Genetic Architecture of Drosophila Lifespan

Reviews

Age mutations

July 1996

Other genetic factors affecting aging include detoxification of reactive oxygen species.

Ageing is a complex phenomenon which remains a major challenge to modern biology. Although the evolutionary biology of ageing is well understood, the mechanisms that limit lifespan are unknown. The isolation and analysis of single- gene mutations which extend lifespan (Age mutations) is likely to reveal processes which influence ageing. Caenorhabditis elegans is the only metazoan in which Age mutations have been identified. The Age mutations not only prolong life, but also confer a complex array of other phenotypes. Some of these phenotypes provide clues to the evolutionary origins of these genes while others allude to mechanisms of lifespan-extension. Many of the Age genes interact and share a second common phenotype, that of stress resistance. Rather than invertebrate ageing being determined by a ‘clock mechanism’, a picture is emerging of ageing as a non-adaptive process determined, in part, by resistance to intrinsic stress mediated by stress-response genes.

Lithgow, G. J. Invertebrate gerontology: the age mutations of Caenorhabditis elegans. Bioessays 18, 809– 815 (1996).

http://onlinelibrary.wiley.com/doi/10.1002/bies.950181007/abstract

Sir2

2001

Other genetic factors affecting aging include chromatin silencing.

The model organism Saccharomyces cerevisiae is providing new insights into the molecular and cellular changes that are related to aging. The yeast protein Sir2p (Silent Information Regulator 2) is a histone deacetylase involved in transcriptional silencing and the control of genomic stability. Recent results have led to the identification of Sir2p as a crucial determinant of yeast life span. Dosage, intracellular localization, and activity of Sir2p all have important effects on yeast longevity. For instance, calorie restriction apparently increases yeast life span by increasing Sir2p activity. Since Sir2p-related proteins have been identified in many prokaryotic and eukaryotic organisms, the fundamental principles derived from the studies in yeast may prove valuable in directing our future research toward an understanding of the mechanisms of aging in higher eukaryotes.

Defossez, P. A., Lin, S. J. & McNabb, D. S. Sound silencing: the Sir2 protein and cellular senescence. Bioessays 23, 327–332 (2001).

http://onlinelibrary.wiley.com/doi/10.1002/bies.1047/abstract

The Genetics of Aging

2001

Lifespan varies in natural populations due to the segregation of multiple genetic factors as well as exposure to different environmental conditions.

Finch, C. & Ruvkun, G. The Genetics of Aging. Annu. Rev. Genomics Hum. Genet. (2001). http://www.annualreviews.org/doi/abs/10.1146/annurev.genom.2.1.435.

http://www.annualreviews.org/doi/full/10.1146/annurev.genom.2.1.435

Complex Genetic Architecture

2002

Quantitative trait loci (QTLs) associated with lifespan in D. melanogaster have been mapped in linkage mapping populations.

Continuous phenotypic variation in life span results from segregating genetic variation at multiple loci, the environmental sensitivity of expression of these loci, and the history of environmental variation experienced by the organism throughout its life. We have mapped quantitative trait loci (QTL) that produce variation in the life span of mated Drosophila melanogaster using a panel of recombinant inbred lines (RIL) that were backcrossed to the parental strains from which they were derived. Five QTL were identified that influence mated life span, three were male-specific, one was female-specific, and one affected life span in both sexes. The additive allelic effects and dominance of QTL were highly sex-specific. One pair of QTL also exhibited significant epistatic effects on life span. We summarize all of the QTL mapping data for Drosophila life span, and outline future prospects for disentangling the genetic and environmental influences on this trait.

Leips, J. & Mackay, T. The complex genetic architecture of Drosophila life span. Exp. Aging Res. (2002). doi:10.1080/0361073029008039

http://www.tandfonline.com/doi/abs/10.1080/03610730290080399

Mechanisms of Ageing: Public or Private

March 2002

Many genes affecting aging are ‘public’, or shared across evolutionary lineages.

Partridge, L. & Gems, D. Mechanisms of ageing: public or private? Nat. Rev. Genet. 3, 165–175 (2002).

http://www.nature.com/nrg/journal/v3/n3/full/nrg753.html

APOE genotype effects on Alzheimer’s disease

July 2004

Approaches to explore the genetic basis of aging in humans are linkage mapping, and more recently, genome wide association (GWA) mapping, to identify polymorphisms affecting diseases and disorders associated with aging, such as Alzheimer’s disease.

The risk of developing Alzheimer’s disease (AD) is tied most closely to age and appears to follow Gompertzian kinetics. However, specific genetic factors are also linked closely to AD, and the apolipoprotein E (APOE) geno- type accounts for as much of 50% of the attributable risk for AD in many populations. This paper reviews the onset, diagnosis, and epidemiology of AD, specifically with regard to the APOE genotype and the interaction of the genotype with age.

apolipoprotein E (APOE) genotype accounts for as much of 50% of the attributable risk for AD in many populations.

Alzheimer’s disease (AD) has been defined clinically as a disease that causes a dementia with an insidious onset and slowly progressing course. Fundamentally affects memory storage processing. Estimating AD onset is to ask individuals who have known the patient when they first became aware of any of the symptoms that subsequently developed into the dementia. symptoms such as anger, depression, anxiety, or inattention might be the symptoms first recalled.

dementia, mild or greater, affects about 15% of the population over 60 yr of age. AD is considered to account for two-thirds of the diagnoses of dementia, thus 10% of the over-60 population. These estimates are the basis for the statement that AD affects about 4 million people in the United States.

Although it has been stated frequently that AD is not part of normal aging, AD is actually related more closely to age than mortality, which doubles in incidence about every 8 yr.

Gompertzian kinetics - fundamental model of aging is the Gompertz survival function. Gompertz curve depends on knowledge of an initial rate of mortality and the doubling time of the rate. Accounts for more than 99.7% of the variance in mortality after 30 yr of age in the United States for the year 2000.

Gompertz survival curve is best explained by a theory that the organism is composed of a number of sub- systems that have evolved in a coordinated fashion to manage environmental stresses with optimal energy efficiency.

incidence rates of AD start much lower than mortality rates but reach 1/1000 by age 62, 1/100 by age 79, and 1/10 by age 94 and approach the mor- tality rate around age 105. one-third of all men and two-thirds of all women will contract AD before they die.

senile plaques (SP): beta-amyloid (Aβ) aggregates, which seem to be closely related to disease causation
neurofibrillary tangles (NFTs): composed of hyperphosphorylated tau protein and are closely related to dementia severity

amyloid pre- cursor protein (APP) and the microtubule-associated protein tau, both have important roles in neuroplasticity and memory function.

pathophysiology suggests that primary AD causation is linked to the processing of the APP.

first-degree relatives of persons with AD at autopsy have a substantially increased risk of AD relative to controls, and twin studies indicate that the heritability of AD exceeds 70%.

individuals who carry one or more apolipoprotein E (APOE) ε4 alleles for AD are more likely to stop their education earlier in life.

genetic factors influence dietary preferences. An environmental factor that has been associated with AD is dietary cholesterol. dietary habits established early in life could mimic genetic influences, and genetic factors could influence dietary preferences.
hypertension, hypercholesterolemia

ε4 allele by itself is likely responsible for 50% of the nonfamilial AD cases in US. APOEε4/4 indi- viduals having an estimated 50% chance of AD onset at 68.4 yr of age, APOEε3/4 individuals at 75.5, and APOE ε3/3 individuals at 84.3. no ε4/4 carrier has been shown to reach age 90 without having AD. ε2 carriers are overly represented among centenarians.

The gene coding for APOE is by far the clearest of the genetic factors that has been associated with relatively later-onset AD, and variations in this gene appear to account for as much as 50% of the population-attributable risk in the United States.

APOEε4 allele is most common in African pygmies (41%), least common in Sardinians (5%), and intermediate in most Western populations (9−19%). TheAPOEε4 allele has a rate of 8% in India and China, which might account for the lower rate of AD found in India and China compared with Western popula- tions, as this allele seems to have the same association with AD in these countries as it does in Western countries

Ashford, J. W. APOE genotype effects on Alzheimer’s disease onset and epidemiology. J. Mol. Neurosci. 23, 157–165 (2004).

http://link.springer.com/article/10.1385%2FJMN%3A23%3A3%3A157

The lesson of centenarians

February 2005

Approaches to explore the genetic basis of aging in humans are linkage mapping, and more recently, genome wide association (GWA) mapping, to identify polymorphisms affecting extreme longevity.

In this paper, we review data of recent literature on the distribution in centenarians of candidate germ-line polymorphisms that likely affect the individual chance to reach the extreme limit of human life. On the basis of previous observations on the immunology, endocrinology and cellular biology of centenarians we focused on genes that regulate immune responses and inflammation (IL-6, IL-1 cluster, IL-10), genes involved in the insulin/IGF-I signalling pathway and genes that counteract oxidative stress (PON1). On the whole, data indicate that polymorphisms of these genes likely contribute to human longevity, in accord with observations emerging from a variety of animal models, and suggest that a common core of master genes and metabolic pathways are responsible for aging and longevity across animal species. Moreover, in the concern of our plan to discover new genetic factors related to longevity, we explored the possibility to by-pass the need of an a-priori choice of candidate genes, extending the search to genes and genomic regions of still unknown function. Alu sequences may be considered as good markers of highly variable and potentially unstable loci in functionally important genomic regions. We extensively screened Alu-rich genomic sites and found a new genomic region associated with longevity.

immune response and inflammation (IL-6, IL-1 cluster, IL-10)
insulin/IGF-I signaling pathway
oxidative stress (PON1)

immune response and inflammation (IL-6, IL-1 cluster, IL-10)
inflamm-aging: chronic inflammation (activated by antigenic/psychological stress)
immune response, stress response, and inflammation constitute an integrated, evolutionary conserved defense network, and the antigens are nothing else than particular type of stressors.

An inflammatory status is compatible with extreme longevity in good health, and pro-inflammatory characteristics have been documented in healthy centenarians. Aids in response to chronic antigenic stressors. However, excessive responsiveness due to an inflammatory genotype can become detrimental by inducing a marked pro-inflammatory status. Hypo-responsiveness to antigenic stress can also be detrimental.

beneficial effects of inflammation, devoted to the neutralization of dangerous/harmful agents early in life and in adulthood, turn to be detrimental late in life, in a period largely not foreseen by evolution, in accord with the prediction of the antagonistic pleiotropy theory of aging.

IL-6: pro-inflammatory cytokine; pleiotropic; regulates proliferation, differentiation, and activity of many cell types; pivotal role in euro-endocrine and immune system homeostasis; major role in acute phase response (balancing the pro-inflammatory/anti-inflammatory pathways; stress response). Age-related increase IL-6 identified in older individuals (starting around 30-40 years; prominent among men and centenarians). Serum level a reliable marker for functional disability, predictor of disability and mortality among elderly. Dysregulation: diabetes, atherosclerosis. (IL-6 -`74 C/G locus variability, 5' upstream). Variation leads to varied susceptibility to type 2 diabetes, cardiovascular diseases, dementia. Gender dependent (specific to males). Capability to produce low levels of IL-6 throughout lifespan (C+ individuals) appears to be beneficial for longevity, in men. "Men depend more on genetics than women to attain longevity. Female longevity depends more on environmental and lifestyle factors or that in women genetic factors become important later in life."

This decrease of C+ carrier frequency in the elderly might be the consequence of the increased risk of cardiovascular disease and AD reported in people with such a genotype.

pleiotropic antagonism: The concept of a Darwinian-evolutionary basis for the development of age-related diseases postulates that genetic traits that are beneficial in younger years to allow for successful reproduction may become deleterious in the elderly, i.e., when selective pressure does not seem to be effective anymore.

inflammatory cytokine IL-1 is a primary mediator of
systemic inflammatory responses and consists of three closely related proteins (IL-1a, IL-1b and IL-1 receptor antagonist-Ra) whose genes are located on the long arm of chromosome 2 (2q13) within a region of 430 kb.
Finnish study, 250 nonagenarians have been recruited and no statistically significant differences have been observed in the allelic frequencies for IL-1a, IL-1b and IL-1RA gene polymorph- isms in nonagenarian subjects compared to 400 younger subjects.
in a very large Italian study of 1131 subjects, including 688 young subjects, 309 elderly subjects and 134 centenarian subjects, we did not observe any significant difference in IL-1a, IL-1b and IL-1Ra allelic, genotypic and haplotypic frequencies neither among the age groups considered nor between males or females.
The Italian and the Finnish studies have investigated a large number of nonagenarians and centenarians (a total of 1780 subjects) of different genetic backgrounds and life styles and no evidence of changing in IL-1 gene cluster frequencies in the different age groups was observed, suggesting that IL-1 genetic variability does not affect longevity.

IL-10, a cytokine with anti-inflammatory and B cell
stimulating activity, is produced by activated T cells, Bcells, monocytes/macrophages and dendritic cells.

IL-10 -1082A/G polymorphism has been reported to be a male-specific marker for longevity. 190 Italian centenarians (159 women and 31 men) and 260 control subjects (99 women and 161 men) were
analysed for the three biallelic polymorphisms at -1082 (G/A), -819 (C/T) and -592 (C/A) nucleotides. An increase of subjects carrying the -1082G homozygous genotype was observed in centenarian men (p < 0.025), but not in centenarian women. No difference was found between centenarians and control subjects regarding the ?819 and
?592 polymorphisms. -1082GG genotype, associated with high IL-10
production, was argued to confer an anti-inflammatory status, thus enhancing the possibility to attain extreme longevity. IL-10 ?1082 GG
genotype; that likely confers an advantage to become a centenarian, is much less frequent in patients affected by Alzheimer’s disease (GG carriers 24%, in comparison with 40% in healthy controls). Accordingly,we can speculate that a lower propensity to produce the anti-inflammatory cytokine IL-10 could increase the risk of Alzheimer’s disease.

oxidative stress (PON1)
enzyme capable to remove lipoperoxides from oxidized lowdensity lipoproteins (LDL). Serum PON1 is a high-density lipoprotein (HDL)- bound arylesterase responsible for the protective effects of HDL on the peroxidation of LDL, by reducing their pro- atherogenic effects. PON1 has at least two amino acid polymorphisms; one located at amino acid position 192 in exon 6 is due to a glutamine (A allele) and arginine (B allele) interchange. The second at amino acid position 55 in exon 3 is due to a leucine (L allele) and methionine (M allele) interchange. These polymorphisms have been associated with the enzymatic activity and concentration of the enzyme, respectively. Analysed a sample of 579 young Italian individuals and 308 Italian centenarians and we have found that the frequency of B allele, and consequently of B+ carriers (AB + BB individuals), significantly increased from young people to centenarians, thus indicating that this allele decreases mortality in carriers. proportion of B +M+ sub- jects in centenarians was 26.3% versus 17.3% of young
subjects whilst the proportion of B +M? individuals did not change appreciably (27.5% in controls versus 27.6% in centenarians).

insulin/IGF-I signaling pathway
model systems from invertebrates to mammals (yeast, worms, fruit flies, and rodents), data indicate that mutations in genes that share similarities with the human genes involved in the insulin/IGF-I signal response pathway are capable of extended lifespan. Centenarians have decreased plasma IGF-I levels and preserved insulin action, thus indicating that insulin responsiveness impacts on human longevity.
long-lived people (aged >85 years of age) have lower IGF-I plasma levels and that these levels are affected by the polymorphisms at IGF-IR (insulin-like growth factor type 1 receptor) and phosphoinositide 3-kinase genes (PI3KCB). G to A transition at codon 1013 in exon 16 for IGF-IR and a T to C transition located 359 bp upstream from the starting codon for phosphoinosi- tide 3-kinase gene. individuals bearing at least oneA allele at the IGF-IR locus (IGF-IR A+) have lower plasma IGF-I levels. IGF- IR A+ subjects are increased in long-lived individuals and the IGF-IR/PI3KCB-A?/T? individuals have the highest free IGF-I plasma levels and the proportion of IGF-IR/ PI3KCB-A+/T+ carriers significantly increased among long- lived individuals.
IGF-IR inactivation prolongs the life span in mice and increases their resistance to oxidative stress.
older women having low serum levels of IGF-I and high serum levels of IL-6 have the highest risk of disability and mortality, in comparison with women who have lowlevels of IL-6 and high levels of IGF-1.

genetics of aging and longevity has its own peculiarities, most probably because it is a post- reproductive genetics, regarding decades of life where the force of selection progressively fades.

Franceschi, C. et al. Genes involved in immune response/inflammation, IGF1/insulin pathway and response to oxidative stress play a major role in the genetics of human longevity: the lesson of centenarians. Mech. Ageing Dev. 126, 351–361 (2005).

http://www.sciencedirect.com/science/article/pii/S0047637404002118

Genetic Determinants of Human Health Span and Life Span

July 2007

Approaches to explore the genetic basis of aging in humans are linkage mapping, and more recently, genome wide association (GWA) mapping, to identify polymorphisms affecting extreme longevity.

We review three approaches to the genetic analysis of the biology and pathobiology of human aging. The first and so far the best-developed is the search for the biochemical genetic basis of varying susceptibilities to major geriatric disorders. These include a range of progeroid syndromes. Collectively, they tell us much about the genetics of health span. Given that the major risk factor for virtually all geriatric disorders is biological aging, they may also serve as markers for the study of intrinsic biological aging. The second approach seeks to identify allelic contributions to exceptionally long life spans. While linkage to a locus on Chromosome 4 has not been confirmed, association studies have revealed a number of significant polymorphisms that impact upon late-life diseases and life span. The third approach remains theoretical. It would require longitudinal studies of large numbers of middle-aged sib-pairs who are extremely discordant or concordant for their rates of decline in various physiological functions. We can conclude that there are great opportunities for research on the genetics of human aging, particularly given the huge fund of information on human biology and pathobiology, and the rapidly developing knowledge of the human genome.

‘‘public’’ mechanism - biochemical genetic pathway, modulation of which can alter the life spans of diverse species.
‘‘r’’ selected species - rapid rates of development, high degrees of fecundity, and short life spans.
‘‘K’’ selected - long periods of development, comparatively few progeny and long life spans.
health span - the period of life during which an individual is free of chronic illness and substantial functional decrements.
long life span cannot be equated with long health span. autosomal dominantly inherited frontotemporal dementia: Affected subjects may exhibit up to a 26-year history of personality disorders, cognitive decline and, eventually, overt dementia. The underlying mutation in these particular pedigrees involves mutations at the tau locus, leading to a greatly accelerated rate of accumulation of neurofibrillary tangles, lesions that are commonly observed, in much smaller numbers, in the brains of much older individuals with wild-type versions of that gene.

classical evolutionary biological theory of aging tells us
that senescence occurs in age-structured populations because of the decline in the force of natural selection with age.

(1) ‘‘longevity assurance,’’ genes that enhance structure and function of the organism throughout the life span
(2) ‘‘antagonistic pleiotropy,’’ alleles selected because of enhanced reproductive fitness early in the life span, but with negative effects late in the life span, when those effects will have escaped the force of natural selection
(3) ‘‘mutation accumulation,’’ constitutional mutations that do not reach a level of phenotypic expression until late in the life span—once again, when they will have escaped the force of natural selection, and thus could not be purged from the population.

Werner syndrome (WS), caused by mutation at a member (WRN) of the RecQ family of helicases, exhibit accelerating rates of development of all forms of arteriosclerosis, type 2 diabetes mellitus, gonadal atrophy, skin atrophy, hair loss and hair greying, regional loss of subcutaneous tissue, osteoporosis, ocular cataracts, and neoplasia.

no convincing evidence of an acceleration in the rates of development of synaptic loss, beta amyloidosis of blood vessels or parenchyma, granulovacuolar degenerations, or neurofibrillary lesions, markers that accumulate, to varying degrees, in the aging brains of many aging human subjects, with or without a clinical or neuropathological diagnosis of Alzheimer disease. Those lesions are very abundant in that disorder, which exhibits exponential increases after the age of 65, with prevalence rates 25%–48% for persons over age 85.

complement factor H and a predicted gene on Chromosome 10q, LOC387715, modulate susceptibility to age-related macular degeneration. ~50-fold increase (95% CI: 10.8–237) in the risk of age-related macular degeneration for subjects who are homozogous for both risk alleles.

With the exception of the important risk factor of the epsilon 4 allele at the APOE locus, numerous candidate loci remain to be fully validated as contributors to the much more common late onset forms of late-life dementia.

Since the structure of our proteins and those of chimpanzees is very nearly identical, our enhanced life spans are probably related primarily to regulatory RNA species, has yet to be applied to the study of evolution of varying longevities.

Parents of centenarians (born in ~1870) were shown to have approximately nine times the odds of living to the tenth decade as compared to controls. Siblings of centenarians were shown to have up to an ~18-fold increase in the chance of achieving a similar age.

New England Centenarian Study recruited long-lived sib-ships for a genome-wide scan of linkage to exceptional longevity. A region on Chromosome 4 was implicated. By high density SNP analysis an exonic genotype in microsomal transfer protein was thought to be the locus associated with the exceptional longevity. Could not be replicated, population stratification?

CETP VV, the protection provided by certain genotypes can extend beyond a known role in a disease entity associated with its ascertainment (in this example, cardiovascular disease). The CETP VV genotype is also associated with enhanced insulin sensitivity and lower risk for hypertension, the metabolic syndrome, and diabetes [87]. Moreover, the CETP VV genotype protects against age-related cognitive decline and Alzheimer’s disease [91], although the role of particular haplotypes at that region may interact with polymorphic alleles at the APOE locus.

apoC III promoter variant. This variant is associated with significant declines in plasma levels of apoC III and a phenotype of large lipoprotein particles. apoC III is under the control of FOXO-1, a transcription factor homologous to the DAF16 gene of Caenorhabditis elegans. DAF16 is a key regulator of a downstream suite of genes that are thought to protect the organism from macromolecular damage and thus enhance life span.

uncensored pairs of Danish human twins has indicated that only about one quarter of the heritability of life span can be attributed to the constitutional genotype.

The sirtuin family of histone deacetylases represents a potential causal link between epigenetic regulation, caloric restriction, and longevity in a number of organisms, including fruit flies.

Martin, G. M., Bergman, A. & Barzilai, N. Genetic determinants of human health span and life span: progress and new opportunities. PLoS Genet. 3, 1121-1130 (2007).

http://dx.doi.org/10.1371/journal.pgen.0030125

Quantitative traits: challenges and prospects

2009

To define the genetic variants associated with lifespan in a biologically relevant context and to further establish causality, I will perform a systems genetic analysis.

A major challenge in current biology is to understand the genetic basis of variation for quantitative traits. We review the principles of quantitative trait locus mapping and summarize insights about the genetic architecture of quantitative traits that have been obtained over the past decades. We are currently in the midst of a genomic revolution, which enables us to incorporate genetic variation in transcript abundance and other intermediate molecular phenotypes into a quantitative trait locus mapping framework. This systems genetics approach enables us to understand the biology inside the 'black box' that lies between genotype and phenotype in terms of causal networks of interacting genes.

Mackay, T. F. C., Stone, E. A. & Ayroles, J. F. The genetics of quantitative traits: challenges and prospects. Nat. Rev. Genet. 10, 565–577 (2009).

http://www.nature.com/nrg/journal/v10/n8/full/nrg2612.html

Quantitative traits: mice, flies, humans

May 2009

Furthermore, the overall understanding of the genetic architecture of quantitative traits will also be advanced through the knowledge acquired from our systems genetic studies on lifespan.

We compare and contrast the genetic architecture of quantitative phenotypes in two genetically well-characterized model organisms, the laboratory mouse, Mus musculus, and the fruit fly, Drosophila melanogaster, with that found in our own species from recent successes in genome-wide association studies. We show that the current model of large numbers of loci, each of small effect, is true for all species examined, and that discrepancies can be largely explained by differences in the experimental designs used. We argue that the distribution of effect size of common variants is the same for all phenotypes regardless of species, and we discuss the importance of epistasis, pleiotropy, and gene by environment interactions. Despite substantial advances in mapping quantitative trait loci, the identification of the quantitative trait genes and ultimately the sequence variants has proved more difficult, so that our information on the molecular basis of quantitative variation remains limited. Nevertheless, available data indicate that many variants lie outside genes, presumably in regulatory regions of the genome, where they act by altering gene expression. As yet there are very few instances where homologous quantitative trait loci, or quantitative trait genes, have been identified in multiple species, but the availability of high-resolution mapping data will soon make it possible to test the degree of overlap between species.

Flint, J. & Mackay, T. F. C. Genetic architecture of quantitative traits in mice, flies, and humans. Genome Res. 19, 723–733 (2009).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647534/

Human

Werner's Syndrome

April 1996

Other genetic factors affecting aging include DNA repair and replication.

Werner's syndrome (WS) is an inherited disease with clinical symptoms resembling premature aging. Early susceptibility to a number of major age-related diseases is a key feature of this disorder. The gene responsible for WS (known as WRN) was identified by positional cloning. The predicted protein is 1432 amino acids in length and shows significant similarity to DNA helicases. Four mutations in WS patients were identified. Two of the mutations are splice-junction mutations, with the predicted result being the exclusion of exons from the final messenger RNA. One of the these mutations, which results in a frameshift and a predicted truncated protein, was found in the homozygous state in 60 percent of Japanese WS patients examined. The other two mutations are nonsense mutations. The identification of a mutated putative helicase as the gene product of the WS gene suggests that defective DNA metabolism is involved in the complex process of aging in WS patients.

Yu, C. E. et al. Positional cloning of the Werner’s syndrome gene. Science 272, 258–262 (1996).

http://science.sciencemag.org/content/272/5259/258.long

Telomere Integrity

January 1998

Other genetic factors affecting aging include telomere integrity.

Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

Bodnar, A. G. et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349–352 (1998).

http://science.sciencemag.org/content/279/5349/349.long

Age-related changes of the 3’APOB-VNTR genotype pool

March 1998

Approaches to explore the genetic basis of aging in humans are linkage mapping, and more recently, genome wide association (GWA) mapping, to identify polymorphisms affecting extreme longevity.

The analysis of seven different age cohorts (697 individuals from 10 to 109 years old) revealed age-related changes in the 3'APOB-VNTR genotype pool. By recoding the 3'APOB-VNTR alleles into three size-classes (small, S, 26-34 repeats; medium, M, 35-39 repeats; large, L, 41-55 repeats), an age-related convex trajectory of the frequency of SS homozygotes was found. The frequency of SS in the genotype pool increased from the group aged 10-19 years (3.06 +/- 1.74%) to that aged 40-49 years (8.51 +/- 4.07%). Then it declined reaching the minimum value in centenarians (1.58 +/- 0.90%). The observed trajectory is in agreement with that expected by assuming crossing of mortality curves relevant to subgroups of individuals having different genotypes.

If a certain locus affects life-expectancy, the gene pool of extremely old individuals should be different from that of younger people.

Protective genotypes will increase with age. OR Age-related changes in the physiological scenario could modify the expected trajectories because a genotype may be protective in youth, but change to being frail in older individuals. (homozygous genotypes of III class INS-VNTR alleles, protective during youth against juvenile diabetes, but frail at older age because of their association with atherosclerosis).

7 different age cohorts (697 individuals, 10-109 years old): age-related changes in the 3' APOB-VNTR genotype pool

Three size-classes: Small - 26-34 repeats, Medium - 35-39 repeats, Large 41-55 repeats. The frequency of SS in the genotype pool increased from the group aged 10-19 (~3.06) to 40-49 (~8.51), then declined, reaching a minimum value in centenarians (~1.58).

3'APOB-VNTR alleles shorter than 35 repeats are significantly less frequent in centenarians than in younger individuals. Analyzed in 7 differently aged groups. Aimed to identify changes in the genotype pool occurring as the population ages and survival selection operates. Survival selection operates on individuals, genotypes analyzed.

Central role of APOB is in cell cholesterol homeostasis (fundamental in membrane synthesis as well as in steroid hormonogenesis). There are small changes in cholesterol homeostasis that accumulate with age.

Copy number itself could affect life-expectancy.

VNTR - variable number tandem repeat

non-monotonous trajectory of 3'APOB-VNTR genotype frequency in a human ageing population, which may be explained by an age-related effect of the APOB locus on health and life-expectancy.

DeBenedictis, G., Carotenuto, L., Carrieri, G., DeLuca, M. & Falcone, E. Age-related changes of the 3’APOB-VNTR genotype pool in ageing cohorts. Ann. Hum. Genet. 62, 115–122 (1998).

http://onlinelibrary.wiley.com/doi/10.1046/j.1469-1809.1998.6220115.x/epdf

Human longevity: Cost reproductive success

December 1998

Other genetic factors affecting aging include reproduction.

The disposable soma theory on the evolution of ageing states that longevity requires investments in somatic maintenance that reduce the resources available for reproduction1,2. Experiments in Drosophila melanogaster indicate that trade-offs of this kind exist in non-human species3, 4, 5, 6, 7. We have determined the interrelationship between longevity and reproductive success in Homo sapiens using a historical data set from the British aristocracy. The number of progeny was small when women died at an early age, increased with the age of death, reaching a plateau through the sixth, seventh and eighth decades of life, but decreased again in women who died at an age of 80 years or over. Age at first childbirth was lowest in women who died early and highest for women who died at the oldest ages. When account was taken only of women who had reached menopause, who were aged 60 years and over, female longevity was negatively correlated with number of progeny and positively correlated with age at first childbirth. The findings show that human life histories involve a trade-off between longevity and reproduction.

Westendorp, R. G. & Kirkwood, T. B. Human longevity at the cost of reproductive success. Nature 396, 743–746 (1998).

http://www.nature.com/nature/journal/v396/n6713/full/396743a0.html

Exceptional longevity: locus on chromosome 4

August 2001

Approaches to explore the genetic basis of aging in humans are linkage mapping, and more recently, genome wide association (GWA) mapping, to identify polymorphisms affecting extreme longevity.

308 individuals belonging to 137 sibships demonstrating exceptional longevity. significant evidence for linkage was noted for chromosome 4 at D4S1564 with a MLS of 3.65 (P = 0.044).

The genome screen of 308 individuals belonging to 137
families identified a region on chromosome 4 between D4S1572 and D4S406 as highly suggestive of linkage (MLS = 2.67). Fine mapping at an average of 1 marker every 3 cMwas performed in a 20-cM region around this peak, resulting in an increased MLS (3.65) at marker D4S1564.

maximum heterogeneity logarithm of odds (hlod) of 3.3 under the dominant model (with 57% of families linked) and a maximum hlod of 2.9 under the recessive model (with 45% of families linked), both occurring at marker D4S1564.

A relative lack of polymorphisms predisposing to age-associated diseases appears to be one prerequisite to achieving exceptional old age. decreased frequency of the apoli- poprotein (apo)E e-4 allele among centenarians exemplifies such a mechanism.

Substantial evidence supports the familial aggregation of exceptional longevity. The existence of rare families demonstrating clustering for this phenotype suggests that a genetic etiology may be an important component. Previous attempts at localizing loci predisposing for exceptional longevity have been limited to association studies of candidate gene polymorphisms. In this study, a genome-wide scan for such predisposing loci was conducted by using 308 individuals belonging to 137 sibships demonstrating exceptional longevity. By using nonparametric analysis, significant evidence for linkage was noted for chromosome 4 at D4S1564 with a MLS of 3.65 (P = 0.044). The analysis was corroborated by a parametric analysis (P = 0.052). These linkage results indicate the likelihood that there exists a gene, or genes, that exerts a substantial influence on the ability to achieve exceptional old age. Identification of the genes in humans that allow certain individuals to live to extreme old age should lead to insights on cellular pathways that are important to the aging process.

Comparing the longevity of siblings of centenarians and siblings of a control group who were from a similar birth cohort, the siblings of the centenarians had a substantially greater chance of surviving to extreme old age compared with the siblings of the controls.

Puca, A. A. et al. A genome-wide scan for linkage to human exceptional longevity identifies a locus on chromosome 4. Proc. Natl. Acad. Sci. U.S.A. 98, 10505–10508 (2001).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC56990/

Paraoxonase (PON1) locus: increased frequency of Arg192 allele in centenarians

March 2002

Approaches to explore the genetic basis of aging in humans are linkage mapping, and more recently, genome wide association (GWA) mapping, to identify polymorphisms affecting extreme longevity.

Human Paraoxonase (PON1) is a High-Density Lipoprotein (HDL)-associated esterase that hydrolyses lipo-peroxides. PON1 has recently attracted attention as a protective factor against oxidative modification of LDL and may therefore play an important role in the prevention of the atherosclerotic process. Two polymorphisms have been extensively studied: a Leucine (L allele) to Methionine (M allele) substitution at codon 55, and a Glutamine (A allele) to Arginine (B allele) substitution at codon 192. We have examined these two aminoacidic changes in 579 people aged 20 to 65 years old, and 308 centenarians. We found that the percentage of carriers of the B allele at codon 192 (B+ individuals) is higher in centenarians than in controls (0.539 vs 0.447), moreover we found that among the B+ individuals, the phenomenon was due to an increase of people carrying M alleles at codon 55 locus. In conclusion, we propose that genetic variability at PON1 locus affects survival at extreme advanced age.

Causes of mortality in elderly (cardiovascular diseases, diabetes): associated with alterations in anti-oxidant, inflammatory and lipidic profile.

Human Paraoxonase (PON1): High-Density Lipoprotein (HDL)-associated esterase that hydrolyses lipo-peroxides. It is a possible protective factor against oxidative modification of LDL and may therefore play an important role in the prevention of the atherosclerotic process (the buildup of fats, cholesterol and other substances in and on the artery walls).

Examined 579 people aged 20-65 and 308 centenarians. The percentage of carriers of the B allele at codon 192 (B+ individuals) is higher in centenarians than in controls (Glutamine (A allele) to Arginine (B allele)). Phenomenon due to an increase of people carrying M alleles at codon 55 locus (Leucine (L allele) to Methionine (M allele)). The genetic variability at PON1 locus affects survival at extreme advanced age.

Bonafè, M. et al. Genetic analysis of Paraoxonase (PON1) locus reveals an increased frequency of Arg192 allele in centenarians. Eur. J. Hum. Genet. 10, 292–296 (2002).

http://www.nature.com/ejhg/journal/v10/n5/pdf/5200806a.pdf

Broken Limits to Life Expectancy

May 2002

Some researchers even predict that the average lifespan will be 100 years by 2065.

Best-practice life expectancy has increased by 2.5 years per decade for a century and a half.

Record life expectancy will reach 100 in about 6 decades. In 2070, female life expectancy would be between 92.5 and 101.5, considerably higher than the Social Security Administration's forecast of 83.9, published in 1999.

Experts have repeatedly asserted that life expectancy is approaching a ceiling: these experts have repeatedly been proven wrong.

The apparent leveling off of life expectancy in various countries is an artifact of laggards catching up and leaders falling behind.

If life expectancy were close to a maximum, then the increase in the record expectation of life should be slowing. It is not. For 160 years, best-performance life expectancy has steadily increased by a quarter of a year per year.

Oeppen, J. & Vaupel, J. W. Broken limits to life expectancy. Science 296, 1029–1031 (2002).

http://www.econ.ku.dk/okocg/VV/VV-Economic%20Growth/articles/artikler-2006/Broken-limits-to-life-expectancy.pdf

GWAS: T2D in Finns

June 2007

Approaches to explore the genetic basis of aging in humans are linkage mapping, and more recently, genome wide association (GWA) mapping, to identify polymorphisms affecting extreme longevity, as well as diseases and disorders associated with aging, such as diabetes.

Identifying the genetic variants that increase the risk of type 2 diabetes (T2D) in humans has been a formidable challenge. Adopting a genome-wide association strategy, we genotyped 1161 Finnish T2D cases and 1174 Finnish normal glucose-tolerant (NGT) controls with >315,000 single-nucleotide polymorphisms (SNPs) and imputed genotypes for an additional >2 million autosomal SNPs. We carried out association analysis with these SNPs to identify genetic variants that predispose to T2D, compared our T2D association results with the results of two similar studies, and genotyped 80 SNPs in an additional 1215 Finnish T2D cases and 1258 Finnish NGT controls. We identify T2D-associated variants in an intergenic region of chromosome 11p12, contribute to the identification of T2D-associated variants near the genes IGF2BP2 and CDKAL1 and the region of CDKN2A and CDKN2B, and confirm that variants near TCF7L2, SLC30A8, HHEX, FTO, PPARG, and KCNJ11 are associated with T2D risk. This brings the number of T2D loci now confidently identified to at least 10.

Genotyped 1161 Finnish T2D cases and 1174 Finnish normal glucose-tolerant (NGT) controls with >315,000 single-nucleotide polymorphisms (SNPs) and imputed genotypes for an additional >2 million autosomal SNPs. genotyped 80 SNPs in an additional 1215 Finnish T2D cases and 1258 Finnish NGT controls. T2D-associated variants in an intergenic region of chromosome 11p12, contribute to the identification of T2D-associated variants near the genes IGF2BP2 and CDKAL1 and the region of CDKN2A and CDKN2B, and confirm that variants near TCF7L2, SLC30A8, HHEX, FTO, PPARG, and KCNJ11 are associated with T2D risk. This brings the number of T2D loci now confidently identified to at least 10.

T2D is a disease characterized by insulin resistance and impaired pancreatic beta-cell function that affects >170 million people worldwide. first-degree relatives having ~3.5 times as much risk as compared to individuals in the general middle- aged population, hereditary factors, together with lifestyle and behavioral factors, play an important role in determining T2D risk.

We observed a modest excess (41 observed versus 31.6 expected; P = 0.19) of SNPs with P values < 10−4. These results argue against the existence of multiple common SNPs with a large impact on T2D disease risk but are consistent with the presence of multiple common SNPs that each confer modest risk.

Scott, L. J. et al. A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants. Science 316, 1341–1345 (2007).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3214617/?tool=pmcentrez

Chromosome 9: Coronary Heart Disease

June 2007

Approaches to explore the genetic basis of aging in humans are linkage mapping, and more recently, genome wide association (GWA) mapping, to identify polymorphisms affecting extreme longevity, as well as diseases and disorders associated with aging, such as coronary heart disease.

Coronary heart disease (CHD) is a major cause of death in Western countries. We used genome-wide association scanning to identify a 58-kilobase interval on chromosome 9p21 that was consistently associated with CHD in six independent samples (more than 23,000 participants) from four Caucasian populations. This interval, which is located near the CDKN2A and CDKN2B genes, contains no annotated genes and is not associated with established CHD risk factors such as plasma lipoproteins, hypertension, or diabetes. Homozygotes for the risk allele make up 20 to 25% of Caucasians and have a ~30 to 40% increased risk of CHD.

McPherson, R. et al. A common allele on chromosome 9 associated with coronary heart disease. Science 316, 1488–1491 (2007).

http://science.sciencemag.org/content/316/5830/1488.full

GWAS: 14,000 cases, 7 common diseases

June 2007

Approaches to explore the genetic basis of aging in humans are linkage mapping, and more recently, genome wide association (GWA) mapping, to identify polymorphisms affecting extreme longevity, as well as diseases and disorders associated with aging (others).

There is increasing evidence that genome-wide association (GWA) studies represent a powerful approach to the identification of genes involved in common human diseases. We describe a joint GWA study (using the Affymetrix GeneChip 500K Mapping Array Set) undertaken in the British population, which has examined approximately 2,000 individuals for each of 7 major diseases and a shared set of approximately 3,000 controls. Case-control comparisons identified 24 independent association signals at P < 5 x 10(-7): 1 in bipolar disorder, 1 in coronary artery disease, 9 in Crohn's disease, 3 in rheumatoid arthritis, 7 in type 1 diabetes and 3 in type 2 diabetes. On the basis of prior findings and replication studies thus-far completed, almost all of these signals reflect genuine susceptibility effects. We observed association at many previously identified loci, and found compelling evidence that some loci confer risk for more than one of the diseases studied. Across all diseases, we identified a large number of further signals (including 58 loci with single-point P values between 10(-5) and 5 x 10(-7)) likely to yield additional susceptibility loci. The importance of appropriately large samples was confirmed by the modest effect sizes observed at most loci identified. This study thus represents a thorough validation of the GWA approach. It has also demonstrated that careful use of a shared control group represents a safe and effective approach to GWA analyses of multiple disease phenotypes; has generated a genome-wide genotype database for future studies of common diseases in the British population; and shown that, provided individuals with non-European ancestry are excluded, the extent of population stratification in the British population is generally modest. Our findings offer new avenues for exploring the pathophysiology of these important disorders. We anticipate that our data, results and software, which will be widely available to other investigators, will provide a powerful resource for human genetics research.

Wellcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447, 661–678 (2007).

http://www.nature.com/nature/journal/v447/n7145/full/nature05911.html

GWA analysis: loci T2D triglyceride levels

June 2007

Approaches to explore the genetic basis of aging in humans are linkage mapping, and more recently, genome wide association (GWA) mapping, to identify polymorphisms affecting diseases and disorders associated with aging, such as diabetes.

386,731 common SNPs in 1464 nations with T2D, 1467 controls (Finland and Sweden; population-based (1022 T2D, 1075 controls): matched on gender, age, BMI, and region of origin and family-based (326 sib-ships discordant for T2D; 442 T2D and 392 controls) characterized for glucose metabolism, lipids, obesity, and blood pressure. Identified 3 loci associated with T2D - noncoding region near CDKN2A and CDKN2B, intron of IGF2BP2, and intron of CDKAL1. Replicated associations near HHEX and SLC30A8. Identified and confirmed SNP in intron of glucokinase regulatory protein (GCKR) with serum triglycerides.

GWAS based-off of Fisher's theory for additive effects at common alleles; human heterozygosity being substantially attributable to common ancestral variants; hypothesis that variants influencing common, late-onset diseases of modernity may not have been subject to purifying selection, and has been made possible by genomic advances such as the human genome sequence, SNP and HapMap databases, and genotyping arrays.

CDKN2A: cyclin-dependent kinase inhibitor-2A, pancreatic islet regenerative capacity

IGF2BP2: insulin-like growth factor 2 binding protein 2, affinity for leader elements in the untranslated regions of IGF-2 transcripts

CDKAL1: homologous to CDK5RAP1, inhibitor of cyclin-dependent kinase CDK5 (transduces glucotoxicity signals in pancreatic beta cells)

TCF7L2: single largest effect of a common SNP on T2D risk in European populations. (Chromosome 10)

GCKR - regulates glucokinase (GCK), the first glycolytic enzyme

The modest effect of each SNP demonstrates that large sample sizes will be required to discover and validate genetic risk factors for common disease.

Diabetes Genetic Initiative. Genome-wide association analysis identifies loci type 2 diabetes triglyceride levels. Science 316 SRC, 1331–1336 (2007).

http://science.sciencemag.org/content/316/5829/1331.full

GWAS: KIAA0350 as a T1D gene

July 2007

Approaches to explore the genetic basis of aging in humans are linkage mapping, and more recently, genome wide association (GWA) mapping, to identify polymorphisms affecting diseases and disorders associated with aging, such as diabetes.

Type 1 diabetes (T1D) in children results from autoimmune destruction of pancreatic beta cells, leading to insufficient production of insulin. A number of genetic determinants of T1D have already been established through candidate gene studies, primarily within the major histocompatibility complex but also within other loci. To identify new genetic factors that increase the risk of T1D, we performed a genome-wide association study in a large paediatric cohort of European descent. In addition to confirming previously identified loci, we found that T1D was significantly associated with variation within a 233-kb linkage disequilibrium block on chromosome 16p13. This region contains KIAA0350, the gene product of which is predicted to be a sugar-binding, C-type lectin. Three common non-coding variants of the gene (rs2903692, rs725613 and rs17673553) in strong linkage disequilibrium reached genome-wide significance for association with T1D. A subsequent transmission disequilibrium test replication study in an independent cohort confirmed the association. These results indicate that KIAA0350 might be involved in the pathogenesis of T1D and demonstrate the utility of the genome-wide association approach in the identification of previously unsuspected genetic determinants of complex traits.

Type 1 diabetes (T1D) in children results from autoimmune destruction of pancreatic beta cells, leading to insufficient production of insulin.

European decent: T1D was significantly associated with variation within a 233-kb linkage disequilibrium block on chromosome 16p13. This region contains KIAA0350, the gene product of which is predicted to be a sugar-binding, C-type lectin. Three common non-coding variants of the gene (rs2903692, rs725613 and rs17673553) in strong linkage dis- equilibrium reached genome-wide significance for association with T1D.

first-degree relatives of patients with T1D being at 15-fold greater risk for developing the condition than the general population.

Variation in four loci already established to account for a significant proportion of the familial clustering of T1D: major histocompatibility complex (MHC) region on chromosome 6p21 (mostly residing in the HLA-DRB1, -DQA1 and -DQB1 genes), the insulin locus (INS) on chromosome 11p15, the protein tyrosine phosphatase-22 (PTPN22) gene on chromosome 1p13 and the gene that encodes the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) on chromosome 2q31. The interleukin-2 receptor alpha (CD25 IL2RA) locus on chromosome 10p15 has also been implicated, and a report that T1D is associated with a non-synonymous variant in the innate immunity gene IFIH1 remains to be independently replicated.

Stage 1: genotyped 550,000 SNPs with the Illumina Human Hap550 Genotyping BeadChip22, from 563 patients with T1D and 1,146 controls of European ancestry (based on self-report) plus 483 complete T1D family trios of the same ancestry. All patients had clinically proven T1D and were using insulin. 534,071 SNPs remained in the analysis.

Three common non-coding variants (rs2903692 allele A, rs725613 allele C and rs17673553 allele G), in strong linkage disequilibrium (LD) in the KIAA0350 gene on chromosome 16p13.13.

KIAA0350 encodes a protein of unknown function and its genomic location is next to the suppressor of cytokine signalling 1 (SOCS1) gene. The almost exclusive expression specificity of KIAA0350 in immune cells, including dendritic cells, B lymphocytes and natural killer (NK) cells, all of which are pivotal in the pathogenesis of T1D, indicates that the variant probably contributes to the disease by modulating immunity. The predicted protein product of KIAA0350 bears similarities to a subset of adhesion and immune function signaling molecules. This gene probably encodes a protein with a calcium-dependent, or C-type, lectin-binding domain structure. Proteins of this type are known to be involved with calcium current flux, and the predicted function of the protein encoded by KIAA0350 includes sugar binding. C-type lectins are known for their recognition of various carbohydrates and are crucial for processes that range from cell adhesion to pathogen recognition.

Hakonarson, H. et al. A genome-wide association study identifies KIAA0350 as a type 1 diabetes gene. Nature 448, 591–594 (2007).

http://www.nature.com/nature/journal/v448/n7153/full/nature06010.html

GWA T1D

July 2007

Approaches to explore the genetic basis of aging in humans are linkage mapping, and more recently, genome wide association (GWA) mapping, to identify polymorphisms affecting extreme longevity, as well as diseases and disorders associated with aging, such as diabetes.

The Wellcome Trust Case Control Consortium (WTCCC) primary genome-wide association (GWA) scan on seven diseases, including the multifactorial autoimmune disease type 1 diabetes (T1D), shows associations at P < 5 x 10(-7) between T1D and six chromosome regions: 12q24, 12q13, 16p13, 18p11, 12p13 and 4q27. Here, we attempted to validate these and six other top findings in 4,000 individuals with T1D, 5,000 controls and 2,997 family trios independent of the WTCCC study. We confirmed unequivocally the associations of 12q24, 12q13, 16p13 and 18p11 (P(follow-up) <or= 1.35 x 10(-9); P(overall) <or= 1.15 x 10(-14)), leaving eight regions with small effects or false-positive associations. We also obtained evidence for chromosome 18q22 (P(overall) = 1.38 x 10(-8)) from a GWA study of nonsynonymous SNPs. Several regions, including 18q22 and 18p11, showed association with autoimmune thyroid disease. This study increases the number of T1D loci with compelling evidence from six to at least ten.

Follow-up analysis of the SNPs showing associations with T1D in the WTCCC GWAS.

The Wellcome Trust Case Control Consortium (WTCCC) primary genome-wide association (GWA) scan on seven diseases, including the multifactorial autoimmune disease T1D, shows associations between T1D and six chromosome regions: 12q24, 12q13, 16p13, 18p11, 12p13 and 4q27. Here, we attempted to validate these and six other top findings in 4,000 individuals with T1D, 5,000 controls and 2,997 family trios independent of the WTCCC study. We confirmed unequivocally the associations of 12q24,
12q13, 16p13 and 18p11, leaving eight regions with small effects or false-positive associations. We also obtained evidence for chromosome 18q22 from a GWA study of nonsynonymous SNPs. Several regions, including 18q22 and 18p11, showed association with autoimmune thyroid disease. This study increases the number of T1D loci with compelling evidence from six to at least ten.

The first, discovered and localized over 20 years ago and having by far the largest effect, are the HLA class II genes on chromosome 6p21 in the major histocompatibility complex (MHC). Other loci are the gene encoding insulin (INS) on 11p15, CTLA4 on 2q33, PTPN22 on 1p13, the interleukin-2 receptor a chain (IL2RA, also known as CD25) region on 10p15 and, most recently, the IFIH1 (also known as MDA5) region on 2q24.

Todd, J. A. et al. Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes. Nat. Genet. 39, 857–864 (2007).

http://www.nature.com/ng/journal/v39/n7/full/ng2068.html

An aging nation: The older population in the United States

Approx. May 2014

In 1900, the human life expectancy was approximately 47 years, and by 2012, it had increased to 78 years. This number is predicted to increase at a steady rate and is likely to reach 83 years by 2050.

Considerable growth in older population. In 2050, the population aged 65+ projected to be 83.7 million, almost double its estimated population of 43.1 million in 2012. (Baby boomers)

Challenges: Social Security, Medicare

U.S. population projected to grow from 314 million (2012) to 400 million (2050) (27% increase)

Ortman, J. M., Velkoff, V. A. & Hogan, H. An aging nation: The older population in the United States. Current Population Reports 1964, (2014).

https://www.census.gov/prod/2014pubs/p25-1140.pdf

M. musculus

O6-methylguanine acceptor protein (Rat)

December 1985

Other genetic factors affecting aging include DNA repair and replication.

N-nitroso compounds react with cellular DNA to produce various damaging adducts, one of the more important being O6-alkylguanine. DNA restoration is accomplished by transfer of the alkyl group to a cysteine residue of an acceptor protein. The levels of acceptor activity were compared in several tissues from well-fed and dietary-restricted inbred SD rats 30-1,194 days of age. Striking and consistent differences were found in the levels of acceptor activity in different tissues from both groups; these levels corresponded to their sensitivity to tumorigenesis by alkylating agents. Acceptor activity levels were highest in the liver and somewhat less in the spleen; there were significantly lower levels in brain and kidney. The random loss with time in the integrity of DNA may cause alterations in cellular function or limit cellular proliferation, thus leading to senescence and death. DNA repair processes may alter the rate of accumulation of damage, thereby affecting potential longevity. There were no significant age-associated changes in the ability of cells from either dietary group to remove DNA adducts and there was no evidence of alterations in the acceptor protein with age that would compromise its functional activity.

Woodhead, A. D. et al. Levels of O6-methylguanine acceptor protein in tissues of rats and their relationship to carcinogenicity and aging. J. Natl. Cancer Inst. 75, 1141–1145 (1985).

http://www.ncbi.nlm.nih.gov/pubmed/3865014

Dwarf mice

November 1996

One well-characterized genetic mechanism affecting aging and lifespan is the insulin-like signaling pathway, which is involved in the regulation of metabolism in M. musculus.

Brown-Borg, H. M., Borg, K. E., Meliska, C. J. & Bartke, A. Dwarf mice and the ageing process. Nature 384, 33 (1996).

http://www.nature.com/nature/journal/v384/n6604/abs/384033a0.html

Motor neuron disease

1998

Other genetic factors affecting aging include detoxification of reactive oxygen species.

Significant progress has been made in the identification of genes and chromosomal loci associated with several types of motor neuron disease. Of particular interest is recent work on the pathogenic mechanisms underlying these diseases, especially studies in in vitro model systems and in transgenic and gene-targeted mice.

Wong, P. C., Rothstein, J. D. & Price, D. L. The genetic and molecular mechanisms of motor neuron disease. Curr. Opin. Neurobiol. 8, 791–799 (1998).

http://www.sciencedirect.com/science/article/pii/S0959438898801232

Defects in growth hormone production

June 2001

One well-characterized genetic mechanism affecting aging and lifespan is the insulin-like signaling pathway, which is involved in the regulation of metabolism in M. musculus.

Single-gene mutations that extend lifespan provide valuable tools for the exploration of the molecular basis for age-related changes in cell and tissue function and for the pathophysiology of age-dependent diseases. We show here that mice homozygous for loss-of-function mutations at the Pit1 (Snell dwarf) locus show a >40% increase in mean and maximal longevity on the relatively long-lived (C3H/HeJ x DW/J)F1 background. Mutant dwJ/dw animals show delays in age-dependent collagen cross-linking and in six age-sensitive indices of immune system status. These findings thus demonstrate that a single gene can control maximum lifespan and the timing of both cellular and extracellular senescence in a mammal. Pituitary transplantation into dwarf mice does not reverse the lifespan effect, suggesting that the effect is not due to lowered prolactin levels. In contrast, homozygosity for the Ghrhrlit mutation, which like the Pit1dw mutation lowers plasma growth hormone levels, does lead to a significant increase in longevity. Male Snell dwarf mice, unlike calorically restricted mice, become obese and exhibit proportionately high leptin levels in old age, showing that their exceptional longevity is not simply due to alterations in adiposity per se. Further studies of the Pit1dw mutant, and the closely related, long-lived Prop-1df (Ames dwarf) mutant, should provide new insights into the hormonal regulation of senescence, longevity, and late life disease.

Flurkey, K., Papaconstantinou, J., Miller, R. A. & Harrison, D. E. Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production. Proc. Natl. Acad. Sci. U.S.A. 98, 6736–6741 (2001).

http://www.pnas.org/content/98/12/6736.long

Deficient in DNA repair and transcription

May 2002

Other genetic factors affecting aging include DNA repair and replication.

De Boer, J. et al. Premature aging in mice deficient in DNA repair and transcription. Science 296, 1276–1279 (2002).

http://science.sciencemag.org/content/296/5571/1276.long

IGF-1

January 2003

One well-characterized genetic mechanism affecting aging and lifespan is the insulin-like signaling pathway, which is involved in the regulation of metabolism in M. musculus.

Studies in invertebrates have led to the identification of a number of genes that regulate lifespan, some of which encode components of the insulin or insulin-like signalling pathways. Examples include the related tyrosine kinase receptors InR (Drosophila melanogaster) and DAF-2 (Caenorhabditis elegans) that are homologues of the mammalian insulin-like growth factor type 1 receptor (IGF-1R). To investigate whether IGF-1R also controls longevity in mammals, we inactivated the IGF-1R gene in mice (Igf1r). Here, using heterozygous knockout mice because null mutants are not viable, we report that Igf1r1/2 mice live on average 26% longer than their wild-type littermates (P < 0.02). Female Igf1r1/2mice live 33% longer than wild-type females (P < 0.001), whereas the equivalent male mice show an increase in lifespan of 16%, which is not statistically significant. Long-lived Igf1r1/2 mice do not develop dwarfism, their energy metabolism is normal, and their nutrient uptake, physical activity, fertility and reproduction are unaffected. The Igf1r1/2 mice display greater resistance to oxidative stress, a known determinant of ageing. These results indicate that the IGF-1 receptor may be a central regulator of mammalian lifespan.

Holzenberger, M. et al. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature 421, 182–187 (2003).

http://www.nature.com/nature/journal/v421/n6919/full/nature01298.html

Lacking insulin receptor

January 2003

One well-characterized genetic mechanism affecting aging and lifespan is the insulin-like signaling pathway, which is involved in the regulation of metabolism in M. musculus.

Caloric restriction has been shown to increase longevity in organisms ranging from yeast to mammals. In some organisms, this has been associated with a decreased fat mass and alterations in insulin/insulin-like growth factor 1 (IGF-1) pathways. To further explore these associations with enhanced longevity, we studied mice with a fat-specific insulin receptor knockout (FIRKO). These animals have reduced fat mass and are protected against age-related obesity and its subsequent metabolic abnormalities, although their food intake is normal. Both male and female FIRKO mice were found to have an increase in mean life-span of ?134 days (18%), with parallel increases in median and maximum life-spans. Thus, a reduction of fat mass without caloric restriction can be associated with increased longevity in mice, possibly through effects on insulin signaling.

Blüher, M., Kahn, B. B. & Kahn, C. R. Extended longevity in mice lacking the insulin receptor in adipose tissue. Science 299, 572–574 (2003).

http://science.sciencemag.org/content/299/5606/572.full

D. melanogaster

Sexual reproduction in males

1981

Other genetic factors affecting aging include reproduction.

Many theories on the evolution of life histories have assumed a physiological cost of reproduction in terms of reduced lifespan1–3. A cost of increased reproduction in terms of reduced longevity has been established experimentally for females, both as an additive genetic4,5 and as a purely phenotypic6,7 effect. Such a physiological cost of reproduction has not been demonstrated for males. The cost of sexual activity has been assumed to be relatively small in those species where the only paternal contribution to an offspring is the gamete8,9. Here we show that increasing sexual activity reduces longevity in the male fruitfly (Drosophila melanogaster) and hence that there is a significant physiological cost of male sexual activity in a species where the father contributes only gametes to his progeny.

Partridge, L. & Farquhar, M. Sexual activity reduces longevity of male fruitflies. Nature 294 SRC, 580–582 (1981).

http://www.nature.com/nature/journal/v294/n5841/abs/294580a0.html

Environmental stress: Postponed senescence

July 1985

Other genetic factors affecting aging include caloric restriction.

Adult Drosophila melanogaster from populations that exhibit postponed senescence were compared to flies from control populations. Male and female flies from populations displaying postponed senescence were significantly more resistant than control flies to desiccation, starvation, and the vapor of a 15% ethanol solution. In the presence of desiccant or when humidity was not controlled, flies from postponed- senescence populations were more resistant than controls to heat stress in the range 37-39 C. Differences in heat tolerance disappeared when experiments were conducted at high humidity. Tolerance to starvation increased with age for females but remained approximately constant for males. Tolerance to desiccation decreased with age for both sexes. Differences between control and postponed-senescence populations with respect to starvation resistance and desiccation resistance were obtained at all ages examined. Females from postponed-senescence populations had lower proportional water content than did females from control populations. Differences in water content cannot explain the greater desiccation resistance of flies from postponed- senescence populations. Genetically increased life span in these D. melanogaster populations appears to result proximally from greater resistance to mortality arising from environmental stress.

Service, P. M., Hutchinson, E. W., MacKinley, M. D. & Rose, M. R. Resistance to environmental stress in Drosophila melanogaster selected for postponed senescence. Physiol. Zool. 58, 380–389 (1985).

http://www.jstor.org/stable/30156013?seq=1#page_scan_tab_contents

Evolution of senescence

1988

Other genetic factors affecting aging include caloric restriction.

We present the results of selection experiments designed to distinguish between antagonistic pleiotropy and mutation accumulation, two mechanisms for the evolution of senescence. Reverse selection for early-life fitness was applied to laboratory populations of Drosophila melanogaster that had been previously selected for late-life fitness. These populations also exhibited reduced early-age female fecundity and increased resistance to the stresses of starvation, desiccation, and ethanol, when compared to control populations. Reverse selection was carried out at both uncontrolled, higher larval rearing density and at controlled, lower larval density. In the uncontrolled-density
selection lines, early-age female fecundity increased to control-population levels in response to the reintroduction of selection for early-age fitness. Concomitantly, resistance to starvation declined in agreement with previous observations of a negative genetic correlation between these two characters and in accordance with the antagonistic-pleiotropy mechanism. However, resistance to stresses of desiccation and ethanol did not decline in the uncontrolled-density lines during 22 generations of reverse selection for early-life fitness. The latter results provide evidence that mutation accumulation has also played a role in the evolution of senescence in this set of Drosophila populations. No significant response in early-age fecundity or starvation resistance was observed in the controlled-density reverse-selection lines, supporting previous observations that selection on Drosophila life-history characters is critically sensitive to larval rearing density.

Service, P. M., Hutchinson, E. W. & Rose, M. R. Multiple genetic mechanisms for the evolution of senescence in Drosophila melanogaster. Evolution (N.Y). 42 SRC, 708–716 (1988).

http://www.jstor.org/stable/2408862?seq=1#page_scan_tab_contents

Postponed Senescence

1991

Other genetic factors affecting aging include resistance to heat, starvation, and other environmental stressors.

Drosophila melanogaster selected for postponed aging have increased resistance to desiccation and increased flight duration. These and other populations were tested for longevity, flight duration, glycogen content, and stress resistance under a variety of conditions. It was found that desiccation resistance waspositively as- sociated with flight duration and glycogen content, while glycogen was ex- hausted in the course of desiccation, as it is in flight Flies with postponed aging also have lower water-loss rates (WLRs) when dead, suggesting that factors other than glycogen content are partly responsible for their increased desiccation resis- tance. However, total epicuticular hydrocarbon does not appear to determine desiccation resistance. Starvation resistance does not vary in a manner that cor- responds with desiccation resistance, under either selection or manipulation, but does vary in association with lipid level This suggests at least two physiological mechanisms by which aging has been postponed in these flies: increased lipid content and increased glycogen content. These mechanisms are at least some- what independent, evolutionarily, genetically, and physiologically.

Graves, J. L., Toolson, E. C., Jeong, C., Vu, N. & Rose, M. R. Desiccation, flight, glycogen, and postponed senescence in Drosophila melanogaster. Physiol. Zool. 65 SRC, 268–286 (1992).

http://www.jstor.org/stable/30158253?seq=1#page_scan_tab_contents

Heat shock proteins

May 1991

Other genetic factors affecting aging include resistance to heat, starvation, and other environmental stressors.

We examined the effect of cellular aging on adult mortality and hsp70 gene expression in Drosophila melanogaster under thermal stress. The results showed that flies exposed to 37 degrees C for various time intervals had reduced survival rate with age. The level of hsp70 mRNA increases in flies up to 23-28 days of age, but then declines as they get older. When flies are shifted to 25 degrees C after 30 min of heat stress, the time-dependent decrease in hsp70 mRNA levels occurs more rapidly in young flies than in old ones. The hsp70 mRNA present during this recovery period is translated into protein, and senescent flies continue to synthesize this protein for up to 5 h after heat shock. The prolonged expression of hsp70 RNA during recovery from heat shock was also observed in young flies fed canavanine, an arginine analogue. These data suggest that in old insects, the accumulation of conformationally altered proteins plays a role in the regulation of hsp70 RNA expression. These results are discussed in relation to the finding that old flies are more sensitive to thermal stress than young ones.

Niedzwiecki, A., Kongpachith, A. M. & Fleming, J. E. Aging affects expression of 70-kDa heat shock proteins in Drosophila. J. Biol. Chem. 266, 9332–9338 (1991).

http://www.jbc.org/content/266/14/9332.long

RNAi

1993

I will use the binary GAL4-UAS expression system to drive expression of RNAi and over- expression constructs.

We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.

Brand, A. H. & Perrimon, N. Targeted gene expresion as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415 (1993).

http://dev.biologists.org/content/118/2/401.long

Acatalasemic mutants

July 1993

Other genetic factors affecting aging include detoxification of reactive oxygen species.

The enzyme catalase protects aerobic organisms from oxygen-free radical damage by converting hydrogen peroxide to molecular oxygen and water before it can decompose to form the highly reactive hydroxyl radical. Hydroxyl radicals are the most deleterious of the activated oxygen inter- mediates found in aerobic organisms. If formed, they can react with biological molecules in their proximity; the ensuing damage has been implicated in the increasing risk of disease and death associated with aging. To study further the regulation and role of catalase we have undertaken a molecular characterization of the Drosophila catalase gene and two potentially acatalasemic alleles. We have demonstrated that a previously existing allele, Catn4, likely contains a null mutation, a mutation which blocks normal translation of the encoded mRNA. The Cat”’ mutation appears to cause a significant change in the protein sequence; however, it is unclear why this change leads to a nonfunctioning protein. Viability of these acatalasemic flies can be restored by transformation with the wild-type catalase gene; hence, we conclude that the lethality of these genotypes is due solely to the lack of catalase. The availability of flies with transformed catalase genes has allowed us to address the effect of catalase levels on aging in Drosophila. Though lack of catalase activity caused decreased viability and life span, increasing catalase activity above wild-type levels had no effect on normal life span

Griswold, C. M., Matthews, A. L., Bewley, K. E. & Mahaffey, J. W. Molecular characterization and rescue of acatalasemic mutants of Drosophila melanogaster. Genetics 134, 781–788 (1993).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1205515/

Cost of mating in females

January 1995

Other genetic factors affecting aging include reproduction.

FEMALE Drosophila melanogaster with environmentally1–3 or genetically4 elevated rates of mating die younger than controls. This cost of mating is not attributable to receipt of sperm5. We demonstrate here that seminal fluid products from the main cells of the male accessory gland are responsible for the cost of mating in females, and that increasing exposure to these products increases female death rate. Main-cell products are also involved in elevating the rate of female egg-laying, in reducing female receptivity to further matings and in removing or destroying sperm of previous mates6–12. The cost of mating to females may therefore represent a side-effect of evolutionary conflict between males.

Chapman, T., Liddle, L. F., Kalb, J. M., Wolfner, M. F. & Partridge, L. Cost of mating in Drosophila melanogaster females is mediated by male accessory gland products. Nature 373, 241–244 (1995).

http://www.nature.com/nature/journal/v373/n6511/abs/373241a0.html

Biochemical and Stress Analysis

1995

Other genetic factors affecting aging include resistance to heat, starvation, and other environmental stressors.

We have performed a comparative analysis of the effects of age of reproduction on the biochemical (protein, lipid, and glycogen content) and stress resistance (ability to survive starvation, desiccation, and exogenous paraquat) parameters on 10 sister lines of five different Drosophila strains. Four pairs of these sister lines were selected under different regimens for either early or delayed reproduction; the fifth pair was maintained in a nonselected state and served as the baseline strain to which all others were compared. It is generally accepted that the early regimens give rise to short-lived phenotypes, whereas the delayed regimens give rise to long-lived phenotypes. Our results suggest that a mechanism involving lipid and starvation resistance is not operative in our long-lived strains. In addition, a mechanism involving glycogen content and desiccation resistance is only weakly supported. Finally, there is strong support for a mechanism that gives rise to enhanced paraquat resistance and therefore may involve regulatory changes in the pattern of ADS gene expression. In addition, the 15-day early age of reproduction regimen (M type) shows qualitatively similar responses to that of the late age at reproduction regimen (L type). These results suggest that correlations between biochemical traits and longevity must be interpreted with caution. We discuss possible reasons for these results, including the possibility of multiple mechanisms, each leading to a different extended longevity phenotype.

Force, A. G., Staples, T., Soliman, S. & Arking, R. Comparative biochemical and stress analysis of genetically selected Drosophila strains with different longevities. Dev. Genet. 17, 340–351 (1995).

http://www.ncbi.nlm.nih.gov/pubmed/8641052

Quantitative Genetics (Book)

1996

Furthermore, inbreeding has increased the genetic variance for quantitative traits to at least double that of the outbred population from which it was derived, which also increases power for GWA mapping.

Falconer, D. S. & Mackay, T. F. C. Introduction to Quantitative Genetics (4th Edition). Trends in Genetics 12, (1996).

Heat-induced

1997

Other genetic factors affecting aging include resistance to heat, starvation, and other environmental stressors.

Survival data were collected on a total of 28,000 Drosophila melanogaster adults in order to investigate mortality patterns and induced physiological responses after a mild thermal stress. A brief, nonlethal heat treatment extends adult life span at normal temperatures by an average of 2 days (64), compared to nontreated controls of the same genotypes. Life expectancy is extended as a demographic consequence of reduced age-specific mortality over a period of up to several weeks after the heat treatment. Heat treatment also increases tolerance to subsequent, more severe thermal stress. Observations on single-sex populations suggest that heat-induced longevity extension is independent of the suppression of reproductive activity.

Khazaeli, A. A., Tatar, M., Pletcher, S. D. & Curtsinger, J. W. Heat-induced longevity extension in Drosophila. I. Heat treatment, mortality, and thermotolerance. J. Gerontol. A. Biol. Sci. Med. Sci. 52, B48–52 (1997).

http://biomedgerontology.oxfordjournals.org/content/52A/1/B48.long

Sex-specific QTLs

September 1997

Quantitative trait loci (QTLs) associated with lifespan in D. melanogaster have been mapped in linkage mapping populations.

Senescence, the decline in survivorship and fertility with increasing age, is a near-universal property of organisms. Senescence and limited lifespan are thought to arise because weak natural selection late in life allows the accumulation of mutations with deleterious late-age effects that are either neutral (the mutation accumulation hypothesis) or beneficial (the antagonistic pleiotropy hypothesis) early in life. Analyses of Drosophila spontaneous mutations, patterns of segregating variation and covariation, and lines selected for late-age fertility have implicated both classes of mutation in the evolution of aging, but neither their relative contributions nor the properties of individual loci that cause aging in nature are known. To begin to dissect the multiple genetic causes of quantitative variation in lifespan, we have conducted a genome-wide screen for quantitative trait loci (QTLs) affecting lifespan that segregate among a panel of recombinant inbred lines using a dense molecular marker map. Five autosomal QTLs were mapped by composite interval mapping and by sequential multiple marker analysis. The QTLs had large sex-specific effects on lifespan and age-specific effects on survivorship and mortality and mapped to the same regions as candidate genes with fertility, cellular aging, stress resistance and male-specific effects. Late age-of-onset QTL effects are consistent with the mutation accumulation hypothesis for the evolution of senescence, and sex-specific QTL effects suggest a novel mechanism for maintaining genetic variation for lifespan.

Nuzhdin, S. V, Pasyukova, E. G., Dilda, C. L., Zeng, Z. B. & Mackay, T. F. Sex-specific quantitative trait loci affecting longevity in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 94, 9734–9739 (1997).

http://www.pnas.org/content/94/18/9734.full

Overexpression: SOD1

June 1998

Other genetic factors affecting aging include detoxification of reactive oxygen species.

Parkes, T. L. et al. Extension of Drosophila lifespan by overexpression of human SOD1 in motorneurons. Nat. Genet. 19, 171–174 (1998).

http://www.nature.com/ng/journal/v19/n2/full/ng0698_171.html

Genotype-environment interaction

January 2000

Quantitative trait loci (QTLs) associated with lifespan in D. melanogaster have been mapped in linkage mapping populations.

The nature of genetic variation for Drosophila longevity in a population of recombinant inbred lines was investigated by estimating quantitative genetic parameters and mapping quantitative trait loci (QTL) for adult life span in five environments: standard culture conditions, high and low temperature, and heat-shock and starvation stress. There was highly significant genetic variation for life span within each sex and environment. In the analysis of variance of life span pooled over sexes and environments, however, the significant genetic variation appeared in the genotype x sex and genotype x environment interaction terms. The genetic correlation of longevity across the sexes and environments was not significantly different from zero in these lines. We estimated map positions and effects of QTL affecting life span by linkage to highly polymorphic roo transposable element markers, using a multiple-trait composite interval mapping procedure. A minimum of 17 QTL were detected; all were sex and/or environment-specific. Ten of the QTL had sexually antagonistic or antagonistic pleiotropic effects in different environments. These data provide support for the pleiotropy theory of senescence and the hypothesis that variation for longevity might be maintained by opposing selection pressures in males and females and variable environments. Further work is necessary to assess the generality of these results, using different strains, to determine heterozygous effects and to map the life span QTL to the level of genetic loci.

Vieira, C. et al. Genotype-environment interaction for quantitative trait loci affecting life span in Drosophila melanogaster. Genetics 154, 213–227 (2000).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1460900/

QTL

August 2000

Quantitative trait loci (QTLs) associated with lifespan in D. melanogaster have been mapped in linkage mapping populations.

The genetic architecture of variation in adult life span was examined for a population of recombinant inbred lines, each of which had been crossed to both inbred parental strains from which the lines were derived, after emergence from both high and low larval density. QTL affecting life span were mapped within each sex and larval density treatment by linkage to highly polymorphic roo-transposable element markers, using a composite interval mapping method. We detected a total of six QTL affecting life span; the additive effects and degrees of dominance for all were highly sex- and larval environment-specific. There were significant epistatic interactions between five of the life span QTL, the effects of which also differed according to genetic background, sex, and larval density. Five additional QTL were identified that contributed to differences among lines in their sensitivity to variation in larval density. Further fine-scale mapping is necessary to determine whether candidate genes within the regions to which the QTL map are actually responsible for the observed variation in life span.

Leips, J. & Mackay, T. F. C. Quantitative trait loci for life span in Drosophila melanogaster: Interactions with genetic background and larval density. Genetics 155, 1773–1788 (2000).

http://www.genetics.org/content/155/4/1773

Deficiency mapping

November 2000

Although the linkage studies lacked the power to resolve QTLs to individual genes and the association studies were at the level of candidate genes implicated by deficiency mapping,

In a previous study, sex-specific quantitative trait loci (QTL) affecting adult longevity were mapped by linkage to polymorphic roo transposable element markers, in a population of recombinant inbred lines derived from the Oregon and 2b strains of Drosophila melanogaster. Two life span QTL were each located on chromosomes 2 and 3, within sections 33E–46C and 65D–85F on the cytological map, respectively. We used quantitative deficiency complementation mapping to further resolve the locations of life span QTL within these regions. The Oregon and 2b strains were each crossed to 47 deficiencies spanning cytological regions 32F–44E and 64C–76B, and quantitative failure of the QTL alleles to complement the deficiencies was assessed. We initially detected a minimum of five and four QTL in the chromosome 2 and 3 regions, respectively, illustrating that multiple linked factors contribute to each QTL detected by recombination mapping. The QTL locations inferred from deficiency mapping did not generally correspond to those of candidate genes affecting oxidative and thermal stress or glucose metabolism. The chromosome 2 QTL in the 35B–E region was further resolved to a minimum of three tightly linked QTL, containing six genetically defined loci, 24 genes, and predicted genes that are positional candidates corresponding to life span QTL. This region was also associated with quantitative variation in life span in a sample of 10 genotypes collected from nature. Quantitative deficiency complementation is an efficient method for fine-scale QTL mapping in Drosophila and can be further improved by controlling the background genotype of the strains to be tested.

Pasyukova, E. G., Vieira, C. & Mackay, T. F. C. Deficiency mapping of quantitative trait loci affecting longevity in Drosophila melanogaster. Genetics 156, 1129–1146 (2000).

http://www.genetics.org/content/156/3/1129

Loss of CHICO

April 2001

One well-characterized genetic mechanism affecting aging and lifespan is the insulin-like signaling pathway, which is involved in the regulation of metabolism in D. melanogaster.

The Drosophila melanogaster gene chico encodes an insulin receptor sub- strate that functions in an insulin/insulin-like growth factor (IGF) signaling pathway. In the nematode Caenorhabditis elegans, insulin/IGF signaling regulates adult longevity. We found that mutation of chico extends fruit fly median life-span by up to 48% in homozygotes and 36% in heterozygotes. Extension of life-span was not a result of impaired oogenesis
in chico
females, nor was it consistently correlated with increased stress resistance. The dwarf phenotype of chico homozygotes was also unnecessary for ex- tension of life-span. The role of insulin/IGF signaling in regulating animal aging is therefore evolutionarily conserved.

Clancy, D. J. et al. Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein. Science 292, 104–106 (2001).

http://science.sciencemag.org/content/292/5514/104.full

Insulin receptor homolog mutant

April 2001

One well-characterized genetic mechanism affecting aging and lifespan is the insulin-like signaling pathway, which is involved in the regulation of metabolism in D. melanogaster.

The Drosophila melanogaster gene insulin-like receptor (InR) is homologous to mammalian insulin receptors as well as to Caenorhabditis elegans daf-2, a signal transducer regulating worm dauer formation and adult longevity. We describe a heteroallelic, hypomorphic genotype of mutant InR, which yields dwarf fe- males with up to an 85% extension of adult longevity and dwarf males with reduced late age-specific mortality. Treatment of the long-lived InR dwarfs with a juvenile hormone analog restores life expectancy toward that of wild-type controls.Weconclude that juvenile hormone deficiency, which results from InR signal pathway mutation, is sufficient to extend life-span, and that in flies, insulin-like ligands nonautonomously mediate aging through retardation of growth or activation of specific endocrine tissue.

Tatar, M. et al. A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science 292, 107–110 (2001).

http://science.sciencemag.org/content/292/5514/107.full

Overexpression: Mn-superoxide Dismutase

June 2002

Other genetic factors affecting aging include detoxification of reactive oxygen species.

A transgenic system (“FLP-out”) based on yeast FLP recombinase allowed induced overexpression of MnSOD enzyme in adult Drosophila melanogaster. With FLP-out a brief heat pulse (HP) of young, adult flies triggered the rearrangement and subsequent expression of a MnSOD transgene throughout the adult life span. Control (no HP) and overexpressing (HP) flies had identical genetic backgrounds. The amount of MnSOD enzyme overexpression achieved varied among six independent transgenic lines, with increases up to 75%. Life span was increased in proportion to the increase in enzyme. Mean life span was increased by an average of 16%, with some lines showing 30–33% increases. Maximum life span was increased by an average of 15%, with one line showing as much as 37% increase. Simultaneous overexpression of catalase with MnSOD had no added benefit, consistent with previous observations that catalase is present in excess in the adult fly with regard to life span. Cu/ZnSOD overexpression also increases mean and maximum life span. For both MnSOD and Cu/ZnSOD lines, increased life span was not associated with decreased metabolic activity, as measured by O2
consumption.

Sun, J., Folk, D., Bradley, T. J. & Tower, J. Induced overexpression of mitochondrial Mn-superoxide dismutase extends the life span of adult Drosophila melanogaster. Genetics 161, 661–672 (2002).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1462135/

doxycycline-dependent mutations

2003

Other genetic factors affecting aging include membrane function.

Background: A P-type transposable element called PdL has been engineered with a doxycycline- inducible promoter directed out through the 3? end of the element. Insertion of PdL near the 5? end of a gene often yields doxycycline-dependent overexpression of that gene and a mutant phenotype. This functional genomics strategy allows for efficient screening of large numbers of genes for overexpression phenotypes.

Results: PdL was mobilized to around 10,000 new locations in the Drosophila melanogaster genome and used to search for genes that would extend life span when overexpressed. Six lines were identified in which there was a 5-17% increase in life span in the presence of doxyxcycline. The mutations were molecularly characterized and in each case a gene was found to be overexpressed using northern blots. Two genes did not have previously known phenotypes and are implicated in membrane transport: VhaSFD encodes a regulatory subunit of the vacuolar ATPase proton pump (H+-ATPase), whereas Sugar baby (Sug) is related to a maltose permease from Bacillus. Three PdL mutations identified previously characterized genes: filamin encodes the homolog of an actin-polymerizing protein that interacts with presenilins. four wheel drive (fwd) encodes a phosphatidylinositol-4-kinase (PI 4-kinase) and CTP:phosphocholine cytidylyltransferase-1 (Cct1) encodes the rate-limiting enzyme in phosphatidylcholine synthesis. Finally, an apparently novel gene (Red herring, Rdh) was found in the first intron of the encore gene.

Conclusions: Screening for conditional mutations that increase Drosophila life span has identified genes implicated in membrane transport, phospholipid metabolism and signaling, and actin cytoskeleton organization.

Landis, G. N., Bhole, D. & Tower, J. A search for doxycycline-dependent mutations that increase Drosophila melanogaster life span identifies the VhaSFD, Sugar baby, filamin, fwd and Cctl genes. Genome Biol. 4, R8 (2003).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC151307/?tool=pmcentrez

Dopa decarboxylase (Ddc)

August 2003

Quantitative trait loci (QTLs) associated with lifespan in D. melanogaster have been mapped by association mapping.

Mutational analyses in model organisms have shown that genes affecting metabolism and stress resistance regulate life span, but the genes responsible for variation in longevity in natural populations are largely unidentified. Previously, we mapped quantitative trait loci (QTLs) affecting variation in longevity between two Drosophila melanogaster strains. Here, we show that the longevity QTL in the 36E;38B cytogenetic interval on chromosome 2 contains multiple closely linked QTLs, including the Dopa decarboxylase (Ddc) locus. Complementation tests to mutations show that Ddc is a positional candidate gene for life span in these strains. Linkage disequilibrium (LD) mapping in a sample of 173 alleles from a single population shows that three common molecular polymorphisms in Ddc account for 15.5% of the genetic contribution to variance in life span from chromosome 2. The polymorphisms are in strong LD, and the effects of the haplotypes on longevity suggest that the polymorphisms are maintained by balancing selection. DDC catalyzes the final step in the synthesis of the neurotransmitters, dopamine and serotonin. Thus, these data implicate variation in the synthesis of bioamines as a factor contributing to natural variation in individual life span.

De Luca, M. et al. Dopa decarboxylase (Ddc) affects variation in Drosophila longevity. Nat. Genet. 34, 429– 433 (2003).

http://www.nature.com/ng/journal/v34/n4/full/ng1218.html

Sir2

June 2004

Other genetic factors affecting aging include caloric restriction.

Calorie restriction can extend life span in a variety of species including mammals, flies, nematodes, and yeast. Despite the importance of this nearly universal effect, little is understood about the molecular mechanisms that mediate the life-span-extending effect of calorie restriction in metazoans. Sir2 is known to be involved in life span determination and calorie restriction in yeast mother cells. In nematodes increased Sir2 can extend life span, but a direct link to calorie restriction has not been demonstrated. We now report that Sir2 is directly involved in the calorie-restriction life-span-extending pathway in Drosophila. We demonstrate that an increase in Drosophila Sir2 (dSir2) extends life span, whereas a decrease in dSir2 blocks the life-span-extending effect of calorie reduction or rpd3 mutations. These data lead us to propose a genetic pathway by which calorie restriction extends life span and provides a framework for genetic and pharmacological studies of life span extension in metazoans.

Rogina, B. & Helfand, S. L. Sir2 mediates longevity in the fly through a pathway related to calorie restriction. Proc. Natl. Acad. Sci. U.S.A. 101, 15998–16003 (2004).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC528752/

QTL: Composit interval mapping

June 2004

Quantitative trait loci (QTLs) associated with lifespan in D. melanogaster have been mapped in linkage mapping populations.

Composite interval mapping was used to identify life-span QTL in F2 progeny of three crosses between different pairs of inbred lines. Each inbred line was derived from a different outbred population that had undergone long-term selection for either long or short life span. Microsatellite loci were used as genetic markers, and confidence intervals for QTL location were estimated by bootstrapping. A minimum of 10 QTL were detected, nine of which were located on the two major autosomes. Five QTL were present in at least two crosses and five were present in both sexes. Observation of the same QTL in more than one cross was consistent with the hypothesis that genetic variation for life span is maintained by balancing selection. For all QTL except one, allelic effects were in the direction predicted on the basis of outbred source population. Alleles that conferred longer life were always at least partially dominant.

Forbes, S. N., Valenzuela, R. K., Keim, P. & Service, P. M. Quantitative trait loci affecting life span in replicated populations of Drosophila melanogaster. I. Composite interval mapping. Genetics 168, 301–311 (2004).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1448087/

Overexpression: dFOXO

July 2004

One well-characterized genetic mechanism affecting aging and lifespan is the insulin-like signaling pathway, which is involved in the regulation of metabolism in D. melanogaster.

Glannakou, M. E., Goss, M., Jünger, M.A. Hafen, E. & Leevers, S. J. Long-lived Drosophila with over-expressed dFOXO in adult fat body. Science 305 SRC, 361 (2004).

http://science.sciencemag.org/content/305/5682/361.full

dFOXO

July 2004

One well-characterized genetic mechanism affecting aging and lifespan is the insulin-like signaling pathway, which is involved in the regulation of metabolism in D. melanogaster.

In Drosophila melanogaster, ageing is slowed when insulin-like signalling is reduced: life expectancy is extended by more than 50% when the insulin-like receptor (InR) or its receptor substrate (chico) are mutated, or when insulin-producing cells are ablated. But we have yet to resolve when insulin affects ageing, or whether insulin signals regulate ageing directly or indirectly through secondary hormones. Caenorhabditis elegans lifespan is also extended when insulin signalling is inhibited in certain tissues, or when repressed in adult worms, and this requires the forkhead transcription factor (FOXO) encoded by daf-16. The D. melanogaster insulin-like receptor mediates phosphorylation of dFOXO, the equivalent of nematode daf-16 and mammalian FOXO3a. We demonstrate here that dFOXO regulates D. melanogaster ageing when activated in the adult pericerebral fat body. We further show that this limited activation of dFOXO reduces expression of the Drosophila insulin-like peptide dilp-2 synthesized in neurons, and represses endogenous insulin-dependent signalling in peripheral fat body. These findings suggest that autonomous and non-autonomous.

Hwangbo, D. S. et al. Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body. Nature 429, 562–566 (2004).

http://www.nature.com/nature/journal/v429/n6991/full/nature02549.html

QTL: Response to selection

September 2004

Quantitative trait loci (QTLs) associated with lifespan in D. melanogaster have been mapped in linkage mapping populations.

Three selection experiments were used to identify chromosome regions that contain QTL affecting late-life and early-life fitness in Drosophila melanogaster. The selection experiments were initiated by crossing pairs of inbred lines that had been derived from outbred laboratory populations that had different mean life spans. QTL regions were located by association with microsatellite markers that showed significant selection responses. Regions between recombination map positions 54 and 81 on chromosome 2, between 0 and 30 on chromosome 3, and near locations 49 and 81 on chromosome 3 had the strongest support as locations of life-span QTL. There was good general agreement between the life-span QTL regions that were identified by selection and those that were identified in a companion recombination mapping experiment that used the same fly stocks. Many marker loci responded in opposite directions to selection for late- and early-life fitness, indicating negative genetic correlations or trade-offs between those traits. Indirect evidence suggested that some negative genetic correlations were due to antagonistic pleiotropy.

Valenzuela, R. K., Forbes, S. N., Keim, P. & Service, P. M. Quantitative trait loci affecting life span in replicated populations of Drosophila melanogaster. II. Response to selection. Genetics 168, 313–324 (2004).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1448088/

Innate immune response

February 2005

Other genetic factors affecting aging include immune response.

Increased activation of the innate immune system is a common feature of aging animals, including mammals and Drosophila melanogaster. With age, D. melanogaster progressively express higher levels of many antimicrobial peptides. It is unknown, however, whether this pattern reflects age-dependent changes in the function of the immune system itself or arises simply because aged adults have greater cumulative exposure to pathogens. Here we demonstrate that aged D. melanogaster transcribe more antimicrobial diptericin when experimentally exposed to septic bacterial infections. This strong net response in older females is the result of persistent diptericin transcription upon septic exposure, whereas young females rapidly terminate this induction. In contrast to their response to septic exposure, when exposed to killed bacteria aged females have less capacity to induce diptericin. Because this functional capacity of innate immunity declines with age, we conclude that female Drosophila undergo immune senescence. Furthermore, we show that fecundity is reduced by induction of innate immunity via the immune deficiency pathway. Consequently, maximum reproduction will occur when the immune response is tightly controlled in young females, even if this increases infection risk at later ages.

Zerofsky, M., Harel, E., Silverman, N. & Tatar, M. Aging of the innate immune response in Drosophila melanogaster. Aging Cell 4, 103–108 (2005).

http://onlinelibrary.wiley.com/doi/10.1111/j.1474-9728.2005.00147.x/abstract

Catsup

May 2006

Quantitative trait loci (QTLs) associated with lifespan in D. melanogaster have been mapped by association mapping.

Quantitative traits are shaped by networks of pleiotropic genes [1]. To understand the mechanisms that maintain genetic variation for quantitative traits in natural populations and to predict responses to artificial and natural selection, we must evaluate pleiotropic effects of underlying quantitative trait genes and define functional allelic variation at the level of quantitative trait nucleotides (QTNs). Catecholamines up (Catsup), which encodes a negative regulator of tyrosine hydroxylase [2], the rate-limiting step in the synthesis of the neurotransmitter dopamine, is a pleiotropic quantitative trait gene in Drosophila melanogaster [2-4]. We used association mapping to determine whether the same or different QTNs at Catsup are associated with naturally occurring variation in multiple quantitative traits. We sequenced 169 Catsup alleles from a single population and detected 33 polymorphisms with little linkage disequilibrium (LD). Different molecular polymorphisms in Catsup are independently associated with variation in longevity, locomotor behavior, and sensory bristle number. Most of these polymorphisms are potentially functional variants in protein coding regions, have large effects, and are not common. Thus, Catsup is a pleiotropic quantitative trait gene, but individual QTNs do not have pleiotropic effects. Molecular population genetic analyses of Catsup sequences are consistent with balancing selection maintaining multiple functional polymorphisms.

Carbone, M. A. et al. Phenotypic variation and natural selection at catsup, a pleiotropic quantitative trait gene in Drosophila. Curr. Biol. 16, 912–919 (2006).

http://www.sciencedirect.com/science/article/pii/S0960982206013480

Pathogen resistance

September 2006

Other genetic factors affecting aging include immune response.

The innate immune response protects numerous organ- isms, including humans, from the universe of pathogenic molecules, viruses and micro-organisms. Despite its role in promoting pathogen resistance, inappropriate activa- tion and expression of NFκ B and other immunity-related effector molecules can lead to cancer, inflammation, and other diseases of aging. Understanding the mechanisms leading to immune system activation as well as the short- and long-term consequences of such activation on health and lifespan is therefore critical for the development of beneficial immuno-modulating and longevity-promoting interventions. Mechanisms of innate immunity are highly conserved across species, and we take advantage of genetic tools in the model organism, Drosophila mela- nogaster , to study the effects of acute and chronic acti- vation of immunity pathways on pathogen resistance and general fitness of adult flies. Our findings indicate that fat body specific overexpression of a putative pathogen recognition molecule, peptidoglycan recognition protein (PGRP-LE), is sufficient for constitutive up-regulation of the immune response and for enhanced pathogen resist- ance. Primary components of fitness are unaffected by acute activation, but chronic activation leads to an inflam- matory state and reduced lifespan. These phenotypes are dependent on the NFκ B-related transcriptional factor, Relish, and they establish a mechanistic basis for a link between immunity, inflammation, and longevity.

Libert, S., Chao, Y., Chu, X. & Pletcher, S. D. Trade-offs between longevity and pathogen resistance in Drosophila melanogaster are mediated by NFkappaB signaling. Aging Cell 5, 533–543 (2006).

http://onlinelibrary.wiley.com/doi/10.1111/j.1474-9726.2006.00251.x/abstract;jsessionid=6C393899E7C136AD30EC13E709AEB604.f02t01

Olfaction and food-derived odors

February 2007

Other genetic factors affecting aging include sensory perception.

Smell is an ancient sensory system present in organisms from bacteria to humans. In the nematode Caeonorhabditis elegans, gustatory and olfactory neurons regulate aging and longevity. Using the fruit fly, Drosophila melanogaster, we showed that exposure to nutrient- derived odorants can modulate life span and partially reverse the longevity-extending effects of dietary restriction. Furthermore, mutation of odorant receptor Or83b resulted in severe olfactory defects, altered adult metabolism, enhanced stress resistance, and extended life span. Our findings indicate that olfaction affects adult physiology and aging in Drosophila, possibly through the perceived availability of nutritional resources, and that olfactory regulation of life span is evolutionarily conserved.

Libert, S. et al. Regulation of Drosophila life span by olfaction and food-derived odors. Science 315, 1133– 1137 (2007).

http://science.sciencemag.org/content/315/5815/1133.full

RNAi library

July 2007

I will use lines from the Vienna Drosophila RNAi Collection, which has more than 13,000 available lines.

Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.

Dietzl, G. et al. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, 151–6 (2007).

http://www.nature.com/nature/journal/v448/n7150/full/nature05954.html

QTL: Microarrays

October 2007

I will perform the MMC analyses separately for each sex because previous studies have shown pervasive sex-specificity of gene expression changes with age.

We developed a rapid, economical method for high-resolution quantitative trait locus (QTL) mapping using microarrays for selective genotyping of pooled DNA samples. We generated 21,207 F2 flies from two inbred Drosophila melanogaster strains with known QTLs affecting lifespan, and hybridized DNA pools of young and old flies to microarrays. We used changes of gene frequency of 2,326 single-feature polymorphisms (SFPs) to map previously identified and additional QTLs affecting lifespan.

Lai, C.-Q. et al. Speed-mapping quantitative trait loci using microarrays. Nat. Methods 4, 839–841 (2007).

http://www.nature.com/nmeth/journal/v4/n10/full/nmeth1084.html

Systems genetics

February 2009

To define the genetic variants associated with lifespan in a biologically relevant context and to further establish causality, I will perform a systems genetic analysis.

Determining the genetic architecture of complex traits is challenging because phenotypic variation arises from interactions between multiple, environmentally sensitive alleles. We quantified genome-wide transcript abundance and phenotypes for six ecologically relevant traits in D. melanogaster wild-derived inbred lines. We observed 10,096 genetically variable transcripts and high heritabilities for all organismal phenotypes. The transcriptome is highly genetically intercorrelated, forming 241 transcriptional modules. Modules are enriched for transcripts in common pathways, gene ontology categories, tissue-specific expression and transcription factor binding sites. The high degree of transcriptional connectivity allows us to infer genetic networks and the function of predicted genes from annotations of other genes in the network. Regressions of organismal phenotypes on transcript abundance implicate several hundred candidate genes that form modules of biologically meaningful correlated transcripts affecting each phenotype. Overlapping transcripts in modules associated with different traits provide insight into the molecular basis of pleiotropy between complex traits.

Ayroles, J. F. et al. Systems genetics of complex traits in Drosophila melanogaster. Nat. Genet. 41, 299– 307 (2009).

http://www.nature.com/ng/journal/v41/n3/full/ng.332.html

Odorant binding proteins

August 2011

I expect to validate many of the genes tested by manipulating gene expression since previous functional confirmation analyses for candidate genes identified by GWA analyses in the DGRP using RNAi knockdown had high validation rates.

Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial.

Swarup, S., Williams, T. I. & Anholt, R. R. H. Functional dissection of odorant binding protein genes in Drosophila melanogaster. Genes. Brain. Behav. 10, 648–657 (2013).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3150612/

DGRP

June 2012

Our laboratory developed the Drosophila melanogaster Genetic Reference Panel (DGRP) to facilitate GWA mapping of complex traits. The lines are available to the community and all measured phenotypes are maintained as a public resource on the DGRP website. The lines are genetically variable for all phenotypes measured to date, including lifespan, and phenotypic variation far exceeds what has been observed among common Drosophila laboratory strains.

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype-phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype-phenotype mapping using the power of Drosophila genetics.

Mackay, T. F. C. et al. The Drosophila melanogaster Genetic Reference Panel. Nature 482, 173–178 (2012).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683990/?tool=pmcentrez

Epistasis

September 2012

Common alleles associated with lifespan in the AIP but not the DGRP could be false positives in the AIP or have epistatic effects in the DGRP.

Epistasis-nonlinear genetic interactions between polymorphic loci-is the genetic basis of canalization and speciation, and epistatic interactions can be used to infer genetic networks affecting quantitative traits. However, the role that epistasis plays in the genetic architecture of quantitative traits is controversial. Here, we compared the genetic architecture of three Drosophila life history traits in the sequenced inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and a large outbred, advanced intercross population derived from 40 DGRP lines (Flyland). We assessed allele frequency changes between pools of individuals at the extremes of the distribution for each trait in the Flyland population by deep DNA sequencing. The genetic architecture of all traits was highly polygenic in both analyses. Surprisingly, none of the SNPs associated with the traits in Flyland replicated in the DGRP and vice versa. However, the majority of these SNPs participated in at least one epistatic interaction in the DGRP. Despite apparent additive effects at largely distinct loci in the two populations, the epistatic interactions perturbed common, biologically plausible, and highly connected genetic networks. Our analysis underscores the importance of epistasis as a principal factor that determines variation for quantitative traits and provides a means to uncover genetic networks affecting these traits. Knowledge of epistatic networks will contribute to our understanding of the genetic basis of evolutionarily and clinically important traits and enhance predictive ability at an individualized level in medicine and agriculture.

Huang, W. et al. Inaugural Article: Epistasis dominates the genetic architecture of Drosophila quantitative traits. Proc. Natl. Acad. Sci. 109, 15553–15559 (2012).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465439/

Genomic variation and gene expression

November 2012

Finally, I will integrate the results of Aims 1 and 2 by mapping the genetic variants with significant changes in allele frequency between the young and long-lived individuals from Aim 1 to the promoter and 3’ UTR regions of the transcripts that are significantly different at different ages, since previous analyses have shown that these regions are greatly enriched for cis-expression QTLs.

Understanding the relationship between genetic and phenotypic variation is one of the great outstanding challenges in biology. To meet this challenge, comprehensive genomic variation maps of human as well as of model organism populations are required. Here, we present a nucleotide resolution catalog of single-nucleotide, multi-nucleotide, and structural variants in 39 Drosophila melanogaster Genetic Reference Panel inbred lines. Using an integrative, local assembly-based approach for variant discovery, we identify more than 3.6 million distinct variants, among which were more than 800,000 unique insertions, deletions (indels), and complex variants (1 to 6,000 bp). While the SNP density is higher near other variants, we find that variants themselves are not mutagenic, nor are regions with high variant density particularly mutation-prone. Rather, our data suggest that the elevated SNP density around variants is mainly due to population-level processes. We also provide insights into the regulatory architecture of gene expression variation in adult flies by mapping cis-expression quantitative trait loci (cis-eQTLs) for more than 2,000 genes. Indels comprise around 10% of all cis-eQTLs and show larger effects than SNP cis-eQTLs. In addition, we identified two-fold more gene associations in males as compared to females and found that most cis-eQTLs are sex-specific, revealing a partial decoupling of the genomic architecture between the sexes as well as the importance of genetic factors in mediating sex-biased gene expression. Finally, we performed RNA-seq-based allelic expression imbalance analyses in the offspring of crosses between sequenced lines, which revealed that the majority of strong cis-eQTLs can be validated in heterozygous individuals.

Massouras, A. et al. Genomic Variation and Its Impact on Gene Expression in Drosophila melanogaster. PLoS Genet. 8, (2012).

http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1003055

Response to selection: Postponed senescence

March 2013

I will perform the MMC analyses separately for each sex because previous studies have shown pervasive sex-specificity of gene expression changes with age.

Limited lifespan and senescence are quantitative traits, controlled by many interacting genes with individually small and environmentally plastic effects, complicating genetic analysis. We performed genome wide analysis of gene expression for two Drosophila melanogaster lines selected for postponed senescence and one control, unselected line to identify candidate genes affecting lifespan as well as variation in lifespan. We obtained gene expression profiles for young flies of all lines, all lines at the time only 10% of the control lines survived, and the time at which 10% of the selected lines survived. Transcriptional responses to aging involved 19% of the genome. The transcriptional signature of aging involved the down-regulation of genes affecting proteolysis, metabolism, oxidative phosphorylation, and mitochrondrial function; and the up-regulation of genes affecting protein synthesis, immunity, defense responses, and the detoxification of xenobiotic substances. The transcriptional signature of postponed senescence involved the up-regulation of proteases and phosphatases and genes affecting detoxification of xenobiotics; and the down-regulation of genes affecting immunity, defense responses, metabolism and muscle function. Functional tests of 17 mutations confirmed 12 novel genes affecting Drosophila lifespan. Identification of genes affecting longevity by analysis of gene expression changes in lines selected for postponed senescence thus complements alternative genetic approaches.

Wilson, R. H., Lai, C., Lyman, R. F. & Mackay, T. F. C. Genomic response to selection for postponed senescence in Drosophila. Mech. Ageing Dev. 134, 79–88 (2013).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918434/

205 DGRP Natural Variation

April 2014

Freeze 2.0 sequences for 205 DGRP lines are published, and 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants have been identified. The lines are genetically variable for all phenotypes measured to date, including lifespan, and phenotypic variation far exceeds what has been observed among common Drosophila laboratory strains...Unbiased GWA analyses in the DGRP using full sequence data are underpowered because of the multiple testing penalty due to the small number of genotypes (205) compared to the large number of tests performed (4,853,802 SNPs and 1,296,080 non-SNP variants); further, only common alleles (minor allele frequencies ≥ 0.05) can be evaluated due to spurious long range LD incurred by rare alleles...A GWA analysis was performed on the lifespan phenotypes using the ~2.5 million variants with minor allele frequencies > 0.05 and adjusted for effects of Wolbachia infection, major polymorphic inversions, and polygenic relatedness...Although the DGRP lines have little relatedness, with a mode near 0, lines of the same karyotype for common polymorphic inversions have increased relatedness, and half of the DGRP lines are infected with Wolbachia, which can have subtle effects on some quantitative traits.

The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates, and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed towards larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting their non-random distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia, and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes and analysis and visualization tools are publicly available.

Huang, W., Massouras, A. & Inoue, Y. Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines. Genome. 1193–1208 (2014). doi:10.1101/gr.171546.113.Freely

http://genome.cshlp.org/content/early/2014/04/08/gr.171546.113.abstract

Olfactory behavior

January 2015

I expect to validate many of the genes tested by manipulating gene expression since previous functional confirmation analyses for candidate genes identified by GWA analyses in the DGRP using RNAi knockdown had high validation rates.

The genetic underpinnings that contribute to variation in olfactory perception are not fully understood. To explore the genetic basis of variation in olfactory perception, we measured behavioral responses to 14 chemically diverse naturally occurring odorants in 260400 flies from 186 lines of the Drosophila melanogaster Genetic Reference Panel, a population of inbred wild-derived lines with sequenced genomes. We observed variation in olfactory behavior for all odorants. Low to moderate broad-sense heritabilities and the large number of tests for genotype-olfactory phenotype association performed precluded any individual variant from reaching formal significance. However, the top variants (nominal P < 5×10(-5)) were highly enriched for genes involved in nervous system development and function, as expected for a behavioral trait. Further, pathway enrichment analyses showed that genes tagged by the top variants included components of networks centered on cyclic guanosine monophosphate and inositol triphosphate signaling, growth factor signaling, Rho signaling, axon guidance, and regulation of neural connectivity. Functional validation with RNAi and mutations showed that 15 out of 17 genes tested indeed affect olfactory behavior. Our results show that in addition to chemoreceptors, variation in olfactory perception depends on polymorphisms that can result in subtle variations in synaptic connectivity within the nervous system.

Arya, G. H. et al. The genetic basis for variation in olfactory behavior in Drosophila melanogaster. Chem. Senses 39, 125–132 (2014).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4398050/

Aggressive behavior

May 2015

I expect to validate many of the genes tested by manipulating gene expression since previous functional confirmation analyses for candidate genes identified by GWA analyses in the DGRP using RNAi knockdown had high validation rates.

Aggression is an evolutionarily conserved complex behavior essential for survival and the organization of social hierarchies. With the exception of genetic variants associated with bioamine signaling, which have been implicated in aggression in many species, the genetic basis of natural variation in aggression is largely unknown. Drosophila melanogaster is a favorable model system for exploring the genetic basis of natural variation in aggression. Here, we performed genome-wide association analyses using the inbred, sequenced lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and replicate advanced intercross populations derived from the most and least aggressive DGRP lines. We identified genes that have been previously implicated in aggressive behavior as well as many novel loci, including gustatory receptor 63a (Gr63a), which encodes a subunit of the receptor for CO2, and genes associated with development and function of the nervous system. Although genes from the two association analyses were largely nonoverlapping, they mapped onto a genetic interaction network inferred from an analysis of pairwise epistasis in the DGRP. We used mutations and RNAi knock-down alleles to functionally validate 79% of the candidate genes and 75% of the candidate epistatic interactions tested. Epistasis for aggressive behavior causes cryptic genetic variation in the DGRP that is revealed by changing allele frequencies in the outbred populations derived from extreme DGRP lines. This phenomenon may pertain to other fitness traits and species, with implications for evolution, applied breeding, and human genetics.

Shorter, J. et al. Genetic architecture of natural variation in Drosophila melanogaster aggressive behavior. Proc. Natl. Acad. Sci. 201510104 (2015). doi:10.1073/pnas.1510104112

http://www.pnas.org/content/112/27/E3555

C. elegans

Thermotolerance

August 1995

Other genetic factors affecting aging include resistance to heat, starvation, and other environmental stressors.

We have discovered that three longevity mutants of the nematode Caenorhabditis elegans also exhibit increased intrinsic thermotolerance (Itt) as young adults. Mutation of the age-1 gene causes not only 65% longer life expectancy but also Itt. The Itt phenotype cosegregates with age-1. Long-lived spe-26 and daf-2 mutants also exhibit Itt. We investigated the relationship between increased thermotolerance and increased life-span by developing conditions for environmental induction of thermotolerance. Such pretreatments at sublethal temperatures induce significant increases in thermotolerance and small but statistically highly significant increases in life expectancy, consistent with a causal connection between these two traits. Thus, when an animal's resistance to stress is increased, by either genetic or environmental manipulation, we also observe an increase in life expectancy. These results suggest that ability to respond to stress limits the life expectancy of C. elegans and might do so in other metazoa as well.

Lithgow, G. J., White, T. M., Melov, S. & Johnson, T. E. Thermotolerance and extended life-span conferred by single-gene mutations and induced by thermal stress. Proc. Natl. Acad. Sci. U.S.A. 92, 7540–7544 (1995).

http://www.pnas.org/content/92/16/7540.long

Longevity reduced by mating

February 1996

Other genetic factors affecting aging include reproduction.

THEORIES of life-history evolution propose that trade-offs occur between fitness components, including longevity and maximal reproduction1–3. In Drosophila, female lifespan is shortened by increased egg production4, receipt of male accessory fluid5 and courting6. Male lifespan is also reduced by courting and/or mating7. Here we show that in the nematode Caenorhabditis elegans, mating with males reduces the lifespan of hermaphrodites by a mechanism independent of egg production or receipt of sperm. Conversely, males appear unaffected by mating. Thus, in C. elegans there is no apparent trade-off between longevity and increased egg or sperm production, but there is a substantial cost to hermaphrodites associated with copulation.

Gems, D. & Riddle, D. L. Longevity in Caenorhabditis elegans reduced by mating but not gamete production. Nature 379, 723–725 (1996).

http://www.nature.com/nature/journal/v379/n6567/abs/379723a0.html

Phosphatidylinositol-3-OH kinase family member

August 1996

One well-characterized genetic mechanism affecting aging and lifespan is the insulin-like signaling pathway, which is involved in the regulation of metabolism in C. elegans.

A pheromone-induced neurosecretory pathway in C. elegans triggers developmental arrest and an increase in longevity at the dauer diapause stage. The gene age-1 is required for non-dauer development and normal senescence. age-1 encodes a homologue of mammalian phosphatidylinositol-3-OH kinase (PI(3)K) catalytic subunits. Lack of both maternal and zygotic age-1 activity causes dauer formation, whereas animals with maternal but not zygotic age-1 activity develop as non-dauers that live more than twice as long as normal. These data suggest that phosphatidylinositol signalling mediated by AGE-1 protein controls lifespan and the dauer diapause decision.

Morris, J. Z., Tissenbaum, H. A. & Ruvkun, G. A phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans. Nature 382, 536–539 (1996).

http://www.nature.com/nature/journal/v382/n6591/abs/382536a0.html

daf-2: Insulin receptor-like gene

August 1997

One well-characterized genetic mechanism affecting aging and lifespan is the insulin-like signaling pathway, which is involved in the regulation of metabolism in C. elegans.

A C. elegans neurosecretory signaling system regulates whether animals enter the reproductive life cycle or arrest development at the long-lived dauer diapause stage. daf-2, a key gene in the genetic pathway that mediates this endocrine signaling, encodes an insulin receptor family member. Decreases in DAF-2 signaling induce metabolic and developmental changes, as in mammalian metabolic control by the insulin receptor. Decreased DAF-2 signaling also causes an increase in life-span. Life-span regulation by insulin-like metabolic control is analogous to mammalian longevity enhancement induced by caloric restriction, suggesting a general link between metabolism, diapause, and longevity.

Kimura, K. D., Tissenbaum, H. A., Liu, Y. & Ruvkun, G. daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277, 942–946 (1997).

http://science.sciencemag.org/content/277/5328/942.full

daf-16: Double lifespan

November 1997

One well-characterized genetic mechanism affecting aging and lifespan is the insulin-like signaling pathway, which is involved in the regulation of metabolism in C. elegans.

The wild-type Caenorhabditis elegans nematode ages rapidly, undergoing development, senescence, and death in less than 3 weeks. In contrast, mutants with reduced activity of the gene daf-2, a homolog of the insulin and insulin-like growth factor receptors, age more slowly than normal and live more than twice as long. These mutants are active and fully fertile and have normal metabolic rates. The life-span extension caused by daf-2 mutations requires the activity of the gene daf-16. daf-16 appears to play a unique role in life-span regulation and encodes a member of the hepatocyte nuclear factor 3 (HNF-3)/forkhead family of transcriptional regulators. In humans, insulin down-regulates the expression of certain genes by antagonizing the activity of HNF-3, raising the possibility that aspects of this regulatory system have been conserved.

Lin, K., Dorman, J. B., Rodan, a & Kenyon, C. daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322 (1997).

http://science.sciencemag.org/content/278/5341/1319.full

Mutation in succinate dehydrogenase cytochrome b: oxidative stress

August 1998

Other genetic factors affecting aging include detoxification of reactive oxygen species.

Much attention has focused on the aetiology of oxidative damage in cellular and organismal ageing. Especially toxic are the reactive oxygen byproducts of respiration and other biological processes. A mev-1(kn1) mutant of Caenorhabditis elegans has been found to be hypersensitive to raised oxygen concentrations. Unlike the wild type, its lifespan decreases dramatically as oxygen concentrations are increased from 1 to 60%. Strains bearing this mutation accumulate markers of ageing (such as fluorescent materials and protein carbonyls) faster than the wild type. We show here that mev-1 encodes a subunit of the enzyme succinate dehydrogenase cytochrome b, which is a component of complex II of the mitochondrial electron transport chain. We found that the ability of complex II to catalyse electron transport from succinate to ubiquinone is compromised in mev-1 animals. This may cause an indirect increase in superoxide levels, which in turn leads to oxygen hypersensitivity and premature ageing. Our results indicate that mev-1 governs the rate of ageing by modulating the cellular response to oxidative stress.

Ishii, N. et al. A mutation in succinate dehydrogenase cytochrome b causes oxidative stress and ageing in nematodes. Nature 394, 694–697 (1998).

http://www.nature.com/nature/journal/v394/n6694/full/394694a0.html

Sensory perception

December 1999

Other genetic factors affecting aging include sensory perception.

Caenorhabditis elegans senses environmental signals through ciliated sensory neurons located primarily in sensory organs in the head and tail. Cilia function as sensory receptors, and mutants with defective sensory cilia have impaired sensory perception. Cilia are membrane-bound microtubule-based structures and in C. elegans are only found at the dendritic endings of sensory neurons. Here we show that mutations that cause defects in sensory cilia or their support cells, or in sensory signal transduction, extend lifespan. Our findings imply that sensory perception regulates the lifespan of this animal, and suggest that in nature, its lifespan may be regulated by environmental cues.

Apfeld, J. & Kenyon, C. Regulation of lifespan by sensory perception in Caenorhabditis elegans. Nature 402, 804–809 (1999).

http://www.nature.com/nature/journal/v402/n6763/full/402804a0.html

Mitochondrial function

December 2002

Other genetic factors affecting aging include mitochondrial function.

To explore the role of mitochondrial activity in the aging process, we have lowered the activity of the electron transport chain and adenosine 5'-triphosphate (ATP) synthase with RNA interference (RNAi) in Caenorhabditis elegans. These perturbations reduced body size and behavioral rates and extended adult life-span. Restoring messenger RNA to near-normal levels during adulthood did not elevate ATP levels and did not correct any of these phenotypes. Conversely, inhibiting respiratory-chain components during adulthood only did not reset behavioral rates and did not affect life-span. Thus, the developing animal appears to contain a regulatory system that monitors mitochondrial activity early in life and, in response, establishes rates of respiration, behavior, and aging that persist during adulthood.

Dillin, A. et al. Rates of behavior and aging specified by mitochondrial function during development. Science 298, 2398–2401 (2002).

http://science.sciencemag.org/content/298/5602/2398.long

RNAi Screen

January 2003

Other genetic factors affecting aging include mitochondrial function.

We report a systematic RNA interference (RNAi) screen of 5,690 Caenorhabditis elegans genes for gene inactiva- tions that increase lifespan. We found that genes important for mitochondrial function stand out as a principal group of genes affecting C. elegans lifespan. A classical genetic screen identified a mutation in the mitochondrial leucyl-tRNA synthetase gene (lrs-2) that impaired mitochondrial function and was associated with longer-lifespan. The long-lived worms with impaired mitochondria had lower ATP content and oxygen consumption, but differen- tial responses to free-radical and other stresses. These data suggest that the longer lifespan of C. elegans with compromised mitochrondria cannot simply be assigned to lower free radical production and suggest a more complex coupling of metabolism and longevity.

Lee, S. S. et al. A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nat. Genet. 33, 40–48 (2003).

http://www.nature.com/ng/journal/v33/n1/full/ng1056.html

S. cerevisiae

Extremely large pools of segregants

2010

To alleviate this problem I propose to use a genetically diverse outbred advanced intercross population (AIP) derived from a subset of DGRP lines to perform extreme QTL mapping to identify potentially causal alleles associated with variation in lifespan.

Most heritable traits, including many human diseases1, are caused by multiple loci. Studies in both humans and model organisms, such as yeast, have failed to detect a large fraction of the loci that underlie such complex traits2, 3. A lack of statistical power to identify multiple loci with small effects is undoubtedly one of the primary reasons for this problem. We have developed a method in yeast that allows the use of much larger sample sizes than previously possible and hence permits the detection of multiple loci with small effects. The method involves generating very large numbers of progeny from a cross between two Saccharomyces cerevisiae strains and then phenotyping and genotyping pools of these offspring. We applied the method to 17 chemical resistance traits and mitochondrial function, and identified loci for each of these phenotypes. We show that the level of genetic complexity underlying these quantitative traits is highly variable, with some traits influenced by one major locus and others by at least 20 loci. Our results provide an empirical demonstration of the genetic complexity of a number of traits and show that it is possible to identify many of the underlying factors using straightforward techniques. Our method should have broad applications in yeast and can be extended to other organisms.

Ehrenreich, I. M. et al. Dissection of genetically complex traits with extremely large pools of yeast segregants. Nature 464, 1039–1042 (2010).

http://www.nature.com/nature/journal/v464/n7291/full/nature08923.html

Chemical resistance

March 2012

To alleviate this problem I propose to use a genetically diverse outbred advanced intercross population (AIP) derived from a subset of DGRP lines to perform extreme QTL mapping to identify potentially causal alleles associated with variation in lifespan.

Many questions about the genetic basis of complex traits remain unanswered. This is in part due to the low statistical power of traditional genetic mapping studies. We used a statistically powerful approach, extreme QTL mapping (X-QTL), to identify the genetic basis of resistance to 13 chemicals in all 6 pairwise crosses of four ecologically and genetically diverse yeast strains, and we detected a total of more than 800 loci. We found that the number of loci detected in each experiment was primarily a function of the trait (explaining 46% of the variance) rather than the cross (11%), suggesting that the level of genetic complexity is a consistent property of a trait across different genetic backgrounds. Further, we observed that most loci had trait-specific effects, although a small number of loci with effects in many conditions were identified. We used the patterns of resistance and susceptibility alleles in the four parent strains to make inferences about the allele frequency spectrum of functional variants. We also observed evidence of more complex allelic series at a number of loci, as well as strain-specific signatures of selection. These results improve our understanding of complex traits in yeast and have implications for study design in other organisms.

Ehrenreich, I. M. et al. Genetic architecture of highly complex chemical resistance traits across four yeast strains. PLoS Genet. 8, (2012).

http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1002570

Dairy Cows

Milk production vs reproductive and energy status

1990

Other genetic factors affecting aging include DNA repair and replication.

Two groups of 10 Holstein cows were chosen by pairs from a 20-yr genetic selection project that used either breed average or breed high sires chosen only for Predicted Differences in milk production. Milk production (305-d mature equivalent) was 10,814 kg and 6912 kg for the high and average groups of cows. Days to first visual estrus and number of ovulations before first visual estrus were greater for the high versus the average group (66 vs. 43 d and 1.6 vs. .7 ovulations). No differences were significant between groups for the interval from parturition to uterine involution or for days to first ovulation. Energy balance was less for the high group during wk 1, 2, 10, and 11. Plasma glucose concentration was lowest during wk 2 for both groups, and nonesterified fatty acids and β-hydroxybutyrate were greatest for both groups during wk 1 and 2. Liver glycogen content was lower at d 15 postpartum for the high group, and liver triglyceride content was greater on d 30 for the high group. The data for reproductive functions support the concept that high milk production is antagonistic to expression of estrous behavior but not to reactivation of ovarian function.

Harrison, R. O., Ford, S. P., Young, J. W., Conley, A. J. & Freeman, A. E. Increased milk production versus
reproductive and energy status of high producing dairy cows. J. Dairy Sci. 73, 2749–2758 (1990).

http://www.journalofdairyscience.org/article/S0022-0302(90)78960-6/abstract

Bioinformatics

DAVID

April 2003

I will also use Gene Ontology.

BACKGROUND:
Functional annotation of differentially expressed genes is a necessary and critical step in the analysis of microarray data. The distributed nature of biological knowledge frequently requires researchers to navigate through numerous web-accessible databases gathering information one gene at a time. A more judicious approach is to provide query-based access to an integrated database that disseminates biologically rich information across large datasets and displays graphic summaries of functional information.
RESULTS:
Database for Annotation, Visualization, and Integrated Discovery (DAVID; http://www.david.niaid.nih.gov) addresses this need via four web-based analysis modules: 1) Annotation Tool - rapidly appends descriptive data from several public databases to lists of genes; 2) GoCharts - assigns genes to Gene Ontology functional categories based on user selected classifications and term specificity level; 3) KeggCharts - assigns genes to KEGG metabolic processes and enables users to view genes in the context of biochemical pathway maps; and 4) DomainCharts - groups genes according to PFAM conserved protein domains.
CONCLUSIONS:
Analysis results and graphical displays remain dynamically linked to primary data and external data repositories, thereby furnishing in-depth as well as broad-based data coverage. The functionality provided by DAVID accelerates the analysis of genome-scale datasets by facilitating the transition from data collection to biological meaning.

Dennis, G., Sherman, B. T., Hosack, D. A. & Yang, J. DAVID: Database for Annotation, Visualization, and Integrated. Genome Biol. 4, R60 (2003).

http://www.ncbi.nlm.nih.gov/pubmed/12734009

Statistical significance

August 2003

I will perform analysis of variance (ANOVA) models on the normalized count data (Y = μ + S + A + T + S×A + S×T + A×T + S×A×T + ε) to separate variation in abundance of each transcript among sexes (S), ages (A) and tissues (T); using an FDR < 0.05

With the increase in genomewide experiments and the sequencing of multiple genomes, the analysis of large data sets has become commonplace in biology. It is often the case that thousands of features in a genomewide data set are tested against some null hypothesis, where a number of features are expected to be significant. Here we propose an approach to measuring statistical significance in these genomewide studies based on the concept of the false discovery rate. This approach offers a sensible balance between the number of true and false positives that is automatically calibrated and easily interpreted. In doing so, a measure of statistical significance called the q value is associated with each tested feature. The q value is similar to the well known p value, except it is a measure of significance in terms of the false discovery rate rather than the false positive rate. Our approach avoids a flood of false positive results, while offering a more liberal criterion than what has been used in genome scans for linkage.

Storey, J. D. & Tibshirani, R. Statistical significance for genomewide studies. Proc. Natl. Acad. Sci. U.S.A. 100, 9440–9445 (2003).

http://www.pnas.org/content/100/16/9440.long

Interpreting genome-wide expression profiles

August 2005

I will also use Gene Set Enrichment.

Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.

Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U.S.A. 102, 15545–50 (2005).

http://www.pnas.org/content/102/43/15545.abstract

Gene ontology

September 2007

I will also use Gene Ontology.

The DAVID Gene Functional Classification Tool http://david.abcc.ncifcrf.gov uses a novel agglomeration algorithm to condense a list of genes or associated biological terms into organized classes of related genes or biology, called biological modules. This organization is accomplished by mining the complex biological co-occurrences found in multiple sources of functional annotation. It is a powerful method to group functionally related genes and terms into a manageable number of biological modules for efficient interpretation of gene lists in a network context.

Huang, D. W., Sherman, B. T. & Tan, Q. The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists. Genome Biol. 8, R183 (2007).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2375021/

TopHat

May 2009

Following preprocessing, rRNA sequences will be removed using TopHat

A new protocol for sequencing the messenger RNA in a cell, known as RNA-Seq, generates millions of short sequence fragments in a single run. These fragments, or 'reads', can be used to measure levels of gene expression and to identify novel splice variants of genes. However, current software for aligning RNA-Seq data to a genome relies on known splice junctions and cannot identify novel ones. TopHat is an efficient read-mapping algorithm designed to align reads from an RNA-Seq experiment to a reference genome without relying on known splice sites.We mapped the RNA-Seq reads from a recent mammalian RNA-Seq experiment and recovered more than 72% of the splice junctions reported by the annotation-based software from that study, along with nearly 20,000 previously unreported junctions. The TopHat pipeline is much faster than previous systems, mapping nearly 2.2 million reads per CPU hour, which is sufficient to process an entire RNA-Seq experiment in less than a day on a standard desktop computer. We describe several challenges unique to ab initio splice site discovery from RNA-Seq reads that will require further algorithm development.TopHat is free, open-source software available from http://tophat.cbcb.umd.edu.Supplementary data are available at Bioinformatics online.

Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2672628/

Modulated Modularity Clustering (MMC)

May 2009

Stone, E. A. & Ayroles, J. F. Modulated modularity clustering as an exploratory tool for functional genomic inference. PLoS Genet. 5, e1000479 (2009).

I will use Modulated Modularity Clustering (MMC) to cluster transcripts significant for the A and A×T terms into co- expression modules associated with lifespan.

In recent years, the advent of high-throughput assays, coupled with their diminishing cost, has facilitated a systems approach to biology. As a consequence, massive amounts of data are currently being generated, requiring efficient methodology aimed at the reduction of scale. Whole-genome transcriptional profiling is a standard component of systems-level analyses, and to reduce scale and improve inference clustering genes is common. Since clustering is often the first step toward generating hypotheses, cluster quality is critical. Conversely, because the validation of cluster-driven hypotheses is indirect, it is critical that quality clusters not be obtained by subjective means. In this paper, we present a new objective-based clustering method and demonstrate that it yields high-quality results. Our method, modulated modularity clustering (MMC), seeks community structure in graphical data. MMC modulates the connection strengths of edges in a weighted graph to maximize an objective function (called modularity) that quantifies community structure. The result of this maximization is a clustering through which tightly-connected groups of vertices emerge. Our application is to systems genetics, and we quantitatively compare MMC both to the hierarchical clustering method most commonly employed and to three popular spectral clustering approaches. We further validate MMC through analyses of human and Drosophila melanogaster expression data, demonstrating that the clusters we obtain are biologically meaningful. We show MMC to be effective and suitable to applications of large scale. In light of these features, we advocate MMC as a standard tool for exploration and hypothesis generation.

Stone, E. A. & Ayroles, J. F. Modulated modularity clustering as an exploratory tool for functional genomic inference. PLoS Genet. 5, e1000479 (2009).

http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1000479

Burrows-Wheeler Aligner (BWA)

July 2009

I will align the sequence reads to the D. melanogaster reference genome using Burrows-Wheeler Aligner (BWA, version 0.6.2).

The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals.We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows-Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is approximately 10-20x faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package.http://maq.sourceforge.net.

Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2705234/

TopHat and Cufflinks

March 2012

the remaining reads will be aligned to the D. melanogaster reference transcriptome and genome using the Tuxedo suite pipeline.

Recent advances in high-throughput cDNA sequencing (RNA-seq) can reveal new genes and splice variants and quantify expression genome-wide in a single assay. The volume and complexity of data from RNA-seq experiments necessitate scalable, fast and mathematically principled analysis software. TopHat and Cufflinks are free, open-source software tools for gene discovery and comprehensive expression analysis of high-throughput mRNA sequencing (RNA-seq) data. Together, they allow biologists to identify new genes and new splice variants of known ones, as well as compare gene and transcript expression under two or more conditions. This protocol describes in detail how to use TopHat and Cufflinks to perform such analyses. It also covers several accessory tools and utilities that aid in managing data, including CummeRbund, a tool for visualizing RNA-seq analysis results. Although the procedure assumes basic informatics skills, these tools assume little to no background with RNA-seq analysis and are meant for novices and experts alike. The protocol begins with raw sequencing reads and produces a transcriptome assembly, lists of differentially expressed and regulated genes and transcripts, and publication-quality visualizations of analysis results. The protocol's execution time depends on the volume of transcriptome sequencing data and available computing resources but takes less than 1 d of computer time for typical experiments and ∼1 h of hands-on time.

Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–78 (2012).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334321/