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PubMed2004

False-positive diagnosis of a single, large-scale mitochondrial DNA deletion by Southern blot analysis: the role of neutral polymorphisms.

Single, large-scale deletions of mitochondrial DNA (mtDNA) are a common finding in the molecular investigation of patients with suspected mitochondrial disorders and are typically detected by Southern blot analysis of muscle DNA that has been linearized by a single cutter enzyme (BamHI or PvuII). We describe our investigations of a 47-year-old woman with exercise intolerance, myalgia, and ptosis who underwent a muscle biopsy for a suspected mitochondrial genetic abnormality. Southern blot analysis after digestion of muscle DNA with BamHI revealed the apparent presence of two mtDNA species, indicative of a heteroplasmic deletion of 2.0-2.5 kb in length involving approximately 50% of all molecules. Contrary to this observation, longrange polymerase chain reaction (PCR) amplified only wild-type mtDNA. Sequence analysis revealed that the patient harbored two previously recognized control region polymorphisms, a homoplasmic 16390G>A variant that introduces a new BamHI site and a heteroplasmic 16390G>A change that abolishes this site, thus explaining the initial false-positive testing for a heteroplasmic mtDNA deletion. Our findings highlight the potential problems associated with the diagnosis of mitochondrial genetic disease and emphasize the need to confirm positive cases of mtDNA deletions using more than one enzyme or an independent method such as long-range PCR amplification.

Biopsy↗

PubMed2006

New variants in the mitochondrial genomes of schizophrenic patients.

The impaired mitochondrial function hypothesis in schizophrenia is based on evidence of altered brain metabolism, morphology, biochemistry and gene expression. Mitochondria have their own genome, which is needed to synthesize some of the subunits of the respiratory chain enzymes. Mitochondrial DNA (mtDNA) is maternally inherited and we observed an excess of maternal transmission of schizophrenia in a set of parent-offspring affected pairs. We therefore hypothesized that mutations in the mtDNA may contribute to the complex genetic basis of schizophrenia. The entire mtDNA of six schizophrenic patients with an apparent maternal transmission of the disease was sequenced and compared to the reference sequence. We have identified 50 variants and among these six have not been previously reported. Three of them were missense variants: MTCO2 7750C>A, MTATP6 8857G>A and MTND4 12096T>A. These were maternally inherited because they were also present in the mtDNA of their respective schizophrenic mothers and none of them were found in 95 control individuals. The MTND4 12096T>A (Leu446His) is a heteroplasmic variant present in five of the six mother-offspring patient pairs that triggers a non-conservative substitution in the ND4 subunit of complex I. Sequence alignment of 110 ND4 peptides from all eukaryotic kingdoms shows that only hydrophobic amino acids are found in this position. Moreover, leucine was conserved or substituted by an isoleucine in all mammalian species. This indicates that the presence of histidine could affect complex I activity in patients with schizophrenia.

Adult↗

PubMed1987

Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified DNA.

A specific segment of mitochondrial DNA from 18 people was examined by two methods of direct DNA sequencing. This segment includes a small noncoding region (V) shown before by restriction analysis to exhibit length polymorphism. All 11 of the human mtDNAs previously reported to have a deletion in this region proved to lack one of the two adjacent copies of a 9-base-pair sequence normally present in human mtDNAs. Phylogenetic analysis suggests that this deletion occurred only once during the evolution of modern types of human mtDNA and that it will be a valuable anthropological marker for peoples of East Asian origin. The one human mtDNA reported to have an addition in region V differs from the wild type by two mutations in the first copy of the 9-base-pair sequence: one transition and an addition of four cytosines, thereby producing a run of 11 cytosines. One of the direct DNA sequencing methods uses a single oligonucleotide primer to facilitate dideoxy sequencing from purified mtDNA templates. The second, more successful, method first amplifies this mtDNA segment enzymatically with two flanking primers (the "polymerase chain reaction") and then uses a third primer for DNA sequencing. This latter method, which works on the DNA extracted from small amounts of blood as well as on purified mtDNA, is shown to be a rapid means of defining sequence variants without purifying and cloning the same DNA segment from many individuals.

Base Sequence↗

PubMed1994

mtDNA variation in the Chibcha Amerindian Huetar from Costa Rica.

The genetic variation in a Chibcha-speaking Amerindian tribe from lower Central America, the Huetar, was analyzed using nucleotide sequences of the hypervariable segments of the mitochondrial DNA (mtDNA) control region, the frequencies of 10 Amerindian-specific mtDNA haplotypes, and the regional distribution of private protein polymorphisms. The sequencing of 713 base pairs (bp) in the control regions of 27 individuals revealed 11 distinct lineages. These were defined by 24 variable sites and a 6-bp deletion between nucleotide pairs (np) 106 and 111. The 6-bp deletion is a new mtDNA marker that will be valuable for Amerindian taxonomic research. Control region sequences and mtDNA haplotype analyses reveal that Huetar mtDNAs are distributed in "Amerindian clusters" A, B, and D. A maximum-likelihood phylogenetic tree suggests a single origin for the 6-bp Huetar deletion in the sample. mtDNA haplotype analysis and the presence of previously characterized private protein variants (PEPA*F, TF*DCHI, and the absence of DI*A) show that the Huetar harbor polymorphisms of considerable antiquity, suggesting an early divergence from the regional founder gene pool for this population. The data also reflect a drastic constriction in population size, an evolutionary event with a proposed central effect on Huetar genetic structure.

Base Sequence↗

PubMed1998

Retinal manifestations in mitochondrial diseases associated with mitochondrial DNA mutation.

PURPOSE: To scrutinize retinal involvement associated with distinct mitochondrial DNA (mtDNA) defects, we reviewed the records of a consecutive series of patients with various mitochondrial diseases. METHODS: Clinical, laboratory and mtDNA studies were performed in: five patients with Kearns-Sayre syndrome (KSS); six patients with chronic progressive external ophthalmoplegia (CPEO); three patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode (MELAS); three patients with myoclonic epilepsy and ragged-red fibers (MERRF); 20 patients with Leber's hereditary optic neuropathy (LHON); 30 patients with simple diabetes mellitus. RESULTS: All KSS patients with neurologic and cardiac symptoms associated with a deletion of mtDNA in muscle biopsy specimens showed widespread retinal pigmentary changes characterized by salt- and pepper-like appearance of the fundus. Three of six patients with CPEO, a mild variant of KSS, showed subtle defects at the level of retinal pigment epithelium of the posterior pole, although mtDNA deletion was similar to that in KSS. Of three patients with MELAS syndrome, one patient showed juvenile cataract and mild retinal pigmentary defect in the posterior pole. Of three patients with MERRF syndrome associated with a mtDNA mutation at nucleotide position (np) 8344, one patient showed mild pigment disorder in the posterior pole in addition to optic neuropathy. Two of 20 patients with LHON associated with a mtDNA mutation at np 11778 showed mild pigmentary defect in the macula together with typical optic neuropathy. In addition, two of 30 patients with isolated diabetes mellitus showed a mtDNA mutation at np 3243 (MELAS mutation), but they did not present with any other neurologic or multisystem disorder. CONCLUSION: The retina, in particular the retinal pigment epithelium, is highly vulnerable to be involved by mtDNA defect, and the retinopathy is phenotypically variable and frequently subclinical, depending to some extent on the type or site of mtDNA defect.

Adolescent↗

PubMed2000

The reductive hotspot hypothesis: an update.

The mitochondrial free radical theory of aging is seriously challenged by the finding that mutant mtDNA never becomes abundant in vivo, a result disputed only in experiments using novel PCR variants whose quantitative accuracy is widely doubted. However, evidence continues to mount that mitochondria are the crucial site of free radical damage in vivo, most notably that mice lacking the nonmitochondrial isoforms of superoxide dismutase are healthy. It is thus important to determine whether a low level of mutant mtDNA could have serious systemic effects. This possibility exists because of the observed mosaic distribution of mutant mtDNA: some cells (or muscle fiber segments) lack any aerobic respiration. Such cells are presumed to satisfy their ATP needs by glycolysis. In vitro, however, NADH recycling by transmembrane pyruvate/lactate exchange does not suffice: cells only survive if they can up-regulate the plasma membrane oxidoreductase (PMOR). The PMOR's physiological electron acceptor is unknown. It was proposed recently (de Grey, A. D. N. J. (1998) J. Anti-Aging Med. 1(1), 53-66) that a prominent in vivo acceptor from these mitochondrially mutant cells may be oxygen, forming extracellular superoxide. The mosaic ("hotspot") distribution of this superoxide would limit its dismutation by extracellular superoxide dismutase; it may thus reduce transition metals leading to oxidation of circulating material, such as LDL. This would raise systemic oxidative stress, greatly amplifying the damage done by the originating mitochondrially mutant cells. This model, now known as the "reductive hotspot hypothesis," has recently gained much indirect experimental support; several direct tests of it are also feasible.

Aging↗

PubMed1994

Low, but strongly structured mitochondrial DNA diversity in root knot nematodes (Meloidogyne).

Root-knot nematodes (genus Meloidogyne) have been the subject of recent and numerous studies of genetic variation because of the need to develop molecular diagnostics for the four globally distributed, parthenogenetic species that are significant agricultural pests. Our analysis of Meloidogyne mtDNA improves on previous studies: (i) by examining restriction site polymorphism among a large number of isolates also characterized for standard morphological, host range and allozyme phenotypes; (ii) by using higher resolution electrophoretic techniques; and (iii) by mapping variable restriction sites with reference to the complete nucleotide sequence. This revealed fivefold less sequence divergence (< 0.6%) between variants than estimated in previous restriction fragment length polymorphism (RFLP) studies, but perfect correspondence between mtDNA haplotype and allozyme (esterase) phenotypes. The mtDNA variation, although limited, is strongly structured with as much divergence between two lineages of Meloidogyne arenaria as between either of these and Meloidogyne javanica. The low diversity of mtDNAs suggests that these parthenogenetic lineages arose from distinct but closely related sexual females, a pattern seen in other parthenogenetic complexes. In contrast to the concordance between mtDNA and allozyme markers, there were several discrepancies between the traditional methods of identification. We suggest that further studies of these nematodes should focus on well defined genetic groups, whether or not these coincide with existing taxonomic units.

Animals↗

PubMed2004

Cosegregation of MIDD and MODY in a pedigree: functional and clinical consequences.

The aim of this study was characterization of a family carrying two mutations known to cause monogenic forms of diabetes, the M626K mutation in the HNF1alpha gene (MODY3) and the A3243G in mtDNA. Beta-cell function and insulin sensitivity were assessed with the Botnia clamp. Heteroplasmy of the A3243G mutation and variants in type 2 diabetes susceptibility genes were determined, and transcriptional activity, DNA binding, and subcellular localization of mutated HNF1alpha were studied. Thirteen family members carried the mutation in mtDNA; 6 of them also had the M626K mutation, whereas none had only the M626K mutation. The protective Ala12 allele in peroxisome proliferator-activated receptor (PPAR)gamma was present in two nondiabetic individuals. Carriers of both mtDNA and HNF1alpha mutations showed an earlier age at onset of diabetes than carriers of only the mtDNA mutation (median 22 vs. 45 years) but no clear difference in beta-cell function or insulin sensitivity. In vitro, the M626K mutation caused a 53% decrease in transcriptional activity in HeLa cells. The mutated protein showed normal nuclear targeting but increased DNA binding. These data demonstrate that several genetic factors might contribute to diabetes risk, even in families with mtDNA and HNF1alpha mutations.

Adolescent↗

PubMed2004

Origin of intra-individual variation in PCR-amplified mitochondrial cytochrome oxidase I of Thrips tabaci (Thysanoptera: Thripidae): mitochondrial heteroplasmy or nuclear integration?

The mitochondrial genome is increasingly being used as a species diagnostic marker in insects. Typically, genomic DNA is PCR amplified and then analysed by restriction analyses or sequencing. This analysis system may cause some serious problems for molecular diagnosis. Besides the errors introduced by the PCR process, mtDNA sequence variation of amplified fragments may originate from mtDNA heteroplasmy or from nuclear integrations of mtDNA fragments, both of which have been shown to occur in insects. Here we document abundant variation in PCR-amplified sequences of the mitochondrial cytochrome oxidase I gene of Thrips tabaci. We confirm that the most common haplotype is of mitochondrial origin. Some of the observed mutations were introduced by the amplification process. However, the occurrence of some haplotypes at elevated frequencies indicates that within-individual variation of the respective fragment exists at low levels in T. tabaci. The frequencies of these sequences are too low to negatively affect mtDNA-based molecular diagnosis of T. tabaci. The possible origin of these variant haplotypes is discussed.

Animals↗

PubMed2006

Global patterns in human mitochondrial DNA and Y-chromosome variation caused by spatial instability of the local cultural processes.

Because of the widespread phenomenon of patrilocality, it is hypothesized that Y-chromosome variants tend to be more localized geographically than those of mitochondrial DNA (mtDNA). Empirical evidence confirmatory to this hypothesis was subsequently provided among certain patrilocal and matrilocal groups of Thailand, which conforms to the isolation by distance mode of gene diffusion. However, we expect intuitively that the patterns of genetic variability may not be consistent with the above hypothesis among populations with different social norms governing the institution of marriage, particularly among those that adhere to strict endogamy rules. We test the universality of this hypothesis by analyzing Y-chromosome and mtDNA data in three different sets of Indian populations that follow endogamy rules to varying degrees. Our analysis of the Indian patrilocal and the matrilocal groups is not confirmatory to the sex-specific variation observed among the tribes of Thailand. Our results indicate spatial instability of the impact of different cultural processes on the genetic variability, resulting in the lack of universality of the hypothesized pattern of greater Y-chromosome variation when compared to that of mtDNA among the patrilocal populations.

Chromosome Mapping↗

PubMed2003

Evidence for recombination of mtDNA in the marine mussel Mytilus trossulus from the Baltic.

A number of studies have claimed that recombination occurs in animal mtDNA, although this evidence is controversial. Ladoukakis and Zouros (2001) provided strong evidence for mtDNA recombination in the COIII gene in gonadal tissue in the marine mussel Mytilus galloprovincialis from the Black Sea. The recombinant molecules they reported had not however become established in the population from which experimental animals were sampled. In the present study, we provide further evidence of the generality of mtDNA recombination in Mytilus by reporting recombinant mtDNA molecules in a related mussel species, Mytilus trossulus, from the Baltic. The mtDNA region studied begins in the 16S rRNA gene and terminates in the cytochrome b gene and includes a major noncoding region that may be analogous to the D-loop region observed in other animals. Many bivalve species, including some Mytilus species, are unusual in that they have two mtDNA genomes, one of which is inherited maternally (F genome) the other inherited paternally (M genome). Two recombinant variants reported in the present study have population frequencies of 5% and 36% and appear to be mosaic for F-like and M-like sequences. However, both variants have the noncoding region from the M genome, and both are transmitted to sperm like the M genome. We speculate that acquisition of the noncoding region by the recombinant molecules has conferred a paternal role on mtDNA genomes that otherwise resemble the F genome in sequence.

Animals↗

PubMed1993

Low levels of intraspecific variation in the mitochondrial DNA of chum salmon (Oncorhynchus keta).

A total of 798 individuals from 42 different populations of chum salmon (Oncorhynchus keta) were examined for mtDNA variation. Populations were sampled across the geographic range of the species, from mainland Japan around the Pacific Rim to the state of Washington in the United States. The entire D-loop region (approximately 1 kb) was sequenced for 16 individuals from representative populations. Subregions (approximately 200 nucleotides each) of the D-loop reported to be rapidly evolving in salmon were sequenced for another 29 individuals. Only 4 nucleotide variants were detected, and they occurred in only 4 individuals. Four coding regions of the mtDNA genome were also examined using restriction fragment analysis of products amplified via the polymerase chain reaction. Only one, the region coding for NADH dehydrogenase subunits 5 and 6, showed any variation at this level. The restriction enzyme AseI revealed a polymorphism where the frequency of haplotypes was correlated geographically. We surveyed all individuals for this polymorphism and documented a cline in frequency of the haplotypes around the Pacific Rim. There was a significant frequency difference between Japan and 3 other major geographic regions (Russia, Alaska/Yukon, and British Columbia/Washington) for the presence of the 2 haplotypes. This marker may prove useful in the identification of continent-of-origin for individual chum salmon caught in the open ocean.

Animals↗

PubMed2003

Partial DAZ deletions in a family with five infertile brothers.

OBJECTIVE: To study the genetic cause of infertility in a family with five infertile brothers. DESIGN: Case report. SETTINGS: Center for reproductive medicine at a university medical center. PATIENT(S): Five brothers presenting with primary infertility due to severely impaired spermatogenesis; also, their parents and two other paternally related family members. INTERVENTION(S): Fluorescence in situ hybridization and sequence family variant analysis was performed in leukocyte DNA to determine the number of deleted in azoospermia (DAZ) genes. Linkage analysis was performed for X chromosome inheritance, and mitochondrial DNA (mtDNA) was screened for mutations. MAIN OUTCOME MEASURE(S): DAZ gene copy number, X chromosome linkage, and mtDNA sequence. RESULT(S): With conventional polymerase chain reaction (PCR) analysis, no deletions of the AZFc region were found, but with fluorescence in situ hybridization and sequence family variant analysis, only two DAZ genes instead of four were detected in all individuals tested. The five brothers did not share an identical X chromosomal locus, and no mutations were found in the mtDNA of the index patient. CONCLUSION(S): A reduced copy number of the DAZ genes is found in five infertile brothers with severely impaired spermatogenesis, as well as in their normospermic father and in two other fertile paternally related family members. This illustrates that the phenotype associated with a reduced copy number of the DAZ genes can be extremely variable.

Adult↗

PubMed1996

Do sequence variants in the major non-coding region of the mitochondrial genome influence mitochondrial mutations associated with disease?

Several different mutations in human mitochondrial DNA (mtDNA) have been associated with disease, but their origins and the basis of the wide phenotypic variability remain to be elucidated. We initially investigated three patients with heteroplasmic disease associated mutations of mtDNA for the presence of cis mutations in the major non-coding region that might influence their origins or pathology. A T --> C transition at nt 16 189 previously identified in one patient with the 3243 G:C mutation was associated with heteroplasmic length variation. Identical length variation was found in patient-derived cybrid lines containing 0-97.5% 3243 G:C. Similarly, heteroplasmic length variation was demonstrated in 2/6 other probands with both the 3243 mutation and the 16,189 polymorphism. The distribution of length variants in probands and in asymptomatic family members was identical in all cases. Thus length variation appears to be independent of the level of 3243 mutant mtDNA and hence probably arose within both 3243 G:C and 3243 A:T mtDNAs. We suggest that the 16,189 polymorphism reflects a predisposition to the formation or fixation of several different mutations in mitochondrial tRNA-LeuUUR.

Base Sequence↗

PubMed1989

Molecular population genetics of mtDNA size variation in crickets.

Nucleotide sequence analysis of a region of cricket (Gryllus firmus) mtDNA showing discrete length variation revealed tandemly repeated sequences 220 base pairs (bp) in length. The repeats consist of 206 bp sequences bounded by the dyad symmetric sequence 5'GGGGGCATGCCCCC3'. The sequence data showed that mtDNA size variation in this species is due to variation in the number of copies of tandem repeats. Southern blot analysis was used to document the frequency of crickets heteroplasmic for two or more different-sized mtDNAs. In New England populations of G. firmus and a close relative Gryllus pennsylvanicus approximately 60% of the former and 45% of the latter were heteroplasmic. From densitometry of autoradiographs the frequencies of mtDNA size classes were determined for the population samples and are shown to very different in the two species. However, in populations where hybridization between the two species has occurred, the frequencies of size classes and cytoplasmic genotypes in each species' distinct mtDNA lineage were shifted in a manner suggesting nuclear-cytoplasmic interactions. The data were applied to reported diversity indices and hierarchical statistics. The hierarchical statistics indicated that the greatest proportion of variation for mtDNA size was due to variation among individuals in their cytoplasmic genotypes (heteroplasmic or homoplasmic state). The diversity indices were used to estimate a per-generation mutation rate for size variants of 10(-4). The data are discussed in light of the relationship between genetic drift and mutation in maintaining variation for mtDNA size.

Animals↗

PubMed1981

Maternal inheritance of mitochondrial DNA polymorphisms in cultured human fibroblasts.

We have isolated the total cellular DNA from the cultured diploid fibroblasts of a six-member, three-generation human family. Using a specific radioactive probe for mitochondrial (mt) sequences we have identified new polymorphic variants in this family for the Hhal restriction endonuclease cleavage pattern of the mtDNA. The inheritance of these cleavage patterns verifies the maternal inheritance of mtDNA through all three generations.

Adolescent↗

PubMed1994

Mitochondrial DNA diversity in the pea aphid Acyrthosiphon pisum.

The pea aphid Acyrthosiphon pisum has been established in North America for at least a century and occurs on a broad range of host plants. Despite its importance as a crop pest, there is little understanding of the genetic structure of its populations or the extent of genetic divergence associated with different host plant utilization. This study examined the extent of mitochondrial DNA (mtDNA) diversity among 35 clones of pea aphids from alfalfa. Polymorphisms were detected at only 2 of 126 restriction sites, suggesting the same impoverished level of genetic diversity found in prior allozyme studies. However, length variation was common and apparently arose as a result of tandem repeats in two regions of the mtDNA molecule. Region 1 occurred in proximity to the control region of the molecule, while region 2 was close to a cluster of tRNA genes flanking the NAD-dehydrogenase subunit 3 gene on the opposite side of the mtDNA molecule. Each of the aphid clones was homoplasmic for a single length variant in region 1, which varied with respect to the number of copies of a 120-bp repeat. By contrast, one-third of the clones were heteroplasmic at region 2, where they possessed a variable number of copies of a 210-bp repeat. Reanalysis of clones after 30 generations of parthenogenetic reproduction established the stability of length variants over this interval, suggesting their value in studies of the genetic structure of aphid populations.

Animals↗

PubMed2006

A case of Kearns-Sayre sindrome with autoimmune thyroiditis and complete atrio-ventricular block.

The Kearns-Sayre syndrome, (characterized by its onset before the age of 20 years, chronic ophthalmoplegia, pigmentary retinal degeneration and at least one of the following symptoms: ataxia, heart block and high protein content in the cerebrospinal fluid) is a severe variant of chronic progressive external ophthalmoplegia with frequent rearrangements of the mitochondrial DNA (mtDNA). The aim of this paper is to report a sporadic paediatric case of Kearns-Sayre syndrome with mtDNA heteroplasmic deletion, absence of cytochrome c-oxidase in many muscle fibers, autoimmune thyroiditis, complete atrio-ventricular heart block in which the diagnosis of subclinical hypothyroiditis associated with autoimmune thyroid disease was made. The subclinical hypothyroidism, more severe in the presence of thyroid antibodies, may have contributed to the pathogenesis of cardiovascular disease. We hypothesized that in this patient, predisposed by mitochondrial deletion, anti-thyroid antibodies may have interfered with the mitochondrial function of conduction heart system, causing atrio-ventricular heart block. It seems important to study anti-thyroid antibodies in every case of Kearn-Sayre syndrome, specially if cardiac rhythm disturbances are present.

Adolescent↗