Multiplex MALDI-TOF MS detection of mitochondrial variants in Brazilian patients with hereditary optic neuropathy.
ABSTRACT: Leber hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by bilateral vision loss. More than 95% of LHON cases are associated with one of the three main mtDNA mutations: G11778A, T14484C, and G3460A. The other 5% of cases are due to other rare mutations related to the disease. The aim of this study was to identify the prevalence and spectrum of LHON mtDNA mutations, including the haplogroup, in a cohort of Brazilian patients with optic neuropathy and to evaluate the usefulness of iPLEX Gold/matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology in detecting LHON mutations.We analyzed a total of 101 patients; 67 had a clinical diagnosis of LHON and 34 had optic neuropathy of unknown etiology. Direct sequencing and iPLEX Gold/MALDI-TOF MS were used to screen for the most common pathogenic point mutations in LHON, together with the rare mutations G3733A, C4171A, T10663C, G14459A, C14482G, A14495G, C14568T, and C14482A.We identified mutations in 36 patients, of whom 83.3% carried the G11778A mutation and 16.7% carried the T14484C mutation. In individuals with mutations, the haplogroups found were L1/L2, L3, C, R, U, D, and H. Rare mutations were not detected in any of the patients analyzed.The frequencies of the main LHON mutations were similar to those previously reported for Latin America. A different frequency was found only for the A3460G mutation. The most frequent haplogroups identified were of African origin. The iPLEX Gold/MALDI-TOF MS technology proved to be highly accurate and efficient for screening mutations and identifying the haplogroups related to LHON. The MassArray platform, combined with other techniques, enabled definitive diagnosis of LHON in 36% (36/101) of the cases studied.
Project description:There are many similarities in the clinical presentation of Leber hereditary optic neuropathy (LHON) and in patients who have optic neuropathy and a history of heavy tobacco and alcohol consumption. The main objective of this study is to investigate the frequency of primary and secondary mitochondrial DNA (mtDNA) mutations for LHON in patients diagnosed as having alcohol and tobacco optic neuropathy (ATON).Twenty-six patients who had a history of heavy alcohol and tobacco consumption and who developed bilateral optic neuropathy were tested for primary mutations (G11778A, T14484C, and G3460A) by restriction analysis, and 14 secondary mutations in the genes mitochondrially encoded NADH dehydrogenase 1 (MT-ND1), mitochondrially encoded NADH dehydrogenase 4 (MT-ND4), mitochondrially encoded NADH dehydrogenase 4L (MT-ND4L), mitochondrially encoded NADH dehydrogenase 5 (MT-ND5), mitochondrially encoded NADH dehydrogenase 6 (MT-ND6), and mitochondrially encoded cytochrome B (MT-CYB) by direct sequencing.Four (15.4%) of 26 patients tested positive for LHON primary mutations, two for the G11778A mutation, and two for the T14484C mutation. No patient tested positive for any of the 14 secondary mutations. Familial recurrence was present in four patients, and only three of these patients have presented the LHON mutation.The diagnosis of LHON should be considered in all patients diagnosed as having optic neuropathy, particularly those with familial recurrence of vision loss.
Project description:Leber hereditary optic neuropathy (LHON) is a mitochondrial genetic disease that preferentially causes blindness in young adult males, affecting about 1 in 25 000 of the British population. It is characterised by bilateral subacute loss of central vision owing to focal degeneration of the retinal ganglion cell layer and optic nerve. Over 95% of LHON cases are primarily the result of one of three mitochondrial DNA (mtDNA) point mutations, G3460A, G11778A, and T14484C, which all involve genes encoding complex I subunits of the respiratory chain. An intriguing feature of LHON is that only approximately 50% of males and approximately 10% of females who harbour a pathogenic mtDNA mutation actually develop the optic neuropathy. This marked incomplete penetrance and gender bias imply that additional mitochondrial and/or nuclear genetic factors must be modulating the phenotypic expression of LHON. It is also likely that environmental factors contribute to the onset of visual failure. However, these secondary precipitating factors remain poorly defined at present. In this review, we describe the natural history of this optic nerve disorder and highlight issues relating to clinical diagnosis, management, and genetic counselling. We also discuss the findings of recently published studies and the light they shed on the complex aetiology and pathophysiology of LHON.
Project description:BACKGROUND:Leber's Hereditary Optic Neuropathy (LHON; MIM 535000) is one of the most commonly inherited optic neuropathies and it results in significant visual morbidity among young adults with a peak age of onset between the ages of 15-30. The worldwide incidence of LHON is approximately 1 in 31,000. 95 % of LHON patients will have one of 3 primary mitochondrial mutations, G3460A (A52T of ND1), G11778A (R340H of ND4) and T14484C (M64V of ND6). There is incomplete penetrance and a marked gender bias in the development of visual morbidity with approximately 50 % of male carriers and 10 % of female carriers developing optic neuropathy. Visual recovery can occur but is dependent on the mutation present with the highest level of visual recovery seen in patients who have the T14484C mutation. The 3 primary mutations are typically identified by individual end-point PCR-restriction fragment length polymorphism (RFLP) or individual targeted bi-directional Sanger sequencing reactions. The purpose of this study was to design a simple multiplex PCR-RFLP that could detect these 3 primary LHON mutations in one assay. METHODS:PCR primers were designed to incorporate a MaeIII restriction site in the presence of 3460A and 14484C mutations with the 11778A mutation naturally incorporating a MaeIII site. A multiplex PCR-RFLP assay was developed to detect the 3 common mutations in a single assay. Synthetic LHON controls based on the mitochondrial genome harbouring the 3 common mutations were synthesized and cloned into plasmids to act as reliable assay controls. DNA from previously tested patients and the synthetic LHON controls were subjected to the multiplex PCR-RFLP assay. The RFLP products were detected by agarose gel electrophoresis. RESULTS:The novel PCR-RFLP assay accurately detects the 3 primary mutations both in patient DNA and in synthesized DNA control samples with a simple visual mutation detection procedure. The synthesized DNA was demonstrated to be a robust control for the detection of LHON Mutations. CONCLUSION:In this paper, we describe a novel, robust and simple PCR-RFLP based method for the detection of mutations causing LHON, and report the generation of a series of LHON DNA controls suitable for all currently published assays.
Project description:Approximately 95% of patients who are diagnosed with Leber's hereditary optic neuropathy (LHON) have one of three mitochondrial point mutations responsible for the disease, G3460A, G11778A, and T14484C. The purpose of this study was to develop a novel multiplex real-time amplification-refractory mutation system (ARMS) PCR combined with high-resolution melt curves to identify the individual mutations involved. The study aimed to provide a more robust, cost- and time-effective mutation detection strategy than that offered with currently available methods. The assay reported in this study will allow diagnostic laboratories to avoid costly next-generation sequencing (NGS) assays for most patients with LHON and to focus resources on patients with unknown mutations that require further analysis.The test uses a combination of multiplex allele-specific PCR (ARMS PCR) in combination with a high-resolution melt curve analysis to detect the presence of the mutations in G3460A, G11778A, and T14484C. PCR primer sets were designed to produce a control PCR product and PCR products only in the presence of the mutations in 3460A, 11778A, and 14484C in a multiplex single tube format. Products produce discrete well-separated melt curves to clearly detect the mutations.This novel real-time ARMS PCR/high-resolution melt curve assay accurately detected 95% of the mutations that cause LHON. The test has proved to be robust, cost- and time-effective with the real-time closed tube system taking approximately 1 h to complete.A novel real-time ARMS PCR/high-resolution melt curve assay is described for the detection of the three primary mitochondrial mutations in LHON. This test provides a simple, robust, easy-to-read output that is cost- and time-effective, thus providing an alternative method to individual endpoint PCR-restriction fragment length polymorphism (RFLP), PCR followed by Sanger sequencing or pyrosequencing, and next-generation sequencing.
Project description: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.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).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.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.
Project description:PURPOSE:The purpose of this study was to investigate the role of modifier factors in the expression of Leber's hereditary optic neuropathy (LHON). METHODS:Thirty-five subjects from two Han Chinese families with maternally transmitted LHON underwent a clinical and genetic evaluation and molecular analysis of mitochondrial (mt)DNA. RESULTS:Matrilineal relatives in the two Chinese families exhibited a wide range of severity in visual impairment, from blindness to nearly normal vision. Very strikingly, all nine affected individuals of 21 matrilineal relatives (13 females/8 males) were female, which translates to 33% and 57% of penetrance for optic neuropathy in the two families. The average age at onset was 22 and 25 years. These observations were in contrast with typical features in many LHON pedigrees that have a predominance of affected males. Molecular analysis of their mtDNAs identified the homoplasmic ND4 G11778A mutation and distinct sets of variants belonging to the Asian haplogroups M1 and M10a. Of other variants, the L175F variant in CO3; the I58V variant in ND6; and the I189V, L292R, and S297A variants in CYTB were located at highly conserved residues of polypeptides. CONCLUSIONS:Only female matrilineal relatives with a wide range of penetrance, severity, and age at onset of optic neuropathy in these two Chinese pedigrees showed the involvement of X-linked or autosomal recessive modifier genes in the phenotypic manifestation of the G11778A mutation. Furthermore, mitochondrial haplogroup-specific variants, together with epigenetic and environmental factors, may contribute to the phenotypic manifestation of the primary LHON-associated G11778A mutation in these pedigrees.
Project description:We report here the clinical, genetics and molecular characterization of a five-generation Han Chinese family with Leber's hereditary optic neuropathy (LHON). Strikingly, this family exhibits very high penetrance and occurrence of optic neuropathy. In particular, 25 (10 males/15 females) of 30 matrilineal relatives exhibited the variable severity, ranging from profound to mild of visual impairment. This penetrance of optic neuropathy in this Chinese family is much higher than those in many families with LHON worldwide. The age-at-onset for visual impairment in matrilineal relatives in this Chinese family varied from 7 to 24years old, with the average of 15 years old. Furthermore, the ratio between affected male and female matrilineal relatives is 1:1.5 in the Chinese family. This observation is in contrast with the typical features in LHON pedigrees that there was predominance of affected males in LHON in many families from different ethnic origins. Molecular analysis of mitochondrial genome identified the known ND4 G11778A mutation and 51 variants, belonging to Asian haplogroup C4a1. The absence of other known secondary LHON-associated and functionally significant mtDNA mutations in this Chinese family suggested that mitochondrial variants may not play an important role in the phenotypic manifestation of the G11778A mutation in this Chinese family. Therefore, nuclear modifier gene(s) may be responsible for very high penetrance and occurrence of optic neuropathy in this Chinese pedigree.
Project description:<h4>Purpose</h4>To investigate the role of mitochondrial haplotypes in the development of Leber's hereditary optic neuropathy (LHON) associated with the ND4 G11778A mutation in Chinese families.<h4>Design</h4>Eight Han Chinese families with maternally transmitted LHON were studied using clinical, genetic, and molecular evaluations.<h4>Participants</h4>One hundred sixty-seven subjects from 8 Chinese families with a wide age range and severity of visual impairment.<h4>Methods</h4>All subjects underwent the clinical and genetic evaluation, as well as molecular analysis of mitochondrial DNA (mtDNA).<h4>Main outcome measures</h4>The ophthalmologic examinations included visual acuity, visual field examination, visual evoked potentials, and fundus photography. Mitochondrial DNA analysis included the polymerase chain reaction amplification of the entire mtDNA and subsequent sequence determination.<h4>Results</h4>Eight families exhibited extremely low penetrance of visual impairment, with the average of 13%. In particular, 14 (12 males and 2 females) of 119 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 17 years. Molecular analysis of mtDNA identified the homoplasimic ND4 G11778A mutation and distinct sets of variants belonging to the Asian haplogroups M8a2, D4g2, B4a1c, B5b, N9a1, D4b2b, C, and M7b1. However, there was an absence of secondary LHON-associated mtDNA mutations in these 8 Chinese families.<h4>Conclusions</h4>The extremely low penetrance of vision loss in these 8 Chinese pedigrees strongly indicates that the G11778A mutation was itself insufficient to produce a clinical phenotype. The absence of secondary LHON mtDNA mutations suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the G11778A mutation in those Chinese families with very low penentrace of vision loss. However, nuclear backgrounds and environmental factors seem to be modifying factors for the phenotypic manifestation of the G11778A mutation in these Chinese families.
Project description:AIM: To investigate mitochondrial factors associated with Leber hereditary optic neuropathy (LHON) through complete sequencing and analysis of the mitochondrial genome of Chinese patients with this disease. METHODS: Two unrelated southern Chinese families with LHON and 10 matched healthy controls were recruited, and their entire mitochondrial DNA (mtDNA) was amplified and sequenced with the universal M13 primer. Then DNA sequence analysis and variation identification were perfomed by DNAssist and Chromas 2 software and compared with authoritative databases such as Mitomap. RESULTS: Mutational analysis of mtDNA in these two Chinese pedigrees revealed one common LHON-associated mutation, G11778A (Arg→His), in the MT-ND4 gene. In addition, there were two secondary mutations in Pedigree 1: C3497T (Ala→Val), and C3571T (Leu→Phe) in the MT-ND1 gene, which have not been reported; and two secondary mutations occurred in Pedigree 2: A10398G (Thr→Ala) in the MT-ND3 gene, and T14502C (Ile→Val) in the MT-ND6 gene. Three polymorphisms, A73G, G94A and A263G in the mtDNA control region, were also found. CONCLUSION: Our study confirmed that the known MT-ND4*G11778A mutation is the most significant cause of LHON. The C3497T and C3571T mutations in Pedigree 1 were also both at hot-spots of MT-ND1; they may affect the respiratory chain in coordination with the primary mutation G11778A. In Pedigree 2, the two secondary mutations A10398G of MT-ND3 and T14502C of MT-ND6 may influence mitochondrial respiratory complex I, leading to the mitochondrial respiratory chain dysfunction which results in optic atrophy together with G11778A. Therefore, not only the common primary LHON mutation is responsible for the visual atrophy, but other secondary mtDNA mutations should also be considered when giving genetic counseling.
Project description:Many incurable mitochondrial disorders result from mutant mitochondrial DNA (mtDNA) and impaired respiration. Leigh's syndrome (LS) is a fatal neurodegenerative disorder of infants, and Leber's hereditary optic neuropathy (LHON) causes blindness in young adults. Treatment of LHON and LS cells harboring G11778A and T8993G mutant mtDNA, respectively, by >90%, with healthy donor mtDNA complexed with recombinant human mitochondrial transcription factor A (rhTFAM), improved mitochondrial respiration by ?1.2-fold in LHON cells and restored >50% ATP synthase function in LS cells. Mitochondrial replication, transcription, and translation of key respiratory genes and proteins were increased in the short term. Increased NRF1, TFAMB1, and TFAMA expression alluded to the activation of mitochondrial biogenesis as a mechanism for improving mitochondrial respiration. These results represent the development of a therapeutic approach for LHON and LS patients in the near future.