These authors have contributed equally to the work.
Genes encoding longevity: from model organisms to humans
Article first published online: 15 JAN 2008
© 2008 The Authors
Volume 7, Issue 2, pages 270–280, April 2008
How to Cite
Kuningas, M., Mooijaart, S. P., Van Heemst, D., Zwaan, B. J., Slagboom, P. E. and Westendorp, R. G. J. (2008), Genes encoding longevity: from model organisms to humans. Aging Cell, 7: 270–280. doi: 10.1111/j.1474-9726.2008.00366.x
- Issue published online: 15 JAN 2008
- Article first published online: 15 JAN 2008
- Accepted for publication 13 December 2007
- model organism
Ample evidence from model organisms has indicated that subtle variation in genes can dramatically influence lifespan. The key genes and molecular pathways that have been identified so far encode for metabolism, maintenance and repair mechanisms that minimize age-related accumulation of permanent damage. Here, we describe the evolutionary conserved genes that are involved in lifespan regulation of model organisms and humans, and explore the reasons of discrepancies that exist between the results found in the various species. In general, the accumulated data have revealed that when moving up the evolutionary ladder, together with an increase of genome complexity, the impact of candidate genes on lifespan becomes smaller. The presence of genetic networks makes it more likely to expect impact of variation in several interacting genes to affect lifespan in humans. Extrapolation of findings from experimental models to humans is further complicated as phenotypes are critically dependent on the setting in which genes are expressed, while laboratory conditions and modern environments are markedly dissimilar. Finally, currently used methodologies may have only little power and validity to reveal genetic variation in the population. In conclusion, although the study of model organisms has revealed potential candidate genetic mechanisms determining aging and lifespan, to what extent they explain variation in human populations is still uncertain.
Over the last century, the mean life expectancy in Western societies has increased dramatically (Oeppen & Vaupel, 2002). In Japan, for instance, the mean life expectancy has increased from 50 years to 80 years in no more than six decades. It is unlikely that changes in the population genome over this time-period can explain for the observed increase in lifespan which is more likely to be attributable to the improvement of environmental conditions and medical care. The increase in mean life expectancy of the total population, however, has left the marked interindividual variance in lifespan unaltered. Socio-economic factors can in part explain this phenomenon, but ample evidence suggests that genetic factors are also at play. Studies of twins and long-lived families have estimated that 20–30% of the variation in human lifespan is determined by genetic factors, which impact becomes more important for survival at older ages (Herskind et al., 1996; Mitchell et al., 2001; Hjelmborg et al., 2006). Furthermore, siblings of centenarians have a significantly higher chance of becoming a centenarian themselves when compared to other members of their birth cohort (Perls et al., 2002). The survival benefits of family members of these long-lived subjects are lifelong and persist up to the highest age categories (Perls et al., 2002; Hjelmborg et al., 2006). Offspring of long-lived sibling pairs have a lower mortality risk already at middle age, whereas their spouses, with whom they have shared in part a common environment, do not show this survival benefit (Schoenmaker et al., 2006).
As for lifespan, aging is under moderate genetic control influencing the rate at which stochastically induced damaged molecules accumulate. Such damage is caused by various endogenous and exogenous biological and biochemical stresses. As a result, over the life course there is a constant rise in vulnerability of the body, leading to a continuously increasing risk of disease and death. Longevity and the maintenance of health in old age can be ensured via two principally different strategies that minimize the risk of permanent damage to occur, that is, by a decrease of environmental hazards or an increase of the durability of the body. Pathways that influence metabolism, maintenance and repair mechanisms, and prevent the accumulation of permanent damage thus represent key molecular candidates for the preservation of health and longevity.
Experiments in model organisms have demonstrated that a series of induced mutations in various genes that make up an integrated molecular pathway can dramatically increase lifespan. The most prominent example includes the Caenorhabditis elegans daf-2 and clk double mutants that live nearly five times longer than wild-type worms (Lakowski & Hekimi, 1996). Most of the genes of model organisms are evolutionarily conserved and present in humans. Here, we will briefly review the genes and mechanisms that have been shown to regulate lifespan in model organisms, but limiting ourselves to those genes, which have human homologues, and have been studied for association with human health and/or longevity (Table 1). In addition, it is addressed whether we can expect to find single genes or molecular pathways that substantially affect lifespan in humans, whether the information obtained from model organisms can be translated to variation in lifespan in humans and whether present genetic surveys are able to pick up this genetic variation. Here, for the first time, we address these questions and suggest approaches for studies in model organisms and in humans that would lead to results that are more easily translatable from model organisms to humans. Results are different for the various species. Therefore, we explore the critical importance of the environment in which organisms live, including humans, because same genes in different environmental settings can lead to different phenotypical effects.
|Sir2||NAD(+)-dependent deacetylase||Regulation of metabolism, stress resistance||Kaeberlein et al., 1999|
|age-1||Phosphatidylinositol kinase||Insulin signalling||Morris et al., 1996|
|daf-2||Insulin receptor-like gene||Insulin signalling||Kimura et al., 1997|
|daf-12||Nuclear hormone receptor||Regulation of metabolic and developmental pathways||Larsen et al., 1995|
|daf-16||Forkhead transcription factor||Regulation of metabolic and developmental pathways||Ogg et al., 1997|
|Cat||Catalase||Antioxidant activity||Orr & Sohal, 1994|
|Chico||Insulin receptor substrate||Insulin signalling||Clancy et al., 2001|
|Sod1||Superoxide dismutase||Antioxidant activity||Parkes et al., 1998|
|Sod2||Superoxide dismutase||Antioxidant activity||Sohal et al., 1995|
|Mei-41||Phosphatidylinositol kinase, ATR kinase othologue||DNA repair||Symphorien & Woodruff, 2003|
|Pcmt||Protein carboxyl methyltransferase||Protein repair||Chavous et al., 2001|
|Gh||Growth hormone||Insulin signalling, tissue proliferation||Bartke, 2005|
|Klotho||Beta-glucuronidase||Inhibits IIS signalling||Kuro-o et al., 1997|
|p53||Tumour protein p53||Tumour suppression||Tyner et al., 2002|
Insulin/IGF-1 signalling (IIS)
The first evidence for genetic regulation of lifespan came from studies with C. elegans. It was discovered that worms with mutations in the dauer formation (Daf) genes, such as daf-2 and age-1, were able to bypass dauer formation, and become long-lived adults (Larsen, 2001). The molecular characterization of the daf-2 and age-1 genes revealed that daf-2 shows homology to the mammalian genes encoding insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-1R) (Kimura et al., 1997); and age-1 to the mammalian phosphatidylinositol-3-OH kinase catalytic subunits, which are located downstream of the IR and IGF-1R (Morris et al., 1996). Next, it was shown that, similar to C. elegans, reduced insulin signalling extends lifespan in Drosophila melanogaster. The increase in lifespan was observed for flies with mutated insulin-like receptor (InR) or its substrate (chico), and for flies with ablated insulin-producing cells (Giannakou & Partridge, 2007). In the latter case, the adult flies also exhibited increased storage of lipids and carbohydrates, reduced fecundity and increased stress and starvation resistance.
In vertebrates, the insulin signalling system is more complex and contains separate receptors for insulin (IR) and IGF-1 (IGF-1R) (Navarro et al., 1999). Data from mice indicate that both these receptors are involved in lifespan regulation. The IGF-1 branch, which acts through growth-hormone-releasing hormone, growth hormone (GH) and IGF-1, influences body composition and is involved in the regulation of gonad function (Bartke, 2005). The Ames and Snell dwarf mice, which are deficient in GH, thyroid hormone and prolactin, are infertile but long lived (Brown-Borg et al., 1996). A similar phenotype is observed for GH receptor knockout mice (Bartke, 2005). Furthermore, mice mutated for the IGF-1 receptor hint at a direct role for reduced IGF-1 signalling in mammalian longevity: Igf1r+/– females, but not males, exhibit a long-lived phenotype (Holzenberger et al., 2003). In contrast, complete disruption of the IR gene leads to insulin resistance, diabetes and shortened lifespan (Okamoto & Accili, 2003). Likewise, tissue-specific IR knockout mouse models develop obesity, insulin resistance and impaired glucose regulation, with the exception of fat-specific IR knockout mice (FIRKO) (Okamoto & Accili, 2003). These mice have reduced fat mass, are protected against age-related obesity and live longer than their littermates. Taken together, the evidence in mouse models shows that reduced IIS can extend lifespan also in mammals.
In humans, defects in GH–GHR–IGF-1 axis result in major pathologies (e.g. Laron Syndrome), but despite the pathologies these patients have a long lifespan, reaching ages of 80–90 years (Laron, 2005). In addition, there is evidence that long-lived subjects, such as centenarians, have decreased plasma IGF-1 levels and preserved insulin action, thus indicating that insulin responsiveness influences human longevity (Paolisso et al., 1997). Furthermore, polymorphisms in the IGF-1R locus, which lower plasma IGF-1 levels, were shown to be enriched among Italian centenarians (Bonafe et al., 2003). This finding was not replicated in a prospective follow-up study of elderly Dutch subjects, but in the same study it was found that a polymorphism in the GH1 gene, which controls IGF-1 activity, associates with longevity (van Heemst et al., 2005a). In addition, a combined effect of variation at the GH1, IGF-1 and IRS1 loci was observed, suggesting an additive effect of multiple variants associated with reduced IIS signalling on human longevity. These data, however, do not reveal a modulation of human lifespan in a magnitude that would come close to what is seen by analogous defects in some of the model organisms.
The Klotho gene, which was identified in mouse models (Kuro-o et al., 1997), encodes a mammalian-specific hormone that negatively regulates the activity of IR and IGF-1R through repressing their autophosphorylation (Kurosu et al., 2005). In mice, genetic variation in the Klotho gene results in an early onset of various age-related disorders, including ectopic calcification, skin and muscle atrophy, osteopenia, medial calcification of the aorta and pulmonary emphysema (Kuro-o et al., 1997). On the other hand, over-expression of Klotho in mice leads to inhibition of insulin and IGF1 signalling and increased lifespan (Kurosu et al., 2005). In humans, a haplotype allele called KL-VS, which contains six sequence variants that are in complete linkage disequilibrium, has been associated with KLOTHO expression and shown to be underrepresented in elderly individuals (Arking et al., 2002). Additional studies have demonstrated that the survival advantage is only present in heterozygous KL-VS allele carriers, whereas in homozygous allele carriers a disadvantage for high-density lipoprotein (HDL) cholesterol levels, systolic blood pressure, stroke and longevity was observed (Arking et al., 2005).
Forkhead transcription factors
In C. elegans, the IIS pathway negatively regulates the activity of DAF-16, which is its main downstream target. The long-lived phenotype of the IIS mutants depends on the presence of an active DAF-16 protein (Mukhopadhyay & Tissenbaum, 2007). In mammals, the DAF-16 homologues are forkhead transcription factors (FOXOs): FOXO1a, FOXO3a, FOXO4 and FOXO6 (Furuyama et al., 2000). Similar to DAF-16 in C. elegans, in mammals the FOXO proteins relay the effects of insulin on lifespan, influence fertility and play a role in complex diseases such as diabetes (Carter & Brunet, 2007). In humans, only few studies have analysed the role of FOXO proteins in the development of age-related diseases, fertility and lifespan. In most studies, no associations between genetic variance in the FOXO1a and FOXO3a genes, and lifespan have been detected (Bonafe et al., 2003; Kojima et al., 2004). These findings, contrast with recent studies where genetic variance in the FOXO1a gene was linked to increased glucose levels (Karim et al., 2006; Kuningas et al., 2007a) increased risk of diabetes and decreased lifespan (Kuningas et al., 2007a). For genetic variance in the FOXO3a gene, associations were observed with increased risks of stroke and mortality, but not with fertility (Kuningas et al., 2007a).
In C. elegans, DAF-12 is a member of the evolutionarily conserved nuclear hormone receptor (NHR) superfamily (Mangelsdorf et al., 1995), and it has been implicated in dauer diapause, developmental timing, metabolism, fertility and longevity. Current data have positioned DAF-12 downstream of the insulin and germline signalling, as the long-lived phenotype of germline-ablated mutants and of some IIS mutants depends on DAF-12 activity, but the exact position is still unknown (Rottiers & Antebi, 2006). In humans, the NHRs most similar to DAF-12 are the liver X receptors (LXRs) (alpha and beta), which have cholesterol breakdown products (oxysterols) as ligands. Upon activation, LXRs regulate various processes that result in cholesterol excretion from the body (Zelcer & Tontonoz, 2006). Recently, a common haplotype of the LXRA gene was associated with increased survival, predominantly because of lower mortality from cardiovascular causes and infection (Mooijaart et al., 2007a). A possible mechanism through which LXR could lead to the observed beneficial effects includes involvement of its target gene apolipoprotein E (APOE). ApoE is an anti-atherosclerotic protein involved in the efflux of cholesterol from macrophages. Genetic variation in APOE has consistently been associated with cognitive decline and cardiovascular disease mortality. Moreover, we have recently shown that independent of genetic variation in APOE, high plasma ApoE levels associate with increased risk of stroke (van Vliet et al., 2007), increased risk of cardiovascular mortality (Mooijaart et al., 2006) and decreased cognitive functioning (Mooijaart et al., 2007b). These data support previous observations that lipoprotein metabolism is critical for exceptional longevity. It has been shown that families of Ashkenazi Jewish centenarians have larger particles of HDL and low-density lipoprotein (LDL), which are associated with a decreased incidence of metabolic syndrome, cardiovascular disease and hypertension (Barzilai et al., 2003). Also, in the Dutch Caucasian population, offspring of long-lived sibling pairs have larger LDL particles than their age-matched partners, again suggesting that larger LDL particles confer a survival benefit (Heijmans et al., 2006).
The Sirtuins represent an evolutionarily conserved family of silent information regulator 2 (Sir2) NAD-dependent protein deacetylases that interact with and influence the activity of various transcription factors and co-regulators (Bordone & Guarente, 2005). Increased expression of the Sir2 gene, either because of an extra copy of the gene or to caloric restriction, has been shown to prolong lifespan in various model organisms (Haigis & Guarente, 2006). Likewise, the administration of resveratrol, which is normally synthesized by plants in response to stress, increases the activity of Sir2, and the lifespan of yeast, worms and fruit flies (Howitz et al., 2003; Wood et al., 2004). However, the effect of resveratrol on lifespan extension in model organisms has been recently put under question (Bass et al., 2007). A study of resveratrol found no significant effects on lifespan in seven independent trials in D. melanogaster and found slight increases in lifespan in some trials but not others in C. elegans (Bass et al., 2007).
In mammals, there are seven Sir2 homologues (SIRT1-7), of which SIRT1 is the most closely related to Sir2 (Frye, 2000). In mouse models, SIRT1 and SIRT3 have been studied the most. SIRT1 has been associated with glucose and fat metabolism, stress resistance and cell survival (Haigis & Guarente, 2006), whereas SIRT3 regulates the activity of acetyl-CoA synthetase, and thereby the entry of carbons from acetate into central metabolism (Haigis & Guarente, 2006). In humans, polymorphisms within SIRT1 and SIRT3 genes have been analysed for association with age-related diseases and longevity. In case of SIRT1, no associations have been found (Flachsbart et al., 2006; Kuningas et al., 2007b), whereas of SIRT3, a G477T polymorphism and a variable number of tandem repeats have been associated with increased lifespan (Rose et al., 2003; Bellizzi et al., 2005). These results suggest that at least one member of the SIRT family is involved in human lifespan regulation.
The life-extending benefits of resveratrol are also being studied in mammals. It has been published that the health and survival of mice on a high-calorie diet can be improved by resveratrol supplementation (Baur et al., 2006). While these results seem promising, there is no evidence that demonstrates the influence of resveratrol supplementation on human health, even though it is being sold as a nutritional supplement.
Antioxidative enzymes, such as catalase and superoxide dismutase (SOD), prevent damage from reactive oxygen species (ROS), but the evidence from model organisms on the beneficial effects of antioxidative enzymes on lifespan has been controversial. Studies with D. melanogaster have demonstrated that over-expression of CuZn–SOD (SOD1), Mn–SOD (SOD2) and catalase leads to lifespan extension (Sohal et al., 1995; Orr & Sohal, 2003). Additional experiments, however, showed that this effect depends on genetic background of the used lines (Orr & Sohal, 2003). Likewise, the extended lifespan of C. elegans by administration of synthetic SOD/catalase mimetics was shown to depend on laboratory conditions (Melov et al., 2000; Keaney & Gems, 2003).
In mammals, one catalase and three SOD genes have been characterized: SOD1, SOD2 and SOD3, of which catalase and SOD2 seem to influence lifespan. In mice, disruption of the SOD2 gene is lethal because of neurodegeneration and damage to the heart (Li et al., 1995; Melov et al., 1998). In contrast, over-expression of SOD2 leads to increased lifespan (Hu et al., 2007), as does over-expression of catalase targeted to mitochondria (Schriner et al., 2005). Mice heterozygous for the mitochondrial form of SOD2 (Sod2 ± mice) showed high levels of DNA oxidation in multiple organs. In spite of their abnormally oxidized DNA, these animals showed no decline in lifespan and no acceleration in the hallmarks of aging, such as cataracts, immune dysfunction and protein modifications (Van Remmen et al., 2003). These data suggest that mice can live reasonably long and healthy lives despite unusually high levels of oxidative damage. On the other hand, a recent study showed that enhanced longevity is associated with low free radical production by mitochondria in vertebrate homeotherms. In that study, no correlations with SOD activity and lifespan were found (Lambert et al., 2007). Altogether, these data illustrate the existence of a balance between the production of free radicals and of antioxidative enzymes.
The evidence for the role of antioxidative enzymes in the preservation of human health is not well established. It has been shown that RNA interference (RNAi) of SOD1 induces senescence in human fibroblasts (Blander et al., 2003), which suggests that SOD1 may play a role in the regulation of cellular lifespan. However, genetic variants in the SOD1 gene have never been studied for that relationship. In contrast, genetic variants in the SOD2 gene have been studied and associated with a number of phenotypes including increased risk for prostate and breast cancer, immunosenescence profile and DNA damage (Liu et al., 2004; Taufer et al., 2005), but not with mortality (De Benedictis et al., 1998; van Heemst et al., 2005a). Likewise, no associations between genetic variants in the catalase gene and mortality have been found (Christiansen et al., 2004).
Macromolecule repair mechanisms
Defects in mechanisms that repair damage to cellular components, such as DNA, proteins and membranes, have been shown to reduce lifespan in various model organisms. Even though these mechanisms are evolutionarily conserved (Eisen & Hanawalt, 1999), systematic comparative genomic analyses across species have not been conducted. In addition, within species, there are many studies demonstrating detrimental effects of impaired repair systems on lifespan, but only few demonstrating beneficial effects of increased repair capacity on lifespan. The only evidence for the latter has come from experiments with D. melanogaster, where the absence of mei-41 excision repair reduces lifespan, whereas flies with one or two extra copies of the gene have significantly increased lifespan (Symphorien & Woodruff, 2003). Likewise, over-expression of protein carboxyl methyltransferase (PCMT), which is a protein repair enzyme, has been correlated with enhanced longevity in a temperature-dependent manner (Chavous et al., 2001). Both of these genes, mei-41 and Pcmt, have homologues in mammals, which are ataxia telangiectasia and Rad3 related (ATR) and PCMT, respectively. In mice, the disruption of the ATR gene leads to chromosomal fragmentation and early embryonic lethality (Brown & Baltimore, 2000), and in humans to a rare Seckel syndrome (Casper et al., 2004). The Pcmt1-null mice, on the other hand, display a fatal seizure disorder and retarded growth (Kim et al., 1999), and die at a mean age of 42 days (Lowenson et al., 2001). In contrast, none of the heterozygous or wild-type littermates died during this period.
Compared to other repair mechanisms, DNA repair has been studied the most in relation to aging and lifespan. The DNA repair-deficient mouse models that have been generated, display a common phenotype of progeria, or cancer predisposition, or both, and have a reduced lifespan (Hasty et al., 2003). Similarly, in humans, all mutations identified in DNA repair genes severely compromise health. For instance, mutations in the components of transcription coupled repair have been associated with the progeroid syndromes of Cokayne syndrome and trichothiodystrophy (Hoeijmakers, 2001; Cleaver, 2005). Likewise, mutations in the RecQ-like DNA helicase genes, WRN, BLM and RecQ4, lead to the progeroid syndromes of Werner, Bloom and Rothmund–Thomson syndrome, respectively (Navarro et al., 2006). In contrast to the strong phenotypes associated with loss-of-function mutations in the RecQ helicases, common polymorphisms with subtle effects on the functionality of these genes do not seem to influence the aging trajectories and survival in the general population (Castro et al., 2000; Bohr et al., 2004; Kuningas et al., 2006). The RecQ helicases are highly conserved throughout evolution, but in higher eukaryotes, the different homologues seem to have distinct functions because failure of one given RecQ gene cannot be complemented by another RecQ gene. These observations underpin the importance of DNA repair in all organisms. The key question that has yet to be answered is whether subtle variants in the DNA repair genes contribute to different lifespans, and whether above-average repair makes for a lifespan extension. In addition, it is still under question, whether subtle variations in the genes that assure genomic integrity influence disease susceptibility and lifespan in the general population. From an aging point of view, their study is important as defective or reduced DNA damage recognition and repair capacity, together with decreased ROS scavenging can greatly compromise human health.
Cellular responses to damage
In response to unrepaired damage, cells trigger either apoptosis or cell cycle arrest. The most well-known protein implicated in the maintenance of genomic stability is p53. Recently, p53 homologues were identified in C. elegans and D. melanogaster. In contrast to mammalian p53, which elicits apoptosis or cell cycle arrest (Attardi, 2005), the p53 in C. elegans and D. melanogaster affects only apoptosis (Derry et al., 2001; Schumacher et al., 2001; Brodsky et al., 2004). Nevertheless, in all of these organisms, reduced p53 activity leads to lifespan extension (Bauer & Helfand, 2006). In mammals, this extension comes at the cost of increased cancer risk (Campisi, 2003). In humans, it has been shown that Pro/Pro carriers of the TP53 codon 72 polymorphism have a significantly lower apoptotic potential than Arg/Arg carriers, both in p53-inducible human cell lines (Dumont et al., 2003; Pim & Banks, 2004; Sullivan et al., 2004) and in normal diploid fibroblasts (Bonafe et al., 2004). Later on, it was shown that despite an increased mortality from cancer, carriers of the same polymorphism have a significantly increased survival at old age in line with the experimental models (van Heemst et al., 2005b). Altogether, these observations support the hypothesis that reduced p53-mediated induction of apoptosis can have beneficial effects on lifespan if tumour formation can be avoided. This might hold true also for other genes that mediate cellular responses to damage.
Experiments in model organisms have demonstrated that changes in genes can dramatically increase their lifespan. In some cases, mean and maximum lifespan is extended up to fivefold. The equivalent life-extending effect in humans would result in an average lifespan of 400 years and a maximum lifespan of over 600 years. Many of the pathways regulating lifespan in model organisms are conserved throughout evolution. Why, then, have we not yet identified genetic determinants that could increase human lifespan by more than a few years? Are we looking at the right genes? Is it fair to expect such dramatic effects? Do we have the tools to observe genetic determinants of human lifespan?
In vertebrate organisms, novel genes and signalling components have appeared during evolution, contributing to increased complexity of the genomes (Long, 2001). Among several molecular mechanisms, gene duplication plays a major role in genome evolution (Long, 2001; Britten, 2006). Often, a mammalian genome contains several homologues of a single invertebrate gene with similar or distinct functions and expression patterns. This can hinder the assessment of the role of a specific candidate gene, because genetic variance in duplicated genes is likely to have less dramatic effects than in the original single gene in invertebrate organisms (Gu et al., 2003; Conant & Wagner, 2004). Furthermore, because vertebrates have acquired pathways that are more diverse, the homologous genes in phylogenetically distant animals could be involved in different pathways.
The genomes of vertebrate organisms also contain genes, which have no homologues in invertebrates. The appearance of novel genes, or ‘add-ons’, probably contributed to the arise of elaborate systems that regulate the activity of the conserved or ‘shared’ genes (Levine & Tjian, 2003). The GH control over IGF-1 and the regulation of IR and IGF1-R activity by KLOTHO have arisen relatively recently in vertebrate evolution (Mian, 1998; Forsyth & Wallis, 2002). Genetic variance in these genes has been associated with lower IIS signalling and with increased lifespan, corroborating the findings from invertebrates. However, in these organisms, this phenotype was attained by modifying the ‘shared’ genes. This demonstrates that in humans, we should not limit ourselves to studying the evolutionarily conserved genes that have been implicated in lifespan regulation in invertebrates, but also include, and maybe even focus on, ‘add-ons’ that regulate their activity. Taken together, the increased complexity of signalling systems in vertebrates adds robustness to the signalling pathways, which is the ability to maintain its functions despite changes in its components or environment (Lenski et al., 2003; Soyer & Bonhoeffer, 2006). In that respect, lifespan regulation can be regarded as a complex genetic trait, for which it is unlikely that single alterations in the genetic machinery will have dramatic effects. Furthermore, genes that have appeared later in evolution, ‘add-ons’, such as microcephalin gene and glucocorticoid receptor, are interesting candidates to determine longevity in vertebrate organisms.
Environmental influences have played a major role in shaping and patterning the genomes of all organisms throughout evolutionary history. Changes in environment can lead to different expressions of genotypic information, and thereby complicate the comparability of results between model organisms and humans (Partridge & Gems, 2007). Most research on model organism has been performed under laboratory conditions, where temperature, presence of pathogens, food availability and population density are tightly controlled. In most cases, these conditions poorly mimic the evolutionary niche in which the genes come to expression, and it may therefore be questioned how well the results obtained in these conditions are applicable to species under ‘natural’ conditions (Walker et al., 2000; Clancy et al., 2001; Marden et al., 2003).
Even for humans, the environment in which the genome effectively evolved has changed. The genes that were originally selected for survival in adverse environments are now expressed under completely new, affluent conditions. For instance, the IIS system was selected and fine tuned in times when food abundance and famine alternated. The genotypes that increased the efficiency to store energy in times of abundance and use these storages in times of famine had a survival advantage. In modern Western societies, where food is constantly abundant, these ‘thrifty genotypes’ lead to increased prevalence of storage diseases, such as obesity and diabetes (Neel, 1962). This reinforces the idea that our genomes have been optimized to increase fitness under adverse conditions and not under modern affluent conditions, resulting in new interactions with outcomes that are both unknown and unpredictable. Taken together, caution should be taken in extrapolating results on genetic variation obtained in model organisms to the human situation, because the environments in which the genes come to expression are often markedly different. Moreover, research into human aging should include various environmental conditions that can explain different phenotypes despite an equal genetic background. Furthermore, several studies have already documented the dominance of nurture over the nature within the context of human lifespan (Fraser & Shavlik, 2001; Peeters et al., 2003).
Mutants versus natural genetic variants
The majority of candidate genes of longevity have been identified by studies with mutant model organisms, such as the worm, fruit fly and mouse models. These approaches are extremely powerful to disentangle biological pathways. However, to date it is largely unknown to what extent these mutations affect fitness in natural environments and whether these candidate loci contain genetic variation, which would contribute to phenotypic variance for lifespan in natural populations (Fig. 1). These questions are of importance because not all candidate loci with major effects on longevity in laboratory conditions may exhibit variation in natural populations. Currently, only few studies have tried to disentangle this question. For instance, it has been shown that the long-lived mutant fruit fly methuselah (mth) underperforms in most cases under conditions that resemble a more natural situation (Baldal et al., 2006). This illustrates that the mth locus would never have been identified to influence lifespan if natural populations of the fruit fly would have been analysed. A similar observation is obtained for the Chico variant that outlives the wild type under food affluence, but becomes short lived when exposed to food-restricted conditions (Clancy et al., 2002). In case of humans, most information on the effect of genetic variation on longevity is being obtained by studying naturally occurring variants in candidate genes identified in mutant model organisms. Hence, the discrepancy between the data from mutant model organism and standing genetic variance in natural populations contributes to the difficulty of translating results from model organisms to humans (Fig. 1). Therefore, to facilitate translation of the results from mutant model organisms to humans, the analysis of standing variation in the same loci in natural populations of model organisms should be encouraged. In addition, in all the studies with model organisms, the genetic background (rest of the genome) should be taken into account, as it has been shown that in some cases lifespan differences disappear when the background is made identical in mutant and controls (Partridge & Gems, 2007).
The lack of very strong effects of the evolutionarily conserved genes in humans can have other reasons than those suggested earlier. Some of these include study design and methodology in human studies. Most of the genetic association studies of longevity have been performed in case–control settings, where genotype or allele frequencies between elderly and a younger population are compared. The main advantage of case–control study design is that cases are readily obtainable and can be efficiently genotyped and compared with control populations. Cases can be nonagenarians, centenarians or long-lived sibpairs. By collecting long-lived sibpairs instead of long-lived singletons, an enrichment of genetic factors contributing to longevity in this population can be expected, while the likelihood of having reached a long life because of exceptional environmental conditions or chance is lower. The difficulty of case–control studies is in selecting controls. In longevity studies, the ideal control group should be composed of participants from the same birth cohort who were not long lived, to minimize the effect of environmental differences caused by cohort differences. However, obtaining such a control group is impossible in a retrospective study design. Such case–control studies are only possible if one is studying in long-lasting population surveys. The longest ongoing studies (e.g. Framingham Heart Study) now allow a difference of 30 years between subjects that survived and those that did not. However, in most studies so far, controls have been selected from the general population of younger generations. This, can lead to biases where allele frequency differences between cases and controls can appear as an association, even if they only reflect the results of changes to the source population because of changes in environment, migratory history, gender differences or other independent processes (Cardon & Palmer, 2003; Manolio et al., 2006). An alternative approach would be prospective cohort studies, which suffer less from population stratification but are more expensive and time consuming (Manolio et al., 2006).
Another consideration for the methodology includes the selection and analysis of genetic variants in the candidate genes. Commonly, a selected number of polymorphisms from the coding region of candidate genes are analysed, leaving aside genetic variants in regulatory regions. In addition, besides analyzing the individual polymorphisms, only few studies have undertaken haplotype analyses. Given the amount of information that has recently become available through the International HapMap project, polymorphisms that tag common haplotypes can easily be identified. The analysis of haplotypes can be more powerful because this analysis captures the joint effect of all unknown gene variants that are in linkage disequilibrium with the markers forming the haplotype (Johnson et al., 2001).
Finally, similar to other association studies, also the results from longevity association studies have been rarely replicated. Explanations for lack of reproducibility include poor study design, small sample size, incorrect assumptions about the underlying genetic architecture and over-interpretation of the data. In addition, for a number of associations (mainly negative), no replication has been undertaken, leaving open the reproducibility. Replication of even negative results is necessary, because the lack of associations in the first study could likewise have been caused by poor study design, population stratification or other reasons. Therefore, before discarding a candidate gene from the list of possible candidates, replication in different cohorts with more thorough genetic analysis is necessary.
Pathway analyses and epigenetic variation
Given the current feasibility of high throughput genotyping and increasing knowledge on cellular mechanisms, pathway analyses instead of analysing individual loci separately could be performed. The appropriate tools are likely to be available soon, because the analysis of complex traits, which are under the influence of multiple and possibly interacting genes, has become a subject of new statistical methodological research (Kristensen et al., 2006). However, besides genetic variation, other mechanisms influence the expression of genomic information. For instance, epigenetic modifications, which are differences in gene expression that cannot be accounted for by changes in the primary DNA sequence, have a significant impact on gene function, and may explain how iso-genetic organisms are phenotypically very distinct. It has been noticed that during aging, epigenetic alterations occur, which could have functional and biological significances. An emerging field, ‘aging epigenetics’, aims to answer these questions (Fraga & Esteller, 2007). Besides epigenetics, different levels of transcriptional and post-transcriptional control through RNA interference, or other mechanisms, can account for phenotypic differences. A family of small, non-coding RNAs, known as microRNAs (miRNA), has recently emerged as sequence-specific regulators of gene expression. miRNAs have been shown to affect a broad spectrum of biological activities, including developmental fate determination, cell signalling, oncogenesis and metabolism (Boehm & Slack, 2006). In addition, miRNAs have arisen as potential modulators of age-related decline. In C. elegans, it was found that the expression of certain miRNAs declines with age, suggesting a general reduction of message-specific translational inhibition during aging (Ibanez-Ventoso et al., 2006; Wang, 2007). Because many miRNAs are conserved across species, such mechanism might be at play also in humans. Altogether, various modifications can contribute to the differences in lifespan between and within species.
Genes and pathways for future analyses
A number of interesting candidate genes, with or without homologues in model organisms, remain to be investigated in humans. The most interesting and so far not very thoroughly analysed genes in respect to longevity include those involved in fertility. In model organisms, fertility and lifespan are closely linked (Partridge et al., 2005). In C. elegans, ablation of germline precursor cells of the gonad abolishes reproduction and extends lifespan (Hsin & Kenyon, 1999), as do mutations that reduce germline proliferation (Arantes-Oliveira et al., 2002). In D. melanogaster, a reduction in fecundity extends lifespan in females (Sgro & Partridge, 1999) and long-lived heterozygous chico mutant females exhibit reduced fecundity, with the homozygotes being almost sterile (Clancy et al., 2001). In mice, Ames and Snell dwarfs are long lived and sterile (Bartke, 2005). The observational studies in historical human populations living under pre-affluent conditions have provided similar evidence. In the English and Finnish aristocracy, women with the longest lifespan had the smallest number of offspring (Westendorp & Kirkwood, 1998; Korpelainen, 2000). Despite this evidence, the genetic determinants for the trade-off between fertility and lifespan are unknown.
This work was supported by an Innovative Orientated Research grant from the Dutch Ministry of Economic Affairs (grant number IGE010114), by the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (NWO) (grant number 911-03-016) and by the Centre for Medical Systems Biology, which is a centre of excellence approved by the NWO. This work was also supported by and carried out within the EU funded Network of Excellence Lifespan (FP6 036894). All co-authors have seen and agreed with the contents of the manuscript, and none of the co-authors has any financial interests to disclose.
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