WO2010030600A2

WO2010030600A2 - Methods and compositions for personalization of dietary supplements based on genetic markers

Info

Publication number: WO2010030600A2
Application number: PCT/US2009/056226
Authority: WO
Inventors: David Bearss; Jason Davis; Dallin Anderson
Original assignee: Code Nutrition, Llc
Priority date: 2008-09-10
Filing date: 2009-09-08
Publication date: 2010-03-18

Description

METHODS AND COMPOSITIONS FOR PERSONALIZATION OF DIETARY SUPPLEMENTS BASED ON GENETIC MARKERS

Technical Field

[0001] The present disclosure relates to methods and compositions for personalized dietary supplements formulated using genetic markers. In particular, methods for determining the genotype of an individual, identifying dietary supplements suited to the individual's genotype, and formulating a personalized dietary supplement based on the individual's genotype are described.

Brief Description of the Drawings

[0002] FIG. 1 provides the results of genotyping performed on samples obtained from 201 subjects, together with recommended supplement strategies based on each subject's genotype.

Detailed Description

Definitions

[0003] The term "dietary supplement", as used herein, is a product that is intended to supplement the diet of a subject and contains one or more dietary ingredients including vitamins, minerals, herbs or other botanicals, amino acids and other substances or their constituents. The term "personalized dietary supplement", refers to a dietary supplement that contains one or more dietary ingredients that have been selected for use by a specific individual or subject.

[0004] The term "linkage disequilibrium", as used herein, is the occurrence in a population of certain combinations of alleles, or markers, at two or more loci in greater proportion than expected from random formation of haplotypes based the allele frequencies at the loci. In some populations, linkage disequilibrium may be generally caused by selection, genetic linkage, the rate of recombination, mutation rate, gene flow, random drift or non-random mating and population structure.

Linkage disequilibrium may be used to observe the association of one or more markers with a gene or trait of interest.

[0005] The term "metabolic deficiency", as used herein, is a defect, shortcoming or disorder relating to the metabolism of an individual. For example, a metabolic deficiency may include a disorder related to any of the biochemical processes that occur in an organism such as a disorder related to the breakdown of food and its transformation into energy. [0006] The term "genotype", as used herein, refers to the genetic makeup of an organism. For example, the genotype of an individual may include the genetic constitution or set of DNA variants at one or more loci of an individual. Methods of Determining and Formulating a Personalized Dietary Supplement [0007] The health of an individual is affected by it's behavior, environment and genes. For example, variations in an individual's genes may control how an individual's body responds to a particular diet and how it processes and metabolizes different nutrients. By understanding the variations in an individual's unique genetic make up, or genotype, a diet and lifestyle program, including personalized dietary supplements, can be created to maximize that individual's health. [0008] Methods and compositions for determining and providing personalized dietary supplements to individuals are provided herein. Generally, the methods described herein include obtaining a sample from an individual, genotyping the sample and matching the genotype of the individual to dietary supplements suited to the individual's genotype. Optionally, the methods described herein may include reporting to the individual the dietary supplements suited to such individual's genotype, formulating a personalized dietary supplement based on the individual's genotype, and providing such personalized dietary supplements to the individual. Additionally, the methods described herein may include obtaining lifestyle information from the individual and matching both the individual's genotype and the individual's lifestyle information to dietary supplements suited to the individual's genotype and lifestyle.

[0009] Once obtained, a sample may be genotyped for the presence of one or more genetic markers in linkage disequilibrium with a desired gene or trait. For example, the sample may be genotyped at one or more genetic markers that are in linkage disequilibrium with at least one gene associated with the health and fitness of an individual, and in one embodiment, the sample collected from an individual may be genotyped at one or more genetic markers that are in linkage disequilibrium with at least one gene associated with tissue function, cellular function, and/or a metabolic function or process, etc. In one such embodiment, the one or more genetic markers may be in linkage disequilibrium with at least one gene that is associated with the utilization of B vitamins. In another such embodiment, the one or more genetic markers may be in linkage disequilibrium with at least one gene associated with cholesterol management. In still another embodiment, the one or more genetic markers may be in linkage disequilibrium with at least one gene associated with fat intake and metabolism. In another embodiment, the one or more genetic markers may be in linkage disequilibrium with at least one gene associated with the utilization of vitamin D and calcium. In yet another embodiment, the one or more genetic markers may be in linkage disequilibrium with at least one gene associated with management of oxidative stress. In another embodiment, the one or more genetic markers may be in linkage disequilibrium with at least one gene associated with at least one trait selected from the utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxjdative stress. The one or more genetic markers selected for genotyping may be one or more single nucleotide polymorphisms, or SNPs. For example, the individual may be genotyped at a particular SNP, or a set of SNPs, which might show genetic variation, or alleles, in the individual's genetic information at those particular SNPs that may be associated with a metabolic deficiency. In one such example, one or more SNPs may have a minor allele, wherein the minor allele is associated with the metabolic deficiency. £0010] Once obtained, the individual's genotype may be matched to one or more dietary supplements suited to the individual's genotype. For example, where an individual's genotype includes one or more genetic markers associated with a deficiency, dietary supplements suited to addressing such deficiency can be identified, reported and formulated into a personalized dietary supplement. A personalized dietary supplement as contemplated herein, may include several different supplements or supplement components and may be specifically formulated for the individual to address one or more traits identified in the individual's genotype. In addition to personalized dietary supplements specifically formulated to an individual's genotype, a personalized dietary supplement as contemplated herein may include one or more pre-formulated dietary supplements that may be used to address at least one trait identified in the individual's genotype. As described herein, in addition to the genetic information obtained from an individual, the methods described herein may additionally include collecting lifestyle information from a subject and matching both the individual's genotype and the individual's lifestyle information to suitable dietary supplements. [0011] In specific embodiments, the methods described herein involve genotyping a sample obtained from an individual for genetic markers exhibiting one or more specific selection criteria. For example, the genetic markers may be SNPs selected for one or more of the presence of such SNPs across diverse populations, the strength of an association between the SNP and an identified deficiency, or the relatively high frequency of a given allele.

1. Obtaining a Sample, Genetic Markers and Genotyping

[0012] In order to genotype an individual, a sample is collected from the individual. The sample to be genotyped, includes the individual's genetic code or genome and can be obtained using any suitable technique. For example, the sample can be collected from any tissue from the individual's body containing sufficient genetic material to genotype the sample at one or more genetic markers. In one such example, the sample is collected using a cheek swab, or buccal swab, by rubbing a cotton swab against the inside of an individual's cheek to gather loose cheek cells. In other embodiments, a sample may include a tissue biopsy or other viable tissue or cell sample or may be collected, for example, from the hair, blood, amniotic fluid, or chorionic villi of an individual. Kits suitable for the collection and transport of samples as described herein are readily commercially available. By way of example the sample may be collected using the Isohelix® range of DNA buccal swabs, the Lightning Powder Buccal Swab DNA Collection Kit and the Oragene® DNA Self-Collection Kit.

[0013] Once obtained, a sample taken from an individual may be genotyped for selected genetic markers. In one embodiment, the genetic markers used to genotype a sample may be selected from genetic markers generally known in the art such as SNPs, microsateliites, restriction fragment length polymorphisms (RFLP)₁ amplified fragment length polymorphism (AFLP), random amplification of polymorphic DNA (RAPD), variable number tandem repeat (VNTR), and short tandem repeat (STR). The genetic marker may be proprietary or commercially available and used with techniques and protocols generally known to those of skill in the art. The genetic markers as described herein may be dominant or co-dominant markers suitable for genotyping and identifying genetic variations within and among individuals and populations that may arise due to one or more mutations of the DNA sequence, or genome, of an individual. [0014] In one embodiment, the genetic markers used to genotype the sample may be selected from SNPs that show linkage disequilibrium (LD) with a desired gene or genetic trait. A SNP is a DNA sequence variation occurring when a single nucleotide — A, T, C, or G — in the genome (or other shared sequence) differs between members of a species (or between paired chromosomes in an individual). For example, two sequenced DNA fragments from different individuals, GCCT to GC TT, contain a difference in a single nucleotide at the third position. Therefore, there are two alleles at this SNP locus: C and T. Within a population, SNPs can be assigned a minor allele frequency — the lowest allele frequency at a locus that is observed in a particular population. This is simply the lesser of the two allele frequencies for a SNP. There are variations in SNP allele frequencies between populations, so a SNP allele that is common in one geographical or ethnic population may be much rarer in another.

[0015] In particular embodiments of the methods described herein, one or more genetic markers are selected from SNP markers associated with one or more metabolic deficiencies. Such SNP markers and their alleles may be used to genotype a sample using techniques and procedures well known by those of skill in the art (KJ Livak et al. (1995). Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization". PCR Methods Appl. 4(6):357-62).

[0016] For example, the SNP genotype of a sample may be detected using standard techniques comprising a polymerase chain reaction (PCR), including synthetic primers and conditions appropriate for PCR, DNA sequencing, and other SNP allele identification methods. In another embodiment, a TaqMan® allelic discrimination assay may be used. Genotyping of samples as described herein may also be performed using other techniques such as conformation-based mutation scanning, direct DNA sequencing, PCR-restriction fragment length polymorphism analysis, oligonucleotide ligation assay genotyping, minisequencing, homogeneous solution hybridization using fluorescence resonance energy transfer detection, molecular beacons, the invader assay, DNA microarray genotyping, electrospray or matrix-assisted laser desorption ionization and ion-trap or time-of-flight mass spectroscopy. 2. Selection of Genetic Markers

[0017] As disclosed herein, a sample may be genotyped with one or more genetic markers, wherein the observed genotype may be associated with at least one metabolic deficiency, and wherein the genotype of the sample may be matched with one or more dietary supplements that may be suitable for administration to a subject with the at least one metabolic deficiency. In one embodiment, a sample is genotyped with one genetic marker associated with a single metabolic deficiency that may be matched with one or more dietary supplements. In yet another embodiment, a sample may be genotyped with one or more genetic markers that are associated with multiple metabolic deficiencies, wherein a single dietary supplement, or a combination of dietary supplements, may be matched with the multiple metabolic deficiencies associated with the sample genotype. In yet another embodiment, multiple genetic markers may be selected that are associated with one or more metabolic deficiencies, wherein one or more dietary supplements may be matched with the one or more metabolic deficiencies.

[0018] The genetic markers used for genotyping are chosen according to their association with genetic traits of interest that may be receptive to, or benefit from, dietary supplementation. In one embodiment, genetic markers are chosen that are in linkage disequilibrium with a gene associated with a deficiency. For example, genetic markers may be chosen that are in linkage disequilibrium, or associated, with one or more genes that are associated with a metabolic deficiency, a tissue function deficiency, cellular function deficiency, and/or other deficiencies related to the health and fitness of a subject. In another embodiment, genetic markers used to genotype a sample may be in linkage disequilibrium with at least one gene that is associated with the utilization of B vitamins. In another such embodiment, the one or more genetic markers may be in linkage disequilibrium with at least one gene associated with cholesterol management. In still another embodiment, the one or more genetic markers may be in linkage disequilibrium with at least one gene associated with fat intake and fat metabolism. In another embodiment, the one or more genetic markers may be in linkage disequilibrium with at least one gene associated with the utilization of vitamin D and calcium. In yet another embodiment, the one or more genetic markers may be in linkage disequilibrium with at least one gene associated with management of oxidative stress. In another embodiment, the one or more genetic markers may be in linkage disequilibrium with at least one gene associated with at least one trait selected from the utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress.

[0019] In one embodiment, the one or more genetic markers genotyped are selected due to the extent to which they exhibit linkage disequilibrium with a particular gene associated with a deficiency. Linkage disequilibrium, or "LD," is the non-random association of alleles at two or more loci, not necessarily on the same chromosome. Whereas, linkage equilibrium is a random association of alleles based on their frequency in the population. For example, loci in linkage equilibrium would simply have a frequency in the population equal to the product of their independent frequencies (p x q = pq). A deviation of the observed frequencies from the expected is referred to as the LD parameter, and is commonly denoted by a capital D. [0020] In another embodiment, the one or more genetic markers selected for genotyping are selected because of their frequency across different populations, i.e. their frequency among populations. For example, genetic markers may be selected because of their frequency in samples taken across geographic regions or across different ethnic groups. In one such example, genetic markers, such as SNP markers, are chosen because they have a major or minor allele frequencies sufficient to facilitate the use of the genetic marker to genotype individuals from a broad geographic and genetic background.

[0021] In another embodiment, genetic association studies may be used to identify the genetic markers for a trait of interest like a disease such as diabetes, obesity, cancer, psychiatric illness and cardiovascular disease. Genetic association studies can be performed to determine whether a genetic variant is associated with a disease or trait. If association is present, a particular allele, genotype or haplotype of a polymorphism or polymorphism(s), i.e. SNPs, will be seen more often than expected by chance in an individual carrying the trait. Thus, a person carrying one or two copies of a high-risk variant is at increased risk of developing the associated disease or having the associated trait.

[0022] In another embodiment, the one or more genetic markers selected for genotyping are selected due to their frequency within a population. In one embodiment, the one or more genetic markers selected for genotyping exhibit a minor allele frequency ("MAF") of between approximately 5% and approximately 50%, or higher. In another embodiment, the one or more genetic markers selected for genotyping exhibit a MAF selected from approximately 5% or higher, 6% or higher, 7% or higher, 8% or higher, 9% or higher, 10% or higher, 11% or higher, 12% or higher, 13% or higher, 14% or higher, 15% or higher, 16% or higher, 17% or higher, 18% or higher, 19% or higher, 20% or higher, 21 % or higher, 22% or higher, 23% or higher, 24% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, and 50% or higher. In yet another embodiment, the one or more genetic markers selected for genotyping exhibit a genetic marker, may have a minor allele frequency of approximately 10% or greater in a population. [0023] In one embodiment, SNP marker selection may be directed to increase the chances that at least one typed SNP would be in LD with the potential causative variant for a trait of interest. In one such embodiment, SNP markers may be selected that would be in LD with genetic variants associated with various traits related to one or more areas of nutritional health. In yet another embodiment, SNP allele frequencies may be estimated by gene counting from published studies and public databases and observed for Hardy-Weinberg equilibrium in each population. In one such embodiment, allele frequencies of SNPs may be compared between populations by chi-square or Fisher's exact tests, where appropriate. In another embodiment, the relative power of SNP markers to detect LD may be determined. For example, the distribution of P-values from LD tests may be screened for marker pairs comprising two or more SNPs. In another example, tests of LD between SNP haplotypes and nearby SNPs may be performed. In yet another embodiment, SNP selection may be determined by several factors including the identification of SNPs being in a coding region of genes and likely having a greater an influence on a phenotype or trait of interest.

[0024] In each embodiment, the genetic markers described herein may be SNP markers exhibiting at least one of the selection criteria described herein. For example, in particular embodiments of the methods described herein, one or more SNPs serve as the genetic markers genotyped, each of such one or more SNPs is associated with at least one metabolic deficiency. In particular, in such an embodiment, each of such one or more SNPs is in LD with at least one gene associated with a metabolic deficiency. In one such embodiment the one or more SNPs selected as the one or more genetic markers to be genotyped exhibit a MAF of 10% or greater, are present across diverse populations. In another embodiment SNPs may be chosen with a logarithm of the odds, or LOD scores greater than 3.0 as evidence for linkage with a trait of interest. A LOD score of 3.0 or greater, represents an odds ratio of 1000:1 in favor of linkage, and may be used as the threshold for significant evidence in favor of linkage; this corresponds to a nominal p- value of about 0.05. Conversely, a LOD score of -2.0 or less, odds of 100:1 against linkage, may constitute exclusion of linkage between the trait and marker. [0025] Where SNP markers associated with at least one metabolic deficiency are used to genotype a sample taken from an individual, a sample may be genotyped as described herein, with one or more SNPs associated with the genes controlling at least one trait selected from the utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress. In one such embodiment, a sample may be genotyped with one or more SNPs associated with the metabolism of thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. In another embodiment, the one or more SNPs may be associated with the metabolism of omega-3 fatty acids and vitamin E. In still another embodiment, the one or more SNPs may be associated with the metabolism of garlic supplements, vitamin B3, fiber, policosanol, and guggulipid. In yet another embodiment, the one or more SNPs may be associated with the metabolism of calcium and vitamin D3. In still yet another embodiment, the one or more SNPs may be associated with the metabolism of antioxidants.

[0026] In addition, where SNP are used to genotype a sample taken from an individual, two or more, three or more, four or more, or five or more SNP markers may be selected for genotyping. In one such embodiment, the SNP markers are selected from one or more SNPs associated with a gene controlling at least one trait selected from the utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress. In another such embodiment, five or more SNP markers may be selected for genotyping, wherein at least one of the five or more SNP markers selected is associated with a gene controlling utilization of B vitamins, at least one of the five or more SNP markers selected is associated with a gene controlling cholesterol management, at least one of the five or more SNP markers selected is associated with a gene controlling fat intake and metabolism, at least one of the five or more SNP markers selected is associated with a gene controlling utilization of vitamin D, and at least one of the five or more SNP markers selected is associated with a gene controlling management of oxidative stress. In another such embodiment, the SNP markers are selected from one or more SNPs associated with the metabolism of thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. In yet another embodiment, the SNP markers are selected from one or more SNPs associated with the metabolism of omega-3 fatty acids and vitamin E. In still another embodiment, the SNP markers are selected from one or more SNPs associated with the metabolism of garlic supplements, vitamin B3, fiber, policosanol, and guggulipid. In yet another embodiment, the one or more SNPs may be associated with the metabolism of calcium and vitamin D3. In still yet another embodiment, the SNP markers are selected from one or more SNPs associated with the metabolism of antioxidants.

[0027] In particular embodiments, genotyping a sample obtained from an individual according to the methods described herein may include genotyping one or more SNPs selected from SNPs in linkage disequilibrium or associated with the human gene SLC19A1 , solute carrier family 19 (folate transporter), member"! , the human gene APOA1 , apolipoprotein A-I, the human gene APOA5, apolipoprotein A- V, gene VDR, vitamin D (1 ,25- dihydroxyvitamin D3) receptor, and the human gene NOS3, nitric oxide synthase 3. In one such embodiment, genotyping a sample obtained from an individual includes genotyping the sample for at least five SNPs, wherein SNPs genotyped include at least one SNP in linkage disequilibrium or associated with each of the human gene SLC19A1 , solute carrier family 19 (folate transporter), memberi , the human gene APOA1 , apolioprotein A-I, the human gene APOA5, apolipoprotein A-V, gene VDR, vitamin D (1 ,25- dihydroxyvitamin D3) receptor, and the human gene NOS3, nitric oxide synthase 3. [0028] Specific SNPs that may be used to genotype a sample according to the methods describe herein include, for example, SNP rs1051266 (SEQ ID NO: 1), which is an SNP marker associated with the human SLC19A1 gene, SNP rs670 (SEQ ID NO: 2), which is an SNP marker associated with the human APOA1 gene, SNP rs662799 (SEQ ID NO: 3), SNP rs731236 (SEQ ID NO: 4), which is an SNP marker associated with the human APOA5 gene, and SNP rs2070744 (SEQ ID NO: 5), which is an SNP marker associated with the human NOS3 gene. In specific embodiments of the methods described herein, the sample obtained from an individual is genotyped for two, three, four, or all five of these referenced SNPs.

3. Matching of Genotype to Dietary Supplements

[0029] Once a sample of the individual has been genotyped, the resulting information can be matched with one or more dietary supplements suited to the individual's genotype. For example, where an individual's genotype is associated with a deficiency in the processing or production of one or more vitamins or nutrients a dietary supplement suitable for administration to the individual and providing the individual with such one or more vitamins or nutrients may be matched for inclusion in a dietary supplement personalized to the individual. Additionally, where an individual's genotype is associated with a deleterious condition or an increased risk for developing a deleterious condition, a dietary supplement suitable for administration to the individual and identified as potentially beneficial in addressing such deleterious condition or reducing the risk of developing such deleterious condition may be matched for inclusion in a dietary supplement personalized to the individual.

[0030] In one embodiment, genotyping a sample obtained from an individual includes genotyping the sample for a genetic marker associated with a deficiency in utilization of B vitamins. For example, genotyping the sample obtained from the individual include genotyping the SNP rs1051266. Where genotyping of the sample reveals the presence of the marker, one or more supplements suited to addressing a deficiency in utilization of B vitamins may be matched to the individual. Dietary supplements suitable for administration to an individual exhibiting a deficiency in utilization of B vitamins include Vitamin B1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine), Vitamin B7 (biotin), Vitamin B9 (folic acid) and Vitamin B12 (cobalamin). [0031] In a specific embodiment, one or more or all of the dietary supplements included in Formula 1 , as detailed in Table 1 , may be matched to an individual having a genotype associated with a deficiency in utilization of B vitamins. Though specific amounts of each of the supplements included in Formula 1 are described in Table 1 , such amounts may vary. Table 1

[0032] In another embodiment, genotyping a sample obtained from an individual includes genotyping the sample for a genetic marker associated with a deficiency in cholesterol management. For example, genotyping the sample obtained from the individual include genotyping the SNP rs670. Where genotyping of the sample reveals the presence of the marker, one or more supplements suited to addressing a deficiency in cholesterol management may be matched to the individual. Dietary supplements suitable for administration to an individual exhibiting a deficiency in cholesterol management include DHA (Cocosahexaenoic Acid - Omega 3) EPA (Eicosapentaenoic Acid - Omega 3) Vitamin B-1 (Thiamin Mononitrate) Riboflavin (Vitamin B-12) Niacin (Niacinamide) Oleic Acid Policosanol Aged Garlic Extract. In a specific embodiment, one or more or all of the dietary supplements included in Formula 2/3, as detailed in Table 2, may be matched to an individual having a genotype associated with a deficiency in cholesterol management. Though specific amounts of each of the supplements included in Formula 2/3 are described in Table 2, such amounts may vary.

Table 2

[0033] In another embodiment, genotyping a sample obtained from an individual includes genotyping the sample for a genetic marker associated with a deficiency in fat intake and metabolism. For example, genotyping the sample obtained from the individual include genotyping the SNP rs66279. Where genotyping of the sample reveals the presence of the marker, one or more supplements suited to addressing a deficiency in fat intake and metabolism may be matched to the individual. Dietary supplements suitable for administration to an individual exhibiting a deficiency in fat intake and metabolism include DHA (Cocosahexaenoic Acid - Omega 3), EPA (Eicosapentaenoic Acid - Omega 3), Vitamin B-1 (Thiamin Mononitrate, Riboflavin (Vitamin B-12), Niacin (Niacinamide), Oleic Acid, Policosanol, Aged Garlic Extract In a specific embodiment, one or more of the dietary supplements included in Formula 2/3, as detailed in Table 2, may be matched to an individual having a genotype associated with a deficiency in fat intake and metabolism. Though specific amounts of each of the supplements included in Formula 2/3 are described in Table 2, such amounts may vary.

[0034] In another embodiment, genotyping a sample obtained from an individual includes genotyping the sample for a genetic marker associated with a deficiency in utilization of vitamin D and calcium. For example, genotyping the sample obtained from the individual include genotyping the SNP rs731236. Where genotyping of the sample reveals the presence of the marker, one or more supplements suited to addressing a deficiency in utilization of vitamin D and calcium may be matched to the individual. Dietary supplements suitable for administration to an individual exhibiting a deficiency in utilization of vitamin D and calcium include Vitamin D (Cholecalciferol) Calcium (Calcium Carbonate) Magnesium (Magnesium Oxide) Vitamin A (50% beta- Carotene, 50% Retinyl Acetate) Vitamin C. In a specific embodiment, one or more or all of the dietary supplements included in Formula 4, as detailed in Table 3, may be matched to an individual having a genotype associated with a deficiency in utilization of vitamin D and calcium. Though specific amounts of each of the supplements included in Formula 4 are described in Table 3, such amounts may vary. Table 3

[0035] In another embodiment, genotyping a sample obtained from an individual includes genotyping the sample for a genetic marker associated with a deficiency in management of oxidative stress. For example, genotyping the sample obtained from the individual include genotyping the SNP rs2070744. Where genotyping of the sample reveals the presence of the marker, one or more supplements suited to addressing a deficiency in management of oxidative stress may be matched to the individual. Dietary supplements suitable for administration to an individual exhibiting a deficiency in management of oxidative stress include Vitamin A, Vitamin C, Vitamin E, Selenium, (Selenium Yeast), Lycopene, Green Tea Leaves Extract (Camellia sinensis), Grape Seed Extract (Vitis vinifera), Coenzyme Q10 (ubiquinone) and Lutein. In a specific embodiment, one or more or all of the dietary supplements included in Formula 5, as detailed in Table 4, may be matched to an individual having a genotype associated with a deficiency in management of oxidative stress. Though specific amounts of each of the supplements included in Formula 5 are described in Table 4, such amounts may vary.

Table 4

[0036] Of course, as described herein, genotyping a sample obtained from an individual may, in some embodiments, involve genotyping the sample for multiple genetic markers. Where multiple genetic markers are genotyped and two or more markers associated with a metabolic deficiency are identified, each of the identified markers may be matched with dietary supplements suited to address the associated deficiency. In one such embodiment, genotyping a sample includes genotyping the sample for at least one SNP associated with each of a deficiency in the utilization of B vitamins, a deficiency in cholesterol management, a deficiency of fat intake and metabolism, a deficiency in utilization of vitamin D and calcium, and a deficiency in management of oxidative stress, and where such genotyping of identifies one or more SNPs associated with a deficiency, the dietary supplements and supplement formulations as described herein may be matched to the identified genotype to address each identified deficiency.

[0037] After matching one or more dietary supplements to an individual's genotype, a dietary supplement personalized to the individual can be provided. In one embodiment, a personalized dietary supplement includes one or more fully customized formulations matched to the genotype of the sample obtained from an individual. In such an embodiment, the dietary supplement is formulated after the individual's genotype is identified and the one or more compositions included in the personalized dietary supplement are formulated to specifically address the genotype of the individual. For example, a fully customized dietary supplement may be created by combining the appropriate individual nutrients together into a single dietary supplement or package to be distributed to an individual. In one such embodiment, the fully customized dietary supplement may be formulated by inputting the genotype or formulation information into a device, which device then automatically collects appropriate individual dietary supplements and combines them into one or more dietary supplement packages or formulations. A customized formulation may be presented to the individual in any forms that is readily ingested by the individual. In one embodiment, the a customized formulation may be presented to the individual as one or more dried powder packets, one or more tablet, one or more gel capsules, or combinations thereof.

[0038] In one embodiment, one or more manufacturers of packaging and dispensing equipment for the Pharmaceutical, Nutraceutical and Medical Industries may be used for formulation. In one such example, a manufacturer may provide individual modules in counting/dispensing/product validation as well as complete automated packaging, sortation and manifest solutions which gives the ability to completely automate customization of combinations of tablets and capsules. In another embodiment, the process of formulation may include order fulfillment software, multiple mixing of tablets in packets and high volume tablet feed systems for larger production including variable product information printed onto each pouch, automatic tablet counting & dispensing, high speed packet registration, security label validation, in-line carton erection and closing, case manifesting with product and/or case sortation, operator reject station and record keeping of all packages filled and manifested.

[0039] In another embodiment, in order to facilitate order fulfillment, reduce costs for the consumer, while still providing a customized nutritional supplement, a selection of pre-made, semi-customizable formulations can be created to fit particular genotypes and metabolic deficiencies. In one such embodiment, a plurality of genetic markers are identified and a selection of pre-made dietary supplement formulations are produced to match the most common combinations of such traits included in a population. Upon genotyping an individual, one or more of the pre-made dietary supplement formulations can then be matched and provided to the individual based on the individual's genotype at the identified plurality of genetic markers.

[0040] In yet another embodiment, a base dietary supplement formulation may be provided that can be combined with pre-made or fully customized dietary supplements matched based on an individual's genotype. For example, a personalized dietary supplement formulation can be combined with a base dietary supplement formulation that includes one or more or all of the dietary supplements included in Base Formula 0, which is detailed in Table 5. Generally, a base formula may include: Biotin, Calcium, Chromium, Copper, Folate (As Folic Acid), Iodine, Iron, Magnesium, Manganese, Molybdenum, Selenium, Vitamin A, Vitamin B12, Vitamin K, Zinc, Vitamin C, Vitamin D, Vitamin E, Thiamin, Riboflavin, Niacin, Vitamin B6, Pantothenic Acid. Though specific amounts of each of the supplements included in Base Formula 0 are described in Table 5, such amounts may vary. Table 5

[0041] Where desired, in addition to the genetic data obtained from an individual, lifestyle data may also be obtained. For example, an individual may be provided with a lifestyle survey, and the information provided in the lifestyle survey may then be used in addition to the individual's genotype to match dietary supplements suited to the individual and provide a personalized dietary supplement. In one such embodiment, a lifestyle survey may be completed by an individual to provide information regarding diet, exercise, and other behaviors that may affect the health and fitness of the individual. The lifestyle survey information may be used in combination with the genotype information to formulate a dietary supplement with maximum benefit for the individual. Examples

Example 1

Selection of Genetic Markers Associated with Metabolic Deficiency [0042] Genetic variation in the form of SNPs may be used as genetic markers to identify individuals with certain traits. For example, the identification of SNPs associated with metabolic deficiencies may be used to formulate a personalized dietary supplement for an individual. The SNPs described herein were selected based on their association with metabolic genes and their frequency in the population. SNPs with MAFs over or equal to 10% were selected for the analyses of SNP transferability across populations.

A. Selection of SNP rs1051266

[0043] The SNP rs1051266 is located in the human gene SLC19A1. The SLC19A1 gene is involved in the transport of folate compounds into mammalian cells via receptor-mediated or carrier-mediated mechanisms. Folate is the anion form of folic acid, also known as vitamin Bg. Folate is necessary for the production and maintenance of new cells. This is especially important during periods of rapid cell division and growth such as infancy and pregnancy. Folate is also needed to synthesize DNA bases (most notably thymine, but also purine bases) needed for DNA replication. Thus folate deficiency, or a deficiency in folate transport into the cells, hinders DNA synthesis and cell division, affecting most notably bone marrow and cancer, both of which participate in rapid cell division.

[0044] The SNP rs1051266 is an 80G/A polymorphism located in exon 2 of SLC19A1. The SNP rs1051266 has a frequency of G/G 28.8%, G/A 55.2% and A/A 16% in a general population study from the United States.

B. Selection of SNP rs670

[0045] The SNP rs670 is a -75G/A SNP in the promoter of the human APOA1 (apolipoprotein A-I) gene. The product of APOA1 is the major protein component of high density lipoprotein (HDL) in plasma. The protein promotes the clearing of cholesterol from tissues to the liver for excretion.

[0046] The SNP rs670 has been reported to be associated with HDL-C concentrations as well as HDL-C response to dietary changes in polyunsaturated fat intake. The amount of the large HDL subfraction has been shown to be higher in G homozygotes and lower in the A carriers. In contrast, the amount of the small HDL subtraction is lower in G homozygotes and higher in A carriers (G. Ruano et al. (2005). "Apolipoprotein A1 genotype affects the change in high density lipoprotein cholesterol subfractions with exercise training". Arthrosclerosis, 185(1): 65-69; C. Lahoz et al. (2003). "Apo A-I promoter polymorphism influences basal HDL- cholesterol and its response to pravastatin therapy". Atherosclerosis 168(2): 289- 95).

C. Selection of SNP rs662799

[0047] The SNP rs662799 is a -1131T/C SNP in the promoter of the human APOA5 gene. The protein encoded by the APOA5 gene is an apolipoprotein and an important determinant of plasma triglyceride levels, a major risk factor for coronary artery disease. It is a component of several lipoprotein fractions including VLDL₁ HDL, chylomicrons. It is believed that APOA5 affects lipoprotein metabolism by interacting with LDL-R gene family receptors.

[0048] The SNP rs662799 has be reported to be associated with triglyceride levels and HDL-C. For example, rs662799 was shown to account for 3.5% of the phenotypic variation in triglyceride levels for a population (Y. Liao et al. (2008). "Multiple genetic determinants of plasma lipid levels in Caribbean Hispanics". Clinical Biochem., 41 (4-5); 306-312).

D. Selection of SNP rs731236

The SNP rs731236 a CTT SNP located in the region of the human vitamin D receptor (VDR) gene. The global allele frequencies for this SNP are C/C 7% C/T 38% and TfT 55%. Vitamin D receptor (VDR), a member of the steroid/thyroid hormone nuclear receptor family, is bound by the steroid hormone 1 ,25-dihydroxyvitamin D₃.

E. Selection of SNP rs2070744

[0049] The SNP rs2070744 is a -786T/C SNP located in the promoter of the human NOS3 gene. Nitric oxide (NO) is produced from L-arginine by the action of the nitric oxide synthase (NOS). There are three NOS isoforms (NOS1 , NOS2, and NOS3). NOS3 (also called endothelial NOS, or eNOS) is the predominant isoform in most tissues. NOS3 is involved in the proliferation of neuronal progenitor cells, which might be implicated in the pathophysiology of depressive disorders. Prior genetic association studies have suggested a possible role of NOS3 gene polymorphisms in affective disorders. [0050] The SNP rs2070744 has been associated with oxidative stress. Haplotype analysis showed a statistically significant association between NOS3 gene variants and features of metabolic syndrome mediated by oxidative stress (Clinical Chemistry. 2007;53:91-97). Global allele frequencies of C/C 11% CfT 50% TfT 39%.

Example 2

Genotypinq of Samples at the Selected SNPs

[0051] Genetic samples were collected from 201 individuals using a buccal swab. The samples were stored at room temperature until genetic analysis was performed. [0052] For each sample, the DNA was extracted with standard techniques. The SNPs rs1051266, rs670, rs66279, rs731236, and rs2070744 were genotyped for each sample according to methods known by those of skill in the art. Briefly, specific oligonucleotide primers were used in a standard PCR reaction to amplify DNA fragments with the target SNPs. The SNP genotype of the amplified fragments were identified using the TaqMan® Allelic Discrimination real-time PCR assay and performed according to the TaqMan® instructions. More particularly, real-time PCR was performed in 5.0 μl mixtures containing 2.55 μl of TaqMan® genotyping master mix, 0.25 μl of TaqMan® SNP assay mix and 2.20 μl of sample DNA (1-10 ng). Thermocycling was performed on the GeneAmp® PCR System 9700 (Applied Biosystems) and consisted generally of 10 min at 95°C and 40 cycles of 95⁰C for 15 s and 6O⁰C for 1 min, followed by a -4°C hold. More specifically, for the SNP rs1051266, primers FN12S and FN12A were used with a 59⁰C annealing temperature. For rs670, suitable primers were used with an annealing temperature appropriate for TaqMan®. For rs662799, suitable primers were used with a 70⁰C annealing temperature. For rs731236, suitable primers were used with a 57°C annealing temperature. For rs2070744, suitable primers were used with a 58°C annealing temperature. Analysis was performed using SDS, version 2.3 software (Applied Biosystems). The results of the genotyping performed on each of the samples obtained are provided in FIG. 1. In addition, as provided in the last column, recommended supplement strategies are also provided in FIG. 1 based on the subjects' genotype, with the numbers reflected in the last column correlating with the numbers of the formulations described herein.

[0053] The TaqMan® Allelic Discrimination assay uses the 5' nuclease activity of Taq polymerase to detect a fluorescent reporter signal generated during or after PCR reactions. For SNP genotyping, one pair of TaqMan® probes and one pair of PCR primers are used. The assay uses two TaqMan® probes that differ at the polymorphic site, with one probe complementary to the major allele and the other to the minor allele. A 5' reporter dye and a 3' quencher dye are covalently linked to the major or minor allele probes. When the probes are intact, fluorescence is quenched because of the physical proximity of the reporter and quencher dyes. During the PCR annealing step, the TaqMan® probes hybridize to the targeted polymorphic site. During the PCR extension phase, the 5¹ reporter dye is cleaved by the 5¹ nuclease activity of the Taq polymerase, leading to an increase in the characteristic fluorescence of the reporter dye. Specific genotyping is determined by measuring the signal intensity of the two different reporter dyes after the PCR reaction. [0054] It will be obvious to those having skill in the art that many changes may be made to the details of the above-described embodiments without departing from the underlying principles of the invention. The scope of the present invention should, therefore, be determined only by the following claims.

Claims

1. A method of determining a personalized dietary supplement regimen for a subject, the method comprising:

obtaining a sample from the individual;

genotyping the sample for the presence of at least one genetic marker linked to a gene associated with at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress; and

identifying one or more dietary supplements suitable for administration to the subject, wherein each of the one or more identified dietary supplements corresponds to at least one of the at least one genetic marker present in the sample.

2. The method of claim 1 , further comprising reporting to the subject one or more dietary supplements corresponding to each of the at least one genetic marker present in the sample.

3. A method according to claim 1 or claim 2, further comprising:

obtaining lifestyle information from the subject; and

identifying one or more lifestyle recommendation in response to the lifestyle information received from the subject.

4. A method according to claim 3, wherein identifying one or more dietary supplements suitable for administration to the subject comprises identifying one or more identified dietary supplements that corresponds to at least one of the at least one genetic marker present in the sample and at least one of the one or more dietary supplements is identified in response to the lifestyle information received from the subject.

5. A method according to any preceding claim, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for at least one Single Nucleotide Polymorphism (SNP) associated with at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress.

6. A method according to any preceding claim, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for at least one SNP associated with a deficiency in at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress.

7. A method according to any preceding claim, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for at least one SNP associated with at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress, wherein each of said at least one SNP exhibits a minor allele frequency of about 10% or greater.

8. A method according to any preceding claim, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for at least one SNP associated with at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress, wherein each of said at least one SNP is present in diverse populations

9. A method according to any preceding claim, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for at least one SNP associated with at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress, wherein each of said at least one SNP shows a linkage with the trait with a LOD score of 3.0 or greater.

10. A method according to any preceding claim, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for two or more SNPs associated with at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress.

11. A method according to any preceding claim, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for three or more SNPs associated with at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress.

12. A method according to any preceding claim, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for four or more SNPs associated with at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress.

13. A method according to any preceding claim, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for five or more SNPs associated with at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress.

14. A method according to any preceding claim, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for an SNP selected from the group consisting of rs1051266, rs670, rs662799, rs731236, and rs2070744.

15. A method according to any preceding claim, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for each of rs1051266, rs670, rs662799, rs731236, and rs2070744.

16. A method of formulating a nutritional supplement personalized for a subject, the method comprising:

obtaining a sample from the individual;

genotyping the sample for the presence of at least one genetic marker linked to a gene associated with a deficiency in at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress;

matching one or more dietary supplements suitable for administration to the subject with the deficiency corresponding to each of the at least one genetic marker present in the sample; and

formulating a nutritional supplement including each of the one or more dietary supplements matched to the deficiency corresponding to each of the each of the at least one genetic marker present in the sample.

17. The method of claim 16, further comprising providing the formulated nutritional supplement to the subject.

18. A method according to claim 16 or claim 17, further comprising:

obtaining lifestyle information from the subject; and

19. A method according to any of claims 16 through 18, wherein matching one or more dietary supplements suitable for administration to the subject further comprises matching one or more dietary supplements to the lifestyle information received from the subject and formulating the nutritional supplement comprises formulating a nutritional supplement including the one or more dietary supplements matched to the lifestyle information received from the subject.

20. A method according to any of claims 16 through 19, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for at least one SNP associated with a deficiency in at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress, wherein each of said at least one SNP exhibits a minor allele frequency of about 10% or greater.

21. A method according to any of claims 16 through 20, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for at least one SNP associated with a deficiency in at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress, wherein each of said at least one SNP is present in diverse populations.

22. A method according to any of claims 16 through 20, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for at least one SNP associated with a deficiency in at least one trait selected from utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress, wherein each of said at least one SNP shows a linkage with the at least one trait with a LOD score of 3.0 or greater

23. A method according to any of claims 16 through 22, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for an SNP selected from the group consisting of rs1051266, rs670, rs662799, rs731236, and rs2070744.

24. A method according to any claims 16 through 23, wherein genotyping the sample for the presence of at least one genetic marker comprises genotyping the sample for each of rs1051266, rs670, rs662799, rs731236, and rs2070744.

25. A method according to any of claims 16 through 24, wherein matching one or more dietary supplements suitable for administration to the subject with the deficiency corresponding to each of the at least one genetic marker present in the sample comprises at least one of identifying one or more dietary supplements corresponding to a deficiency in utilization of B vitamins, identifying one or more dietary supplements corresponding to a deficiency in cholesterol management, identifying one or more dietary supplements corresponding to a deficiency in fat intake and metabolism, identifying one or more dietary supplements corresponding to a deficiency in utilization of vitamin D or calcium, and identifying one or more dietary supplements corresponding to a deficiency in management of oxidative stress.

26. A method according to claim 25, wherein identifying one or more dietary supplements corresponding to a deficiency in utilization of B vitamins comprises identifying a dietary supplement formulation comprising one or more constituents identified in Formula 1.

27. A method according to claim 25, wherein identifying one or more dietary supplements corresponding to a deficiency in cholesterol management comprises identifying a dietary supplement formulation comprising one or more constituents identified in Formula 2/3.

28. A method according to claim 25, wherein identifying one or more dietary supplements corresponding to a deficiency in fat intake and metabolism comprises identifying a dietary supplement formulation comprising one or more constituents identified in Formula 2/3.

29. A method according to claim 25, wherein identifying one or more dietary supplements corresponding to a deficiency in utilization of vitamin D or calcium comprises identifying a dietary supplement formulation comprising one or more constituents identified in Formula 4.

30. A method according to claim 25, wherein identifying one or more dietary supplements corresponding to a deficiency in management of oxidative stress comprises identifying a dietary supplement formulation comprising one or more constituents identified in Formula 5.

31. A method of formulating a nutritional supplement personalized for a subject, the method comprising:

obtaining a sample from the individual;

genotyping the sample for the presence of single nucleotide polymorphisms (SNPs) associated with deficiencies in utilization of B vitamins, cholesterol management, fat intake and metabolism, utilization of vitamin D and calcium, and management of oxidative stress, wherein the SNPs comprise rs1051266, rs670, rs662799, rs731236, and rs2070744;

providing a plurality of pre-made dietary supplement formulations matched to one or more deficiencies associated with the presence of rs1051266, rs670, rs662799, rs731236, and rs2070744; and

providing the subject with one or more of the plurality of pre-made dietary supplement formulations according to the results of the genotyping.

2. A method according to claim 31 , wherein providing a plurality of pre-made dietary supplement formulations comprises providing a formulation selected from a formulation matched to the presence of rs1051266, wherein the formulation comprises one or more constituents identified in Formula 1 , a formulation matched to the presence of rs670, wherein the formulation comprises one or more constituents identified in Formula 2/3, a formulation matched to the presence of rs662799, wherein the formulation comprises one or more constituents identified in Formula 2/3, a formulation matched to the presence of rs731236, wherein the formulation comprises one or more constituents identified in Formula 4, and a formulation matched to the presence of rs2070744, wherein the formulation comprises one or more constituents identified in Formula 5.