Amanda Ramos

Human mitochondrial DNA complete amplification and sequencing: a new validated primer set that prevents nuclear DNA sequences of mitochondrial origin co-amplification.

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.

Frequency and pattern of heteroplasmy in the complete human mitochondrial genome.

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.

Frequency and Pattern of Heteroplasmy in the Control Region of Human Mitochondrial DNA

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.

Mitochondrial DNA Damage Patterns and Aging: Revising the Evidences for Humans and Mice

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.

Nuclear insertions of mitochondrial origin: Database updating and usefulness in cancer studies.

Nuclear insertions of mitochondrial origin (NUMTs) can be useful tools in evolution and population studies. However, due to their similarity to mitochondrial DNA (mtDNA), NUMTs may also be a source of contamination in mtDNA studies. The main goal of this work is to present a database of NUMTs, based on the latest version of the human genome-GRCh37 draft. A total of 755 insertions were identified. There are 33 paralogous sequences with over 80% sequence similarity and of a greater length than 500bp. The non-identical positions between paralogous sequences are listed for the first time. As an application example, the described database is used to evaluate the impact of NUMT contamination in cancer studies. The evaluation reveals that 220 positions from 256 with zero hits in the current mtDNA phylogeny could in fact be traced to one or more nuclear insertions of mtDNA. This is due to they are located in non-identical positions between mtDNA and nuclear DNA (nDNA). After in silico primer validation of each revised cancer study, risk of co-amplification between mtDNA and nDNA was detected in some cases, whereas in others no risk of amplification was identified. This approach to cancer studies clearly proves the potential of our NUMT database as a valuable new tool to validate mtDNA mutations described in different contexts. Moreover, due to the amount of information provided for each nuclear insertion, this database should play an important role in designing evolutionary, phylogenetic and epidemiological studies.

Validated primer set that prevents nuclear DNA sequences of mitochondrial origin co-amplification : a revision based on the New Human Genome Reference Sequence (GRCh37)

A new human genome reference sequence – GRCh37 – was recently generated and made available by the Genome Reference Consortium. Since the prior disposable human reference sequence – hg18 – was previously used for the mitochondrial DNA primer BLAST validation, a revision of those previously published primer pairs is required. Thus, the aim of this Short Communication is to perform an in silico BLAST test of the published disposable nine primer pairs using the new human reference sequence and to report the pertinent modifications. The new analysis showed that one of the tested primer pairs requires a revision. Therefore, a new validated primer pair, which specifically amplifies the mitochondrial region located between positions 6520 and 9184, is presented.

Mitochondrial DNA patterns in the Iberian Northern plateau: population dynamics and substructure of the Zamora province

Several studies have shown the importance of recent events in the configuration of the genetic landscape of a specific territory. In this context, due to the phenomena of repopulation and demographic fluctuations that took place in recent centuries, the Iberian Northern plateau is a very interesting case study. The main aim of this work is to check if recent population movements together with existing boundaries (geographical and administrative) have influenced the current genetic composition of the area. To accomplish this general purpose, mitochondrial DNA variations of 214 individuals from a population located in the Western region of the Iberian Northern plateau (the province of Zamora) were analyzed. Results showed a typical Western European mitochondrial DNA haplogroup composition. However, unexpected high frequencies of U5, HV0, and L haplogroups were found in some regions. The analyses of microdifferentiation showed that there are differences between regions, but no geographic substructure organization can be noticed. It can be stated that the differences observed in the genetic pool of the sampled area at regional level results from the mixture of different populations carrying new lineages into this area at different points in history.

Differential mtDNA Damage Patterns in a Transgenic Mouse Model of Machado–Joseph Disease (MJD/SCA3)

Mitochondrial dysfunction has been associated with late onset neurodegenerative disorders, among which is Machado–Joseph disease (MJD/SCA3). In a previous study, using a transgenic mouse model of MJD, we reported a decrease in mitochondrial DNA (mtDNA) copy number and an accumulation of the 3876-bp deletion with age and with phenotype development. We extended this study by analyzing the pattern of mtDNA depletion and the accumulation of the 3876-bp deletion in 12 older transgenic (TG) and 4 wild-type (wt) animals, and by investigating the accumulation of somatic mutations in the D-loop region in 76 mice (42 TG and 34 wt). mtDNA damage was studied in TG and wt mice at different ages and tissues (blood, pontine nuclei, and hippocampus). Results for older mice demonstrate an accumulation of the mtDNA 3867-bp deletion with age, which was more pronounced in TG animals. Furthermore, the tendency for mtDNA copy number decrease with age, in all analyzed tissues of TG and wt animals, was also confirmed. No point mutations were detected in the D-loop, neither in TG nor wt animals, in any of the tissues analyzed. Due to the absence of mtDNA somatic mutations, we can suggest that mtDNA point mutation accumulation cannot be used to monitor the development and progression of the phenotype in this mouse model and likely in any MJD mice model. The present results further confirm not only the association between mtDNA alterations (copy number and deletions) and age, but also between such alterations and the expression of the mutant ataxin-3 in TG mice.

Novel candidate blood-based transcriptional biomarkers of Machado-Joseph disease.

Machado‐Joseph disease (or spinocerebellar ataxia type 3) is a late‐onset polyglutamine neurodegenerative disorder caused by a mutation in the ATXN3 gene, which encodes for the ubiquitously expressed protein ataxin‐3. Previous studies on cell and animal models have suggested that mutated ataxin‐3 is involved in transcriptional dysregulation. Starting with a whole‐transcriptome profiling of peripheral blood samples from patients and controls, we aimed to confirm abnormal expression profiles in Machado‐Joseph disease and to identify promising up‐regulated genes as potential candidate biomarkers of disease status.

Replicating studies of genetic modifiers in spinocerebellar ataxia type 3: can homogeneous cohorts aid?

Sir, We read with interest the paper by Tezenas du Montcel and colleagues (2014), which identified modifiers of the age at onset in spinocerebellar ataxias (SCAs). In polyglutamine (polyQ) SCAs, the size of the CAG expansion is incompletely correlated with the age at onset of the disease, suggesting the involvement of additional genetic factors. The identification of modifier genes for polyQ SCAs should not only contribute to more precise onset predictions, of relevance for genetic counselling, but also unveil aspects of the underlying pathogenesis, and pinpoint possible therapeutic targets (Genin et al. , 2008). Proposed modifiers for SCAs have often failed to be validated in independent studies, a fact that has been mostly attributed to the lack of statistical power (as discussed, for example, in Pulst et al. , 2005). The report by Tezenas du Montcel et al. (2014) represents therefore an important contribution to the problematic modifier genes in polyQ SCAs; this study includes the largest cohorts of patients for SCA1, 2, 3, 6, 7, 17 and dentato-rubro-pallydoluysian atrophy (DRPLA) analysed so far, which were recruited under the scope of the European Consortium on Spinocerebellar Ataxias (EUROSCA). Moreover, main findings from the EUROSCA series were tested in four independent smaller cohorts (three Caucasian cohorts from the USA, France and Italy, as well as one Asian from Japan). SCA3, also named Machado-Joseph disease, is the most frequent polyQ SCA, reaching in the Azores Islands (Portugal), the highest value of prevalence worldwide (Araujo, 2012). This particular epidemiological situation has allowed us to thoroughly follow …