Circulating miR-122-5p, miR-92a-3p, and miR-18a-5p as Potential Biomarkers in Human Liver Transplantation Follow-Up.

The requirement of blood-circulating sensitive biomarkers for monitoring liver transplant (LT) is currently a necessary step aiming at the reduction of standard invasive protocols, such as liver biopsy. In this respect, the main objective of this study is to assess circulating microRNA (c-miR) changes in recipients' blood before and after LT and to correlate their blood levels with gold standard biomarkers and with outcomes such as rejection or complications after graft. An miR profile was initially performed; then, the most deregulated miRs were validated by RT-qPCR in 14 recipients pre- and post-LT and compared to a control group of 24 nontransplanted healthy subjects. MiR-122-5p, miR-92a-3p, miR-18a-5p, and miR-30c-5p, identified in the validation phase, were also analyzed considering an additional 19 serum samples collected from LT recipients and focusing on different follow-up (FU) times. The results showed significant, FU-related changes in c-miRs. In particular, miR-122-5p, miR-92a-3p, and miR-18a-5p revealed the same trend after transplantation and an increase in their level was found in patients with complications, independently from FU times. Conversely, the variations in the standard haemato-biochemical parameters for liver function assessment were not significant in the same FU period, confirming the importance of c-miRs as potential noninvasive biomarkers for monitoring patients' outcomes.

HAPLOFIND: a new method for high-throughput mtDNA haplogroup assignment.

Deep sequencing technologies are completely revolutionizing the approach to DNA analysis. Mitochondrial DNA (mtDNA) studies entered in the "postgenomic era": the burst in sequenced samples observed in nuclear genomics is expected also in mitochondria, a trend that can already be detected checking complete mtDNA sequences database submission rate. Tools for the analysis of these data are available, but they fail in throughput or in easiness of use. We present here a new pipeline based on previous algorithms, inherited from the "nuclear genomic toolbox," combined with a newly developed algorithm capable of efficiently and easily classify new mtDNA sequences according to PhyloTree nomenclature. Detected mutations are also annotated using data collected from publicly available databases. Thanks to the analysis of all freely available sequences with known haplogroup obtained from GenBank, we were able to produce a PhyloTree-based weighted tree, taking into account each haplogroup pattern conservation. The combination of a highly efficient aligner, coupled with our algorithm and massive usage of asynchronous parallel processing, allowed us to build a high-throughput pipeline for the analysis of mtDNA sequences that can be quickly updated to follow the ever-changing nomenclature. HaploFind is freely accessible at the following Web address: https://haplofind.unibo.it.

Epigenetic aging differences between Wichí and Criollos from Argentina: Insights from genomic history and ecology.

Background and objectives: Epigenetic estimators based on DNA methylation levels have emerged as promising biomarkers of human aging. These estimators exhibit natural variations across human groups, but data about indigenous populations remain underrepresented in research. This study aims to investigate differences in epigenetic estimators between two distinct human populations, both residing in the Gran Chaco region of Argentina, the Native-American Wichí, and admixed Criollos who are descendants of intermarriages between Native Americans and the first European colonizers, using a population genetic approach. Methodology: We analyzed 24 Wichí (mean age: 39.2 ± 12.9 yo) and 24 Criollos (mean age: 41.1 ± 14.0 yo) for DNA methylation levels using the Infinium MethylationEPIC (Illumina) to calculate 16 epigenetic estimators. Additionally, we examined genome-wide genetic variation using the HumanOmniExpress BeadChip (Illumina) to gain insights into the genetic history of these populations. Results: Our results indicate that Native-American Wichí are epigenetically older compared to Criollos according to five epigenetic estimators. Analyses within the Criollos population reveal that global ancestry does not influence the differences observed, while local (chromosomal) ancestry shows positive associations between specific SNPs located in genomic regions over-represented by Native-American ancestry and measures of epigenetic age acceleration (AgeAccelHannum). Furthermore, we demonstrate that differences in population ecologies also contribute to observed epigenetic differences. Conclusions and implications: Overall, our study suggests that while the genomic history may partially account for the observed epigenetic differences, non-genetic factors, such as lifestyle and ecological factors, play a substantial role in the variability of epigenetic estimators, thereby contributing to variations in human epigenetic aging.

Circulating perilipin 2 levels are associated with fat mass, inflammatory and metabolic markers and are higher in women than men.

Perilipin 2 (PLIN2) is a protein involved in lipid storage and metabolism in non-adipose tissues. Detectable levels of circulating PLIN2 (cPLIN2) have been reported to be associated with some types of cancer, but no systematic analysis of age-related modifications in cPLIN2 levels has ever been performed. We measured serum cPLIN2 in a group of old people including centenarians in comparison with young subjects and tested possible correlations with parameters of body composition, fat and glucose metabolism, and inflammation. We found that: i. levels of cPLIN2 do not change with age, but women have higher levels of cPLIN2 with respect to men; ii. cPLIN2 levels strongly correlate to BMI, as well as fat and lean mass; iii. cPLIN2 levels strongly correlate with the proinflammatory adipokine leptin. Due to the adipogenic activity of leptin, it is hypothesized that cPLIN2 is affected and possibly regulated by this pleiotropic adipokine. Moreover, these results suggest that cPLIN2 (possibly together with leptin) could be assumed as a proxy for body adiposity.

Circulating miR-19a-3p and miR-19b-3p characterize the human aging process and their isomiRs associate with healthy status at extreme ages.

Blood circulating microRNAs (c-miRs) are potential biomarkers to trace aging and longevity trajectories to identify molecular targets for anti-aging therapies. Based on a cross-sectional study, a discovery phase was performed on 12 donors divided into four groups: young, old, healthy, and unhealthy centenarians. The identification of healthy and unhealthy phenotype was based on cognitive performance and capabilities to perform daily activities. Small RNA sequencing identified 79 differentially expressed c-miRs when comparing young, old, healthy centenarians, and unhealthy centenarians. Two miRs, that is, miR-19a-3p and miR-19b-3p, were found increased at old age but decreased at extreme age, as confirmed by RT-qPCR in 49 donors of validation phase. The significant decrease of those miR levels in healthy compared to unhealthy centenarians appears to be due to the presence of isomiRs, not detectable with RT-qPCR, but only with a high-resolution technique such as deep sequencing. Bioinformatically, three main common targets of miR-19a/b-3p were identified, that is, SMAD4, PTEN, and BCL2L11, converging into the FoxO signaling pathway, known to have a significant role in aging mechanisms. For the first time, this study shows the age-related increase of plasma miR-19a/b-3p in old subjects but a decrease in centenarians. This decrease is more pronounced in healthy centenarians and was confirmed by the modified pattern of isomiRs comparing healthy and unhealthy centenarians. Thus, our study paves the way for functional studies using c-miRs and isomiRs as additional parameter to track the onset of aging and age-related diseases using new potential biomarkers.

Human Aging and Longevity Are Characterized by High Levels of Mitokines.

Mitochondrial stress elicits the production of stress response molecules indicated as mitokines, including fibroblast growth factor 21 (FGF21), growth differentiation factor 15 (GDF15), and humanin (HN). Many diseases are characterized by progressive mitochondrial dysfunction with alterations of mitokine secretion. It is still controversial whether healthy aging and extreme longevity are accompanied by an altered production of mitokines. We analyzed FGF21, HN, and GDF15 plasma levels in 693 subjects aged from 21 to 113 years, and the association of these mitokines with parameters of health status. FGF21, HN, and GDF15 resulted increased in old age, with the highest levels found in centenarians. These molecules are associated with worsened parameters (such as handgrip strength, insulin sensitivity, triglycerides), particularly in 70-year-old persons, and their levels are inversely correlated with survival in the oldest subjects. Considering the positive biological effect of these molecules, our results can be interpreted in the framework of the hormetic paradigm as an attempt of the cells/tissues to cope with a stress that can have beneficial or detrimental effects depending on its intensity. Finally, persons with Down Syndrome (characterized by accelerated aging) have higher levels of GDF15 and HN with respect to their siblings, suggesting that these molecules, especially GDF15, could be considered markers of biological age.

Mitochondrial DNA involvement in human longevity.

The main message of this review can be summarized as follows: aging and longevity, as complex traits having a significant genetic component, likely depend on a number of nuclear gene variants interacting with mtDNA variability both inherited and somatic. We reviewed the data available in the literature with particular attention to human longevity, and argued that what we hypothesize for aging and longevity could have a more general relevance and be extended to other age-related complex traits such as Alzheimer's and Parkinson's diseases. The genetics which emerges for complex traits, including aging and longevity, is thus even more complicated than previously thought, as epistatic interactions between nuclear gene polymorphisms and mtDNA variability (both somatic and inherited) as well as between mtDNA somatic mutations (tissue specific) and mtDNA inherited variants (haplogroups and sub-haplogroups) must be considered as additional players capable of explaining a part of the aging and longevity phenotype. To test this hypothesis is one of the main challenge in the genetics of aging and longevity in the next future.

Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans.

A large part of the aging phenotype, including immunosenescence, is explained by an imbalance between inflammatory and anti-inflammatory networks, which results in the low grade chronic pro-inflammatory status we proposed to call inflammaging. Within this perspective, healthy aging and longevity are likely the result not only of a lower propensity to mount inflammatory responses but also of efficient anti-inflammatory networks, which in normal aging fail to fully neutralize the inflammatory processes consequent to the lifelong antigenic burden and exposure to damaging agents. Such a global imbalance can be a major driving force for frailty and common age-related pathologies, and should be addressed and studied within an evolutionary-based systems biology perspective. Evidence in favor of this conceptualization largely derives from studies in humans. We thus propose that inflammaging can be flanked by anti-inflammaging as major determinants not only of immunosenescence but eventually of global aging and longevity.

Association studies on human mitochondrial DNA: methodological aspects and results in the most common age-related diseases.

Mitochondrial DNA (mtDNA) follows direct maternal inheritance and, as such, can be used in phylogenetic studies to determine a human lineage tree. The presence of common polymorphisms allows a classification of mtDNA in haplogroups and sub-haplogroups, according to the branch they belong to. Thanks to the rapidly growing number of mtDNA sequences available, this classification is being corrected and redefined to be more accurate. In parallel with this process, several studies are trying to identify an association between common mtDNA polymorphisms and common complex traits, as hypothesized by the common disease-common variant theory. Here we review the associations already reported with the main age-related complex diseases and we identify the critical points (sample size, size of the recruiting area, careful matching between cases and controls regarding geographical origin and ethnicity, data quality checking) to be taken in account in planning such studies. On the whole, this research area is opening a new perspective as an important component of "mitochondrial medicine", capable of identifying new molecular targets for the diagnosis, prevention and treatment of common complex diseases.

Role of mitochondrial DNA in longevity, aging and age-related diseases in humans: a reappraisal.

The genetic variability of H. sapiens mitochondrial DNA (mtDNA) can be either germ-line inherited or somatically acquired, and its effect on aging and longevity as well as on the pathogenesis of complex age-related diseases is a hot topic. Here we illustrate the complexity of such studies, related to the large genetic variability of mtDNA in different populations and the fact that the rate of the aging process is different in different cells, tissues and organs. As far as concern Alzheimer's disease, the accumulation of somatic mutations in several tissues have been investigated, as well as the inherited mtDNA variability. However, the issue is still controversial and further studies are needed to clarify the role of mtDNA variants in Alzheimer's disease. This review is aimed to summarize the most recent advances in this field. By high throughput mtDNA sequencing and the study of large cohorts of ethnically homogeneous subjects/patients, it is now possible to perform high dimensionality studies in order to clarify the genetic associations among several inherited mtDNA variants and longevity or age-associated diseases in humans.

Massive parallel sequencing of human whole mitochondrial genomes with Ion Torrent technology: an optimized workflow for Anthropological and Population Genetics studies.

Investigation of human mitochondrial DNA variation patterns and phylogeny has been extensively used in Anthropological and Population Genetics studies and sequencing the whole mitochondrial genome is progressively becoming the gold standard. Among the currently available massive parallel sequencing technologies, Ion Torrent™ semiconductor sequencing represents a promising approach for such studies. Nevertheless, an experimental protocol conceived to enable the achievement of both as high as possible yield and of the most homogeneous sequence coverage through the whole mitochondrial genome is still not available. The present work was thus aimed at improving the overall performance of whole mitochondrial genomes Ion Torrent™ sequencing, with special focus on the capability to obtain robust coverage and highly reliable variants calling. For this purpose, a series of cost-effective modifications in standard laboratory workflows was fine-tuned to optimize them for medium- and large-scale population studies. A total of 54 human samples were thus subjected to sequencing of the whole mitochondrial genome with the Ion Personal Genome Machine™ System in four distinct experiments and using Ion 314 chips. Seven of the selected samples were also characterized by means of conventional Sanger sequencing for the sake of comparison. Obtained results demonstrated that the implemented optimizations had definitely improved sequencing outputs in terms of both variants calling efficiency and coverage uniformity, enabling to setup an effective and accurate protocol for whole mitochondrial genome sequencing and a considerable reduction in experimental time consumption and sequencing costs.

The genetics of human longevity.

Aging is due to a complex interaction of genetic, epigenetic, and environmental factors, but a strong genetic component appears to have an impact on survival to extreme ages. In order to identify "longevity genes" in humans, different strategies are now available. In our laboratory, we performed association studies on a variety of "candidate" polymorphisms in Italian centenarians. Many genes/polymorphisms gave negative results, while others showed a positive association with human longevity and a sometimes-positive association with unsuccessful aging (myocardial infarction, Alzheimer's disease, and type 2 diabetes). Results regarding genes involved in inflammation (IL-1 cluster, IL-6, IL-10, TNF-alpha, TGF-beta, TLR-4, PPARgamma), insulin/IGF-1 signaling pathway and lipid metabolism (apolipoproteins, CETP, PON1), and oxidative stress (p53, p66(shc)) will be described. In addition, a strong role of the interaction between nuclear and mitochondrial genomes (mtDNA haplogroups and the C150T mutation) emerged from our findings. Thus, the genetics of human longevity appears to be quite peculiar in a context where antagonistic pleiotropy can play a major role and genes can have a different biological role at different ages.

Age-dependent effects of in vitro radiofrequency exposure (mobile phone) on CD95+ T helper human lymphocytes.

Recent studies on "nonthermal" effects of mobile phone radiofrequency (RF) suggest that RF can interact with cellular functions and molecular pathways. To study the possible RF effects on human lymphocyte activation, we analyzed CD25, CD95, CD28 molecules in unstimulated and stimulated CD4+ e CD8+ T cells in vitro. Peripheral blood mononuclear cells (PBMCs) from young and elderly donors were exposed or sham-exposed to RF (1,800 MHz, Specific Absorption Rate 2 W/kg) with or without mitogenic stimulation. No significant changes in the percentage of these cell subsets were found between exposed and sham-exposed lymphocytes in both young and elderly donors. Nevertheless, after RF exposure we observed a slight, but significant, downregulation of CD95 expression in stimulated CD4+ T lymphocytes from elderly, but not from young donors. This age-related result is noteworthy given the importance of a such molecule in regulation of the immune response.

Fine Dissection of Human Mitochondrial DNA Haplogroup HV Lineages Reveals Paleolithic Signatures from European Glacial Refugia.

Genetic signatures from the Paleolithic inhabitants of Eurasia can be traced from the early divergent mitochondrial DNA lineages still present in contemporary human populations. Previous studies already suggested a pre-Neolithic diffusion of mitochondrial haplogroup HV*(xH,V) lineages, a relatively rare class of mtDNA types that includes parallel branches mainly distributed across Europe and West Asia with a certain degree of structure. Up till now, variation within haplogroup HV was addressed mainly by analyzing sequence data from the mtDNA control region, except for specific sub-branches, such as HV4 or the widely distributed haplogroups H and V. In this study, we present a revised HV topology based on full mtDNA genome data, and we include a comprehensive dataset consisting of 316 complete mtDNA sequences including 60 new samples from the Italian peninsula, a previously underrepresented geographic area. We highlight points of instability in the particular topology of this haplogroup, reconstructed with BEAST-generated trees and networks. We also confirm a major lineage expansion that probably followed the Late Glacial Maximum and preceded Neolithic population movements. We finally observe that Italy harbors a reservoir of mtDNA diversity, with deep-rooting HV lineages often related to sequences present in the Caucasus and the Middle East. The resulting hypothesis of a glacial refugium in Southern Italy has implications for the understanding of late Paleolithic population movements and is discussed within the archaeological cultural shifts occurred over the entire continent.

MARK-AGE standard operating procedures (SOPs): A successful effort.

Within the MARK-AGE project, a population study (3337 subjects) was conducted to identify a set of biomarkers of ageing which, as a combination of parameters with appropriate weighting, would measure biological age better than any single marker. The MARK-AGE project involves 14 European countries and a total of 26 research centres. In such a study, standard operating procedures (SOPs) are an essential task, which are binding for all MARK-AGE Beneficiaries. The SOPs cover all aspects of subject's recruitment, collection, shipment and distribution of biological samples (blood and its components, buccal mucosa cells or BMC and urine) as well as the anthropometric measurements and questionnaires.

mtDNA mutations in human aging and longevity: controversies and new perspectives opened by high-throughput technologies.

The last 30 years of research greatly contributed to shed light on the role of mitochondrial DNA (mtDNA) variability in aging, although contrasting results have been reported, mainly due to bias regarding the population size and stratification, and to the use of analysis methods (haplogroup classification) that resulted to be not sufficiently adequate to grasp the complexity of the phenomenon. A 5-years European study (the GEHA EU project) collected and analyzed data on mtDNA variability on an unprecedented number of long-living subjects (enriched for longevity genes) and a comparable number of controls (matched for gender and ethnicity) in Europe. This very large study allowed a reappraisal of the role of both the inherited and the somatic mtDNA variability in aging, as an association with longevity emerged only when mtDNA variants in OXPHOS complexes co-occurred. Moreover, the availability of data from both nuclear and mitochondrial genomes on a large number of subjects paves the way for an evaluation at a very large scale of the epistatic interactions at a higher level of complexity. This scenario is expected to be even more clarified in the next future with the use of next generation sequencing (NGS) techniques, which are becoming applicable to evaluate mtDNA variability and, then, new mathematical/bioinformatic analysis methods are urgently needed. Recent advances of association studies on age-related diseases and mtDNA variability will also be discussed in this review, taking into account the bias hidden by population stratification. Finally, very recent findings in terms of mtDNA heteroplasmy (i.e. the coexistence of wild type and mutated copies of mtDNA) and aging as well as mitochondrial epigenetic mechanisms will also be discussed.

The three genetics (nuclear DNA, mitochondrial DNA, and gut microbiome) of longevity in humans considered as metaorganisms.

Usually the genetics of human longevity is restricted to the nuclear genome (nDNA). However it is well known that the nDNA interacts with a physically and functionally separated genome, the mitochondrial DNA (mtDNA) that, even if limited in length and number of genes encoded, plays a major role in the ageing process. The complex interplay between nDNA/mtDNA and the environment is most likely involved in phenomena such as ageing and longevity. To this scenario we have to add another level of complexity represented by the microbiota, that is, the whole set of bacteria present in the different part of our body with their whole set of genes. In particular, several studies investigated the role of gut microbiota (GM) modifications in ageing and longevity and an age-related GM signature was found. In this view, human being must be considered as "metaorganism" and a more holistic approach is necessary to grasp the complex dynamics of the interaction between the environment and nDNA-mtDNA-GM of the host during ageing. In this review, the relationship between the three genetics and human longevity is addressed to point out that a comprehensive view will allow the researchers to properly address the complex interactions that occur during human lifespan.

Development of a pilot project on data sharing among partners of the Italian Hub of Population Biobanks (HIBP): association between lipid profile and socio-demographic variables.

The Italian Hub of Population Biobanks (HIBP) includes both ongoing and completed studies that are heterogeneous in both their purpose and in the specimens collected. The heterogeneity in starting conditions makes sharing study data very difficult because of technical, ethical, and collection rights issues that hamper collaboration and synergy. With the aim of overcoming these difficulties and establishing the "proof-of-concept" that sharing studies is achievable among Italian collections, a data-sharing pilot project has been agreed to by HIBP members. Participants agreed to the general methodology and signed a shared Data Transfer Agreement. The biobanks involved were: EURAC (Micros study), CIG (GEHA project), CNESPS (FINE, MATISS, MONICA, OEC1998, ITR (Italian Twin Register), and IPREA studies, and MOLIBANK (Moli-Sani project). Biobank data were uploaded into a common database using a dedicated informatics infrastructure. Demographic data, and anthropometric and hematochemical parameters were shared for each record. Each biobank uploaded into the common database a dataset with a minimum of 1000 subjects, for a total of 5071 records. After a harmonization process, the final dataset included 3882 records. Subjects were grouped into three main geographic areas of Italy (North, Center, and South) and separate analyses were performed for men and women. The 3882 records were analyzed through multivariate logistic regression analysis. Results were expressed as odds ratios with 95% confidence interval. Results show several geographical differences in the lipidemic pattern, mostly regarding cholesterol-HDL, which represents a strong basis for further, deeper sample-based studies. This HIBP pilot study aimed to prove the feasibility of such collaborations and it provides a methodological prototype for future studies based on the participation in the partnership of well-established quality collections.

The co-occurrence of mtDNA mutations on different oxidative phosphorylation subunits, not detected by haplogroup analysis, affects human longevity and is population specific.

To re-examine the correlation between mtDNA variability and longevity, we examined mtDNAs from samples obtained from over 2200 ultranonagenarians (and an equal number of controls) collected within the framework of the GEHA EU project. The samples were categorized by high-resolution classification, while about 1300 mtDNA molecules (650 ultranonagenarians and an equal number of controls) were completely sequenced. Sequences, unlike standard haplogroup analysis, made possible to evaluate for the first time the cumulative effects of specific, concomitant mtDNA mutations, including those that per se have a low, or very low, impact. In particular, the analysis of the mutations occurring in different OXPHOS complex showed a complex scenario with a different mutation burden in 90+ subjects with respect to controls. These findings suggested that mutations in subunits of the OXPHOS complex I had a beneficial effect on longevity, while the simultaneous presence of mutations in complex I and III (which also occurs in J subhaplogroups involved in LHON) and in complex I and V seemed to be detrimental, likely explaining previous contradictory results. On the whole, our study, which goes beyond haplogroup analysis, suggests that mitochondrial DNA variation does affect human longevity, but its effect is heavily influenced by the interaction between mutations concomitantly occurring on different mtDNA genes.

Analysis of population substructure in two sympatric populations of Gran Chaco, Argentina.

Sub-population structure and intricate kinship dynamics might introduce biases in molecular anthropology studies and could invalidate the efforts to understand diseases in highly admixed populations. In order to clarify the previously observed distribution pattern and morbidity of Chagas disease in Gran Chaco, Argentina, we studied two populations (Wichí and Criollos) recruited following an innovative bio-cultural model considering their complex cultural interactions. By reconstructing the genetic background and the structure of these two culturally different populations, the pattern of admixture, the correspondence between genealogical and genetic relationships, this integrated perspective had the power to validate data and to link the gap usually relying on a singular discipline. Although Wichí and Criollos share the same area, these sympatric populations are differentiated from the genetic point of view as revealed by Non Recombinant Y Chromosome genotyping resulting in significantly high Fst values and in a lower genetic variability in the Wichí population. Surprisingly, the Amerindian and the European components emerged with comparable amounts (20%) among Criollos and Wichí respectively. The detailed analysis of mitochondrial DNA showed that the two populations have as much as 87% of private haplotypes. Moreover, from the maternal perspective, despite a common Amerindian origin, an Andean and an Amazonian component emerged in Criollos and in Wichí respectively. Our approach allowed us to highlight that quite frequently there is a discrepancy between self-reported and genetic kinship. Indeed, if self-reported identity and kinship are usually utilized in population genetics as a reliable proxy for genetic identity and parental relationship, in our model populations appear to be the result not only and not simply of the genetic background but also of complex cultural determinants. This integrated approach paves the way to a rigorous reconstruction of demographic and cultural history as well as of bioancestry and propensity to diseases of Wichí and Criollos.