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.     

Leber遗传性视神经病变家系的线粒体基因突变分析

为探讨Leber遗传性视神经病变（Leber′s hereditary optic neuropathy,LHON）家系线粒体DNA（mtDNA）常见致病原发突变的频谱,用聚合酶链反应（polymerase chain reaction,PCR）和单链构象多态性（single-stranded conformational polymorphism,SSCP）以及DNA测序的方法,对13个家系22位临床诊断为LHON的患者及其母系亲属21人的线粒体DNA进行检测,同时检测71例正常人作为对照。临床拟诊为LHON的13个家系中,11个家系存在mtDNA位点11778 G→A突变,另2个家系存在14484位点T→C突变。说明中国LHON病人存在线粒体DNA 11778或14484位点突变,其中14484位点突变在国内尚未见报道。 Abstract:The purpose of the study is to investigate the frequency of common pathogenic primary mitochondrial DNA mutations in pedigrees of Leber′s hereditary optic neuropathy (LHON).Mutations were determined by polymerase chain reaction (PCR),single-stranded conformational polymorphism (SSCP) and DNA sequencing.Twenty-two patients with suspicion of LHON and twenty-one their maternal relatives underwent molecular genetic evaluation.Seventy-one normal individuals underwent molecular genetic evaluation as control at the same time.Members from 13 families with suspicion of LHON,11 families had nucleotide position nt11778 G→A mutations.Another 2 families had nt14484 T→C mutations.It is concluded that the point mutations at nucleotides 11778 and 14484 are primary LHON mutations,but the point mutation of nt14484 is rare in Chinese.     

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.     

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.     

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.     

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.     

The pedigree rate of sequence divergence in the human mitochondrial genome: there is a difference between phylogenetic and pedigree rates

We have extended our previous analysis of the pedigree rate of control-region divergence in the human mitochondrial genome. One new germline mutation in the mitochondrial DNA (mtDNA) control region was detected among 185 transmission events (generations) from five Leber hereditary optic neuropathy (LHON) pedigrees. Pooling the LHON pedigree analyses yields a control-region divergence rate of 1.0 mutation/bp/10(6) years (Myr). When the results from eight published studies that used a similar approach were pooled with the LHON pedigree studies, totaling >2,600 transmission events, a pedigree divergence rate of 0.95 mutations/bp/Myr for the control region was obtained with a 99.5% confidence interval of 0.53-1.57. Taken together, the cumulative results support the original conclusion that the pedigree divergence rate for the control region is approximately 10-fold higher than that obtained with phylogenetic analyses. There is no evidence that any one factor explains this discrepancy, and the possible roles of mutational hotspots (rate heterogeneity), selection, and random genetic drift and the limitations of phylogenetic approaches to deal with high levels of homoplasy are discussed. In addition, we have extended our pedigree analysis of divergence in the mtDNA coding region. Finally, divergence of complete mtDNA sequences was analyzed in two tissues, white blood cells and skeletal muscle, from each of 17 individuals. In three of these individuals, there were four instances in which an mtDNA mutation was found in one tissue but not in the other. These results are discussed in terms of the occurrence of somatic mtDNA 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.     

Fifteen novel mutations in the mitochondrial NADH dehydrogenase subunit 1, 2, 3, 4, 4L, 5 and 6 genes from Iranian patients with Leber’s hereditary optic neuropathy (LHON)

Leber’s hereditary optic neuropathy (LHON) is an optic nerve dysfunction resulting from mutations in mitochondrial DNA (mtDNA), which is transmitted in a maternal pattern of inheritance. It is caused by three primary point mutations: G11778A, G3460A and T14484C; in the mitochondrial genome. These mutations are sufficient to induce the disease, accounting for the majority of LHON cases, and affect genes that encode for the different subunits of mitochondrial complexes I and III of the mitochondrial respiratory chain. Other mutations are secondary mutations associated with the primary mutations. The purpose of this study was to determine MT-ND variations in Iranian patients with LHON. In order to determine the prevalence and distribution of mitochondrial mutations in the LHON patients, their DNA was studied using PCR and DNA sequencing analysis. Sequencing of MT-ND genes from 35 LHON patients revealed a total of 44 nucleotide variations, in which fifteen novel variations—A14020G, A13663G, C10399T, C4932A, C3893G, C10557A, C12012A, C13934T, G4596A, T12851A, T4539A, T4941A, T13255A, T14353C and del A 4513—were observed in 27 LHON patients. However, eight patients showed no variation in the ND genes. These mutations contribute to the current database of mtDNA polymorphisms in LHON patients and may facilitate the definition of disease-related mutations in human mtDNA. This research may help to understand the disease mechanism and open up new diagnostic opportunities for LHON.     

Rare Primary Mitochondrial DNA Mutations and Probable Synergistic Variants in Leber's Hereditary Optic Neuropathy

Background

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited blinding disorder, which in over 90% of cases is due to one of three primary mitochondrial DNA (mtDNA) point mutations (m.11778G>A, m.3460G>A and m.14484T>C, respectively in MT-ND4, MT-ND1 and MT-ND6 genes). However, the spectrum of mtDNA mutations causing the remaining 10% of cases is only partially and often poorly defined.

Methodology/Principal Findings

In order to improve such a list of pathological variants, we completely sequenced the mitochondrial genomes of suspected LHON patients from Italy, France and Germany, lacking the three primary common mutations. Phylogenetic and conservation analyses were performed. Sixteen mitochondrial genomes were found to harbor at least one of the following nine rare LHON pathogenic mutations in genes MT-ND1 (m.3700G>A/p.A132T, m.3733G>A-C/p.E143K-Q, m.4171C>A/p.L289M), MT-ND4L (m.10663T>C/p.V65A) and MT-ND6 (m.14459G>A/p.A72V, m.14495A>G/p.M64I, m.14482C>A/p.L60S, and m.14568C>T/p.G36S). Phylogenetic analyses revealed that these substitutions were due to independent events on different haplogroups, whereas interspecies comparisons showed that they affected conserved amino acid residues or domains in the ND subunit genes of complex I.

Conclusions/Significance

Our findings indicate that these nine substitutions are all primary LHON mutations. Therefore, despite their relative low frequency, they should be routinely tested for in all LHON patients lacking the three common mutations. Moreover, our sequence analysis confirms the major role of haplogroups J1c and J2b (over 35% in our probands versus 6% in the general population of Western Europe) and other putative synergistic mtDNA variants in LHON expression.     

The spectrum of mitochondrial DNA mutations in families with Leber hereditary optic neuroretinopathy

The mitochondrial complex I genes were sequenced in seven Leber hereditary optic neuroretinopathy (LHON) families without the ND4/11778 and ND1/3460 mutations. Four replacement mutations restricted only to LHON families were found, one in the ND1 gene at nt 4025, and three in the ND5 gene at nt 12811, 13637, and 13967. The mutations did not change evolutionarily conserved amino acids suggesting that they are not primary LHON mutations in these families. They may be considered as secondary LHON mutations serving as exacerbating factors in an appropriate genetic background. A complex III mutation, cyt b/15257, has been suggested to be one of the primary mutations causing LHON. Its presence was determined for 23 Finnish LHON families, and it was detected in two families harboring the ND4/11778 mutation. Similarly, complex IV mutation COI/7444 was screened in Finnish LHON families, and it was found in one family carrying the ND1/3460 mutation.     

Complete mitochodrial genome of the crab spider Ebrechtella tricuspidata (Araneae: Thomisidae): A novel tRNA rearrangement and phylogenetic implications for Araneae

Mitochondrial genomes are widely used for phylogenetic and phylogeographic analyses among arthropods, but there is a lack of sufficient mitochondrial genome sequence data for spiders. Herein, we sequenced and characterized the complete mitochondrial genome of a crab spider Ebrechtella tricuspidata (Araneae: Thomisidae). The circular mitochondrial genome is 14,352 bp long, including a standard set of 37 genes and an A + T-rich region. Nucleotide composition is highly biased toward A + T nucleotides (77.3%). A novel gene order rearrangement is detected by a tRNA (trnL1) translocation. Tandem repeats are not identified in the A + T-rich region. Most of the tRNAs are greatly reduced in size and cannot be folded into typical cloverleaf-shaped secondary structures. The phylogenetic analysis confirms that the mitochondrial genome sequences are useful in resolving higher-level relationship of Araneae. Overall, our data present in this study will elevate our knowledge on the architecture and evolution of spider mitochondrial genome.     

Mitochondrial tRNA mutations in Chinese children with tic disorders

Aim: To conduct the clinical, genetic, and molecular characterization of 494 Han Chinese subjects with tic disorders (TD).Methods: In the present study, we performed the mutational analysis of 22 mitochondrial tRNA genes in a large cohort of 494 Han Chinese subjects with TD via Sanger sequencing. These variants were then assessed for their pathogenic potential via phylogenetic, functional, and structural analyses.Results: A total of 73 tRNA gene variants (49 known and 24 novel) on 22 tRNA genes were identified. Among these, 18 tRNA variants that were absent or present in <1% of 485 Chinese control patient samples were localized to highly conserved nucleotides, or changed the modified nucleotides, and had the potential structural to alter tRNA structure and function. These variants were thus considered to be TD-associated mutations. In total, 25 subjects carried one of these 18 putative TD-associated tRNA variants with the total prevalence of 4.96%.Limitations: The phenotypic variability and incomplete penetrance of tic disorders in pedigrees carrying these tRNA mutations suggested the involvement of modifier factors, such as nuclear encoded genes associated mitochondrion, mitochondrial haplotypes, epigenetic, and environmental factors.Conclusion: Our data provide the evidence that mitochondrial tRNA mutations are the important causes of tic disorders among Chinese population. These findings also advance current understanding regarding the clinical relevance of tRNA mutations, and will guide future studies aimed at elucidating the pathophysiology of maternal tic disorders.     

两种毛蚜线粒体基因组比较分析

【目的】线粒体基因组分析已被应用于昆虫系统发育研究。本研究以蚜科Aphididae重要类群毛蚜亚科物种为代表,测定并比较分析了该类蚜虫的线粒体基因组特征,探讨了基于线粒体基因组信息的蚜虫系统发育关系重建。【方法】以毛蚜亚科三角枫多态毛蚜Periphyllus acerihabitans Zhang和针茅小毛蚜Chaetosiphella stipae Hille Ris Lambers,1947为研究对象,利用长短PCR相结合的方法测定线粒体基因组的序列,分析了基因组的基本特征;基于在线t RNAscan-SE Search Server搜索方法预测了t RNA的二级结构;基于12个物种(本研究获得的2个物种和10个Gen Bank上下载的物种数据)的蛋白编码基因(PCGs)序列,利用最大似然法和贝叶斯法重建了蚜科的系统发育关系。【结果】两种毛蚜均获得了约94%的线粒体基因组数据,P.acerihabitans获得了14 908 bp,控制区为1 205 bp;C.stipae获得了13 893 bp,控制区为609 bp。两种毛蚜同时获得33个基因,包含接近完整的13个蛋白编码基因(PCGs)(nad5不完整),18个tRNA,2个rRNA基因;ka/ks值表明,C.stipae的进化速率更快。从基因组组成、基因排列顺序、核苷酸组成分析、密码子使用情况、t RNA二级结构等特征来分析,两种蚜虫线粒体基因组基本特征相似。系统发育重建结果表明毛蚜亚科、蚜亚科的单系性得到了支持,毛蚜亚科位于蚜科的基部位置。【结论】两种毛蚜线粒体基因组的基本特征相似,符合蚜虫线粒体基因组的一般特征,两种线粒体基因组的长度差异主要来自控制区长度的不同;系统发育重建支持毛蚜亚科与蚜亚科的单系性,毛蚜亚科位于蚜科较为基部的位置。研究结果为蚜虫类系统发育重建提供了参考。     

Applications of the method of high resolution melting analysis for diagnosis of Leber's disease and the three primary mutation spectrum of LHON in the Han Chinese population

Current screening methods, such as single strand conformational polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC) and direct DNA sequencing that are used for detecting mutation in Leber's hereditary optic neuropathy (LHON) subjects are time consuming and costly. Here we tested high-resolution melt (HRM) analysis for mtDNA primary mutations in LHON patients. In this study, we applied the high resolution melting (HRM) technology to screen mtDNA primary mutations in 50 LHON patients from their peripheral blood. In order to evaluate the reliability of this technique, we compared the results obtained by HRM and direct mtDNA sequencing. We also investigated the spectrum of three most common mtDNA mutations implicated in LHON in the Han Chinese population. The results showed HRM analysis differentiated all of the mtDNA primary mutations and identified 4 additional mtDNA mutations from 50 patients in the blind study. The prevalence of three primary mutations were 11778G>A (87.9%), 14484T>C (6.5%) and 3460G>A (1.7%) in the Han Chinese population. In conclusion, HRM analysis is a rapid, reliable, and low-cost tool for detecting mtDNA primary mutations and has practical applications in molecular genetics.     

Sequence analysis of the entire mitochondrial genome in Parkinson's disease

The pathogenesis of Parkinson's disease (PD) is largely unknown. Indirect evidence suggests that mutations in mitochondrial DNA (mtDNA) might play a role, but previous studies have not consistently associated any specific mutations with PD. However, these studies have generally been confined to limited areas of the mitochondrial genome. We therefore sequenced the entire mitochondrial genome from substantia nigra of 8 PD and 9 control subjects. Several sequence variants were distributed differently between PD and control subjects, but all were previously reported polymorphisms. Several secondary LHON mutations were found, as well as a number of novel missense mutations, but all were rare and did not differ between PD and control subjects. Finally, PD and control subjects did not differ in the total number of all mutations, nor the total number of missense mutations. Thus, mtDNA involvement in PD, if any, is likely to be complex and should be reconsidered carefully.     

High penetrance of sequencing errors and interpretative shortcomings in mtDNA sequence analysis of LHON patients

For identifying mutation(s) that are potentially pathogenic it is essential to determine the entire mitochondrial DNA (mtDNA) sequences from patients suffering from a particular mitochondrial disease, such as Leber hereditary optic neuropathy (LHON). However, such sequencing efforts can, in the worst case, be riddled with errors by imposing phantom mutations or misreporting variant nucleotides, and moreover, by inadvertently regarding some mutations as novel and pathogenic, which are actually known to define minor haplogroups. Under such circumstances it remains unclear whether the disease-associated mutations would have been determined adequately. Here, we re-analyse four problematic LHON studies and propose guidelines by which some of the pitfalls could be avoided.     

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.