Pathogenic Mechanisms of Somatic Mutation and Genome Mosaicism in Aging.

Age-related accumulation of postzygotic DNA mutations results in tissue genetic heterogeneity known as somatic mosaicism. Although implicated in aging as early as the 1950s, somatic mutations in normal tissue have been difficult to study because of their low allele fractions. With the recent emergence of cost-effective high-throughput sequencing down to the single-cell level, enormous progress has been made in our capability to quantitatively analyze somatic mutations in human tissue in relation to aging and disease. Here we first review how recent technological progress has opened up this field, providing the first broad sets of quantitative information on somatic mutations in vivo necessary to gain insight into their possible causal role in human aging and disease. We then propose three major mechanisms that can lead from accumulated de novo mutations across tissues to cell functional loss and human disease.

SIRT6 Is Responsible for More Efficient DNA Double-Strand Break Repair in Long-Lived Species.

DNA repair has been hypothesized to be a longevity determinant, but the evidence for it is based largely on accelerated aging phenotypes of DNA repair mutants. Here, using a panel of 18 rodent species with diverse lifespans, we show that more robust DNA double-strand break (DSB) repair, but not nucleotide excision repair (NER), coevolves with longevity. Evolution of NER, unlike DSB, is shaped primarily by sunlight exposure. We further show that the capacity of the SIRT6 protein to promote DSB repair accounts for a major part of the variation in DSB repair efficacy between short- and long-lived species. We dissected the molecular differences between a weak (mouse) and a strong (beaver) SIRT6 protein and identified five amino acid residues that are fully responsible for their differential activities. Our findings demonstrate that DSB repair and SIRT6 have been optimized during the evolution of longevity, which provides new targets for anti-aging interventions.

Nuclear Genomic Instability and Aging.

The nuclear genome decays as organisms age. Numerous studies demonstrate that the burden of several classes of DNA lesions is greater in older mammals than in young mammals. More challenging is proving this is a cause rather than a consequence of aging. The DNA damage theory of aging, which argues that genomic instability plays a causal role in aging, has recently gained momentum. Support for this theory stems partly from progeroid syndromes in which inherited defects in DNA repair increase the burden of DNA damage leading to accelerated aging of one or more organs. Additionally, growing evidence shows that DNA damage accrual triggers cellular senescence and metabolic changes that promote a decline in tissue function and increased susceptibility to age-related diseases. Here, we examine multiple lines of evidence correlating nuclear DNA damage with aging. We then consider how, mechanistically, nuclear genotoxic stress could promote aging. We conclude that the evidence, in toto, supports a role for DNA damage as a nidus of aging.

The central role of DNA damage in the ageing process.

Ageing is a complex, multifaceted process leading to widespread functional decline that affects every organ and tissue, but it remains unknown whether ageing has a unifying causal mechanism or is grounded in multiple sources. Phenotypically, the ageing process is associated with a wide variety of features at the molecular, cellular and physiological level-for example, genomic and epigenomic alterations, loss of proteostasis, declining overall cellular and subcellular function and deregulation of signalling systems. However, the relative importance, mechanistic interrelationships and hierarchical order of these features of ageing have not been clarified. Here we synthesize accumulating evidence that DNA damage affects most, if not all, aspects of the ageing phenotype, making it a potentially unifying cause of ageing. Targeting DNA damage and its mechanistic links with the ageing phenotype will provide a logical rationale for developing unified interventions to counteract age-related dysfunction and disease.

A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A.

Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in aging and metabolism regulation. Targeted sequencing of SIRT6 locus in a population of 450 Ashkenazi Jewish (AJ) centenarians and 550 AJ individuals without a family history of exceptional longevity identified enrichment of a SIRT6 allele containing two linked substitutions (N308K/A313S) in centenarians compared with AJ control individuals. Characterization of this SIRT6 allele (centSIRT6) demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double-strand break repair, and more robustly kill cancer cells compared with wild-type SIRT6. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD+ concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which was correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA.

Somatic Mutagenesis in Mammals and Its Implications for Human Disease and Aging.

DNA mutations as a consequence of errors during DNA damage repair, replication, or mitosis are the substrate for evolution. In multicellular organisms, mutations can occur in the germline and also in somatic tissues, where they are associated with cancer and other chronic diseases and possibly with aging. Recent advances in high-throughput sequencing have made it relatively easy to study germline de novo mutations, but in somatic cells, the vast majority of mutations are low-abundant and can be detected only in clonal lineages, such as tumors, or single cells. Here we review recent results on somatic mutations in normal human and animal tissues with a focus on their possible functional consequences.

Chromosome instability and aneuploidy in the mammalian brain.

This review investigates the role of aneuploidy and chromosome instability (CIN) in the aging brain. Aneuploidy refers to an abnormal chromosomal count, deviating from the normal diploid set. It can manifest as either a deficiency or excess of chromosomes. CIN encompasses a broader range of chromosomal alterations, including aneuploidy as well as structural modifications in DNA. We provide an overview of the state-of-the-art methodologies utilized for studying aneuploidy and CIN in non-tumor somatic tissues devoid of clonally expanded populations of aneuploid cells.CIN and aneuploidy, well-established hallmarks of cancer cells, are also associated with the aging process. In non-transformed cells, aneuploidy can contribute to functional impairment and developmental disorders. Despite the importance of understanding the prevalence and specific consequences of aneuploidy and CIN in the aging brain, these aspects remain incompletely understood, emphasizing the need for further scientific investigations.This comprehensive review consolidates the present understanding, addresses discrepancies in the literature, and provides valuable insights for future research efforts.

Do DNA Double-Strand Breaks Drive Aging?

DNA double-strand breaks (DSBs) are rare, but highly toxic, lesions requiring orchestrated and conserved machinery to prevent adverse consequences, such as cell death and cancer-causing genome structural mutations. DSBs trigger the DNA damage response (DDR) that directs a cell to repair the break, undergo apoptosis, or become senescent. There is increasing evidence that the various endpoints of DSB processing by different cells and tissues are part of the aging phenotype, with each stage of the DDR associated with specific aging pathologies. In this Perspective, we discuss the possibility that DSBs are major drivers of intrinsic aging, highlighting the dynamics of spontaneous DSBs in relation to aging, the distinct age-related pathologies induced by DSBs, and the segmental progeroid phenotypes in humans and mice with genetic defects in DSB repair. A model is presented as to how DSBs could drive some of the basic mechanisms underlying age-related functional decline and death.

SomaMutDB: a database of somatic mutations in normal human tissues.

De novo mutations, a consequence of errors in DNA repair or replication, have been reported to accumulate with age in normal tissues of humans and model organisms. This accumulation during development and aging has been implicated as a causal factor in aging and age-related pathology, including but not limited to cancer. Due to their generally very low abundance mutations have been difficult to detect in normal tissues. Only with recent advances in DNA sequencing of single-cells, clonal lineages or ultra-high-depth sequencing of small tissue biopsies, somatic mutation frequencies and spectra have been unveiled in several tissue types. The rapid accumulation of such data prompted us to develop a platform called SomaMutDB (https://vijglab.einsteinmed.org/SomaMutDB) to catalog the 2.42 million single nucleotide variations (SNVs) and 0.12 million small insertions and deletions (INDELs) thus far identified using these advanced methods in nineteen human tissues or cell types as a function of age or environmental stress conditions. SomaMutDB employs a user-friendly interface to display and query somatic mutations with their functional annotations. Moreover, the database provides six powerful tools for analyzing mutational signatures associated with the data. We believe such an integrated resource will prove valuable for understanding somatic mutations and their possible role in human aging and age-related diseases.

Aging: a sirtuin shake-up?

The yeast sirtuin (Sir2) is a histone deacetylase that modulates yeast replicative life span by suppressing genome instability through chromatin modification. In this issue, Oberdoerffer et al. (2008) report that SIRT1, the mammalian ortholog of Sir2, is involved in DNA damage-induced chromatin reorganization, which promotes genome stability in mammalian cells.

Evidence for a limit to human lifespan.

Driven by technological progress, human life expectancy has increased greatly since the nineteenth century. Demographic evidence has revealed an ongoing reduction in old-age mortality and a rise of the maximum age at death, which may gradually extend human longevity. Together with observations that lifespan in various animal species is flexible and can be increased by genetic or pharmaceutical intervention, these results have led to suggestions that longevity may not be subject to strict, species-specific genetic constraints. Here, by analysing global demographic data, we show that improvements in survival with age tend to decline after age 100, and that the age at death of the world's oldest person has not increased since the 1990s. Our results strongly suggest that the maximum lifespan of humans is fixed and subject to natural constraints.

Single-cell whole-genome sequencing reveals the functional landscape of somatic mutations in B lymphocytes across the human lifespan.

Accumulation of mutations in somatic cells has been implicated as a cause of aging since the 1950s. However, attempts to establish a causal relationship between somatic mutations and aging have been constrained by the lack of methods to directly identify mutational events in primary human tissues. Here we provide genome-wide mutation frequencies and spectra of human B lymphocytes from healthy individuals across the entire human lifespan using a highly accurate single-cell whole-genome sequencing method. We found that the number of somatic mutations increases from <500 per cell in newborns to >3,000 per cell in centenarians. We discovered mutational hotspot regions, some of which, as expected, were located at Ig genes associated with somatic hypermutation (SHM). B cell-specific mutation signatures associated with development, aging, or SHM were found. The SHM signature strongly correlated with the signature found in human B cell tumors, indicating that potential cancer-causing events are already present even in B cells of healthy individuals. We also identified multiple mutations in sequence features relevant to cellular function (i.e., transcribed genes and gene regulatory regions). Such mutations increased significantly during aging, but only at approximately one-half the rate of the genome average, indicating selection against mutations that impact B cell function. This full characterization of the landscape of somatic mutations in human B lymphocytes indicates that spontaneous somatic mutations accumulating with age can be deleterious and may contribute to both the increased risk for leukemia and the functional decline of B lymphocytes in the elderly.

Accurate identification of single-nucleotide variants in whole-genome-amplified single cells.

Mutation analysis in single-cell genomes is prone to artifacts associated with cell lysis and whole-genome amplification. Here we addressed these issues by developing single-cell multiple displacement amplification (SCMDA) and a general-purpose single-cell-variant caller, SCcaller (https://github.com/biosinodx/SCcaller/). By comparing SCMDA-amplified single cells with unamplified clones from the same population, we validated the procedure as a firm foundation for standardized somatic-mutation analysis in single-cell genomics.

Comparative genetics of longevity and cancer: insights from long-lived rodents.

Mammals have evolved a remarkable diversity of ageing rates. Within the single order of Rodentia, maximum lifespans range from 4 years in mice to 32 years in naked mole rats. Cancer rates also differ substantially between cancer-prone mice and almost cancer-proof naked mole rats and blind mole rats. Recent progress in rodent comparative biology, together with the emergence of whole-genome sequence information, has opened opportunities for the discovery of genetic factors that control longevity and cancer susceptibility.

Turning anti-ageing genes against cancer.

Recent studies in diverse organisms implicate proto-oncogenic pathways, including insulin-like growth factor-I (IGF-I), Ras and AKT/protein kinase B in the ageing process. Although IGF-I is thought to contribute to cancer by promoting growth and preventing apoptosis, evidence from model organisms suggests that proto-oncogene homologues might contribute to the DNA mutations and chromosomal damage that are observed in tumour cells by increasing DNA damage, in both dividing and non-dividing cells, and involving error-prone systems in DNA repair. This raises the possibility that cancer can be reduced by chronic downregulation of pro-ageing pathways.

Quantitative detection of low-abundance somatic structural variants in normal cells by high-throughput sequencing.

The detection and quantification of low-abundance somatic DNA mutations by high-throughput sequencing is challenging because of the difficulty of distinguishing errors from true mutations. There are several approaches available for analyzing somatic point mutations and small insertions or deletions, but an accurate genome-wide assessment of somatic structural variants (somSVs) in bulk DNA is still not possible. Here we present Structural Variant Search (SVS), a method to accurately detect rare somSVs by low-coverage sequencing. We demonstrate direct quantitative assessment of elevated somSV frequencies induced by known clastogenic compounds in human primary cells.