Maternal inheritance and mitochondrial DNA variants in familial Parkinson's disease

Background  

Mitochondrial function is impaired in Parkinson's disease (PD) and may contribute to the pathogenesis of PD, but the causes of mitochondrial impairment in PD are unknown. Mitochondrial dysfunction is recapitulated in cell lines expressing mitochondrial DNA (mtDNA) from PD patients, implicating mtDNA variants or mutations, though the role of mtDNA variants or mutations in PD risk remains unclear. We investigated the potential contribution of mtDNA variants or mutations to the risk of PD.     

唐氏综合征患儿线粒体基因组突变的分析

目的 研究唐氏综合征中线粒体DNA突变情况.方法 采用高通量测序和焦磷酸测序检测7个唐氏综合征(Down's syndrome,DS)家系中的患儿和母亲的线粒体基因组序列,分析线粒体基因组序列的变化情况.结果 ①DS患儿中检测到36个与其母亲中不同的线粒体DNA突变,其中14个位点是首次在唐氏综合征样本中发现;②36个线粒体DNA突变主要发生于D-Loop区和线粒体复合物Ⅰ中;③ 线粒体基因组13个编码基因中,有11个基因检测到线粒体DNA的突变;④ 焦磷酸测序对线粒体基因组杂合突变频率的检测结果和高通量测序结果吻合.结论 DS患儿中广泛存在线粒体DNA的突变,这些突变可能与唐氏综合征的线粒体功能异常相关.     

Mitochondrial DNA Polymerase γ Mutations and Their Implications in mtDNA Alterations in Colorectal Cancer

Mitochondrial DNA was found to be highly mutated in colorectal cancer cells. One of the key molecules involved in the maintenance of the mitochondrial genome is the nuclear‐encoded polymerase gamma. The aim of our study was to determine if there is a link between polymorphisms within the polymerase gamma gene (POLG) and somatic mutations within the mitochondrial genome in cancer cells. We investigated POLG sequence variability in 50 colorectal cancer patients whose complete mitochondrial genome sequences were determined. Relative mtDNA copy number was also determined. We identified 251 sequence variants in the POLG gene. Most of them were germline‐specific (～92%). Twenty‐one somatic changes in POLG were found in 10 colorectal cancer patients. We have found no association between the occurrence of mtDNA somatic mutations and the somatically occurring variants in POLG. MtDNA content was reduced in patients carrying somatic variants in POLG or germline nucleotide variants located in the region encoding the POLG polymerase domain, but the difference did not reach statistical significance. Our findings suggest that somatic mtDNA mutations occurring in colorectal cancer are not a consequence of somatic mutations in POLG. Nevertheless, POLG nucleotide variants may lead to a decrease in mtDNA content, and consequently result in mitochondrial dysfunction.     

MPV17‐related mitochondrial DNA maintenance defect: New cases and review of clinical,biochemical, and molecular aspects

Mitochondrial DNA (mtDNA) maintenance defects are a group of diseases caused by deficiency of proteins involved in mtDNA synthesis, mitochondrial nucleotide supply, or mitochondrial dynamics. One of the mtDNA maintenance proteins is MPV17, which is a mitochondrial inner membrane protein involved in importing deoxynucleotides into the mitochondria. In 2006, pathogenic variants in MPV17 were first reported to cause infantile‐onset hepatocerebral mtDNA depletion syndrome and Navajo neurohepatopathy. To date, 75 individuals with MPV17‐related mtDNA maintenance defect have been reported with 39 different MPV17 pathogenic variants. In this report, we present an additional 25 affected individuals with nine novel MPV17 pathogenic variants. We summarize the clinical features of all 100 affected individuals and review the total 48 MPV17 pathogenic variants. The vast majority of affected individuals presented with an early‐onset encephalohepatopathic disease characterized by hepatic and neurological manifestations, failure to thrive, lactic acidemia, and mtDNA depletion detected mainly in liver tissue. Rarely, MPV17 deficiency can cause a late‐onset neuromyopathic disease characterized by myopathy and peripheral neuropathy with no or minimal liver involvement. Approximately half of the MPV17 pathogenic variants are missense. A genotype with biallelic missense variants, in particular homozygous p.R50Q, p.P98L, and p.R41Q, can carry a relatively better prognosis.     

Novel non‐neutral mitochondrial DNA mutations found in childhood acute lymphoblastic leukemia

Mitochondria produce adenosine triphosphate (ATP) for energy requirements via the mitochondrial oxidative phosphorylation (OXPHOS) system. One of the hallmarks of cancer is the energy shift toward glycolysis. Low OXPHOS activity and increased glycolysis are associated with aggressive types of cancer. Mitochondria have their own genome (mitochondrial DNA [mtDNA]) encoding for 13 essential subunits of the OXPHOS enzyme complexes. We studied mtDNA in childhood acute lymphoblastic leukemia (ALL) to detect potential pathogenic mutations in OXPHOS complexes. The whole mtDNA from blood and bone marrow samples at diagnosis and follow‐up from 36 ALL patients were analyzed. Novel or previously described pathogenic mtDNA mutations were identified in 8 out of 36 patients. Six out of these 8 patients had died from ALL. Five out of 36 patients had an identified poor prognosis genetic marker, and 4 of these patients had mtDNA mutations. Missense or nonsense mtDNA mutations were detected in the genes encoding subunits of OXPHOS complexes, as follows: MT‐ND1, MT‐ND2, MT‐ND4L and MT‐ND6 of complex I; MT‐CO3 of complex IV; and MT‐ATP6 and MT‐ATP8 of complex V. We discovered mtDNA mutations in childhood ALL supporting the hypothesis that non‐neutral variants in mtDNA affecting the OXPHOS function may be related to leukemic clones.     

Parental diabetes status reveals association of mitochondrial DNA haplogroup J1 with type 2 diabetes

Background  

Although mitochondrial dysfunction is consistently manifested in patients with Type 2 Diabetes mellitus (T2DM), the association of mitochondrial DNA (mtDNA) sequence variants with T2DM varies among populations. These differences might stem from differing environmental influences among populations. However, other potentially important considerations emanate from the very nature of mitochondrial genetics, namely the notable high degree of partitioning in the distribution of human mtDNA variants among populations, as well as the interaction of mtDNA and nuclear DNA-encoded factors working in concert to govern mitochondrial function. We hypothesized that association of mtDNA genetic variants with T2DM could be revealed while controlling for the effect of additional inherited factors, reflected in family history information.     

Deciphering exome sequencing data: Bringing mitochondrial DNA variants to light

The expanding use of exome sequencing (ES) in diagnosis generates a huge amount of data, including untargeted mitochondrial DNA (mtDNA) sequences. We developed a strategy to deeply study ES data, focusing on the mtDNA genome on a large unspecific cohort to increase diagnostic yield. A targeted bioinformatics pipeline assembled mitochondrial genome from ES data to detect pathogenic mtDNA variants in parallel with the “in‐house” nuclear exome pipeline. mtDNA data coming from off‐target sequences (indirect sequencing) were extracted from the BAM files in 928 individuals with developmental and/or neurological anomalies. The mtDNA variants were filtered out based on database information, cohort frequencies, haplogroups and protein consequences. Two homoplasmic pathogenic variants (m.9035T>C and m.11778G>A) were identified in 2 out of 928 unrelated individuals (0.2%): the m.9035T>C (MT‐ATP6) variant in a female with ataxia and the m.11778G>A (MT‐ND4) variant in a male with a complex mosaic disorder and a severe ophthalmological phenotype, uncovering undiagnosed Leber's hereditary optic neuropathy (LHON). Seven secondary findings were also found, predisposing to deafness or LHON, in 7 out of 928 individuals (0.75%). This study demonstrates the usefulness of including a targeted strategy in ES pipeline to detect mtDNA variants, improving results in diagnosis and research, without resampling patients and performing targeted mtDNA strategies.     

Mitochondrial genome architecture in non‐alcoholic fatty liver disease

Non‐alcoholic fatty liver disease (NAFLD) is associated with mitochondrial dysfunction, a decreased liver mitochondrial DNA (mtDNA) content, and impaired energy metabolism. To understand the clinical implications of mtDNA diversity in the biology of NAFLD, we applied deep‐coverage whole sequencing of the liver mitochondrial genomes. We used a multistage study design, including a discovery phase, a phenotype‐oriented study to assess the mutational burden in patients with steatohepatitis at different stages of liver fibrosis, and a replication study to validate findings in loci of interest. We also assessed the potential protein‐level impact of the observed mutations. To determine whether the observed changes are tissue‐specific, we compared the liver and the corresponding peripheral blood entire mitochondrial genomes. The nuclear genes POLG and POLG2 (mitochondrial DNA polymerase‐γ) were also sequenced. We observed that the liver mtDNA of patients with NAFLD harbours complex genomes with a significantly higher mutational (1.28‐fold) rate and degree of heteroplasmy than in controls. The analysis of liver mitochondrial genomes of patients with different degrees of fibrosis revealed that the disease severity is associated with an overall 1.4‐fold increase in mutation rate, including mutations in genes of the oxidative phosphorylation (OXPHOS) chain. Significant differences in gene and protein expression patterns were observed in association with the cumulative number of OXPHOS polymorphic sites. We observed a high degree of homology (～98%) between the blood and liver mitochondrial genomes. A missense POLG p.Gln1236His variant was associated with liver mtDNA copy number. In conclusion, we have demonstrated that OXPHOS genes contain the highest number of hotspot positions associated with a more severe phenotype. The variability of the mitochondrial genomes probably originates from a common germline source; hence, it may explain a fraction of the ‘missing heritability’ of NAFLD. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Mitochondrial DNA (mtDNA) in brain samples from patients with major psychiatric disorders: Gene expression profiles,MtDNA content and presence of the MtDNA common deletion

Several lines of evidence support a mitochondrial dysfunction in major psychiatric disorders. The objective of this study was to determine whether mitochondrial DNA (mtDNA) expression or content are implicated in the mitochondrial dysfunction observed in schizophrenia (SCH), bipolar disorder (BD), and major depressive disorder (MDD). MtDNA gene expression and mtDNA content (including the MT‐ND4 deletion) were measured by RT‐qPCR and qPCR, respectively. Post‐mortem brain tissue from 60 subjects, divided evenly into four diagnostic groups (SCH, BD, MDD, and control (C)), was analyzed. MT‐ND1 gene expression was significantly increased in the BD group compared with the C group. MDD and SCH patients showed a similar pattern of mtDNA expression, which was different from that in BD patients. Similarly, a larger number of MDD and SCH patients tended to have the MT‐ND4 gene deleted compared with BD and C subjects. However, no other significant differences were observed in mtDNA gene expression and mtDNA content. Notably, high variability was observed in the mtDNA gene expression and content in each diagnostic group. Previous studies and the present work provide evidence for a role of mtDNA in SCH, BD and MDD. However, further studies with larger patient and control groups as well as by analyzing distinct brain regions are needed to elucidate the role of mtDNA in major psychiatric disorders. © 2013 Wiley Periodicals, Inc.     

Glutathione peroxidase 1 protects mitochondria against hypoxia/reoxygenation damage in mouse hearts

Glutathione peroxidase 1 (GPx1) plays an important role in preventing cardiac dysfunction following ischemia-reperfusion injury. However, its role in protecting cardiac mitochondria against reoxygenation-induced reactive oxygen species (ROS) generation in vivo is unclear. We examined the role of GPx1 in protecting cardiac mitochondria against hypoxia–reoxygenation (HR) damage by testing for alterations in cardiac mitochondrial function. We used a two-dimensional gel electrophoresis proteomics analysis to examine the effects of reoxygenation on cardiac protein in wild-type (GPx1^+/+) and GPx1 knockout (GPx1^?/?) mouse hearts. We identified 42 protein spots showing differential expression in the two groups. Sixteen of the proteins identified were located in mitochondria and were involved in a number of key metabolic pathways. To verify our proteomics findings functionally, we performed NADH autofluorescence measurements and ATP production assays. The reduced expression of oxidative phosphorylation proteins in GPx1^?/? mice following HR treatment resulted in loss of the mitochondrial membrane potential and decreased mitochondrial respiration. Mitochondrial ROS production and oxidative mtDNA damage were increased markedly during reoxygenation in GPx1^?/? hearts. We also found morphological abnormalities in cardiac mitochondria and myocytes in HR-treated GPx1^?/?. This is the first report of the role of GPx1 in protecting cardiac mitochondria against reoxygenation damage in vivo. These findings will help clarify the mechanisms of HR injury and will aid in the development of antioxidant therapies to prevent cardiac mitochondrial dysfunction associated with reoxygenation.     

Capture of Somatic mtDNA Point Mutations with Severe Effects on Oxidative Phosphorylation in Synaptosome Cybrid Clones from Human Brain

Mitochondrial DNA (mtDNA) is replicated throughout life in postmitotic cells, resulting in higher levels of somatic mutation than in nuclear genes. However, controversy remains as to the importance of low‐level mtDNA somatic mutants in cancerous and normal human tissues. To capture somatic mtDNA mutations for functional analysis, we generated synaptosome cybrids from synaptic endings isolated from fresh hippocampus and cortex brain biopsies. We analyzed the whole mtDNA genome from 120 cybrid clones derived from four individual donors by chemical cleavage of mismatch and Sanger sequencing, scanning around two million base pairs. Seventeen different somatic point mutations were identified, including eight coding region mutations, four of which result in frameshifts. Examination of one cybrid clone with a novel m.2949_2953delCTATT mutation in MT‐RNR2 (which encodes mitochondrial 16S rRNA) revealed a severe disruption of mtDNA‐encoded protein translation. We also performed functional studies on a homoplasmic nonsense mutation in MT‐ND1, previously reported in oncocytomas, and show that both ATP generation and the stability of oxidative phosphorylation complex I are disrupted. As the mtDNA remains locked against direct genetic manipulation, we demonstrate that the synaptosome cybrid approach can capture biologically relevant mtDNA mutants in vitro to study effects on mitochondrial respiratory chain function.     

Mitochondrial cardiomyopathies: how to identify candidate pathogenic mutations by mitochondrial DNA sequencing, MITOMASTER and phylogeny

Pathogenic mitochondrial DNA (mtDNA) mutations leading to mitochondrial dysfunction can cause cardiomyopathy and heart failure. Owing to a high mutation rate, mtDNA defects may occur at any nucleotide in its 16 569 bp sequence. Complete mtDNA sequencing may detect pathogenic mutations, which can be difficult to interpret because of normal ethnic/geographic-associated haplogroup variation. Our goal is to show how to identify candidate mtDNA mutations by sorting out polymorphisms using readily available online tools. The purpose of this approach is to help investigators in prioritizing mtDNA variants for functional analysis to establish pathogenicity. We analyzed complete mtDNA sequences from 29 Italian patients with mitochondrial cardiomyopathy or suspected disease. Using MITOMASTER and PhyloTree, we characterized 593 substitution variants by haplogroup and allele frequencies to identify all novel, non-haplogroup-associated variants. MITOMASTER permitted determination of each variant''s location, amino acid change and evolutionary conservation. We found that 98% of variants were common or rare, haplogroup-associated variants, and thus unlikely to be primary cause in 80% of cases. Six variants were novel, non-haplogroup variants and thus possible contributors to disease etiology. Two with the greatest pathogenic potential were heteroplasmic, nonsynonymous variants: m.15132T>C in MT-CYB for a patient with hypertrophic dilated cardiomyopathy and m.6570G>T in MT-CO1 for a patient with myopathy. In summary, we have used our automated information system, MITOMASTER, to make a preliminary distinction between normal mtDNA variation and pathogenic mutations in patient samples; this fast and easy approach allowed us to select the variants for traditional analysis to establish pathogenicity.     

A potential impact of DNA repair on ageing and lifespan in the ageing model organism Podospora anserina: Decrease in mitochondrial DNA repair activity during ageing

The free radical theory of ageing states that ROS play a key role in age-related decrease in mitochondrial function via the damage of mitochondrial DNA (mtDNA), proteins and lipids. In the sexually reproducing ascomycete Podospora anserina ageing is, as in other eukaryotes, associated with mtDNA instability and mitochondrial dysfunction. Part of the mtDNA instabilities may arise due to accumulation of ROS induced mtDNA lesions, which, as previously suggested for mammals, may be caused by an age-related decrease in base excision repair (BER).Alignments of known BER protein sequences with the P. anserina genome revealed high homology. We report for the first time the presence of BER activities in P. anserina mitochondrial extracts. DNA glycosylase activities decrease with age, suggesting that the increased mtDNA instability with age may be caused by decreased ability to repair mtDNA damage and hence contribute to ageing and lifespan control in this ageing model.Additionally, we find low DNA glycosylase activities in the long-lived mutants grisea and ΔPaCox17::ble, which are characterized by low mitochondrial ROS generation.Overall, our data identify a potential role of mtDNA repair in controlling ageing and life span in P. anserina, a mechanism possibly regulated in response to ROS levels.     

Assembly of the mitochondrial membrane system. Organization of yeast mitochondrial DNA in the Oli1 region

Summary The mitochondrial DNA (mtDNA) of a cytoplasmic petite mutant (DS401) of Saccharomyces cerevisiae genetically marked for the ATPase proteolipid, serine tRNA and varl genes has been characterized by restriction endonuclease analysis and DNA sequencing. The DS401 mtDNA segment is 5.3 kb long spanning the region between 79.1 and 86.8 units of the wild type genome. Most of the DS401 mtDNA consists of A+T rich sequences. In addition, however, there are ten short sequences with a high content of G+C and two sequences that have been identified as the ATPase proteolipid and the serine tRNA genes. The two genes map at 81 and 83 units and are transcribed from the same DNA strand. Even though there are other possible coding sequences in the DNA segment, none are sufficiently long to code for a gene product of the size of the varl protein. Based on the relative organization of the G+C rich clusters and genes, a model has been proposed for the processing of mitochondria) RNA. This model postulates the existence of mitochondrial double strand specific RNases that cleave the RNA at the G+C clusters.     

Rare Pathogenic Variants in Mitochondrial and Inflammation-Associated Genes May Lead to Inflammatory Cardiomyopathy in Chagas Disease

Abstract

Cardiomyopathies are an important cause of heart failure and sudden cardiac death. Little is known about the role of rare genetic variants in inflammatory cardiomyopathy. Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory cardiomyopathy prevalent in Latin America, developing in 30% of the 6 million patients chronically infected by the protozoan Trypanosoma cruzi, while 60% remain free of heart disease (asymptomatic (ASY)). The cytokine interferon-γ and mitochondrial dysfunction are known to play a major pathogenetic role. Chagas disease provides a unique model to probe for genetic variants involved in inflammatory cardiomyopathy.

Methods

We used whole exome sequencing to study nuclear families containing multiple cases of Chagas disease. We searched for rare pathogenic variants shared by all family members with CCC but absent in infected ASY siblings and in unrelated ASY.

Results

We identified heterozygous, pathogenic variants linked to CCC in all tested families on 22 distinct genes, from which 20 were mitochondrial or inflammation-related – most of the latter involved in proinflammatory cytokine production. Significantly, incubation with IFN-γ on a human cardiomyocyte line treated with an inhibitor of dihydroorotate dehydrogenase brequinar (enzyme showing a loss-of-function variant in one family) markedly reduced mitochondrial membrane potential (ΔψM), indicating mitochondrial dysfunction.

Conclusion

Mitochondrial dysfunction and inflammation may be genetically determined in CCC, driven by rare genetic variants. We hypothesize that CCC-linked genetic variants increase mitochondrial susceptibility to IFN-γ-induced damage in the myocardium, leading to the cardiomyopathy phenotype in Chagas disease. This mechanism may also be operative in other inflammatory cardiomyopathies.

     

Increased salivary level of 8-hydroxydeoxyguanosine is a marker of premature oxidative mitochondrial DNA damage in gingival tissue of patients with periodontitis

Introduction:  

Oxidative stress may contribute to the pathogenesis of periodontitis. However, the detailed molecular mechanism remains unclear. Both 8-hydroxydeoxyguanosine (8-OHdG) and mitochondrial DNA (mtDNA) deletion have been reported as early oxidative DNA damage markers. In this study, 8-OHdG levels in saliva and mtDNA deletions in gingival tissue of patients with chronic periodontitis (CP) were evaluated.     

Mutational analysis of the mitochondrial tRNA genes and flanking regions in umbilical cord tissue from uninfected infants receiving AZT‐based therapies for prophylaxis of HIV‐1

A sensitive vertical denaturing gradient gel electrophoresis (DGGE) method, using 13 unipolar psoralen‐clamped PCR primer pairs, was developed for detecting sequence variants in the 22 tRNA genes and flanking regions (together spanning ～21%) of the human mitochondrial genome. A study was conducted to determine (i) if mitochondrial DNA (mtDNA) polymorphisms and/or mutations were detectable in healthy newborns and (ii) if prepartum 3′‐azido‐2′,3′‐dideoxythymidine (AZT) based HIV‐1 prophylaxis was associated with significant increases in mtDNA mutations and changes in the degree of heteroplasmy of sequence variants in uninfected infants born to HIV‐1‐infected mothers. DGGE analysis of umbilical cord tissue (where vascular endothelium and smooth muscle cells are the major source of mtDNA) showed that mtDNA sequence variants were significantly elevated by threefold in AZT‐treated infants compared with unexposed controls (P < 0.001), with 24 changes observed in 19/52 (37%) treated newborns (averaging 0.46 changes/subject) versus only eight changes found in 7/55 (13%) unexposed newborns (averaging 0.15 changes/subject). Six distinct sequence variants occurring in unexposed controls were predominately synonymous and homoplasmic, representing previously reported polymorphisms. Uninfected infants exposed to a combination of AZT and 2′,3′‐dideoxy‐3′‐thiacytidine and “maternal HIV‐1” had a significant shift in the spectrum of mutations (P = 0.04) driven by increases in nonsynonymous heteroplasmic sequence variants at polymorphic sites (10 distinct variants) and novel sites (four distinct variants). While the weight of evidence suggests that prepartum AZT‐based prophylaxis produces mtDNA mutations, additional research is needed to determine the degree to which fetal responses to maternal HIV‐1 infection, in the absence of antiretroviral treatment, contribute to prenatal mtDNA mutagenesis. Environ. Mol. Mutagen., 2009. © 2008 Wiley‐Liss, Inc.     

Phylogenetic analysis of the mitochondrial genome indicates significant differences between patients with Alzheimer disease and controls in a French-Canadian founder population

The activity of cytochrome oxidase (CO), the terminal enzyme of the mitochondrial electron transport chain, has been reported to be lower in the brains of Alzheimer disease (AD) patients. This suggests that a modification of mitochondrial DNA (mtDNA) may be responsible for this decrease of CO activity. Many mtDNA variants were found by different studies at a higher frequency in AD patients, suggesting that mtDNA variants could confer a genetic susceptibility to AD. In this study, we sequenced the entire mitochondrial genome region that encompasses the three CO genes and the 22 mitochondrial tRNA in 69 AD patients and 83 age-matched controls. We detected a total of 95 mtDNA variants. The allele frequencies of the majority of these variants were similar in patients and controls. However, a haplotype composed of three different modifications (positions: 5633, 7476, and 15812) was present in three of the 69 late-onset AD patients (4.3%) and also in 1 of 16 early-onset AD patients (6.2%) but not in control individuals. Given that one of these variants (15812) has already been shown to be associated with another neurodegenerative disease and that all three modifications are relatively conserved and their frequencies in the general population is only 0.1%, our data suggest that the presence of this haplotype may represent a risk factor for AD. We also found a significant association (P < 0.05) of two other variants at positions 709 (rRNA 12S) and 15928 (tRNA^Thr). These two mtDNA variants are three times more frequent in control individuals compared with AD patients, suggesting that they may be protective against AD. Am. J. Med. Genet 85:20–30, 1999. © 1999 Wiley-Liss, Inc.