Genome-wide approaches to understanding human ageing

The use of genomic technologies in biogerontology has the potential to greatly enhance our understanding of human ageing. High-throughput screens for alleles correlated with survival in long-lived people have uncovered novel genes involved in age-associated disease. Genome-wide longevity studies in simple eukaryotes are identifying evolutionarily conserved pathways that determine longevity. It is hoped that validation of these 'public' aspects of ageing in mice, along with analyses of variation in candidate human ageing genes, will provide targets for future interventions to slow the ageing process and retard the onset of age-associated pathologies.


Introduction
The study of the biology of ageing (biogerontology) has seen ar eawakening in recent years. Modernm oleculart echniques are being applied in an effortt ou nderstand both the changes that occur as people age and,p erhaps more importantly,t o identifyt he genes that determine howq uickly thesea geassociated changes progress. In addition, as life expectancy has increased in developed nations, an ageing population has contributed to ag reat societal (and financial) interest in understandingt he humana geing process and in ameliorating age-associated physicala nd cognitive declines.
The genomics revolution, in particular,has begun to have a profoundi mpact on the wayt hat biogerontologists approach the study of ageing. Global geneexpression profilingh as been used to characterise transcriptional changesa ssociated with age and longevity,a sd iscussed in several recent reviews. [1][2][3][4][5][6] Proteomics and metabolomics technologies area lso now being applied to ageing-related problems 7,8 and,a st hese technologies continue to mature,w ill certainly be used more extensively. One particularly important application of these technologies will be the identification of diagnostic biomarkers of ageing and ageing rate. [9][10][11][12] This review will describe theu se of genomics methods to identifyg enes that influence human ageing. Tw ot ypes of approaches will be discussed: genome-wide studies of allelic variants that correlate withl ongevity in people and highthroughput life span studies in lowereukaryotes. The synthesis of these approaches is beginning to uncoverh ighly conserved aspectso ft he ageing process and to identify candidate gene targetsf or future interventioni nto human ageing and age-associated disease.

Searching for allelic variants that determine longevity in humans
The long life span enjoyedb ymost people presents adifficulty for researchersw ishingt os tudy theg enetic and environmental factorst hat influence ageing and age-associated disease in people.T he lack of accepted biomarkerso fa geing rate means that there is no diagnostic test which can be used to determine whether ap articular mutation or environmental change is likely to have an impact on longevity. 13 The search for such biomarkersi sa no ngoing process.F or now, however, alternative, methods areb eing developed to address these questions.
One approach for identifying genetic features that influence longevity is thes tudy of individuals that achievee xtreme longevity. 14,15 Centenarians represent just such ag roup, with approximately 1i n1 0,000 people reaching their 100th birthday. 16 There is substantial evidence that genetic components influence humanl ongevity and that centenarians are people who have escaped the common age-associated diseases which account for al arge fraction of the mortality in the overall population. 15,17,18 In apioneering efforttoidentify genetic polymorphisms over-represented among centenarians, Puca and colleagues used linkage analysist os can the genomes of 308 individuals belonging to 137 sibshipsd isplaying extreme longevity. 19  wasn oted for al ocus on chromosome 4, 19 which was subsequently mapped to theg ene coding for microsomal triglyceride transfer protein ( MTP). 20 Alleles of MTP are associated with abetalipoproteinaemia and familial hypobetalipoproteinaemia in humans. 21,22 It therefores eems likely that the longevity-associated allele identifiedb yP uca and colleagues represents an allele that is protectivea gainst heartd isease. 20 An alternativet os tudying extremely long-lived cohorts is to identify genetic polymorphisms that change in frequency across ap opulation as af unction of age.F or example,ahighthroughput single nucleotide polymorphism (SNP)-typing approach -where allele frequencies aredetermined for alarge number of SNPs from individuals of many different ageshas the potential to uncovera lleles that influence longevity. Under such ad esign, alleles that result in disease susceptibility should decrease in frequency with donor age,w hile alleles that are important for longevity should increase in frequency. This approach wasu sed successfully to identify an isoleucine to valine polymorphism in the protein kinase A( PKA) anchoring protein AKAP2, which correlates with decreased longevity and cardiac disease. 23 PKA activity has been linked to ageing in simple eukaryotes. 10,24 -26 Micewithaltered levels of PKA have phenotypes consistent with an ageing-related role,s uch as decreased adipose tissue,p rotection against obesity and elevated expression of uncoupling proteins. 27 -29 Surprisingly,n oa dditional large-scale searches for age-related changesi nS NP frequencies have been described. As technologies for SNP discovery and quantitativea nalysis continue to improve, this approach mayw arrant further attention.

Difficulties in using humans to study human longevity
Both examples of age-correlated polymorphismsh ighlighted above ( AKAP2 and MTP)d emonstrate one of the difficulties associated with identifying genes that influence the rate of ageing from genetic studies of longevity in people: thep rofound effect of ar elatively small number of age-associated diseases on human mortality.Amajority of deaths in developed nations occur as ar esult of ar elatively small number of age-associated diseases, including cardiovasculard isease, cancer,s trokea nd diabetes. Thus, one of the prerequisites for achieving extreme longevity is ar educed risk for these diseases, and polymorphisms conferring reduced risk to one or more of these diseases can have as ignificante ffect on individual longevity.Inthe examples of MTP and AKAP2,the observede ffects on longevity area lmost certainly due to an altered risk of cardiovasculard isease. 20,23 Does this meant hat individuals with the longevity-associated allele of MTP are ageing more slowly? Not necessarily. The risk for one or more age-associated phenotypesi sr educed; however, there is no evidence that many or all age-associated phenotypes area lso retarded in suchi ndividuals. Other alleles have also been correlated with life span in people (Table 1), and in almost everyc asec an be attributed to delayed onset of one,o ra few,a ge-associated disease(s).
As econd difficulty in population-based studies of human longevity is controlling for population-specific factors. 50 For example,t he prevalence of specific age-associated diseases is variable in different populations. This can be due to any combination of cultural (eg diet), historical (eg famine), environmental (eg exposure to toxic chemicals) or genetic components, and can have ap rofoundi mpact on the types of genotypic variants that influencesurvival to old age. Recently, it has been suggested the role of MTP as al ongevity-related locus mayb es pecific for the cohortu sed in the study from which it wasi nitially identified. 50,51 Simply designing an appropriately controlled human longevity study is challenging, and care must be taken to avoid population stratification. Thus, it will be important to test any candidate human longevity locus in multiple populations to determine the generality of thec orrelation.
At hird potentialb arrier to identifying genetic variants that have as ignificant impact on humana geing mayb et hat such variants carry al arge selective disadvantage and have been culled from the gene pool. To date,n og enetic variant has been definitively shown to slowt he rate of human ageing, although rare mutations can acceleratea tl east some aspects of the ageing process,r esulting in progeria syndromes. 52 Abundant evidence from model organisms, however, suggests that mutations in single genes can dramatically slowt he rate of ageing and the onset of many (perhaps all) age-associated phenotypes. On the surface,i tm ay seem surprising that, if such 'master regulators' of ageing exist in people,n oa lleles have been identifiedt hat confer extreme longevity.T he examplesf roms impler eukaryotes, however, alsod emonstrate quite clearly that these types of mutations often come with significantfi tness and reproductivec osts. 53 -55 Thus, strong longevity-enhancinga lleles in genes that influence ageing rate are likely to have been selected against during recent human evolution, perhaps makingt heir detection by largescale polymorphism studies impossible.
Genome-wide approaches to identifying 'public' pathways of ageing The use of model systems for ageing-related research provides an avenue for getting around many of the difficulties associated with human studies. Both micea nd rats arec ommonly-used mammalian models for ageing and longevity studies. Several simplee ukaryotic models have also been developed, including the budding yeast Saccharomycesc erevisiae,t he nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. 56 Perhaps the greatest advantage afforded by model organismsf or ageing-related studies is that the relatively short life spans of these organisms allows for controlled longevity studies to determine whether ap articular mutationo r environmental change altersa geing rate.I np articular,i nterventions that increase populationl ife span are of interest because they either slowt he rate of ageing, delayt he onset of ageing or both. Several dozen single-gene mutations that significantly increase life span have been identifiedfromstudies in model organisms. 57 More recently,g enome-wide screens for longevity have been carried out in both yeast and worms, resulting in aw ealth of data and ab etter understanding of the degree to which theg enetic basis of ageing has been conserved. Aq uestiont hat is often raised when considering the usefulness of model organismsi na geing research is whether the ageing process has been sufficiently evolutionarily conserved that the mechanisms of ageing are shared between lower eukaryotes and people,orevenbetween non-human mammals and people.W hile this question is impossible to answer at this time,t here is reason to think that at least somea spects of ageing are highly conserved. 58 -60 For example,a ll of the model organismsu sed for ageing-related research displaya n approximatelye xponentiali ncrease in mortality witha ge (Gompertz-Makeham-likem ortality), consistent with the idea that important aspects of the underlying ageing process is conserved. 61 Perhaps the most compelling reason to think that ageing is highly conservedi st he recent identification of several conserveddeterminants of longevity -orthologous genes (or similar environmental changes) that determine ageing rate in evolutionarily divergent organisms ( Table 2). These conservedl ongevity determinants arel ikelyt or egulate 'public mechanisms of ageing' 84  Genome-wide studies of ageing in worms C. elegans has proven to be one of the most important model organismsfor ageing-related research, providing the first wellcharacterised model for the role of insulin/insulin-like growth factor I(IGF-1) in ageing. Several components of this pathway have been shown to regulate longevity in worms, including an insulin-likereceptor (Daf-2), 64,65 aphosphatidylinositol 3-kinase (Age-1), 85,86 proteins orthologous to Akt kinases(Akt-1, Akt-2 and Sgk-1) 72 -74 and aFOXO-family transcription factor (Daf-16). 87,88 Global geneexpression profilingbymicroarrayhas further elucidated someofthe downstreamcomponents of this pathway,which are involved in antimicrobial, oxidativeand other stress responses. 89,90 The true powero f C. elegans as am odelf or ageing-related research has become apparent witht he developmento fR NA interference (RNAi) as at echnology for genee xpression knock-down in worms. Thet ypicalf ood source provided to C. elegans is live Escherichia coli grownonasolid medium. E. coli expressing ap lasmid-encoded double-stranded RNAc orresponding to a C. elegans open reading frame (ORF) knocks down expression of the targeted gene. 91 An RNAi library corresponding to 17,000 unique genes ( , 85 per cent of C. elegans ORFs) has been constructed, 92 and twoi ndependent genome-wide RNAi screens have been carried out for genes that influencel ongevity in worms. 73,93 -95 Genes that increase life span when knocked down can be grouped into functional categories, the largest being genes important for mitochondrial respiration and genes involved in insulin/IGF-I signalling. Several uncharacterised genes were identifieda s well, suggestingt hat important aspectso ft he ageing process remain uncharacterised, even in simplee ukaryotes.
Am ulti-organism approach to identifying public pathways regulating longevity Recently,agenomic approach to uncovering genetic determinants of longevity that have been conservedf romy east to mammals has been described, based on the hypothesis that protein families which function to determine ageing rate in both yeast and wormsa re likelyt op layasimilar role in mammals. 10,96 The first phase of this proposal involves genomic analysiso fa geing in yeast. 97 Tw ot ypes of ageing are commonly studied in yeast:r eplicative life span, which refers to the number of times ay east cell can divide prior to senescence, 98 and chronological life span, measured by the length of time ac ell can survivei nanon-dividing state. 99 High-throughput assays for both replicativeand chronological ageing have been developed, 26,100 which will allowf or life span determination of approximately 4,800 single-gene deletion strains contained in the yeast ORF deletion collection. 101 For each yeast ageing geneidentifiedfromthese screens, the homologous genes in C. elegans (if any) will be examined by measuring life span in response to RNAi-mediated knockdown. Those orthologue pairst hat determine longevity in both yeast and C. elegans will then be candidates for further study in am ammalians ystem.C onditional and tissue specific knockout of mouse genes orthologous to conservedy east and C. elegans ageing genes will be carried out and selected lines subjectedt ol ife span analysis. Due to the costly nature of rodent longevity studies, this is an exceptional method for identifying high-interest candidates. Clearly,a ny gene family found to determine life span in yeast,w orms and mice will be of great interesta salikelyd eterminant of humanl ongevity.
Even prior to life span studies in mice,t he identification of longevity determinants conservedi ns imple eukaryotes, such as yeast and worms, will allowfor SNP analysisofhuman orthologues to determine whether there is ac orrelation of certain alleles with longevity or age-associated disease.F or example,increased expression of Sir2-family proteinsincreases life span in yeast, 75 worms 76 and flies, 77 and alleles of ahuman homolog, SIRT3, arer eported to correlate withl ongevity in people 78 (Table 1). Likewise,m utationo ft he insulin/IGF-I receptor homologues increase life span in worms 64,65 and flies, 66 and it has been suggested that allelic variants in the human IGF-I receptor correlate with longevity. 36 The nutrient-responsivek inases TOR( target of rapamycin),P KA and Sch9/Akt represent additionalhigh-interest candidates, for which humanl ongevity data have not been reported. More subtle effects on human ageing, which mayb em asked by disease alleles in genome-wide scans, can potentially be uncovered by this type of targeted approach based on knowledge gleaned from model organisms.

Conclusion
The further development and application of genomicsmethods toward biogerontology has the potentialt od ramatically enhance understandingofhumanageing. Genomic approaches have already uncovered genes important in the onset of human age-associated disease.I ns impler eukaryotes, genome-wide studies arerapidlyproviding adetailed picture of themolecular pathwayst hat regulate ageing. The moste ffective future studies mayc omef romc ombining the genes identified in simpler organisms with technologies to rapidly uncover allelic variationi nh uman orthologues which mayi nfluence longevity and disease.There is ample reason for optimism that these approaches will enhance our understanding of the molecularbiology of ageing and, ultimately, our ability to treat age-associated disease.