined, 56 of which carried one of the classic LHON mutations at nucleotide (nt) 3460, 11778, or 14484. Analysis of these sequences indicated that there were several instances in which the mtDNAs were either identical or related by descent. The most striking example was a haplogroup J mtDNA that carried the 14484 LHON mutation. Four different but related mitochondrial genotypes were identified in seven of the Dutch pedigrees with LHON, including six of those described by van Senus. The control region of the founder sequence for these Dutch pedigrees with LHON matches the control-region sequence that Macmillan and colleagues identified in the founder mtDNA of French Canadian pedigrees with LHON. In addition, we obtained a perfect match between the Dutch 14484 founder sequence and the complete mtDNA sequences of two Canadian pedigrees with LHON. Those results indicate that these Dutch and French Canadian 14484 pedigrees with LHON share a common ancestor, that the single origin of the 14484 mutation in this megalineage occurred before the year 1600, and that there is a 14484/haplogroup J founder effect. We estimate that this lineage--including the 14484 LHON mutation--arose 900-1,800 years ago. Overall, the phylogenetic analyses of these mtDNA sequences conservatively indicate that a LHON mutation has arisen at least 42 times in the Dutch population. Finally, analysis of the mtDNA sequences from those pedigrees that did not carry classic LHON mutations suggested candidate pathogenic mutations at nts 9804, 13051, and 14325.     

The role of mtDNA background in disease expression: a new primary LHON mutation associated with Western Eurasian haplogroup J

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted form of blindness caused by mitochondrial DNA (mtDNA) mutations. Approximately 90% of LHON cases are caused by 3460A, 11778A, or 14484C mtDNA mutations. These are designated "primary" mutations because they impart a high risk for LHON expression. Although the 11778A and 14484C mutations unequivocally predispose carriers to LHON, they are preferentially associated with mtDNA haplogroup J, one of nine Western Eurasian mtDNA lineages, suggesting a synergistic and deleterious interaction between these LHON mutations and haplogroup J polymorphism(s). We report here the characterization of a new primary LHON mutation in the mtDNA ND4L gene at nucleotide pair 10663. The homoplasmic 10663C mutation has been found in three independent LHON patients who lack a known primary mutation and all of which belong to haplogroup J. This mutation has not been found in a large number of haplotype-matched or non-haplogroup-J control mtDNAs. Phylogenetic analysis with primarily complete mtDNA sequence data demonstrates that the 10663C mutation has arisen at least three independent times in haplogroup J, indicating that it is not a rare lineage-specific polymorphism. Analysis of complex I function in patient lymphoblasts and transmitochondrial cybrids has revealed a partial complex I defect similar in magnitude to the 14484C mutation. Thus, the 10663C mutation appears to be a new primary LHON mutation that is pathogenic when co-occurring with haplogroup J. These results strongly support a role for haplogroup J in the expression of certain LHON mutations.     

Clustering of Caucasian Leber hereditary optic neuropathy patients containing the 11778 or 14484 mutations on an mtDNA lineage.

Leber hereditary optic neuropathy (LHON) is a type of blindness caused by mtDNA mutations. Three LHON mtDNA mutations at nucleotide positions 3460, 11778, and 14484 are specific for LHON and account for 90% of worldwide cases and are thus designated as "primary" LHON mutations. Fifteen other "secondary" LHON mtDNA mutations have been identified, but their pathogenicity is unclear. mtDNA haplotype and phylogenetic analysis of the primary LHON mutations in North American Caucasian patients and controls has shown that, unlike the 3460 and 11778 mutations, which are distributed throughout the European-derived (Caucasian) mtDNA phylogeny, patients containing the 14484 mutation tended to be associated with European mtDNA haplotype J. To investigate this apparent clustering, we performed chi2-based statistical analyses to compare the distribution of LHON patients on the Caucasian phylogenetic tree. Our results indicate that, unlike the 3460 and 11778 mutations, the 14484 mutation was not distributed on the phylogeny in proportion to the frequencies of the major Caucasian mtDNA haplogroups found in North America. The 14484 mutation was next shown to occur on the haplogroup J background more frequently that expected, consistent with the observation that approximately 75% of worldwide 14484-positive LHON patients occur in association with haplogroup J. The 11778 mutation also exhibited a moderate clustering on haplogroup J. These observations were supported by statistical analysis using all available mutation frequencies reported in the literature. This paper thus illustrates the potential importance of genetic background in certain mtDNA-based diseases, speculates on a pathogenic role for a subset of LHON secondary mutations and their interaction with primary mutations, and provides support for a polygenic model for LHON expression in some cases.     

Co-occurrence of A1555G and G11778A in a Chinese family with high penetrance of Leber's hereditary optic neuropathy

Co-occurrence of double pathogenic mtDNA mutations with different claimed pathological roles in one mtDNA is infrequent. It is tentative to believe that each of these pathogenic mutations would have its own deleterious effect. Here we reported one three-generation Chinese family with a high penetrance of LHON (78.6%). Analysis of the complete mitochondrial genome in the proband revealed the presence of the LHON primary mutation G11778A in the NADH dehydrogenase 4 (ND4) gene and a deafness-associated mutation A1555G in the 12S rRNA gene. The other mtDNA variants in this family suggested a haplogroup status G2b. Although A1555G has long been confirmed to be a primary mutation for aminoglycoside-induced and non-syndromic hearing loss, none of the maternally related members in this family showed hearing impairment. It thus seems that the occurrence of A1555G in this family had no pathological manifestation. However, whether A1555G has a synergistic effect with G11778A and contribute to the high penetrance of LHON remained an open question. To our knowledge, this is the first report that identified the co-existence of a deafness mutation A1555G and a primary LHON mutation G11778A in one family.     

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。     

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.     

Evidence against an X-linked visual loss susceptibility locus in Leber hereditary optic neuropathy.

Pedigree analysis of British families with Leber hereditary optic neuropathy (LHON) closely fits a model in which a pathogenic mtDNA mutation interacts with an X-linked visual loss susceptibility locus (VLSL). This model predicts that 60% of affected females will show marked skewing of X inactivation. Linkage analysis in British and Italian families with genetically proven LHON has excluded the presence of such a VLSL over 169 cM of the X chromosome both when all families were analyzed together and when only families with the bp 11778 mutation were studied. Further, there was no excess skewing of X inactivation in affected females. There was no evidence for close linkage to three markers in the pseudoautosomal region of the sex chromosomes. The mechanism of incomplete penetrance and male predominance in LHON remains unclear.     

Multiple Origins of a Mitochondrial Mutation Conferring Deafness

A point mutation (1555G) in the smaller ribosomal subunit of the mitochondrial DNA (mtDNA) has been associated with maternally inherited traits of hypersensitivity to streptomycin and sensorineural deafness in a number of families from China, Japan, Israel, and Africa. To determine whether this distribution was the result of a single or multiple mutational events, we carried out genetic distance analysis and phylogenetic analysis of 10 independent mtDNA D-loop sequences from Africa and Asia. The mtDNA sequence diversity was high (2.21%). Phylogenetic analysis assigned 1555G-bearing haplotypes at very divergent points in the human mtDNA evolutionary tree, and the 1555G mutations occur in many cases on race-specific mtDNA haplotypes, both facts are inconsistent with a recent introgression of the mutation into these races. The simplest interpretation of the available data is that there have been multiple origins of the 1555G mutation. The genetic distance among mtDNAs bearing the pathogenic 1555G mutation is much larger than among mtDNAs bearing either evolutionarily neutral or weakly deleterious nucleotide substitutions (such as the 4336G mutation). These results are consistent with the view that pathogenic mtDNA haplotypes such as 1555G arise on disparate mtDNA lineages which because of negative natural selection leave relatively few related descendants. The co-existence of the same mutation with deafness in individuals with very different nuclear and mitochondrial genetic backgrounds confirms the pathogenicity of the 1555G mutation.     

Phylogeography of pipistrelle-like bats within the Canary Islands,based on mtDNA sequences

Evolution of three Canary Island Vespertilionid bat species, Pipistrellus kuhlii, Pipistrellus maderensis, and Hypsugo savii was studied by comparison of approximately 1 kbp of mtDNA (from cytochrome b and 16S rRNA genes) between islands. mtDNA reveals that both P. kuhlii and P. maderensis exist in sympatry on Tenerife (and possibly other islands). Their morphological similarity explains why their co-occurrence had not been detected previously. Levels of sequence divergence are quite low within P. maderensis. Haplotypes were either identical or separated by /=12 mutational steps) indicating colonization of the latter from the former sometime during the last approximately 1.2 Ma, with low subsequent gene flow. Unlike P. maderensis the El Hierro population alone appears to represent an ESU. The H. savii haplotypes detected in Gran Canaria and Tenerife are identical or separated by 1 mutational step.     

Novel mtDNA mutations and oxidative phosphorylation dysfunction in Russian LHON families

Leber's hereditary optic neuropathy (LHON) is characterized by maternally transmitted, bilateral, central vision loss in young adults. It is caused by mutations in the mitochondrial DNA (mtDNA) encoded genes that contribute polypeptides to NADH dehydrogenase or complex I. Four mtDNA variants, the nucleotide pair (np) 3460A, 11778A, 14484C, and 14459A mutations, are known as "primary" LHON mutations and are found in most, but not all, of the LHON families reported to date. Here, we report the extensive genetic and biochemical analysis of five Russian families from the Novosibirsk region of Siberia manifesting maternally transmitted optic atrophy consistent with LHON. Three of the five families harbor known LHON primary mutations. Complete sequence analysis of proband mtDNA in the other two families has revealed novel complex I mutations at nps 3635A and 4640C, respectively. These mutations are homoplasmic and have not been reported in the literature. Biochemical analysis of complex I in patient lymphoblasts and transmitochondrial cybrids demonstrated a respiration defect with complex-I-linked substrates, although the specific activity of complex I was not reduced. Overall, our data suggests that the spectrum of mtDNA mutations associated with LHON in Russia is similar to that in Europe and North America and that the np 3635A and 4640C mutations may be additional mtDNA complex I mutations contributing to LHON expression.     

No genetic differences between affected and unaffected members of a German family with Leber's hereditary optic neuropathy (LHON) with respect to ten mtDNA point mutations associated with LHON.

In order to investigate possible synergistic influences of different mtDNA mutations on penetrance and severity of Leber's hereditary optic neuropathy (LHON), a large German LHON pedigree is characterized with respect to 10 different mutations associated with LHON. All members of the family carry three different mtDNA mutations (at nucleotide 4,216, 11,778 and 13,708) in a homoplasmic form, regardless of whether or not they are clinically affected. Testing for another 7 mutations reveals negative results in all family members. Hence, the variable disease expression of the family members cannot be explained by varying combinations of LHON-associated mtDNA mutations.     

Clinical evaluation and mitochondrial DNA sequence analysis in three Chinese families with Leber's hereditary optic neuropathy

We report here the clinical, genetic, and molecular characterization of three Chinese families (WZ4, WZ5, and WZ6) with Leber's hereditary optic neuropathy (LHON). Clinical and genetic evaluations revealed the variable severity and age-of-onset in visual impairment in these families. Penetrances of visual impairment in these Chinese families were 33.3%, 35.7%, and 35.5%, respectively, with an average 34.8%. Furthermore, the average age-at-onset in those Chinese families was 17, 20, and 18 years. In addition, the ratios between affected male and female matrilineal relatives in these Chinese families were 3:0, 1:1, and 1.2:1, respectively. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the distinct sets of mtDNA polymorphism, in addition to the identical G11778A mutation associated with LHON in many families. The fact that mtDNA of those pedigrees belonged to different haplogroups F1, D4, and M10 suggested that the G11778A mutation occurred sporadically and multiplied through evolution of the mtDNA in China. However, there was the absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in these Chinese families. The I187T mutation in the ND1, the S99A mutation in the A6, the V254I in CO3, and I58V in ND6 mutation, showing high evolutional conservation, may contribute to the phenotypic expression of the G11778A mutation in the WZ6 pedigree. By contrast, none of mtDNA variants are evolutionarily conserved and implicated to have significantly functional consequence in WZ4 and WZ5 pedigrees. Apparently, these variants do not have a potential modifying role in the development of visual impairment associated with G11778A mutation in those two families. Thus, nuclear modifier gene(s) or environmental factor(s) seem to account for the penetrance and expressivity of LHON in these three Chinese families carrying the G11778A mutation.