We analyzed the control region of the mitochondrial DNA (mtDNA) from maternally related individuals originating from the Azores Islands (Portugal) in order to estimate the mutation rate of mtDNA and to gain insights into the process by which a new mutation arises and segregates into heteroplasmy. Length and/or point heteroplasmies were found at least in one individual of 72% of the studied families. Eleven new point substitutions were found, all of them in heteroplasmy, from which five appear to be somatic mutations and six can be considered germinal, evidencing the high frequency of somatic mutations in mtDNA in healthy young individuals. Different values of the mutation rate according to different assumptions were estimated. When considering all the germinal mutations, the value of the mutation rate obtained is one of the highest reported so far in family studies. However, when corrected for gender (assuming that the mutations present in men have the same evolutionary weight of somatic mutations because they will inevitably be lost) and for the probability of intraindividual fixation, the value for the mutation rate obtained for HVRI and HVRII (0.2415 mutations/site/Myr) was in the upper end of the values provided by phylogenetic estimations. These results indicate that the discrepancy, that has been reported previously, between the human mtDNA mutation rates observed along evolutionary timescales and the estimations obtained using family pedigrees can be minimized when corrections for gender proportions in newborn individuals and for the probability of intraindividual fixation are introduced. The analyses performed support the hypothesis that (1) in a constant, tight bottleneck genetic drift alone can explain different patterns of heteroplasmy segregation and (2) in neutral conditions, the destiny of a new mutation is strictly related to the initial proportion of the new variant. Another important point arising from the data obtained is that, even in the absence of a paternal contribution of mtDNA, recombination may occur between mtDNA molecules present in an individual, which is only observable if it occurs between mtDNA types that differ at two or more positions.

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.

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SummaryThe Azores islands (Portugal), uninhabited when discovered by Portuguese navigators in the fifteenth century, are located in the Atlantic Ocean 1500 km from the European mainland. The archipelago is formed by nine islands of volcanic origin that define three geographical groups: Eastern (S. Miguel and Sta. Maria), Central (Terceira, Faial, Pico, Graciosa and S. Jorge) and Western (Flores and Corvo). To improve the genetic characterisation of the Azorean population, and to clarify some aspects related to the history of settlement, a study of mtDNA was conducted in the population of the archipelago. The HVRI region was sequenced and specific RFLPs were screened in 146 samples obtained from unrelated individuals with Azorean ancestry (50 from the Eastern group, 60 from the Central group, and 37 from the Western group). Samples were classified into haplogroups based on the information obtained from both sequencing and RFLP analysis.All the analyses performed support the idea that, in the whole group of islands, the majority of mtDNA lineages originated from the Iberian Peninsula, mainly from Portugal (mainland). However contributions from other European populations, especially from Northern Europe, cannot be disregarded. The values obtained for the various diversity parameters in the Azores archipelago indicate that the Azorean population, as a whole, does not exhibit the typical characteristics of an isolated population. The analysis of genetic data by groups of islands showed that the Western group exhibited particular features. The distribution of haplogroups in the Western group is very atypical, being significantly different from what is observed in the Eastern and Central groups. Furthermore, the diversity values are, in general, lower than those observed in other populations used for comparison. African haplogroups were found in all the groups of islands. Therefore the presence of Moorish and African slaves on the islands, as reported in historical sources, is supported by the mtDNA genetic data, especially in the Eastern group. The presence of Jews in the Central group is also supported by the mtDNA data. Neither historical nor genetic data (phylogeography of mtDNA) supports the idea of a differential settlement history for the Western group; however, it is represented in the phylogenies as an isolated branch. The effect of genetic drift, induced by the reduced population size since peopling occurred, has led to a very atypical distribution of haplogroups/haplotypes in this group of islands.We cannot ignore the influence of biodemographic and genetic processes, namely founder effect, genetic drift, migration, and even recent mutational events in the mtDNA lineages of the Azorean populations. Nevertheless, a great part of the variation in the Azorean mtDNA can be explained by the settlement history.

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.

Lewy body diseases include dementia with Lewy bodies and Parkinson's disease. Whereas dementia with Lewy bodies and Parkinson's disease can be distinguished as separate clinical entities, the pathological picture is very often identical. α-synuclein aggregation is a key event in the pathogenesis of Lewy body diseases and β-synuclein inhibits α-synuclein aggregation in vitro and in vivo. Recently, β-synuclein has been shown to interact directly with α-synuclein, regulating its functionality and preventing its oligomerization. In this study, we analysed the expression of two β-synuclein transcript variants and the main α-synuclein transcript SNCA140, in frozen samples of three areas from brains of patients with (i) pure diffuse Lewy body pathology; (ii) pure Alzheimer's disease pathology; (iii) diffuse Lewy body pathology and concomitant Alzheimer's disease pathology and (iv) controls. Relative messenger RNA expression was determined by real-time polymerase chain reaction, expression changes were evaluated by the ΔΔC(t) method and messenger RNA expression data were confirmed at the protein level. A drastic diminution of β-synuclein expression was observed in cortical areas of all samples that presented neuropathological features corresponding to pure diffuse Lewy body pathology and the clinical phenotype of dementia with Lewy bodies, but not in those with neuropathological features corresponding to diffuse Lewy body pathology and concomitant Alzheimer's disease pathology or the clinical phenotype of Parkinson's disease with dementia. The correlation of expression data with the clinical phenotype and neuropathological diagnosis of the patients suggested the existence of a specific molecular subtype of dementia with Lewy bodies, characterized by a strong decrease of β-synuclein in the frontal and temporal cortices. Furthermore, our findings provide new insights into the pathogenesis of Lewy body diseases that may be important for the understanding of molecular mechanisms involved in these complex diseases.

GBA mutations are also an important risk factor for DLB development in the Spanish population, are associated with earlier disease onset, and are more prevalent in men. © 2015 International Parkinson and Movement Disorder Society.