“Method of Using Foxo3a Polymorphisms and Haplotypes to Predict and Promote Healthy Aging and Longevity” in Patent Application Approval Process…

"Method of Using Foxo3a Polymorphisms and Haplotypes to Predict and Promote Healthy Aging and Longevity" in Patent Application Approval Process (USPTO 20150337383)

By a News Reporter-Staff News Editor at Politics & Government Week -- A patent application by the inventors Donlon, Timothy Atchison (Honolulu, HI); Wilcox, Bradley John (Honolulu, HI); Curb, David J. (Honolulu, HI), filed on August 10, 2015, was made available online on December 3, 2015, according to news reporting originating from Washington, D.C., by VerticalNews correspondents.

This patent application is assigned to Kuakini Medical Center.

The following quote was obtained by the news editors from the background information supplied by the inventors: "The FOXO3A gene belongs to the forkhead family of transcription factors which are characterized by a distinct forkhead domain. This gene likely functions as a trigger for apoptosis through expression of genes necessary for cell death. Translocation of this gene with the MLL gene is associated with secondary acute leukemia. Alternatively spliced transcript variants encoding the same protein have been observed.

"The FOXO3A gene is one of the human homologs of DAF-16, a gene that has been described to extend lifespan in the model organisms C. elegans (Murphy C T (2006) The search for DAF-16/FOXO transcriptional targets: approaches and discoveries. Exp Gerontol 41:910-921) and D. melanogaster. (Giannakou M E et al. (2007) Dynamics of the action of dFOXO on adult mortality in Drosophila. Aging Cell 6:429-438).

"The FOXO3A gene is located on human chromosome 6q21, from position 108,987,719 to 109112664 (NCBI ver. 36), is composed of four (4) exons that can be alternately expressed, which results in the same protein (variant #1 is described by file NM.sub.--001455.3; variant #2 is described by file NM.sub.--201559.2. The FOXO3A protein is composed of 673 amino acids and is 71,277 Da in size. The amino acid sequence of FOXO3A, as defined by the file 'NP.sub.--963853' at the NCBI is identified as SEQ ID No. 1 and is the following:

"TABLE-US-00001 MAEAPASPAPLSPLEVELDPEFEPQSRPRSCTWPLQRPELQASPAKPSGE TAADSMIPEEEDDEDDEDGGGRAGSAMAIGGGGGSGTLGSGLLLEDSARV LAPGGQDPGSGPATAAGGLSGGTQALLQPQQPLPPPQPGAAGGSGQPRKC SSRRNAWGNLSYADLITRAIESSPDKRLTLSQIYEWMVRCVPYFKDKGDS NSSAGWKNSIRHNLSLHSRFMRVQNEGTGKSSWWIINPDGGKSGKAPRRR AVSMDNSNKYTKSRGRAAKKKAALQTAPESADDSPSQLSKWPGSPTSRSS DELDAWTDFRSRTNSNASTVSGRLSPIMASTELDEVQDDDAPLSPMLYSS SASLSPSVSKPCTVELPRLTDMAGTMNLNDGLTENLMDDLLDNITLPPSQ PSPTGGLMQRSSSFPYTTKGSGLGSPTSSFNSTVFGPSSLNSLRQSPMQT IQENKPATFSSMSHYGNQTLQDLLTSDSLSHSDVMMTQSDPLMSQASTAV SAQNSRRNVMLRNDPMMSFAAQPNQGSLVNQNLLHHQHQTQGALGGSRAL SNSVSNMGLSESSSLGSAKHQQQSPVSQSMQTLSDSLSGSSLYSTSANLP VMGHEKFPSDLDLDMFNGSLECDMESIIRSELMDADGLDFNFDSLISTQN VVGLNVGNFTGAKQASSQSWVPG

"FOXO3A interacts with YWHAB/14-3-3-beta and YWHAZ/14-3-3-zeta, UniProt: the Universal Protein Resource (www.uniprot.org), which is required for cytosolic sequestration. Upon oxidative stress, interacts with STK4, which disrupts interaction with YWHAB/14-3-3-beta and leads to nuclear translocation. The subcellular location of FOXO3A is in the cytoplasm, and cytosol. It translocates to the nucleus upon oxidative stress and in the absence of survival factors. In the presence of survival factors such as IGF-1, FOXO3A is phosphorylated on Thr-32 and Ser-253 by AKT1/PKB. This phosphorylated form then interacts with 14-3-3 proteins and is retained in the cytoplasm. Survival factor withdrawal induces dephosphorylation and promotes translocation to the nucleus where the dephosphorylated protein induces transcription of target genes and triggers apoptosis. Although AKT1/PKB doesn't appear to phosphorylate Ser-315 directly, it may activate other kinases that trigger phosphorylation at this residue. FOXO3A is phosphorylated by STK4 on Ser-209 upon oxidative stress, which leads to dissociation from YWHAB/14-3-3-beta and nuclear translocation.

"Human longevity is a complex phenotype with multiple determinants. While non-genetic factors, including diet, physical activity, health habits and psychosocial factors are important, up to 50% of the variation in human lifespan might be explained by genetic differences..sup.1-5 Several studies suggest that about 25% of the variation in human lifespan in average-lived populations can be explained by genetic factors but in populations with larger numbers of exceptional survivors the genetic contribution to lifespan may be much higher. For example, family studies of nonagenarians and centenarians show that sibling relative risk, a common method for assessing potential genetic contribution to a complex phenotype.sup.6 is particularly high and grows with increasing age of the proband..sup.7-10 However, studies of candidate 'longevity-associated' genes in humans, hereafter referred to as 'longevity genes,' have generally been disappointing. Few replications have been observed across populations, with the exception of the ApoE gene..sup.3

"In contrast, there have been several robust genetic findings in model organisms of aging..sup.11-13 For example, variation in single genes can result in substantial differences in lifespan in model organisms, particularly with genes that are considered part of the insulin/IGF-1 (HS) signaling pathway..sup.14-18

"Mutations that increase SIR-2 activity or that decrease insulin/IGF-1 signaling both increase the lifespan of C. elegans by activating the DAF-16/FOXO protein..sup.19,20 In mammalian cells, a Sir2 homolog 'SIRT1,' influences several downstream transcription events affecting lifespan, including the cellular response to stress. STRT1 accomplishes this by regulating the FOXO (Forkhead box transcription) factors, a family of proteins that function as sensors in the IIS pathway and are also regulators of longevity in several mammals..sup.17

"Genetic knock-out models in mammals (and other species) have also supported the HS hypothesis. For example, mice with a fat-specific insulin receptor knockout (FIRKO) have reduced fat mass, protection against age-related obesity and have extended longevity..sup.21 Many other mutations in the HS pathway appear to impact longevity in mice. These include mutations in the IGF-1 receptor.sup.22 IRS-1.sup.22 IRS-2.sup.23 PAPP-A.sup.24 and the Ames Dwarf mouse mutation..sup.22

"The basic molecular pathway of insulin signaling is conserved through evolution, evidence of which can be seen in yeast, flies, worms, rodents and humans..sup.25 A key regulator of this pathway in worms is the transcription factor DAF-16 (abnormal DAuer Formation-16), which is required for the large lifespan extension produced in C. elegans by inhibiting insulin/IGF-1 signaling..sup.16 A number of factors appear to extend lifespan in C. elegans in a daf-16 dependent manner, such as AMP kinase.sup.26 14-3-3 proteins.sup.27 the lin-4 microRNA.sup.28 and heat shock factor..sup.29 Homologues of DAF-16 in several species have been linked to aging phenotypes and longevity..sup.30 For example, the stress responsive Jun-N-K terminal Kinase (INK) pathway appears to require FOXO to prolong lifespan in Drosophila.sup.31 and when flies over express dFOXO, the DAF-16 ortholog, it can markedly increase lifespan..sup.32 The remarkable convergence of such a diverse array of signals on DAF-16/FOXO suggests that this protein may be an important, evolutionarily conserved 'node' in a signaling network that impacts aging and longevity.

"The human homologue of DAF-16 includes four FOXOs: FOXO1, FOXO3A, FOXO4 and FOXO6. Therefore, it is tempting to hypothesize that common, natural variation in the form of single nucleotide polymorphisms (SNPs) in FOXO and related genes might influence human longevity. 'FOXO3' is synonymous with 'FOXO3A' since FOXO3B is a pseudo-gene on chromosome 17.

"This is an appealing hypothesis. A connection between insulin, FOXO, oxidative stress and human longevity would be particularly interesting since oxidative stress has long been a favorite putative mechanism of aging. Since 1956, the free radical theory of aging has hypothesized that aging results partly from damage to DNA, cells and tissues from cumulative exposure to reactive oxygen molecules.sup.33 and although not yet universally accepted, supportive evidence has accumulated over the years..sup.34,35 Thus, FOXO may provide a potential branch-point or bridge between insulin signaling, free radicals and human aging/longevity.

"There has been some prior work linking genes in the IIS pathway to human longevity.sup.36,37 including an interesting recent report by Suh et al.sup.38 which links functionally significant IGF-1 receptor mutations to exceptional longevity, but we have not found any published reports of association between FOXO genes and human longevity. Prior studies have found links between FOXO genes and other aging phenotypes, including 4-year survival and stroke risk.sup.39 as well as premature menopause..sup.40

"Human longevity, however, is a complex phenotype that encompasses disease-specific risks as well as the individual rate of aging. The study of its genetic antecedents is challenging. The study of longevity may be affected by small genetic effect sizes, population stratification artifact, population heterogeneity, lack of sufficient numbers of long-lived study participants, and other problems..sup.3,4,41 Therefore, in order to assess potential genetic contributions to human longevity from genes linked to HS signaling, we chose a large, homogeneous, long-lived population of men well characterized for aging phenotypes and we performed a nested-case control study of 5 candidate longevity genes with links to the IIS pathway. These genes were chosen based on prior associations with aging phenotypes principally from gene knockout, transgenic, mutant and other model organisms of aging..sup.3,4,14-17,16,42 Priority was given to genes that are involved in insulin sensitivity and glucose (energy) homeostasis.

"The rapid aging of the population will place unprecedented challenges on society due to increased prevalence of chronic disease and disability..sup.45 Better understanding of mechanisms of aging, including biological pathways that may have widespread influence on how we age, could have important implications for lowering our risk for age-related disease and disability. There are many biologically plausible candidate genes for human longevity but only one finding has so far been widely replicated in multiple populations, that of the ApoE gene..sup.3 This gene has widespread effects on aging phenotypes, particularly cardiovascular disease and dementia, and as such influences the ability to achieve a long and healthy life."

In addition to the background information obtained for this patent application, VerticalNews journalists also obtained the inventors' summary information for this patent application: "The challenge in finding genes that have widespread effects on human aging phenotypes and longevity suggests that it may be helpful to use model organisms to identify a priori potential candidates before conducting human studies. Therefore, we chose to study several candidate genes within the human insulin/IGF-1 signaling pathway and/or oxidative stress response system on the basis of sequence and/or functional homology with model organisms of aging or prior human studies. We constructed a list of human candidate genes from these signaling pathways and assessed variations in these candidate genes occurring at a frequency of approximately 10% or greater in the Japanese population. Due to limited resources, only three SNPs were chosen from each gene for analysis. SNPs were selected from regions with linkage disequilibrium (LD), when possible, in order to provide maximal coverage of each gene.

"In general the invention provides compositions and methods for detecting the FOXO3A 'GCC' haplotype (e.g. a FOXO3A haplotype associated with an increased longevity, defined herein as the likelihood of a human subject living an additional 15 or more years). In preferred embodiments, the detected FOXO3A haplotypes are associated with either an increased likelihood or a decreased likelihood of living longer, however the invention necessarily encompasses materials and methods for detecting a FOXO3A haplotype associated with neither an increased nor a decreased likelihood of living longer and/or minimizing risk for age-associated diseases (e.g. a 'normal' or 'wt' genotype). Age-associated diseases refers to coronary heart disease (CHD), also known as coronary artery diseases, stroke, cancer, chronic obstructive pulmonary disease (COPD) or other chronic lung disease, Parkinson's disease, diabetes, obesity, dementia (and general cognitive function), frailty (ability to walk) or other age-related disease or physical and or cognitive impairment. There may also be an association with obesity in humans.

"The 'GCC' haplotype encompasses tens of kilobases of DNA. Other SNPs in this region demonstrate linkage disequilibrium with the three SNPs described herein. It is anticipated that additional SNPs will be identified within this GCC haplotype that also have an association with longevity and healthy aging, and may be useful of predicting age-associated diseases. The 'GCC haplotype' can serve as a surrogate for other types of alteration of DNA, either within or adjacent to the FOXO3A gene, that is ultimately found to be the 'functional variant' that leads to the prediction of exceptional longevity and/or healthy aging. These other alterations may be in the form of inversions, duplications, deletions, and may include other genes or transcripts that were previously unknown, for example, the gene 'LOC100130966'. LOC100130966 is similar to SMT3 suppressor of mif two 3 homolog 2 has been identified to lie within exon 2 of the FOXO3A gene, which is within the 'GCC' haplotype. The DNA sequence for LOC100130966 is described by the GenBank accession ID# 'XM.sub.--001725519' and the predicted amino acid sequence of LOC100130966 is described by GenBank file 'XP.sub.--001725571'.

"Haplotype analysis may be used to potentially predict which patients would benefit by aggressive wellness or disease prevention/treatment interventions. Haplotype analysis may be provided in a kit form. Risk calculators could use such information for purposes of assessing likelihood of disease, disability or death or determining how many years of survival or disease-free survival a person has. Such information would be important to patients, health insurance companies, long term care insurance companies and physicians or other health care providers in order to provide some guidance as to the patient's long-term needs. Pharmaceuticals could be developed that modify the action of the FOXO3A gene, modify the cellular location of the FOXO3A protein and/or its interactions with other proteins, or modify the amount or type of protein produced by the gene in order to impact health or diseases related to aging.

"Homologous sequences in mice may be associated with premature ovarian failure. Castrillon D H, Miao L, Kollipara R, Horner J W, DePinho R A. Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science. 2003 Jul. 11; 301(5630):215-8. Consequently, similar haplotype analysis can be useful in veterinary applications.

"Further features of the invention will now become apparent from the following description, by way of example only, with reference to the accompanying Figures and Tables.

BRIEF DESCRIPTION OF THE FIGURES

"FIG. 1 shows the results of an ARMS-PCR assay to detect the FOXO3A G/T variants using the primers and conditions described in Table 10.

"FIG. 2 illustrates the schematic outline of the ARMS-PCR assay to detect the FOXO3A G/T variants using the primers described in Table 10.

"The following tables are part of the description:

"Table 1. Baseline Characteristics of the HHP/HAAS Cohort in 1991-93 (n=3,741)

"Table 2. Baseline Characteristics by Case-Control Status

"Table 3. Candidate Genes for Human Longevity and the MAF in Cases and Controls

"Table 4. FOXO 3A3 Genotype by Case-Control Status

"Table 5. Difference in Health Status between Genotype Groups at Baseline

"Table 6. Insulin Sensitivity Phenotypes According to FOXO3A Genotype

"Table 7. Prevalence of Aging-related Phenotypes in Relation to FOXO3A3 Genotype

"Table 8. Genotype Distribution by Maximum Attained Age

"Table 9. Primers for Identification of the rs2802292 G-T Polymorphism Table 10. PCR Conditions for Identification of rs2802292 G-T Polymorphism"

URL and more information on this patent application, see: Donlon, Timothy Atchison; Wilcox, Bradley John; Curb, David J. Method of Using Foxo3a Polymorphisms and Haplotypes to Predict and Promote Healthy Aging and Longevity. Filed August 10, 2015 and posted December 3, 2015. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=4723&p=95&f=G&l=50&d=PG01&S1=20151126.PD.&OS=PD/20151126&RS=PD/20151126

Keywords for this news article include: Kinase, Proinsulin, Peptide Hormones, Enzymes and Coenzymes, Transcription Factors, Kuakini Medical Center.

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