Ancient mtDNA Genetic Variants Modulate mtDNA Transcription and Replication

Although the functional consequences of mitochondrial DNA (mtDNA) genetic backgrounds (haplotypes, haplogroups) have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are “evolutionary silent hitchhikers”. We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by in vitro transcription, hypothesizing that haplogroup-defining mutations occurring within regulatory motifs of mtDNA could affect these processes. We thus screened >2500 complete human mtDNAs representing all major populations worldwide for natural variation in experimentally established protein binding sites and regulatory regions comprising a total of 241 bp in each mtDNA. Our screen revealed 77/241 sites showing point mutations that could be divided into non-fixed (57/77, 74%) and haplogroup/sub-haplogroup-defining changes (i.e., population fixed changes, 20/77, 26%). The variant defining Caucasian haplogroup J (C295T) increased the binding of TFAM (Electro Mobility Shift Assay) and the capacity of in vitro L-strand transcription, especially of a shorter transcript that maps immediately upstream of conserved sequence block 1 (CSB1), a region associated with RNA priming of mtDNA replication. Consistent with this finding, cybrids (i.e., cells sharing the same nuclear genetic background but differing in their mtDNA backgrounds) harboring haplogroup J mtDNA had a >2 fold increase in mtDNA copy number, as compared to cybrids containing haplogroup H, with no apparent differences in steady state levels of mtDNA-encoded transcripts. Hence, a haplogroup J regulatory region mutation affects mtDNA replication or stability, which may partially account for the phenotypic impact of this haplogroup. Our analysis thus demonstrates, for the first time, the functional impact of particular mtDNA haplogroup-defining control region mutations, paving the path towards assessing the functionality of both fixed and un-fixed genetic variants in the mitochondrial genome

Mitochondrial DNA Haplogroup D4a Is a Marker for Extreme Longevity in Japan

We report results from the analysis of complete mitochondrial DNA (mtDNA) sequences from 112 Japanese semi-supercentenarians (aged above 105 years) combined with previously published data from 96 patients in each of three non-disease phenotypes: centenarians (99–105 years of age), healthy non-obese males, obese young males and four disease phenotypes, diabetics with and without angiopathy, and Alzheimer's and Parkinson's disease patients. We analyze the correlation between mitochondrial polymorphisms and the longevity phenotype using two different methods. We first use an exhaustive algorithm to identify all maximal patterns of polymorphisms shared by at least five individuals and define a significance score for enrichment of the patterns in each phenotype relative to healthy normals. Our study confirms the correlations observed in a previous study showing enrichment of a hierarchy of haplogroups in the D clade for longevity. For the extreme longevity phenotype we see a single statistically significant signal: a progressive enrichment of certain “beneficial” patterns in centenarians and semi-supercentenarians in the D4a haplogroup. We then use Principal Component Spectral Analysis of the SNP-SNP Covariance Matrix to compare the measured eigenvalues to a Null distribution of eigenvalues on Gaussian datasets to determine whether the correlations in the data (due to longevity) arises from some property of the mutations themselves or whether they are due to population structure. The conclusion is that the correlations are entirely due to population structure (phylogenetic tree). We find no signal for a functional mtDNA SNP correlated with longevity. The fact that the correlations are from the population structure suggests that hitch-hiking on autosomal events is a possible explanation for the observed correlations

The Mitochondrial Genome Is a “Genetic Sanctuary” during the Oncogenic Process

Since Otto Warburg linked mitochondrial physiology and oncogenesis in the 1930s, a number of studies have focused on the analysis of the genetic basis for the presence of aerobic glycolysis in cancer cells. However, little or no evidence exists today to indicate that mtDNA mutations are directly responsible for the initiation of tumor onset. Based on a model of gliomagenesis in the mouse, we aimed to explore whether or not mtDNA mutations are associated with the initiation of tumor formation, maintenance and aggressiveness. We reproduced the different molecular events that lead from tumor initiation to progression in the mouse glioma. In human gliomas, most of the genetic alterations that have been previously identified result in the aberrant activation of different signaling pathways and deregulation of the cell cycle. Our data indicates that mitochondrial dysfunction is associated with reactive oxygen species (ROS) generation, leading to increased nuclear DNA (nDNA) mutagenesis, but maintaining the integrity of the mitochondrial genome. In addition, mutational stability has been observed in entire mtDNA of human gliomas; this is in full agreement with the results obtained in the cancer mouse model. We use this model as a paradigm of oncogenic transformation due to the fact that mutations commonly found in gliomas appear to be the most common molecular alterations leading to tumor development in most types of human cancer. Our results indicate that the mtDNA genome is kept by the cell as a “genetic sanctuary” during tumor development in the mouse and humans. This is compatible with the hypothesis that the mtDNA molecule plays an essential role in the control of the cellular adaptive survival response to tumor-induced oxidative stress. The integrity of mtDNA seems to be a necessary element for responding to the increased ROS production associated with the oncogenic process

Absence of pathogenic mitochondrial DNA mutations in mouse brain tumors

BACKGROUND: Somatic mutations in the mitochondrial genome occur in numerous tumor types including brain tumors. These mutations are generally found in the hypervariable regions I and II of the displacement loop and unlikely alter mitochondrial function. Two hypervariable regions of mononucleotide repeats occur in the mouse mitochondrial genome, i.e., the origin of replication of the light strand (O(L)) and the Arg tRNA. METHODS: In this study we examined the entire mitochondrial genome in a series of chemically induced brain tumors in the C57BL/6J strain and spontaneous brain tumors in the VM mouse strain. The tumor mtDNA was compared to that of mtDNA in brain mitochondrial populations from the corresponding syngeneic mouse host strain. RESULTS: Direct sequencing revealed a few homoplasmic base pair insertions, deletions, and substitutions in the tumor cells mainly in regions of mononucleotide repeats. A heteroplasmic mutation in the 16srRNA gene was detected in a spontaneous metastatic VM brain tumor. CONCLUSION: None of the mutations were considered pathogenic, indicating that mtDNA somatic mutations do not likely contribute to the initiation or progression of these diverse mouse brain tumors

Mutations of SURF-1 in Leigh Disease Associated with Cytochrome c Oxidase Deficiency

Leigh disease associated with cytochrome c oxidase deficiency (LD[COX-]) is one of the most common disorders of the mitochondrial respiratory chain, in infancy and childhood. No mutations in any of the genes encoding the COX-protein subunits have been identified in LD(COX-) patients. Using complementation assays based on the fusion of LD(COX-) cell lines with several rodent/human rho0 hybrids, we demonstrated that the COX phenotype was rescued by the presence of a normal human chromosome 9. Linkage analysis restricted the disease locus to the subtelomeric region of chromosome 9q, within the 7-cM interval between markers D9S1847 and D9S1826. Candidate genes within this region include SURF-1, the yeast homologue (SHY-1) of which encodes a mitochondrial protein necessary for the maintenance of COX activity and respiration. Sequence analysis of SURF-1 revealed mutations in numerous DNA samples from LD(COX-) patients, indicating that this gene is responsible for the major complementation group in this important mitochondrial disorder

Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs

Mitochondrial diseases are commonly caused by mutations in the mitochondrial DNA (mtDNA), which in most cases co–exists with the wild–type (mtDNA heteroplasmy). We have engineered TAL–effector nucleases (TALENs) to localize to mitochondria and cleave different classes of pathogenic mtDNA mutations. MitoTALEN expression led to permanent reductions in deletion or point mutant mtDNA in patient–derived cells, raising the possibility that they can be curative to some of these diseases

The use of common mitochondrial variants to detect and characterise population structure in the Australian population: Implications for genome-wide association studies

There is an increasing recognition of the potential role of variants in mitochondrial DNA and nuclear-encoded mitochondrial proteins in modifying disease risk. This has led to a rise in the number of mitochondrial association studies being undertaken. The unique inheritance pattern of mitochondria makes mitochondrial DNA variation susceptible to having geographical structure. Such a structure may have a dramatic impact on mitochondrial association studies, particularly in heterogeneous populations. By combining self-reported ancestry data and mitochondrial genotype data for a sample of 3839 individuals from 1037 Australian families, population substructure is tested by looking for evidence of differences in mitochondrial haplogroup and single nucleotide polymorphism (SNP) frequencies between different ancestral groups in Australia. In addition, the substructure within ancestral groups is tested by comparing the similarity of mates to randomly drawn pairs of individuals from the same ancestral group. It is shown that there are significant differences in the frequency of variants both between European and non-European groups, and within Europe. This agrees with previous studies of European mitochondrial variation. No evidence was found for structure within ancestral groups. These results have implications for future association studies in the Australian population, and other populations of heterogeneous ancestry

Delivery of mtZFNs into early mouse embryos

Mitochondrial diseases often result from mutations in the mitochondrial genome (mtDNA). In most cases, mutant mtDNA coexists with wild-type mtDNA, resulting in heteroplasmy. One potential future approach to treat heteroplasmic mtDNA diseases is the specific elimination of pathogenic mtDNA mutations, lowering the level of mutant mtDNA below pathogenic thresholds. Mitochondrially targeted zinc-finger nucleases (mtZFNs) have been demonstrated to specifically target and introduce double-strand breaks in mutant mtDNA, facilitating substantial shifts in heteroplasmy. One application of mtZFN technology, in the context of heteroplasmic mtDNA disease, is delivery into the heteroplasmic oocyte or early embryo to eliminate mutant mtDNA, preventing transmission of mitochondrial diseases through the germline. Here we describe a protocol for efficient production of mtZFN mRNA in vitro, and delivery of these into 0.5 dpc mouse embryos to elicit shifts of mtDNA heteroplasmy