Nonsynonymous site heteroplasmy in fish mitochondrial DNA

Heteroplasmic nucleotide polymorphisms are rarely observed in wild animal mitochondrial DNA. The occurrence of such site heteroplasmy is expected to be extremely rare at nonsynonymous sites where the number of nucleotide substitutions per site is low due to functional constraints. This report deals with nonsynonymous mitochondrial heteroplasmy from two wild fish species, chum salmon and Japanese flounder. We detected an A/C nonsynonymous heteroplasmic site corresponding to putative amino acids, Ile or Met, in NADH dehydrogenase subunit-5 (ND5) region of chum salmon. The heteroplasmic site was at the 3rd position of 58th codon. As for Japanese flounder we detected a C/T nonsynonymous heteroplasmic site corresponding to putative amino acids, Leu or Pro, in ND4 region. The heteroplasmic site was at the 2nd position of 450th codon. We also verified heteroplasmy at these sites by sequencing cloned fragments.     

A new mitochondrial disease associated with mitochondrial DNA heteroplasmy.

A variable combination of developmental delay, retinitis pigmentosa, dementia, seizures, ataxia, proximal neurogenic muscle weakness, and sensory neuropathy occurred in four members of a family and was maternally transmitted. There was no histochemical evidence of mitochondrial myopathy. Blood and muscle from the patients contained two populations of mitochondrial DNA, one of which had a previously unreported restriction site for AvaI. Sequence analysis showed that this was due to a point mutation at nucleotide 8993, resulting in an amino acid change from a highly conserved leucine to arginine in subunit 6 of mitochondrial H(+)-ATPase. There was some correlation between clinical severity and the amount of mutant mitochondrial DNA in the patients; this was present in only small quantities in the blood of healthy elderly relatives in the same maternal line.     

Naturally occurring mitochondrial DNA heteroplasmy in the MRL mouse

The MRL/MpJ mouse is an inbred laboratory strain of Mus musculus, known to exhibit enhanced autoimmunity, increased wound healing, and increased regeneration properties. We report the full-length mitochondrial DNA (mtDNA) sequence of the MRL mouse (Accession # EU450583), and characterize the discovery of two naturally occurring heteroplasmic sites. The first is a T3900C substitution in the TPsiC loop of the tRNA methionine gene (tRNA-Met; mt-Tm). The second is a heteroplasmic insertion of 1-6 adenine nucleotides in the A-tract of the tRNA arginine gene (tRNA-Arg; mt-Tr) at positions 9821-9826. The level of heteroplasmy varied independently at these two sites in MRL individuals. The length of the tRNA-Arg A-tract increased with age, but heteroplasmy at the tRNA-Met site did not change with age. The finding of naturally occurring mtDNA heteroplasmy in an inbred strain of mouse makes the MRL mouse a powerful new experimental model for studies designed to explore therapeutic measures to alter the cellular burden of heteroplasmy.     

Screening for mitochondrial DNA heteroplasmy in children at risk for mitochondrial disease

Temporal temperature gradient gel electrophoresis was used to screen 70% of the mtDNA, including all 22 tRNA genes, for heteroplasmy in 75 children with neuromuscular and/or multi-system dysfunction and elevated lactate levels, and in 95 controls. Standard PCR/ASO (allele specific oligonucleotide) and Southern analyses were also employed. Excluding common length variants, heteroplasmy was found in 22 patients and two controls (P < 0.001), with four patients demonstrating heteroplasmy in two locations each. Of the 23 heteroplasmic variants sequenced among the patients, 17 were novel point variants in the control region (CR) and only two involved tRNA genes. Heteroplasmy is highly associated with the disease group, and is predominately found in the CR, an area rarely studied in patient populations. These variants may be pathological mutations or disease markers.     

Size polymorphism and heteroplasmy in the mitochondrial DNA of lower vertebrates

The mitochondrial DNA of the bowfin fish and each of two species of treefrogs displays large-scale size variation. Within each species, mitochondrial genomes span more than a 700 base pair range, and the size polymorphism is localized to one portion of the genome. In addition, about 5 percent of the total 357 individuals surveyed were observed to carry two size classes of mtDNA. These findings are among the few documented instances of extensive within-species mtDNA size polymorphism and individual heteroplasmy, and constitute exceptions to previously reached generalizations about the molecular basis of mtDNA variation.     

RecA-dependent DNA repair results in increased heteroplasmy of the Arabidopsis mitochondrial genome

Plant mitochondria have very active DNA recombination activities that are responsible for its plastic structures and that should be involved in the repair of double-strand breaks in the mitochondrial genome. Little is still known on plant mitochondrial DNA repair, but repair by recombination is believed to be a major determinant in the rapid evolution of plant mitochondrial genomes. In flowering plants, mitochondria possess at least two eubacteria-type RecA proteins that should be core components of the mitochondrial repair mechanisms. We have performed functional analyses of the two Arabidopsis (Arabidopsis thaliana) mitochondrial RecAs (RECA2 and RECA3) to assess their potential roles in recombination-dependent repair. Heterologous expression in Escherichia coli revealed that RECA2 and RECA3 have overlapping as well as specific activities that allow them to partially complement bacterial repair pathways. RECA2 and RECA3 have similar patterns of expression, and mutants of either display the same molecular phenotypes of increased recombination between intermediate-size repeats, thus suggesting that they act in the same recombination pathways. However, RECA2 is essential past the seedling stage and should have additional important functions. Treatment of plants with several DNA-damaging drugs further showed that RECA3 is required for different recombination-dependent repair pathways that significantly contribute to plant fitness under stress. Replication repair of double-strand breaks results in the accumulation of crossovers that increase the heteroplasmic state of the mitochondrial DNA. It was shown that these are transmitted to the plant progeny, enhancing the potential for mitochondrial genome evolution.     

The distribution of mitochondrial DNA heteroplasmy due to random genetic drift

Cells containing pathogenic mutations in mitochondrial DNA (mtDNA) generally also contain the wild-type mtDNA, a condition called heteroplasmy. The amount of mutant mtDNA in a cell, called the heteroplasmy level, is an important factor in determining the amount of mitochondrial dysfunction and therefore the disease severity. mtDNA is inherited maternally, and there are large random shifts in heteroplasmy level between mother and offspring. Understanding the distribution in heteroplasmy levels across a group of offspring is an important step in understanding the inheritance of diseases caused by mtDNA mutations. Previously, our understanding of the heteroplasmy distribution has been limited to just the mean and variance of the distribution. Here we give equations, adapted from the work of Kimura on random genetic drift, for the full mtDNA heteroplasmy distribution. We describe how to use the Kimura distribution in mitochondrial genetics, and we test the Kimura distribution against human, mouse, and Drosophila data sets.     

Expansion/contraction of mammalian mitochondrial DNA repeats in Escherichia coli mimics the mitochondrial heteroplasmy.

Length polymorphism due to tandem repeats is a common feature in animal mitochondrial DNA. The rabbit mitochondrial genome contains a 20 bp repeat domain, which generates a general heteroplasmic state. The observed polymorphic patterns suggest a dynamic equilibrium between gain and loss of units that maintains the copy number in the range 3-19 repeat units. In the apparent absence of recombination, slipped-strand mispairing during replication appears to be the primary cause of additions and deletions. To investigate this hypothesis we have set up a plasmid assay in Escherichia coli. A variable number of repeat units was inserted into a plasmid in both orientations relative to the colE1 origin of replication. Our data show that (i) a minimum unit number (>3) is necessary to generate length polymorphs, (ii) the number of events increases with the length tract, (iii) an excess of additions over deletions is found when the copy number is less than 10 and the trend is reversed when it is over 10, (iv) the frequency of deletions-additions is dependent on the orientation, (v) the polymorphism patterns are different according to the orientation. The length polymorphic pattern generated in the bacteria, in one orientation, mimics that observed in the mitochondria, suggesting that slipped mispairing between repeated sequences during DNA replication is responsible for the mitochondrial heteroplasmic state.     

Individual human hair mitochondrial DNA control region heteroplasmy proportions in mothers and children

Due to maternal inheritance, lack of recombination and a high polymorphic density, the mtDNA control region hypervariable (HV) regions are well suited for forensic identification using a maternal relative as the known sample. This analysis can be performed in hair, however, heteroplasmy in this tissue is not rare and can result in an apparent sequence mismatch that complicates this application. There is little data comparing mother and child mtDNA-CR heteroplasmic proportions in hair. In this study, we assayed four hairs per individual in 26 mother-child pairs by TTGE for heteroplasmy across HV1. Single nucleotide heteroplasmy was detected in seven families, and in four families at least two hairs were heteroplasmic. In each of the latter families, sequencing and PCR-RFLP confirmed single nucleotide heteroplasmy in proportions of the variant ranging from < or =10 to > or =90% in the mothers, with far less variability in their children. Sequencing alone would have revealed apparent homoplasmic differences at one nucleotide in these families, possibly resulting in an 'inconclusive' verdict for relatedness of child and mother. However, mother-child heteroplasmic variability did not exceed intra-individual variability in the mothers alone.     

Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes.

Many human mitochondrial diseases are associated with defects in the mitochondrial DNA (mtDNA). Mutated and wild-type forms of mtDNA often coexist in the same cell in a state called heteroplasmy. Here, we report the isolation of a Caenorhabditis elegans strain bearing the 3.1-kb uaDf5 deletion that removes 11 genes from the mtDNA. The uaDf5 deletion is maternally transmitted and has been maintained for at least 100 generations in a stable heteroplasmic state in which it accounts for approximately 60% of the mtDNA content of each developmental stage. Heteroplasmy levels vary between individual animals (from approximately 20 to 80%), but no observable phenotype is detected. The total mtDNA copy number in the uaDf5 mutant is approximately twice that of the wild type. The maternal transmission of the uaDf5 mtDNA is controlled by at least two competing processes: one process promotes the increase in the average proportion of uaDf5 mtDNA in the offspring, while the second promotes a decrease. These two forces prevent the segregation of the mtDNAs to homoplasmy.     

Induction of mitochondrial DNA heteroplasmy by intra- and interspecific transplantation of germ plasm in Drosophila

A new experimental system for inducing mitochondrial DNA heteroplasmy in Drosophila was developed. By transplanting the germ plasm of Drosophila melanogaster and Drosophila mauritiana into the posterior pole of the recipient eggs of D. melanogaster, it was possible to introduce foreign mitochondria into the recipient female germline. Heteroplasmic individuals containing both donor and recipient mtDNA were obtained in intra- and interspecific combinations at similar frequencies. The proportion of donor-derived mtDNA in the heteroplasmic individuals varied considerably from individual to individual irrespective of the donor species used. No significant decrease in or elimination of donor mtDNA was observed, and the heteroplasmic state in female germlines persisted for several generations. The present system should serve very much to promote the study and clarification of the transmission genetics of mtDNA in insects.     

MitoBamAnnotator: A web-based tool for detecting and annotating heteroplasmy in human mitochondrial DNA sequences

The use of Next-Generation Sequencing of mitochondrial DNA is becoming widespread in biological and clinical research. This, in turn, creates a need for a convenient tool that detects and analyzes heteroplasmy. Here we present MitoBamAnnotator, a user friendly web-based tool that allows maximum flexibility and control in heteroplasmy research. MitoBamAnnotator provides the user with a comprehensively annotated overview of mitochondrial genetic variation, allowing for an in-depth analysis with no prior knowledge in programming.     

Neutral mitochondrial heteroplasmy alters physiological function in mice

Cytoplasmic transfer is an assisted reproductive technique that involves the infusion of ooplasm from a donor oocyte into a recipient oocyte of inferior developmental competence. Although this technique has shown some success for couples with recurrent in vitro fertilization failure, it results in mitochondrial heteroplasmy in the offspring, defined as the presence of two different mitochondrial genomes in the same individual. Because the long-term health consequences of mitochondrial heteroplasmy are unknown, there is a need for appropriate animal models to evaluate any physiological changes of dual mtDNA genotypes. This longitudinal study was designed as a preliminary screen of basic physiological functions for heteroplasmic mice (NZB mtDNA on a BALB/cByJ background). The mice were tested for cardiovascular and metabolic function, hematological parameters, body mass analysis, ovarian reserve, and tissue histologic abnormalities over a period of 15 mo. Heteroplasmic mice developed systemic hypertension that corrected over time and was accompanied by cardiac changes consistent with pulmonary hypertension. In addition, heteroplasmic animals had increased body mass and fat mass compared with controls at all ages. Finally, these animals had abnormalities in electrolytes and hematological parameters. Our findings suggest that there are significant physiological differences between heteroplasmic and control mice. Because ooplasm transfer appears to be consistently associated with mitochondrial heteroplasmy, children conceived through ooplasm transfer should be closely followed to determine if they are at risk for any health problems.     

Restriction site heteroplasmy in the mitochondrial DNA of the marine fish Sciaenops ocellatus (L.)

Restriction site heteroplasmy involving the enzymes NcoI and XbaI was detected in the mitochondrial DNAs of two individuals of the marine fish Sciaenops ocellatus. This represents only the sixth documented example of mitochondrial DNA restriction site heteroplasmy in animals. Two heteroplasmic individuals were found in a survey of nearly 750 individuals, suggesting that in most studies the incidence of mitochondrial DNA site heteroplasmy may be too low to be routinely detected.     

Length and restriction site heteroplasmy in the mitochondrial DNA of american shad (alosa sapidissima)

Restriction endonuclease analysis was used to assess mitochondrial DNA (mtDNA) variation in American shad (Alosa sapidissima) collected from 14 rivers ranging from Florida to Quebec. Two types of heteroplasmy were observed, one involving a major length polymorphism and the other a single restriction site. Shad mtDNA occurred in two principal size classes, 18.3 and 19.8 kb. Of 244 shad examined, 30 were heteroplasmic and carried both size classes of mtDNA in varying proportions; the remainder were homoplasmic for the smaller size class of mtDNA. The large mtDNA variant occurred most frequently at the southern end of the range, and except for two individuals from Nova Scotia, was not detected among shad from rivers north of the Delaware. In contrast, ten shad heteroplasmic for a SalI restriction site originated from rivers ranging from South Carolina to Nova Scotia. DNA mapping and hybridization experiments indicated that the length polymorphism is in the D-loop-containing region and consists of a tandemly repeated 1.5-kb DNA sequence occurring in two and three copies, respectively, in the two major size classes of shad mtDNA. Continuous length variation up to approximately 40 bp occurs among copies of the repeat both within and among individuals. Restriction site data support the conclusion that both forms of heteroplasmy in shad mtDNA have originated more than once.     

A new method for analysis of mitochondrial DNA point mutations and assess levels of heteroplasmy

Determination of mitochondrial DNA (mtDNA) heteroplasmy for the diagnosis of patients with mitochondrial disorders is a difficult task due to the coexistence of wild-type and mutant genomes. We have developed a new method for genotyping and quantification of heteroplasmic point mutations in mtDNA based on the SNaPshot technology. We compared the data of this method with the widely used "last hot-cycle" PCR-RFLP method by studying 15 patients carrying mtDNA mutations. We showed that SNaPshot is an accurate, reproducible, and sensitive technique for the determination of heteroplasmic mtDNA mutations in different tissues from patients, and it is a promising system to be used in prenatal and postnatal diagnosis of mtDNA-associated disorders.     

Nuclear mitochondrial interplay in the modulation of the homopolymeric tract length heteroplasmy in the control (D-loop) region of the mitochondrial DNA

We have studied the genetic characteristics of a homopolymeric tract length heteroplasmy associated with the 16189C variant in the mtDNA D-loop control region to identify the factor(s) involved in the generation of the length heteroplasmy. The relative proportion of the various lengths of the polycytosines (i.e., the pattern of the length heteroplasmy) is maintained in an individual, and previous evidence shows that it is regenerated de novo following cell divisions. The pattern is maintained in maternally related individuals, suggestive of mtDNA determinants. Of the 38 individuals with the 16189C variant studied, 39% were found to exhibit the (16180)AAACCCCCCCCCCC(16193) variant associated with A16183C polymorphism [(11C)-group], while 53% showed the (16180)AACCCCCCCCCCCC(16193) variant associated with a further A16182C polymorphism [(12C)-group]. Haplotype analysis of the mtDNA revealed a specific association of the longer mean length of the poly[C] in the (12C)-group with haplogroup B. A similar association was also observed in the (11C)-group, but with a novel haplogroup. Cybrid constructions revealed that the involvement of nuclear factor(s) in the generation of the length heteroplasmy is prominent in homopolymeric tract of eight cytosines. The nuclearly coded factor(s) is/are presumably related to the fidelity of the nuclearly coded components of the mitochondrial DNA replication machinery.     

Degree of heteroplasmy reflects oxidant damage in a large family with the mitochondrial DNA A8344G mutation

Mitochondria are the source of most oxygen-derived free radicals. Mutations in mitochondrial DNA can impair mitochondrial electron transport resulting in decreased ATP production and increased free radical-induced oxidant injury. The specific mitochondrial DNA mutation A8344G alters the TPsiC loop or the mitochondrial tRNA for lysine. We investigated a large five-generational family harboring this mutation to determine whether the degree of heteroplasmy (proportion of mutated mitochondrial genomes) for the mtA8344G mutation correlated with a marker of oxidant damage. We measured F2-isoprostanes because they are specific and reliable markers of oxidant injury formed when free radicals attack esterified arachidonate in cell membranes. Family members with high heteroplasmy (>40%) had significantly higher F2-isoprostane levels (62 +/- 39 pg/ml) than those with lower heteroplasmy (33 +/- 13 pg/ml, P < 0.001). The degree of heteroplasmy for the mtA8344G mutation in this family correlated positively with F2-isoprostane levels (P = 0.03). This study highlights the underappreciated role free radicals play in the complex pathophysiology of inherited mitochondrial DNA disorders. The most important novel finding from this family is that some currently asymptomatic individuals with moderate heteroplasmy have evidence of ongoing free-radical mediated oxidant injury.