Human Longevity Alleles

- Naftali Horwitz, Scientific Venture consultant LTF & Jyothi Devakumar, CSO LTF

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This is a part of a blog series on the exciting landscape of longevity. We, at LTF, are creating an investment map as we raise our fund 2.0.

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Some of the interesting questions the aging/longevity field is trying to address include:

"Is Aging malleable or is it a fixed program?"

"Is this process linked to specific genes?"

"Can tweaking these genes impact the aging process?"

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Around 30 years ago, the scientific field was largely in agreement that aging was a fixed process that couldn’t be altered. This thought process was directly challenged by the discovery of C.elegans worms with mutations in several key genes (age-1, daf-2, daf-16) involved in growth and nutrient sensing pathways that exhibited both mean and maximal lifespans increases compared to wild-type control worms. These model organisms provided clear evidence that genetic perturbations could have direct effects on the aging trajectory of an organism and as such, disproved the theory that aging could not be genetically encoded. Since then, scientists have better characterised several pathways (Growth Factor/Insulin-IGF signalling, mTOR, Sirtuin, Heat shock proteins, etc.) that play a role in longevity and health. Most of these studies however have revolved around the use of model organisms (i.e. worms, flies, fish, mice, non-human primates) and as such, their relevance to human aging isn’t entirely clear. 

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Evolution however has already performed what are known as “experiments of nature” where specific stochastic mutations in humans exhibit enrichment in older individuals as well as enrichment in long-lived and geographically distinct populations. Here we highlight certain case studies whereby human mutations in pathways also identified as longevity promoting in model organisms, exhibit beneficial effects in humans, thereby reinforcing the importance of these pathways as viable targets for aging therapeutics. 

In order to identify common genetic changes that underlie extreme longevity in humans, scientists have begun sequencing the genomes of thousands or even hundreds of thousands of elderly and young individuals and examined their genomes either based on candidate approaches where they look at specific regions of interest or genome wide association studies (GWAS) where they look at millions of germline variants at once to determine differences in enrichment of genetic variants between long lived individuals and their younger counterparts. 

One example are variants in the FOXO3A gene, the homologue of daf-16 in worms, and one of the key downstream modulators of the insulin-IGF signalling pathway that influences metabolism. In worms and flies signalling downstream of the insulin receptor (daf-2/InR), naturally leads to phosphorylation of FOXO and sequestration from the nucleus. Mutations in the insulin receptor lead to the activation of FOXO, which in turn induces the expression of factors that retard cell growth and proliferation and promote longevity. These mutations extend lifespan in these model organisms by up to 50%. In mice, FOXO protein overexpression has shown enhancement in key longevity promoting pathways like autophagy, mitophagy, and proteasomal degradation. In humans, approximately 17 single nucleotide polymorphisms (SNPs) have been identified in the FOXO3A locus, and have been associated with increased longevity as they appear to be more enriched in centenarian populations in Germany, Japan, France and the US. Although in model organisms, elevated FOXO expression is linked to longevity, in humans, functional annotations of these SNPs are limited. 

Similarly, another family of genes and their variants have captured the attention of scientists and longevity enthusiasts alike. These are genetic variants in the loci of sirtuins, a family of NAD+ - dependent deacetylase proteins, that link energy metabolism, genome maintenance, and aging. Originally identified in yeast as crucial for maintaining genomic integrity and inhibiting the formation of toxic extrachromosomal rDNA circles (ERCs), overexpressing these proteins could extend yeast lifespan by over 70%. In mammals, several sirtuin family members have been implicated with longevity and health. Mice with an extra copy of SIRT1 gene are characterised by a lower level of DNA damage and of p16, both of which are hallmarks of ageing. Mice lacking SIRT3, the mitochondrial specific sirtuin critical for proper mitochondrial biogenesis and reactive oxygen species detoxification, are characterised by decreased oxygen consumption and simultaneous increase in reactive oxygen species. These mice develop several diseases of aging at an accelerated pace, such as cancer, metabolic syndrome, cardiovascular disease, and neurodegenerative diseases. Male mice overexpressing SIRT6 exhibit lifespan increases of ~15% and display lower serum levels of insulin-like growth factor 1 (IGF1), higher levels of IGF-binding protein 1 and altered phosphorylation levels of major components of IGF1 signalling. In humans, various allelic variants in sirtuin genes have been identified in centenarians. For example, targeted sequencing of SIRT6 locus in populations of centenarian jews revealed two linked substitutions (N308K/A313S). Characterization of this allele demonstrated it to be a stronger suppressor of retrotransposons, confer enhanced stimulation of DNA repair, and better protection against cancer compared with wild-type SIRT6. This variant also displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD+ concentrations and substrates. SIRT3, a separate sirtuin family member, also shows variant alleles that correlate with longevity and increased expression of SIRT3 protein likely due to changes in enhancer elements that regulate expression of SIRT3. 

Intriguingly, the genetic loci found to have the strongest association with longevity to date, APOE haplotypes, have the least understood mechanism of all. Apolipoprotein E (APOE) is a protein involved in the transport of cholesterol and other lipids to cells and mediates the clearance of triglyceride-rich lipoproteins by binding to hepatic APOE receptors. Mice devoid of APOE display poor lipoprotein clearance with subsequent accumulation of cholesterol ester-enriched particles in the blood, which promote the development of atherosclerotic plaques. Human carriers of the APOE ε2 allele have lower levels of total cholesterol and LDL, and higher levels of HDL and triglycerides (TGs) in the plasma as well as lower risk of developing Alzheimer's and cardiovascular disease. This allele is also enriched in long-lived individuals. Carriers of the APOE ε4 allele on the other hand show an increased risk of all cause mortality along with Alzheimers and Cardiovascular disease and exhibit higher levels of total cholesterol, LDL, and triglycerides (TGs), and lower levels of HDL. It remains unclear however, how changes in lipid levels translate to the development of Alzheimer’s and the effects of longevity. Laboratory experiments using APOE-targeted replacement (APOE-TR) mice in which the murine Apoe gene locus is replaced with human APOE alleles are ongoing to try to determine a mechanistic understanding of this allele and longevity. 

In addition, several other genetic loci, previously associated with aging and longevity in model organisms, have also been identified in long-lived human populations including genes regulating mitochondrial OXPHOS complexes, DNA damage repair mechanisms, telomeric maintenance machinery, heat shock proteins, and translation machinery. However, each of these polymorphisms turns out to explain only a very small fraction of the longevity variability. From GWAS studies only a few number of variants exhibit multiple test significance and are successfully replicated in different studies and across different populations. Additionally, none of the variants identified thus far as being associated with longevity seem to be shared among all, or most centenarians, and none seem sufficient to achieve longevity (all are fairly common in groups who die earlier). 

These findings have led some in the field to conclude that perhaps genetics may not be as strong a driver of longevity as previously thought (estimates range between 10-25%), while others have pointed to issues with methodologies of these experiments like inadequate sample sizes or population stratification as well as the need to validate rare variants among multiple cohorts and ensure the presence of appropriate control populations. Furthermore, these findings underscore the need for better functional characterization of longevity-associated genes and variants on various parameters relevant to health and longevity, including cellular and organismal resistance to stress and improved fitness. There are two distinct human populations however, that have been functionally characterised longitudinally and who’s mutations definitively alter their health in advanced age. 

One such population, a group of growth hormone receptor deficient ecuadorians (n=99) ( found to harbour either a splice site mutation on exon 6 leading to a misfolded and degraded growth hormone receptor or a truncated receptor due to an early stop codon) were followed for 22 years. Originally identified due to their short stature, these remarkable individuals, over the course of the study, exhibited only one incidence of nonlethal malignancy and no cases of diabetes, in contrast to a prevalence of 17% for cancer and 5% for diabetes in control subjects. This diabetic protection is conferred in spite of the fact that these individuals had higher incidences of obesity (21% vs 13.4% in the general Ecuadorian population). Finally, these individuals continually exhibited lower levels of IGF-1/IGF-2 in serum.

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Unlike mouse models where deletion of the insulin receptor (particularly in fat; FIRKO mice) leads to increase in mean life-span of approximately 18%, this human population didn’t exhibit longer lifespans. This could be due to a number of factors including environmental and societal implications given their physical limitations. However, these findings are consistent with previous reports of patients with congenital IGF-I deficiency (Laron syndrome, GH gene deletion, GHRH receptor defects and IGF-I resistance) having no incidences of cancer. Likewise, genomic analyses have identified clusters of functional mutations in the IGF-1 receptor in centenarians as well as epidemiological analyses of centenarians with lower levels of IGF-1 displaying significantly longer survival. Finally, a recent discovery in French, American and Amish populations harbouring a growth hormone receptor exon 3 deletion (d3-GHR), showed that male individuals homozygous for this variant exhibited longer lifespans with a decreased incidence of diabetes. These individuals however did not display any changes in circulating IGF-1 levels. 

More recently, among an isolated Berne amish community in Indiana, a mutation was identified in SERPINE1, which codes for Plasminogen activator inhibitor–1 (PAI-1), a key component of the senescence-related secretome and a direct mediator of cellular senescence. This mutation which consists of a dinucleotide insertion (TA) within the coding sequence of the PAI-1 gene, resulting in a frameshift mutation and formation of a premature stop codon and the synthesis of a truncated, nonfunctional PAI-1 protein, has several positive phenotypic consequences. Heterozygosity was associated with significantly longer leukocyte telomere length, lower fasting insulin levels, and lower prevalence of diabetes mellitus. Meanwhile, carriers of the null SERPINE1 allele had a longer life span. These results align well with animal models of PAI-1 inhibition which show extended median survival, and protection against the development of hypertension and arteriosclerosis. Although exciting, this population needs to be followed longitudinally especially given the small population size, low genetic diversity, and homogenous environmental conditions to ensure that this genetic change is truly driving aging associated phenotypes. The scientists who pioneered this exciting discovery in the Amish community, have now started a company, Zoe Biosciences, to develop therapies aimed to block PAI-1 activity. We at LTF have now partnered with Zoe Biosciences in their efforts to extend healthy life and we are excited to share in their forthcoming developments. 

While it may be tempting to propose gene editing approaches to recapitulate these rare mutations in the general population (and some groups are working to make this a reality), this may be a rather misguided approach. For starters, longevity is a polygenic trait and therefore one single variant change may result in a rather small impact on the magnitude of longevity. Likewise, based on the sensitivity of our assays and the rarity of our long lived populations, we can’t be sure that the allelic changes picked up in centenarians are firmly linked to longevity. Finally, even in the case of growth hormone mutations where clear phenotypic changes on health can be observed, therapeutic approaches like small molecule inhibitors that allow for temporal control would be preferred given that these pathways are still needed for proper growth and development. 

Gene editing approaches are most likely only appropriate for rare diseases of early life such as progerias. These diseases manifest due to rare mutations in DNA repair machinery and nuclear integrity and due to the high unmet need and the specificity of the genetic lesion, would greatly benefit from a permanent gene editing approach. However, since these mutations are rarely seen in physiological aging, these gene editing approaches would not be applicable to the general population. 

Overall, these examples of human genetic variation and their associations with longevity should be met with enthusiasm especially given that they are largely recapitulated in model organisms. Although the effect size of these genetic changes might decrease as you go from worms, to mice, to humans, these mutations may still impact key components of the aging pathologies and therefore may greatly impact the way that elderly populations live out their final years. We should eagerly continue in our efforts to develop therapeutic strategies to modulate the pathways associated with these genetic variants as well as continuing to uncover more variants enriched in rare populations and centenarians. It’s a big world out there and there’s no telling what experiment nature has already conducted. 

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