Determining the levels of human mitochondrial heteroplasmy is of utmost importance in several fields. In spite of this, there are currently few published works that have focused on this issue. In order to increase the knowledge of mitochondrial DNA (mtDNA) heteroplasmy, the main goal of this work is to investigate the frequency and the mutational spectrum of heteroplasmy in the human mtDNA genome. To address this, a set of nine primer pairs designed to avoid co-amplification of nuclear DNA (nDNA) sequences of mitochondrial origin (NUMTs) was used to amplify the mitochondrial genome in 101 individuals. The analysed individuals represent a collection with a balanced representation of genders and mtDNA haplogroup distribution, similar to that of a Western European population. The results show that the frequency of heteroplasmic individuals exceeds 61%. The frequency of point heteroplasmy is 28.7%, with a widespread distribution across the entire mtDNA. In addition, an excess of transitions in heteroplasmy were detected, suggesting that genetic drift and/or selection may be acting to reduce its frequency at population level. In fact, heteroplasmy at highly stable positions might have a greater impact on the viability of mitochondria, suggesting that purifying selection must be operating to prevent their fixation within individuals. This study analyses the frequency of heteroplasmy in a healthy population, carrying out an evolutionary analysis of the detected changes and providing a new perspective with important consequences in medical, evolutionary and forensic fields.

To date, there are no published primers to amplify the entire mitochondrial DNA (mtDNA) that completely prevent the amplification of nuclear DNA (nDNA) sequences of mitochondrial origin. The main goal of this work was to design, validate and describe a set of primers, to specifically amplify and sequence the complete human mtDNA, allowing the correct interpretation of mtDNA heteroplasmy in healthy and pathological samples. Validation was performed using two different approaches: (i) Basic Local Alignment Search Tool and (ii) amplification using isolated nDNA obtained from sperm cells by differential lyses. During the validation process, two mtDNA regions, with high similarity with nDNA, represent the major problematic areas for primer design. One of these could represent a non-published nuclear DNA sequence of mitochondrial origin. For two of the initially designed fragments, the amplification results reveal PCR artifacts that can be attributed to the poor quality of the DNA. After the validation, nine overlapping primer pairs to perform mtDNA amplification and 22 additional internal primers for mtDNA sequencing were obtained. These primers could be a useful tool in future projects that deal with mtDNA complete sequencing and heteroplasmy detection, since they represent a set of primers that have been tested for the non-amplification of nDNA.

A significant body of work, accumulated over the years, strongly suggests that damage in mitochondrial DNA (mtDNA) contributes to aging in humans. Contradictory results, however, are reported in the literature, with some studies failing to provide support to this hypothesis. With the purpose of further understanding the aging process, several models, among which mouse models, have been frequently used. Although important affinities are recognized between humans and mice, differences on what concerns physiological properties, disease pathogenesis as well as life-history exist between the two; the extent to which such differences limit the translation, from mice to humans, of insights on the association between mtDNA damage and aging remains to be established. In this paper we revise the studies that analyze the association between patterns of mtDNA damage and aging, investigating putative alterations in mtDNA copy number as well as accumulation of deletions and of point mutations. Reports from the literature do not allow the establishment of a clear association between mtDNA copy number and age, either in humans or in mice. Further analysis, using a wide spectrum of tissues and a high number of individuals would be necessary to elucidate this pattern. Likewise humans, mice demonstrated a clear pattern of age-dependent and tissue-specific accumulation of mtDNA deletions. Deletions increase with age, and the highest amount of deletions has been observed in brain tissues both in humans and mice. On the other hand, mtDNA point mutations accumulation has been clearly associated with age in humans, but not in mice. Although further studies, using the same methodologies and targeting a larger number of samples would be mandatory to draw definitive conclusions, the revision of the available data raises concerns on the ability of mouse models to mimic the mtDNA damage patterns of humans, a fact with implications not only for the study of the aging process, but also for investigations of other processes in which mtDNA dysfunction is a hallmark, such as neurodegeneration.

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In this work, we present the results of the screening of human mitochondrial DNA (mtDNA) heteroplasmy in the control region of mtDNA from 210 unrelated Spanish individuals. Both hypervariable regions of mtDNA were amplified and sequenced in order to identify and quantify point and length heteroplasmy. Of the 210 individuals analyzed, 30% were fully homoplasmic and the remaining presented point and/or length heteroplasmy. The prevalent form of heteroplasmy was length heteroplasmy in the poly(C) tract of the hypervariable region II (HVRII), followed by length heteroplasmy in the poly(C) tract of hypervariable region I (HVRI) and, finally, point heteroplasmy, which was found in 3.81% of the individuals analyzed. Moreover, no significant differences were found in the proportions of the different kinds of heteroplasmy in the population when blood and buccal cell samples were compared. The pattern of heteroplasmy in HVRI and HVRII presents important differences. Moreover, the mutational profile in heteroplasmy seems to be different from the mutational pattern detected in population. The results suggest that a considerable number of mutations and, particularly, transitions that appear in heteroplasmy are probably eliminated by drift and/or by selection acting at different mtDNA levels of organization. Taking as a whole the results reported in this work, it is mandatory to perform a broad-scale screening of heteroplasmy to better establish the heteroplasmy profile which would be important for medical, evolutionary, and forensic proposes.

Our findings support the hypothesis that mutated ataxin-3 is associated with transcription dysregulation, detectable in peripheral blood cells. Furthermore, this is the first report suggesting a pool of up-regulated genes in Machado-Joseph disease that may have the potential to be used for fine phenotyping of this disease. © 2015 International Parkinson and Movement Disorder Society.

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