Complete mitochondrial DNA profile in stroke: A geographical matched case-control study in Spanish population.

INTRODUCTION: Stroke, the second leading cause of death worldwide, is a complex disease influenced by many risk factors among which we can find reactive oxygen species (ROS). Since mitochondria are the main producers of cellular ROS, nowadays studies are trying to elucidate the role of these organelles and its DNA (mtDNA) variation in stroke risk. The aim of the present study was to perform a comprehensive evaluation of the association between mtDNA mutations and mtDNA content and stroke risk. MATERIAL AND METHODS: Homoplasmic and heteroplasmic mutations of the mtDNA were analysed in a case-controls study using 110 S cases and their corresponding control individuals. Mitochondrial DNA copy number (mtDNA-CN) was analysed in 73 of those case-control pairs. RESULTS: Our results suggest that haplogroup V, specifically variants m.72C > T, m.4580G > A, m.15904C > T and m.16298 T > C have a protective role in relation to stroke risk. On the contrary, variants m.73A > G, m.11719G > A and m.14766C > T appear to be genetic risk factors for stroke. In this study, we found no statistically significant association between stroke risk and mitochondrial DNA copy number. CONCLUSIONS: These results demonstrate the possible role of mtDNA genetics on the pathogenesis of stroke, probably through alterations in mitochondrial ROS production.

Mitochondrial DNA haplogroups J and T increase the risk of glioma.

The presence of different sets of mitochondrial polymorphisms generated by the accumulation of mutations in different maternal lineages has allowed differentiating mitochondrial haplogroups in human populations. These polymorphisms, in turn, may have effects at the phenotypic level, considering a possible contribution of these germinal mutations to the development of certain diseases such as cancer. The main goal of the present study is to establish a possible association between mitochondrial haplogroups and the risk of suffering glioma. Blood samples were obtained from 32 patients from Catalonia (Spain) diagnosed with different grades of glioma (II, III and IV), according to the World Health Organization. The mitochondrial genome was amplified and sequenced using MiSeq 2000 (Illumina). The HaploGrep tool implemented in mtDNA-Server v.1.0.5 was used for the identification of mitochondrial haplogroups. Data obtained in the present study was further pooled with data from previous European studies including glioma patients from Galicia (Spain) and Italy. Results for the Catalonian samples showed an association between individuals with haplogroup J and the increased risk of suffering glioma, with a significant increase of the frequency of individuals with this haplogroup (25%) regarding the general population (7%). Combining different sets of patients with European origin, it appears that individuals with haplogroups J and T have a significantly higher risk of suffering glioma (p < 0.001; OR: 2.407 and p = 0.007; OR: 1.82, respectively). This is the first study that establishes an association between different mitochondrial haplogroups and the risk of suffering glioma, highlighting the role of mitochondrial variants in this disease.

The role of control region mitochondrial DNA mutations in cardiovascular disease: stroke and myocardial infarction.

Recent studies associated certain type of cardiovascular disease (CVD) with specific mitochondrial DNA (mtDNA) defects, mainly driven by the central role of mitochondria in cellular metabolism. Considering the importance of the control region (CR) on the regulation of the mtDNA gene expression, the aim of the present study was to investigate the role of mtDNA CR mutations in two CVDs: stroke and myocardial infarction (MI). MtDNA CR mutations (both fixed and in heteroplasmy) were analysed in two demographically-matched case-control samples, using 154 stroke cases, 211 MI cases and their corresponding control individuals. Significant differences were found, reporting mutations m.16145 G > A and m.16311 T > C as potential genetic risk factors for stroke (conditional logistic regression: p = 0.038 and p = 0.018, respectively), whereas the m.72 T > C, m.73 A > G and m.16356 T > C mutations could act as possible beneficial genetic factors for MI (conditional logistic regression: p = 0.001, p = 0.009 and p = 0.016, respectively). Furthermore, our findings also showed a high percentage of point heteroplasmy in MI controls (logistic regression: p = 0.046; OR = 0.209, 95% CI [0.045-0.972]). These results demonstrate the possible role of mtDNA mutations in the CR on the pathogenesis of stroke and MI, and show the importance of including this regulatory region in genetic association studies.

Sensitivity of mitochondrial DNA heteroplasmy detection using Next Generation Sequencing.

Although the use of Next Generation Sequencing (NGS) in mitochondrial DNA (mtDNA) studies related to forensic and human genetics has contributed to the report of heteroplasmy at very low levels (lower than 1% and even 0.5%), their detection is not a straightforward process. Our purpose is to establish mitochondrial heteroplasmy detection limits, generating mixed bases at low frequencies by the PCR co-amplification of mtDNA and a nuclear insertion of mitochondrial origin (NUMT). A primer set that presents a perfect annealing with both mitochondrial and nuclear DNA was used to amplify the mitochondrial DNA region located between positions 6739 and 8910 and the corresponding region located inside a NUMT located in chromosome 1 (GRCh38.p12 Primary Assembly 631910-634079) that presents an identity of 98% with the corresponding region of mtDNA in two samples. Independent Nextera XT® (Illumina) NGS libraries were generated for each sample and sequenced in different MiSeq (Illumina) runs. Non-identical and identical positions between individual mtDNA and NUMT sequences were detected, and heteroplasmy detections limits were established: a) with a minor allele frequency <1.5%, false positive and negative can occur, even with a depth higher than 3000X; b) with a minor allele frequency >3%, no presence of false positive and negative were detected with a depth of ~1000X; and c) with a minor allele frequency between 1.5% and 3%, a minimal depth of 3000X was necessary to avoid false positives or negatives detection. Our results demonstrate an alternative strategy to establish a reliable limit of mitochondrial heteroplasmy detection.

Involvement of mitochondrial haplogroups in myocardial infarction and stroke: A case-control study in Castile and Leon (Spain) population.

There are strong evidences that common mitochondrial DNA (mtDNA) haplogroups may influence the pathogenesis of cardiovascular diseases (CVDs). In this matched case-control study, we investigate the association between mtDNA haplogroups and two CVDs, myocardial infarction (MI) and stroke, and classical cardiovascular risk factors. Data obtained show that haplogroup H constitute a susceptibility risk factor for MI (p=0.001; OR=2.379, 95% CI [1.440-3.990]). Otherwise, our data also suggest a beneficial role of haplogroup J against hypertension (p=0.019; OR=0.348, 95% CI [0.144-0.840]). These results may provide some guidance for predicting the genetic risk of these diseases in different human populations through the differences in energy efficiency between haplogroups.

Y-chromosome analysis in a Northwest Iberian population: unraveling the impact of Northern African lineages.

OBJECTIVES: To provide new clues about the genetic origin, composition and structure of the population of the Spanish province of Zamora, with an emphasis on the genetic impact of the period of Islamic rule in the Iberian Peninsula. METHODS: Polymorphisms in the paternally inherited Y-chromosome, Single Nucleotide Polymorphisms and Short Tandem Repeats, were analyzed in 235 unrelated males born in six different regions in the Zamora province. RESULTS: A relatively homogenous Y-chromosome haplogroup composition was observed in the Zamora province. Haplogroups R1b1-P25 and I-M170, widespread in European populations, accounted for 64.9% of the total sample. Moreover, all of the observed African lineages, accounting for 10.2% of the total variability, belonged to haplogroups having Northwest African origin (E1b1b1b-M81, E1b1b1a-ß-M78, and J1-M267). CONCLUSIONS: No differences between regions or sub-structure due to geographical boundaries were detected. The specific Northwest African male lineages observed contrast with the mitochondrial DNA data, where the majority of African lineages were found to be sub-Saharan. This work made it possible to study the impact of recent historical events in the male gene pool in the province of Zamora in Spain.

Molecular polymorphism of the ABO blood group: a study in Poland, Spain, and Andorra.

OBJECTIVES: The main goal of this study is to increase knowledge on the molecular level of the ABO blood group system in Europe by providing data for Poland, Spain, and Andorra populations. METHODS: A total of 172 oral scrapings samples from individuals of Polish origin, 108 peripheral blood samples of autochthonous individuals from the province of Zamora (Spain), and 81 peripheral blood samples from individuals with Andorran origin, were analyzed. Molecular characterization of the allelic variants was performed by the analysis of exons 6 and 7 of the ABO gene. RESULTS: Seven common alleles were identified, namely: A101, A102, A201, B101, O01, O02, and O03. Less common variants (O05, O09, O21, O26, O06, O11, and O12), were also detected. CONCLUSIONS: The results obtained contribute to the knowledge of the molecular European ABO map, and are discussed in regard to the allelic frequency reported by other Caucasian and Asian populations.

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.

Linguistic isolates in Portugal: insights from the mitochondrial DNA pattern.

Miranda do Douro, located in the northeastern region of Portugal, has notable characteristics not only from a geographic or naturalistic point of view, but also from a cultural perspective. A remarkable one is the coexistence of two different languages: Portuguese and Mirandese, the second being an Astur-Leonese dialect. The current persistence of the Astur-Leonese dialect in this population falls on the singularity of the region: relative isolation, implying difficulties to communicate with other Portuguese regions, while the same location facilitated the establishment of social and commercial relationships with adjacent Spanish territories, origin of the Astur-Leonese language. The objective of this study was to characterize the population from Miranda through the analysis of maternal lineages in order to evaluate whether its mitochondrial DNA diversity fitted the patterns previously reported for other populations from the Iberian Peninsula. Viewing that, the entire control region of mitochondrial DNA from 121 individuals was examined. Miranda showed a haplogroup composition usual for a Western European population, in the sense that as high as 63.6% of sequences belonged to macro-haplogroup R0. Lineages ascribed to have an African (L2a and L1b) origin, were detected, but reaching an amount commonly found in Portugal. Miranda also presented a few haplogroups typically found in Jewish populations, while rarely observed in other Iberian populations. The finding can be explained by gene flow with crypto-Jew communities that since long are known to be established in the region where Miranda is located. In Miranda, both genetic and nucleotide diversities presented low values (0.9292 ± 0.0180 and 0.01101 ± 0.00614 respectively) when compared to populations from its micro-geographical framework, which constitute a sign of population isolation that certainly provided conditions for the survival of the Astur-Leonese dialect in the region.

Primer effect in the detection of mitochondrial DNA point heteroplasmy by automated sequencing.

The correct detection of mitochondrial DNA (mtDNA) heteroplasmy by automated sequencing presents methodological constraints. The main goals of this study are to investigate the effect of sense and distance of primers in heteroplasmy detection and to test if there are differences in the accurate determination of heteroplasmy involving transitions or transversions. A gradient of the heteroplasmy levels was generated for mtDNA positions 9477 (transition G/A) and 15,452 (transversion C/A). Amplification and subsequent sequencing with forward and reverse primers, situated at 550 and 150 bp from the heteroplasmic positions, were performed. Our data provide evidence that there is a significant difference between the use of forward and reverse primers. The forward primer is the primer that seems to give a better approximation to the real proportion of the variants. No significant differences were found concerning the distance at which the sequencing primers were placed neither between the analysis of transitions and transversions. The data collected in this study are a starting point that allows to glimpse the importance of the sequencing primers in the accurate detection of point heteroplasmy, providing additional insight into the overall automated sequencing strategy.

Tracing the origin of the east-west population admixture in the Altai region (Central Asia).

A recent discovery of Iron Age burials (Pazyryk culture) in the Altai Mountains of Mongolia may shed light on the mode and tempo of the generation of the current genetic east-west population admixture in Central Asia. Studies on ancient mitochondrial DNA of this region suggest that the Altai Mountains played the role of a geographical barrier between West and East Eurasian lineages until the beginning of the Iron Age. After the 7th century BC, coinciding with Scythian expansion across the Eurasian steppes, a gradual influx of East Eurasian sequences in Western steppes is detected. However, the underlying events behind the genetic admixture in Altai during the Iron Age are still unresolved: 1) whether it was a result of migratory events (eastward firstly, westward secondly), or 2) whether it was a result of a local demographic expansion in a 'contact zone' between European and East Asian people. In the present work, we analyzed the mitochondrial DNA lineages in human remains from Bronze and Iron Age burials of Mongolian Altai. Here we present support to the hypothesis that the gene pool of Iron Age inhabitants of Mongolian Altai was similar to that of western Iron Age Altaians (Russia and Kazakhstan). Thus, this people not only shared the same culture (Pazyryk), but also shared the same genetic east-west population admixture. In turn, Pazyryks appear to have a similar gene pool that current Altaians. Our results further show that Iron Age Altaians displayed mitochondrial lineages already present around Altai region before the Iron Age. This would provide support for a demographic expansion of local people of Altai instead of westward or eastward migratory events, as the demographic event behind the high population genetic admixture and diversity in Central Asia.

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.

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.

Y-chromosome variation in South Iberia: insights into the North African contribution.

Population of Pedroches Valley, a hypothetical Berber settlement, located in the northwest portion of Córdoba province (Andalusia, Spain), had been analyzed for its Y-chromosome diversity. Moreover, to contextualize this population, 127 Y-chromosomes from a general Andalusia sample and a North African Berber community (Marrakech, Morocco) were also typed. For all samples, 24 single nucleotide polymorphisms of the non-recombining portion of the Y-chromosome (NRY) were analyzed and those samples described as belonging to E3b1b-M81 haplogroup were also typed for 16 Y-chromosome short tandem repeats. Our analysis showed low levels of North African E3b1b-M81 haplogroup in the Pedroches Valley population (1.5%), which is a lower contribution than would be expected. This result rejects the hypothesis of a gradual genetic assimilation of Berber settlers during the Islamic period.

Genetic structure of the Azores Islands: a study using 15 autosomal short tandem repeat loci.

The Azores archipelago (Portugal), located in the Atlantic Ocean, 1,500 km from the European mainland, is formed by nine islands of volcanic origin. The relative position of these islands allows the definition of three geographical groups: Eastern, Central and Western. Previous studies of the Azores using Short Tandem Repeats (STRs) have highlighted differences in the frequencies of several loci, when compared to Mainland Portugal or Madleira Island. Furthermore, linkage disequilibrium (LD), described for Azorean samples has been tentatively explained as reflecting the presence of genetic sub-structuring in the archipelago. To provide information concerning the genetic profile of the Azores Islands and to evaluate the presence of substructuring we have determined the allelic frequencies of 15 autosomal STR loci, using the AmpFlSTR Identifiler Kit, in representative samples from the Azorean Islands. Either considering the Azores as a whole, or analysing by island all the loci were in conformity with Hardy-Weinberg equilibrium. Average gene diversity ranged from 0.7669 in Corvo to 0.7972 in Terceira Island. Allelic independence between loci, tested for the global sample, detected significant LD (after correction for multiple tests) for pairs D21S11/D7S820 and D3S1358/D5S818. The exact test of population differentiation, combining the information of the 15 markers analysed, revealed significant differences between the three groups of islands, and between islands. Inter-island analysis reinforces the previous data that suggested the existence of sub-structuring in the Azores archipelago. Moreover, the data generated by this study can be used in a future forensic genetic database of the Azores after the appropriate enlacement of sample size by island, preventing, in that way, misinterpretations caused by population substructuring and small sample sizes.

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.

Mutation patterns of mtDNA: empirical inferences for the coding region.

BACKGROUND: Human mitochondrial DNA (mtDNA) has been extensively used in population and evolutionary genetics studies. Thus, a valid estimate of human mtDNA evolutionary rate is important in many research fields. The small number of estimations performed for the coding region of the molecule, showed important differences between phylogenetic and empirical approaches. We analyzed a portion of the coding region of mtDNA (tRNALeu, ND1 and tRNAIle genes), using individuals belonging to extended families from the Azores Islands (Portugal) with the main aim of providing empirical estimations of the mutation rate of the coding region of mtDNA under different assumptions, and hence to better understand the mtDNA evolutionary process. RESULTS: Heteroplasmy was detected in 6.5% (3/46) of the families analyzed. In all of the families the presence of mtDNA heteroplasmy resulted from three new point mutations, and no cases of insertions or deletions were identified. Major differences were found in the proportion and type of heteroplasmy found in the genes studied when compared to those obtained in a previous report for the D-loop. Our empirical estimation of mtDNA coding region mutation rate, calculated taking into account the sex of individuals carrying new mutations, the probability of intra-individual fixation of mutations present in heteroplasmy and, to the possible extent, the effect of selection, is similar to that obtained using phylogenetic approaches. CONCLUSION: Based on our results, the discrepancy previously reported between the human mtDNA coding region mutation rates observed along evolutionary timescales and estimations obtained using family pedigrees can be resolved when correcting for the previously cited factors.