US007970552B1

(12) United States Patent (10) Patent No.: US 7,970,552 B1

Stefanon et al. 45) Date of Patent: Jun. 28,9 2011

(54) DIAGNOSTIC SYSTEM FOR SELECTING 3. R 238, E. 1 NUTRITION AND PHARMACOLOGICAL 6,493,641- - w B1 12/2002 Singheblak et et al. al. PRODUCTS FOR ANIMALS 6,537,213 B2 3/2003 Dodds 6,730,023 B1 5, 2004 Dodds (76) Inventors: Bruno Stefanon, Martignacco (IT): W. 7,029.441 B2 4/2006 Dodds Jean Dodds, Santa Monica, CA (US) 7,296,5377,134,995 B2 12/200611/2006 BurghardiDodds et al. (*) Notice: Subject to any disclaimer, the term of this (Continued) patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS WO WO99-67642 A2 12/1999 (21) Appl. No.: 12/927,769 (Continued) (22) Filed: Nov. 19, 2010 OTHER PUBLICATIONS Related U.S. Application Data Swanson et al., “Nutritional Genomics: Implication for Companion Animals'. The American Society for Nutritional Sciences, (2003).J. (63) Continuation of application No. 12/316,824, filed on Nutr. 133:3033-3040 (18 pages). Dec. 16, 2008, now Pat. No. 7,873,482. (Continued) (51) Int. Cl. G06F 9/00 (2006.01) Primary Examiner — Edward Raymond (52) U.S. Cl...... 702/19 (74) Attorney, Agent, or Firm — Greenberg Traurig, LLP 58) Field of Classification Search ...... 702/19, (58) 702/182 185 (57) ABSTRACT See application file for complete search history. An analysis of the profile of a non-human animal comprises: a) providing a genotypic database to the species of the non (56) References Cited human animal Subject or a selected group of the species; b) obtaining animal data; c) correlating the database of a) with U.S. PATENT DOCUMENTS the data ofb) to determine a relationship between the database 3,995,019 A 11, 1976 Jerome of a) and the data of b); c) determining the profile of the 5,691,157 A 1 1/1997 Gong et al. animal based on the correlating step; and d) determining a 5,817,025 A 10, 1998 Alekseev et al. genetic profile based on the molecular dietary signature, the 5,830,709 A 11/1998 Benson et al. 5,911,687 A 6, 1999 Sato et al. molecular dietary signature being a variation of expression of 5,954,640 A 9, 1999 Szabo a set of which may differ for the genotype of each 6,018,786 A 1/2000 Kricket al. animal or a group of animals Nutrition and pharmalogical 6,063,028 A 5, 2000 Luciano assessments are made. Reporting the determination is by the 6,081,786 A 6/2000 Barry et al. 6,135,055 A 10, 2000 Pratt Internet, and payment for the report is obtained through the 6,136,055 A 10, 2000 Stanek Internet. 6,156,355 A 12/2000 Shields, Jr. et al. 6,218,122 B1 4/2001 Friend et al. 24 Claims, 23 Drawing Sheets 6,232,522 B1 5/2001 Harley et al. (6 of 23 Drawing Sheet(s) Filed in Color)

A-38 US 7,970,552 B1 Page 2

U.S. PATENT DOCUMENTS Swanson et al., “Canine Nutritional Model: Influence of Age, Diet, 2002/0022772 A1 2/2002 Dodds and Genetics on Health and Well-Being'. Current Nutrition & Food 2003. O135096 A1 7, 2003 Dodds Science, vol. 2, No. 2, May 2006, pp. 115-126(12) (1 page). 2003/0233984 A1 12/2003 van de Ligt et al. M.F. Böttcher et al., “Total and allergen-specific immunoglobulin A 2005/00907 18 A1* 4/2005 Dodds ...... 600/300 levels in Saliva in relation to the development of allergy in infants up 2006, OO45909 A1 3, 2006 Friesen et al. to 2 years of age” Clin. Exp. Allergy Sep. 2002, vol. 32, No. 9, pp. 2006, OO64250 A1 3, 2006 Goldstein 1293-1298. 2006/0200320 A1 9, 2006 Al-Murrani 2006/0283393 Al 12/2006 Burghardi et al. A.P. Foster et al., "Serum IgE and IgG responses to food antigens in 2007/0118295 A1 5, 2007 Al-Murrani normal and atopic dogs, and dogs with gastrointestinal disease' Vet. Immunol. Immunopathol. May 2003, vol. 92, pp. 113-124. FOREIGN PATENT DOCUMENTS Michael J. Day, “The Canine Model of Dietary Hypersensitivity” WO WOO1,28415 A1 4/2001 Proc. Nutr. Soc. Nov. 2005, vol. 64, No. 4, pp. 458-464. WO WOO1,69487 A1 9, 2001 Dodds (Veterinary Practice, 1992, vol. 4. No. 2, p. 25-31). WO WO 2004f1 1357O A2 12/2004 Hetzel et al., (The Journal of Nutrition, 1989, vol. 119, p. 145-151). WO WO 2010/075.007 A3 T 2010 Williams et al., (Nucleic Acids Research, 1990, vol. 18, No. 22, p. WO WO 2010/075,009 A3 T 2010 6531-6535). OTHER PUBLICATIONS Dodds (Advances in Veterinary Science and Comparative Medicine, Swanson et al., “Diet Affects Nutrient Digestibility, Hematology, and 1995, vol.39, p. 29-95). Serum Chemistry of Senior and Weanling Dogs”, Journal of Ameri R. Wolter, “La nutrition de l'animal de sport” Laboratoire de Nutri can Science, (2004) J. Anim. Sci. 2004. 82:1713-1724 (19 pages). tion sportive (INRA) Ecole Nationale Veterinaired Alfort, Science & Kato et al., “A Perspective on DNA Microarray Technology in Food Sports 2, (1987) p. 63-93. and Nutritional Science'. Current Opinion in Clinical Nutrition and Harrison et al., The Journal of Nutrition, 1987, vol. 117, p. 376-382. Metabolic Care, (2005) 8:516-522 (7 pages). K. McNutt, Phd., J.D., “The Individualized Prescriptive Foods Era Spielbauer et al., “Impact of Microarray Technology in Nutrition and Has Dawned'. Nutrition Today, May/Jun. 1993, p. 43-47, USA. Food Research”. (2005) Mol. Nutr. Food Res., 49, 908-917 (10 pages). * cited by examiner U.S. Patent Jun. 28, 2011 Sheet 1 of 23 US 7,970,552 B1

38. • registists, SN's * ixitxeities: * sistice ixitxxix.

* xxxxiigists&state:

* g ex38xisg, sixxiiity

Nutriest sex-axwww.www.wawawawawawao

tis 888& health and receix iggeraxtix: production sexes kxxxixi, sigs: taxsixties: * 88xyge regixties: *ish::is raisixties: * taxesgxi regisixties * gags: 8 gigs intexxsetix8.

MRTAssoirrg" ssex sistics

F.G. 1

U.S. Patent Jun. 28, 2011 Sheet 3 of 23 US 7,970,552 B1

EEE CE AAS: A3, AASE SE CY8. GENOTYPE AND FUNCTIONAL {x^i} ^{{^A. (Ex: { ECC 30: - 3.x: - 3\{Xiaxi NORMAL (HEALTHY) ANIMALS (JN-EA... Y. ANiviAS

-arror

x 3Ex AASE

U.S. Patent Jun. 28, 2011 Sheet 4 of 23 US 7,970,552 B1

£Nia 3-8 fri: 888 NESS 8: 88,3-33

– EEE-388: 83C: Rikas. 3888 -80. 8Y 83&. 8NA ANAYS$8 e.g., 88:8AR8AY

C:\;x&S R388 C38A-33. 388. A 3888 & ES fro- Ex; 28:88 ER

N. & ASE EC38:38.8 WAKA8. CSS is 83- 3 {Chi8:ENS is SEN: YE

GENOvic PROFES OF ANMAI.S wir-i : FERENT 3ENory:RES, IE&Y }}E N:3 A. 8 PATHOLOGY OF THE AN-MA. SENSYE

ker------T- EFFECT ON THE FunCEONA. GENOMiC C. C. E. N TRENT A-A.

AA3 from s------Exi Y SER

------aa--- a--- h;$8.

ESR; Nixi:NS

U.S. Patent Jun. 28, 2011 Sheet 7 of 23 US 7,970,552 B1

01| 01+vo|| 1+o-go|1

V7|1

w c:

-: Y:

re U.S. Patent Jun. 28, 2011 Sheet 8 of 23 US 7,970,552 B1

KEEE CE SA38 xxi.A. fxxix.3 TESTx £xSAMPLE {{- OF{* AN{x^{S ANIMAL

xasy-awa'assassaxxasaaxa-swaxxYaxxas:

{ i Niš (E^{i 38:

NORRA -EA-Y. ABKOREA. N-EATY

:^COxi.A. (Ex.: FuNCTIONAL GENOMC 88: 3.

wwaawaawaawwaawa.

EN AAS:

a 3&

FG. 6A U.S. Patent Jun. 28, 2011 Sheet 9 of 23 US 7,970,552 B1

NDvDJALTARGET SAMPLE O. As Akitiki. (8 Ki-C3, fx {-Yo

ES

pro

A&Y C. Ng's SCC Sf

Foops

ES S :- $8 38-3

SEC his S$8

FiG, 63 U.S. Patent Jun. 28, 2011 Sheet 10 of 23 US 7,970,552 B1

ki. 8888: 88

88.88: 880

friC. 7 U.S. Patent Jun. 28, 2011 Sheet 11 of 23 US 7,970,552 B1

********* 8(8)*** U.S. Patent Jun. 28, 2011 Sheet 12 of 23 US 7,970,552 B1

dieckia sigraise of 83rograxxxiiie

sciectar signatire of circuit

:

w: 3& 88 s & 8 'ss : ** s & xx 8'* 8 8as x x& * 8c s s x &^ && &8° && &8 U.S. Patent Jun. 28, 2011 Sheet 13 of 23 US 7,970,552 B1

:ww.

8. : s: a;

8. i. :

i

g

Sf s

ar,

: U.S. Patent Jun. 28, 2011 Sheet 14 of 23 US 7,970,552 B1

U.S. Patent Jun. 28, 2011 Sheet 15 of 23 US 7,970,552 B1

TNFAIP6

TNFRSF13C NRSA NBS 8.

U.S. Patent Jun. 28, 2011 Sheet 17 Of 23 US 7,970,552 B1

§§ sae

& : s:

-eodz/so»na

sanae

%%%&&&&&&&&&&&&&&********** saman? ********!

} *****************

U.S. Patent Jun. 28, 2011 Sheet 18 of 23 US 7,970,552 B1

FIG 15

- f x:

88:

U.S. Patent Jun. 28, 2011 Sheet 19 Of 23 US 7,970,552 B1

§?

xx

| t U.S. Patent Jun. 28, 2011 Sheet 20 of 23 US 7,970,552 B1

60 www-was

xas------

&;3. ~i

8-i 8 8. M

------Maxw Assi8.333 s

process recistration

;: ww -'s-----awasepowawa wax-axaawaamamamasa-Ya-Yama'awa'a-o-ra, P--- U.S. Patent Jun. 28, 2011 Sheet 21 of 23 US 7,970.552 B1

Xe

*********** U.S. Patent Jun. 28, 2011 Sheet 22 of 23 US 7,970,552 B1

(x3,..??**********

US 7,970,552 B1 1. 2 DAGNOSTIC SYSTEM FOR SELECTING also been proposed, but these are not definitive when applied NUTRITION AND PHARMACOLOGICAL to different animals or species of animals. PRODUCTS FOR ANIMALS The fields of nutrigenetics and nutrigenomics have opened the way in humans for “personalized nutrition', as pharma RELATED APPLICATION cogenetics and pharmacogenomics have led to the concept of “personalized medicine” and “designer drugs'. Similar sci This application is a continuation of application Ser. No. entific advances and concepts are being applied to the nutri 12/316,824, filed Dec. 16, 2008. The content of that applica genetics and nutrigenomics of animals. In other words, by tion is incorporated by reference herein. understanding animal nutritional needs, animal nutritional 10 status, animal physiological or pathophysiological condi BACKGROUND tions, animal functional genomic profiles and animal geno types, nutrigenetics and nutrigenomics should enable better This disclosure is concerned with animal nutrition diagno management or control of the health and well-being of indi sis. More particularly the disclosure is directed to the testing vidual animals or a group of animals by precisely matching and diagnosis of genetic issues relating to nutrition issues and 15 their nutrient needs or dietary composition with their unique disorders of companion animals, for instance dogs and cats. genetic makeup. Further this disclosure relates to a method, system and “DNA polymorphisms” (i.e. SNPs) have been used for apparatus for the management of nutrition assessment in rela animal genotyping, in order to identify breed characteristics, tion to animals worldwide. In particular, the disclosure relates or disease Susceptibility, or have been applied to group animal to a bioinformatics system and its implementation in relation populations by one or more phenotypic traits according to the to animal biological, genetic and nutrition data. frequency of a set of geneticalleles. More specifically the disclosure is directed to animal nutri The “functional genomic profile' is another technique used tion diagnosis and care, animal well-being and nutrition, and to identify breed characteristics, or disease susceptibility or is methods and systems for enhanced determination of these applied to group animal populations or an individual animal factors. 25 one or more by several phenotypic traits according to the Breeders, owners, and caregivers of animals which can be pattern of gene expressions (genomics), or expres companions, such as dogs, cats, horses, farm, food, or Zoo sions (proteomics) or metabolites (metabolomics). animals, and wildlife, have a need to understand the nutrition The specific interaction between the nutritional environ and biological attributes, and related genetic makeup and ment and the genome of an individual has been termed the disorder background, particularly in relation to nutritional 30 molecular dietary signature of that individual features. It is important for nutritionists or other animal food pro Substantial investments in time, effort and financial fessionals to prescribe or recommend nutrient needs or diets resources are made by the breeders, owners, and caregivers of on the basis of more precise knowledge of how nutrients or these animals, particularly purebred animals, to characterize food components interact at the level of the genome, where their nutrition state. There is also a need to conduct periodic 35 these constituents act by “up- or down-regulating a set of comprehensive nutrition assessments of animals. target genes. Animal nutritionists or other animal food pro The probability that an individual animal will develop a fessionals should design nutrients or foods tailored to the specific nutrition-related condition in its lifetime is a product genome or genomic profile or to prescribe or recommend the of complex interactions between its genetic makeup, environ inclusion of specific molecules in the diets of animals to mental influences including diet, and agents of disease (e.g., 40 optimize physiological homeostasis, disease prevention and chemical, physical, or biological) that the animal encounters. treatment, and productive or reproductive performances. The physical attributes and other descriptive and nutrition Individualized nutrition requires an even more refined tech assessment information are generally termed in this applica nique or approach than is currently available or applied. tion as the phenotypic information. Genetic trait or disorder The disclosure also relates to the application of pharmaco information is termed in this application as the genotypic 45 genetics to animals, namely the pharmacology dealing with information. Generally, these are two distinct and differing the influence of genetic variation on the drug response of sets of information. individual animals. It correlates the expression of genes or Current laboratory and research systems and computeriza single-nucleotide genetic polymorphisms (called SNPs) with tion have not achieved comprehensive nutrition assessments the efficacy or toxicity of a pharmaceutical product. The of animals, and nor have communication protocols been used 50 pharmacogenomics application of the disclosure relates to the effectively in this technological area to facilitate such a rela identification and development to design and/or optimize tionship, or to provide relational bioinformatics database sys pharmaceutical product therapy by taking the animal tems for management and dissemination of comprehensive patient’s genotype into account, and thereby improve phar and cumulative information for individual animals. maceutical product efficacy with minimal adverse effects. More specifically, it is necessary in animal nutrition diag 55 The disclosure also relates to pharmacogenomics as nosis and care for comprehensive nutrition assessments of applied to a single or a few gene interactions with pharma animals for diseases and disorders of animals be achieved in ceutical products. The pharmacogenomics application of the order to reduce morbidity and mortality, and improve the disclosure considers the whole genome application of genetic quality of life and lifespan. Currently this is not done in technologies as they apply to the drugs and characterization relation to the nutrition assessment data of an animal together 60 of pharmaceutical products. with the genetic data related to that same animal. Current tests The development of genotype-specific pharmaceutical do not provide as much data as possible to attain correct product therapy provides individuals with the opportunity for nutritional diagnosis and disorder predictions with the net having increased efficacy and lower toxicity than currently result of an improvement in the quality of life and increased available drugs. Several dozen polymorphisms of human longevity. 65 pharmaceutical product—metabolizing have been Various attempts have been made to customize a nutrientor characterized to date, but this has not been applied to animals food products for a specific animal and various methods have who may have similar polymorphisms. The disclosure relates US 7,970,552 B1 3 4 to the type and dose of pharmaceutical product prescribed for FIG. 8 shows the affect of image acquisition and a data an animal as influenced by genotype. processing system of the microarray. Spots are scanned, and the intensity of the colour converted in digits and then pro SUMMARY cessed with SAM (statistical analysis of microarray) soft ware, and this is illustrated graphically. The disclosure uses genetic information of DNA polymor FIG. 9 represents typical molecular dietary signatures phism, the functional genomic profile, and the different respectively of two compounds, namely andrographolide and response of an individual animal to a biologically active nutri curcumin respectively. ent in order to identify and improve upon or optimize the FIG. 10 is a heat map. The heat map shows the expression nutrient composition of the diet for an individual animal. 10 levels of the genes encoding for individual normal, healthy A unique feature of the disclosure is that the response to a dogs of genotype D1 or D2, and unhealthy individual dogs biologically active nutrient ingestion or exposure is a severely (D1) and mildly (D2) affected with liver disease dynamic event since it depends upon the genetic variants of before and after sylimarin administration for 15 days. Gene nutritionally inducible genes (polymorphisms, as SNPs) that expression values were normalised for the mean value of the can lead to a different effect of the biologically active nutrient 15 row. levels range from negative (green) to in individual animals having different genotypes. positive (red) and the graded intensity of the values are indi Effectively, the genotype of the individual animal is an cated by the line (from -3 to +3). essential component of this disclosure to permit the identifi FIG. 11 is a heat map The heat map shows the expression cation of the biologically active nutrient for that individual levels of the genes encoding for individual normal healthy animal. Sardinian (G1-1a; G1-1b) and Bergamasca (G2-1a; G2-1b) The assessment of the biologically active nutrient compo sheep, individual affected unhealthy Sardinian (G1-2a: sition of the diet arises from using reference data relating to G1-2b) and Bergamasca (G2-2a: G2-2b) sheep, and after healthy animals with different genotypes, plus target data individual treatments with Echinacea angustifolia of Sardin relating to animals affected with different physiological or 25 ian (G1-3a; G1-3b) and Bergamasca (G2-3a; G2-3b) sheep. pathophysiological states termed "unhealthy animals' and Gene expression values were normalised for the mean value having different genotypes, and nutritional data relating to the of the row. Gene expression levels range from negative different effects of nutritional compounds in healthy and (green) to positive (red) and the graded intensity of the values unhealthy animals or groups of animals with different geno are indicated by the line (from -3 to +3). types. 30 FIG. 12 is a heat map. The heat map shows the different The assessment of the pharmacological product arises molecular dietary signatures of Echinacea angustifolia on from using reference data relating to healthy animals with individual sheep of two different genotypes (G1 Sardinian: different genotypes, plus target data relating to animals G2 Bergamasca). affected with different physiological or pathophysiological FIG. 13 is a heat map. The heat map shows the different states termed “unhealthy animals' and having different 35 molecular dietary signatures of Sylimarin in individual dogs genotypes, and pharmacological data relating to the different of two different genotypes (D1 or D2), effects of the pharmacological product in healthy and FIG.14 is an overall view of a web-based system to provide unhealthy animals or groups of animals with different geno access to a database management system of an animal genetic types. database and a nutrition assessment database of the disclo Additional and further objects, features, and advantages of 40 sure, in relation to the Internet. the present disclosure will be readily apparent to those skilled FIG. 15 is a graphical illustration of a computer network, in the art. namely the Internet. FIG. 16 is a block diagram of an exemplary computer BRIEF DESCRIPTION OF THE DRAWINGS system for practicing various aspects of the disclosure. 45 FIG.17 is a view of a browser for the database management The patent or application file contains at least one drawing system for accessing an animal genetic database and a nutri executed in color. Copies of this patent or patent application tion assessment database of the disclosure. publication with color drawing(s) will be provided by the FIG. 18 is a basic flow diagram illustrating an exemplary Office upon request and payment of the necessary fee. process by which an operator of a central data processing FIG.1 describes the effect of a nutrient at different cellular 50 resource (CDPR) receives and transmits data relating to nutri and tissue levels. tion assessment and genetic information. FIG. 2 describes the dynamic integration between nutrige FIG. 19 is a detailed flow diagram of the system steps netic and nutrigenomic systems. employed in one embodiment of the present disclosure, FIG. 3 describes the relationship between nutrigenomics wherein a remote user accesses and outputs data. and nutrigenetics, leading to a molecular dietary signature. 55 FIG. 20 is a detailed flow diagram of the methods and steps FIGS. 4A and 4B describes a flow diagram showing the employed by a remote user to add data to the database. method of dynamic nutrient determination. FIG. 21 is a flow chart illustrating an exemplary process by FIGS. 5A, 5B and 5C describe datasets showing the which the laboratory dynamically contributes, transmits and method of dynamic nutrient determination relative to FIGS. receives data associated with health assessment and genetic 6A and 6B. 60 data to the CDPR. FIGS. 6A and 6B describe flow diagrams showing the method of dynamic nutrient determination using biological DETAILED DESCRIPTION OF THE samples. DISCLOSURE FIG. 7 describes a microarray hybridised with mRNA obtained from blood of a test animal, labelled with Cy3 dye 65 The present disclosure provides methods and compositions (green), and a pool of mRNA of a pool of healthy animals of for improving the health and/or well-being of an animal, in the same genotype, labelled with Cy5 dye (red). particular a companion animal Such as a dog or a cat. The US 7,970,552 B1 5 6 disclosure also provides for manufacturing, composing and The molecular dietary signature relates to the interaction providing the necessary biologically active nutrient or nutri between the nutritional environment and genome in an indi ents for animals. vidual in the sense of nutritional genomics or nutrigenomics. The disclosure is concerned with nutritional genomics or The basic concept is that chemical nutrients affect gene nutrigenomics and nutrigenetics. 5 expressions in a specific mode Switching from health to a The disclosure includes a method of modulating the regu pathophysiological condition or vice versa. The advancement lation of a gene or the protein expression or metabolites in an of knowledge in human and animal genomes and the spread animal by nutritional management, including the step of anal of biotechnology offer the opportunity to individualize ysing the gene or protein expressions or metabolites. Selected dietary intervention to prevent, mitigate or cure chronic dis genes, or metabolites in the samples are identified for 10 eases (i.e. individualized nutrition). The concept applies not a particular phenotypic parameter. The effect of a biologically only to companion pet animals, laboratory animals, but also to nutrient-genome interactions in farm animals. For farm active nutrient varies for different genotypes. A biologically animals, nutrigenomics can be applied for the improvement active nutrient is provided to the animal to modulate the of productive performances, and the control of infectious and selected genes, proteins or metabolites So as to change the 15 metabolic diseases, through the use of appropriated dietary response of the animal to the particular phenotypic parameter compositions or Supplements. in a desirable manner. In companion pet animals, nutrigenomics can be directed Typical genes, proteins and metabolites are, for example, to enhancement or maintenance of health and quality of life those involved in the toxicology and nutrigenomics research through the identification of the most suitable diet or supple (apoptosis, cell cycle, DNA damage signalling pathway, drug mentation to maintain or optimize the physiological health. metabolism phase I and phase II enzymes, PI3K-AKT sig The animal genome and biotechnology systems, such as nalling pathway, toxicology and drug resistance), cytokines microarray platforms, can be used to modify the effect of and inflammatory response (inflammatory cytokines and nutrients on gene and protein expression profiles and the receptors, inflammatory response and autoimmunity, NFKI3 adaptation of animals to nutrient exposure, and as a mecha signalling pathway, TNF ligand and receptor), metabolic dis 25 nism to identify genetic variants with favorable or unfavor eases (diabetes, insulin signalling pathway, obesity, oxidative able traits. Nutrigenomics, namely the integration of func stress and antioxidant defences) and neurological disorders tional genomics, nutrition, health and biological response, (depression, epilepsy, general anxiety disorders and panic and the regulatory role of nutrients on gene expressions is disorders). enabled by microarray technology and integrated on an infor The animals can be selected from livestock, companion, 30 matics platform. Nutrigenetics is the retrospective analysis of sporting, working and different domesticated pet and labora genetic variations among individuals with regard to their tory animals, also including fish. These can include for clinical response to specific nutrients. example the following: birds, cat, cattle, dog, donkey, goat, The high throughput Screening technologies are employed guinea pig, hamster, horse, mouse, pig, poultry, quail, parrots, to identify a large number of markers or target molecules of a rabbit, rat, salmon, sheep, trout and turkey or exotic animals. 35 specific parameter treatment or pathology. This is applied to The phenotypic parameter can be, for example, growth, animal or pet nutrition to identify a set of genes, proteins, reproduction, lactation, maintenance, geriatric, inherited and metabolites or other markers that are unique for a specific acquired diseases, allergic, arthritic, autoimmune, inflamma intake of each nutrient, chemical compound or Xenobiotic. A tory, metabolic and pathopsychological or psychological specific nutrient affects body response in a form that is a conditions. 40 molecular dietary signaturr. The identification of the selected genes, proteins or This same concept as applied to gene expressions, mea metabolites in the sample can be effected by high throughput Sured with microarray technology, leads to the identification screening (HTS) techniques, such as microarray, pathway of a unique molecular dietary signature for each specific specific microarray, serial analysis of gene expression and nutrient. In the case of gene expressions, the utilization of a gene sequencing. Alternative HTS methods to analyse the 45 public data repository allows the identification of a set of sample include proteomic and metabolomic assays. genes involved in biological processes, molecular function or The term “healthy” is a well defined term. In this applica cellular component, or in a mix of them, which are affected by tion the term refers to an individual animal that has been the dietary change or composition. The three main classifica determined to be well on the basis of physical examination, tions of gene functions are incorporated in the laboratory data of blood or other biological fluids or tissues, 50 project, which provides a controlled vocabulary to describe and the information provided by the animals caregiver, gene and gene product attributes in any organism. Other owner or guardian. classifications are (KEGG, Kyoto Encyclopaedia of Gene and The term “unhealthy” is a well defined term. In this appli Genomes; and Biocarta) to identify the unique signature that cation the term refers to an individual animal with physical or a dietary change or composition is able to produce in an physiological or pathological or genetic deviation from the 55 organism. state of health. The signature of a particular nutrient can also vary from The term “biologically active nutrient in this application individual to individual, according to the DNA polymor refers to a compound or composition or ingredient of an phisms of the genes or genome. In the case that the genetic ingested material that has some biological measurable or make-up of the individuals is known, the molecular dietary documented effect in the body of an individual animal. 60 signature of mutant animals compared to that of wild-type The method includes identifying a biologically active animals forms a family of molecular signatures, which are nutrient based on what is termed the “molecular dietary sig used for the identification of the action of the nutrient. nature' that the biologically active nutrient induces in an individual animal, the molecular dietary signature being a Example variation of expression of a set of genes, protein or metabo 65 lites which may differ for the genotype of the individual Compound A is an anti-arthrosis natural plant extract animal. which is fed to a group of 20 dogs, 10 healthy and 10 US 7,970,552 B1 7 8 unhealthy dogs affected by arthrosis. The compound is fed for (b) selecting a “target' dataset containing the functional 15 days. Before and after the period of administration, a blood genomic profile of biological samples of the genotypes of sample is drawn and used for a transcriptome analysis (gene different animals, the different tanimals being unhealthy ani expression) using a commercial oligomicroarray containing mals; 44000 probes. The number of genes which significantly var (c) using a “biologically active nutrient dataset compris ied after the treatment is 73, when compared to those of the ing different effects of biologically active nutritional compo group of healthy animals that received a placebo. nents on functional genomic profiles of the different animals Data mining using a public domain repository database and of different genotypes from those of the target group (b), the Software indicated that the 73-gene variation of gene expres different genotypes being differently responsive to the same sion involved the Gene Ontology pathway response to stress, 10 biologically active nutritional components; and external stimuli, immune system process and cell communi (d) having the reference dataset or target dataset include an cation. The average number of genes involved in each path individual animal for which the biologocally active nutrient way is 15 (10 up-regulated and 5 down-regulated), 10 (5 is to be identified. up-regulated and 5 down-regulated), 23 (18 up-regulated and At least one of the “reference' or “target group' datasets is 5 down-regulated) and 25 (5 up-regulated and 20 down-regu 15 related with the “biologically active nutrient dataset to iden lated), respectively for a total of 73 genes (38 up-regulated tify a biologically active nutrient for the selected animal and 35 down-regulated). These genes form a distinct cluster genotype to prevent, treat, control, or modulate a state of molecular dietary signature, which significantly differs from physiological homeostasis or pathophysiological condition the level of expression of the placebo fed control group of of the individual animal in the reference dataset or target dogs, and represent the action and response of the organism to group. the dietary compound. No other dietary compounds tested The identification is based on the molecular dietary signa will produce the same molecular dietary signature when ture being the expression of a gene or a set of genes which administered to dogs. may differ for the genotypes of different animals of the same species. The nutrient identification includes the molecular 25 dietary signature that the biologically active nutrient induces in the individual animal. Gene Ontology Up-regulated Down-regulated Total The animal can be either a canine or a feline. The canine or Response to stress 10 5 15 feline is from the group consisting of one or more breed type, Response to external 5 5 10 specific breed, chronological age, physiological age, activity stimuli 30 level, healthy, and unhealthy. Immune system process 18 5 23 The pathophysiological phenotypic conditions can be any Cell communication 5 2O 25 one or more examples of any inherited or acquired diseases or Total 38 35 73 conditions such as autoimmunity, anxiety, arthritis, depres Sion, variable body condition score, immune Suppression, 35 inflammation, aural disease, skin, aging and behavioral However, in looking at the individual response for each dog changes, cancer or neoplasia, cardiovascular disease, ocular of the group receiving Compound A, some variations disease, orthopedic disease, endocrine disease, hematogical occurred. In other words, if the average values are 38 genes disease, kidney disease, gastrointestinal disorders including up-regulated and 35 genes down-regulated. Some of these inflammatory bowel disease (IBD), acute or chronic diarrhea, genes will not change expression levels in Some of the dogs 40 exocrine pancreatic insufficiency, mal-digestion and pancre receiving compound A. In the example, 5 of 10 dogs respond atitis, hepatic disorder, liver disease, obesity, dental disease, differently to the dietary administration of compound A. and pulmonary disease. The data of the individual animal can be one or more data items related to genotype, including breed, breed(s) of par Gene Ontology Up-regulated Down-regulated Total 45 ents, pedigree, sex, coat type, and evident hereditary condi tions and disorders. Physiological related conditions include Response to stress 8 5 13 one or more of age, weight, veterinary medical history, repro Response to external 5 2 7 stimuli ductive history, health or unhealthy conditions, appetite, Immune system process 10 5 15 physical activity level, mental acuity, behavioral abnormali Cell communication 4 18 22 50 ties and disposition. The reference data can include one or more data of DNA, Total 27 30 57 RNA, proteins, metabolites and biomarkers selected from an individual animal or groups of animals with different geno In the example, genotyping of these dogs indicated that the types in physiological homeostasis. 5 individuals with a different response to the biologically 55 The target group data can include one or more data of active compound A presented a single nucleotide polymor DNA, RNA, proteins, metabolites and biomarkers selected phism (SNP) of the canine CYP1A2 gene that results in a from an individual animal or groups of animals with different deficiency of cytochrome P450 activity. For the biologically genotypes in non-physiological homeostasis. active compound A, two molecular dietary signatures are The biologocally active nutrient data can include one or reported, one for each genotype. 60 more data of DNA, RNA, proteins, metabolites and biomar There is a method of identifying a biologically active nutri kers selected from an individual animal or groups of animals ent for an individual animal having a genotype, which com with different genotypes, the different genotypes being prises: responsive differently to the same nutritional components. (a) using a “reference' dataset containing functional The data comprise analytical data from a biological sample genomic profiles of biological samples of the genotypes of 65 obtained from an individual animal. different animals of the species, the different animals being The identified nutrient can be one or more of a food, part of healthy animals; a food, a Supplement, a nutraceutical or any biologocally US 7,970,552 B1 9 10 active nutrient selected to enhance an aspect of health of an cesses is a step in the post-genomic research and is a relatively animal. Health can be promoted by preventing, attenuating or new area of knowledge, referred as “nutritional genomics' or eliminating at least one disease state in one or more animals or “nutrigenomics', a discipline aimed at the description of the by restoring physiological homeostasis. global expression pattern of a cell or of tissues in different A food composition is prepared as a result of the identified 5 environmental conditions or the change of the expression nutrient, achieved by this method. patterns of these genes as a consequence of physiological The disclosure also includes a method of diagnosing a cues, nutrition and diseases. The initial concept applied to healthy, unhealthy or physiological disorder, or a predisposi humans, but also has been shown to apply to animals. tion to disease or physiological disorder for an individual While nutrigenomics is the identification of the appropriate animal having a genotype, comprising: 10 nutrient to modify the phenotype, based on nutrient-inducible (a) using a “reference' dataset containing functional genes, nutrigenetics represents the identification of the appro genomic profiles of biological samples of the genotypes of priate nutrient for a defined genotype. Nutrigenetics is an different animals of the species, the different animals being applied Science, driven by the paradigms of nutritional phar healthy animals; macology, the onset of genetic polymorphism, and of clinical (b) selecting a “target' dataset containing the functional 15 experience. Nutrigenomics is a discovery Science, driven by genomic profile of biological samples of the genotypes of the paradigms of molecular biology, enabled by microarray different animals, the animals being unhealthy animals; technology, and integrated on an informatics platform. (c) using a “biologically active nutrient dataset compris The role of gene-nutrient interaction is recognized for ing different effects of biologically active nutritional compo Some monogenic and multi-factorial defects. Monogenic dis nents on functional genomic profiles of the different animals eases are determined by a single gene and multi-factorial of different genotypes from those of the target group (b), the diseases by the combination of several genes with other non different genotypes being differently responsive to the same genetic factors. Sometimes, the classification may be an over biologically active nutritional components; and simplification, since monogenic diseases also may involve (d) having the reference dataset or target dataset include an more than a single gene and environmental factors can modu individual animal for which the biologocally active nutrient 25 late the expression of phenotype. Some classical monogenic is to be identified. diseases in humans are phenylketunuria, galactosemia, lac At least one of the “reference' or “target group' datasets is tose intolerance and celiac disease. In most of the case of related with the “biologically active nutrient dataset to iden monogenic disease, dietary intervention can be used to avoid tify a biologically active nutrient for the selected animal or treat the patients. In the case of phenylketunuria, an auto genotypes to prevent, treat, control, or modulate a state of 30 Somal recessive defect resulting from a deficiency of pheny physiological homeostasis or pathophysiological condition lalanine hydroxylase which leads to mental retardation, a of the individual animal in the reference dataset or target phenylalanine restricted diet avoids the severe consequences group. of the disease. Similarly, galactosemia, an autosomal defect, In another aspect of the disclosure there is a method of is related to the deficiency of one of the three main enzymes identifying a biologically active nutrient for animals, com 35 involved in galactose metabolism (galactose-1-phosphate prising: uridyltransferase, galactokinase, uridine-diphosphate galac (a) using a “reference' dataset containing functional tose-4 epimerase), impairing galactose metabolism, result genomic profiles of biological samples of the genotypes of ing in feeding difficulties, and prolonged conjugated hyper different animals of the species, the different animals being bilirubinemia during neonatal life. Avoidance of breast healthy animals; 40 feeding and galactose in the diet prevent the consequences of (b) selecting a “target group' dataset containing the func this defect. tional genomic profile of biological samples of the genotypes Among the multi-factorial chronic/age-related diseases, of different animals, the animals being unhealthy animals; cardiovascular diseases, and metabolic syndrome, cancer, (c) using a “biologically active nutrient dataset compris osteoporosis and neurological diseases are some classical ing different effects of biologically active nutritional compo 45 examples in humans and these syndromes are generally asso nents on functional genomic profiles of the different animals ciated with the aging process. Senescence is an obligate fate of different genotypes from those of the target group (b), the of cells, but gaining the knowledge of the gene-environment different genotypes being differently responsive to the same interactions can be effective in reducing the gap between biologically active nutritional components; and normal and ideal—healthy—aging. Dietary factors are rel (d) having the reference group or target group include the 50 evant for the onset and progression of degenerative diseases animals. and solid scientific evidence has to be provided to support At least one of the “reference' or “target group' datasets is nutritional intervention. Also the multi-factorial chronic/age related with the “biologically active nutrient dataset to iden related diseases respond in a different way according to the tify a biologically active nutrient for the selected animal genotype of the individual animal, leading to a so-called genotypes to prevent, treat, control, or modulate a state of 55 “individual susceptibility” or “genetic risk factor. physiological homeostasis or pathophysiological condition The disclosure integrates the concepts of nutrigenetics of the animal in the reference dataset or target group. The with that of nutrigenomics, considering: analysis is affected by gene or protein expression or the (a) the different genetic make up of individual animals, or metabolite expression in the biological samples of the target a group of them; dataset. 60 (b) the different functional genomic profile for different The exact number of genes needed to create organisms has phenotypic classes of animals (namely healthy, unhealthy, still to be defined for most of the animal species, and it is affected, not affected, physiological states, pathophysiologi likely that the total number of transcripts ranges from 30,000 cal conditions); and to 100,000. Irrespective of that number, the challenge remains (c) the variable response of an individual animal or group to understand the role of the genes in terms of development, 65 of animals to a nutrient. intake of nutrients, disease and physiological functions. The FIGS. 1 to 13 inclusive represent the concepts of nutrige interaction between nutrients and cellular or genetic pro netics and nutrigenomics. The system and the method of the US 7,970,552 B1 11 12 disclosure permits the design of food and nutrients for an response (nutrigenomic effect) but the level of modulation individual animal, and to diagnose the healthy condition of an depends upon the genotype of individual animals (nutrige animal. netic effect). FIG. 1 shows in detail how a nutrient can affect the bio For instance, the assessment of SNPs of all the genes logical response of an animal at the DNA, RNA, protein or involved in the ADME is more closely related to the nutrige metabolite levels. Nutrients can affect gene transcriptions netic effect of a composition, but does not take into account directly, as ligands for factor receptors, or indi the complex interaction that the composition has at the rectly, as primary or secondary metabolic pathways, thereby molecular level, considering that genes have a multitasking altering concentrations of Substrates or intermediates and action. The activation of the transcription of a gene or of a set signal transduction pathways and signaling. The alteration of 10 of genes determines the activation of other genes, and the expression of a Subset of genes in the genome is achieved by translated proteins can have a positive or negative feedback acting at several levels (Clarke and Kim, 1998; Van Ommen, activity on the same gene from which they originated. 2004), through effector genes, effects on enzymes and modi The integration between nutrigenetic and nutrigenomic fication of metabolites and their concentrations. effects is shown in FIG. 3. This leads to a unique fingerprint The effect of a nutrient is thus related not only to the genetic 15 for each nutrient and for each group of animals sharing an background of the individual, i.e. the polymorphism of the identical genotype, whether this fingerprint is a "molecular DNA, but also to the interaction between nutrients and the dietary signature', in the case of RNA, or a "protein signa coordinated regulation of gene expression, activities ture' in the case of proteome, or a “metabolic signature' in the and metabolites. DNA variability among individuals (SNPs) case of metabolome. is statistically associated with the effect of a nutrient on This fingerprint arises from a retrospective analysis (i.e. groups of animals, but does not consider the variations seen SNPs of DNA) and from a perspective view of the interaction within individuals that relate to the effect that different envi of a nutrient with cell activity at a molecular level and gives ronmental factors have on genotype. the “molecular dietary signature', in the case of RNA, or The analysis of gene or protein expressions or metabolites “protein signature' in the case of proteome, or “metabolic in a biological sample permits accurate description of the 25 signature' in the case of metabolome. physiological or patho-physiological conditions of the ani FIGS. 4A and 4B are flowcharts showing the method for mal, thereby indicating which molecular, cellular or meta designing a nutritional formula. For example, the genotype of bolic pathways need to be considered for dietary intervention. the test animal is analyzed using a DNA microarray with The relevance of using gene expression data in relation to 40,000 SNPs and the functional genomic profile is analyzed functional gene annotation is explained by the Gene Ontol 30 using a RNA microarray with 40000 probes of 60mer. The ogy (GO) project (http://www.geneontoloqV.org/). This functional genomic profile is compared with a reference project provides a controlled vocabulary to describe the gene dataset, containing functional genomic profile for normal and gene product attributes in any organism. The G0 project healthy animals having different genotypes. When the com has developed three structured controlled vocabularies (on parison is a match, a regular diet is designed by considering tologies) that describe gene products in terms of their associ 35 the genotype of the normal healthy animal. When there is no ated biological processes, cellular components and molecular match with any of the existing functional genomic profile, the functions in a species-independent manner. According to GO, functional genomic profile of the test animal is compared a single gene can be associated with different functions, for with a target data set, containing functional genomic profile example Murine PI3K (phosphoinositide-3-kinase) has the for the affected unhealthy animals having different geno following ontologies: biological process, negative regulation 40 types. of apoptosis; biological process, protein amino acid phospho The match of the test animal functional genomic profile rylation; and molecular function, protein binding. with a functional genomic profile of the target dataset permits The multitasking role of this gene, as with many others, the identification of the involved pathological state. Selection requires the understanding of the specific pathway or path of the required or recommended dietary ingredients or biol ways involved in the observed biological response to the 45 ogocally active nutrient is determined by comparing the environment. modification of the functional genomic profile due to the Another example is the V-raf-leukemia viral oncogene 1, specific pathology identified with the data of the nutrient which is associated with: biological process, apoptosis; bio dataset, containing the functional genomic profile of the logical process, cytoskeleton organization; cellular compo nutrient or nutrients for animals having different genotypes. nent, cytosol; and molecular function, protein kinase activity. 50 In this respect, the biological response to a nutrient depends Furthermore, these genes can be regulated from (upwards) or upon the genotype of the animal, and a biologocally active can regulate (downwards) other genes, thus altering one or nutrient could, for example, have a positive effect on a first more biological response, according to the type of environ genotype, a mild effect on a second genotype and no effect on mental stimulus, its intensity and duration. The analysis of the a third genotype. polymorphisms of these multitasking genes indicate their 55 FIGS. 5A, 5B and 5C describe datasets showing the genetic variability and can be statistically associated to a method of dynamic nutrient determination relative to FIG. 4. specific pathological state, which depends upon the design of In the figure, the values of functional genomic profile repre the experiment, but does not identify which pathway is really sent the relative expressions of genes involved in inflamma associated in that particular individual. Instead, the simulta tory process measured with quantitative real time RT-PCR. neous determination of a large number of genes expressed in 60 In the FIG. 5A, the functional genomic profile of two a tissue or biological fluid and the use of appropriated infor animals is compared to the functional genomic profile of matic tools for data mining clearly indicate which molecular, reference data set. The match of the functional genomic pro cellular and metabolic pathway has been invoked by the envi file with that of reference dataset indicates a normal condition ronmental stimulus. (Animal A), and the mismatch an abnormal unhealthy condi FIG. 2 summarizes the dynamic integration between the 65 tion (Animal B). The comparison of functional genomic pro nutritional effect and the genetic variability. Nutrients inter file of the abnormal unhealthy animal (Animal B) with target act with an animal phenotype by modulating the biological data set allows one to identify the type of pathology (Pathol US 7,970,552 B1 13 14 ogy P-A), based on a matched functional genomic profile. a microarray containing 40,000 spots of probes of 60mer. The query of the biologocally active nutrient data set indi After Scanning and data processing the differential functional cates that the appropriated compound is NBC-A, since it has genomic profile of the test animal is compared to that of the a reverse effect of the expression values of target genes. pool of normal healthy animals. The colour of each spot Compound NBC-A is used to supplement the diet of the 5 varies from green to red. A green spot indicates the over animal (Animal A) to restore the animal's physiological expression of test animals profile compared to the results of homeostasis. the normal healthy pool. A red spot indicates under-expres In the FIG. 5B, the functional genomic profile of two sion of the test animal’s profile as compared to results of the animals of different genotype but the same pathology is normal healthy pool. A yellow spot indicates no variations at reported in the reference data set. The functional genomic 10 profile for the same pathology differs between dogs and the the gene expression level of the spot. If the spot is yellow, the relative values are reported in the target data set. Similarly to test animal is considered to be normal and healthy. If the spot FIG. 5A, the match of functional genomic profile of the is green or red, the test animal is considered to be affected and animals with the functional genomic profile of the target data unhealthy. After having recorded all the spots and assigned set indicates the presence of the pathology. Searching the 15 each of them a numeric value according to the intensity of the biologocally active nutrient data set for a biologically active colour of each spot, the relative value of expression of all the nutrient with an functional genomic profile able to counteract genes of the microarray are used for data mining, by means of the pathology, it was determined that genotype A requires bioinformatic tools (http://www.geneontology.org/; http:// biologically active nutrient A and genotype B requires bio www.genome.ad.jp/kegg? http://babelomics.bioinfo.cipf.es/ logically active nutrient B to treat the same pathology. In the index.html; http://david.abcc.nciferf.gov/) for gene func example, a different effect of biologically active nutrient A tional annotation. and biologically active nutrient Bonanimals with genotype A The process enables one to identify a set of genes and gene and B is shown. associated functions which are different or identical to those In the FIG. 5C, the functional genomic profile of two of normal healthy animals. This permits the diagnosis of the animals of different genotype but the same pathology is 25 condition of the test animal. In the case that the functional reported in the reference dataset. The reference dataset con genomic profile indicates a pathological condition in the test tains the functional genomic profile of normal healthy ani animal, the identification of the appropriated biologically mals with different genotypes (symbols). In the example, the active nutrient is achieved by using the cells (i.e. leukocytes) functional genomic profile is considered based on four genes of the test animal in an in vitro assay. (G1, G2. G3 and G4). The functional genomic profile of two 30 Based on the identified pathology, a set of potential bio test animals of known genotypes (square and triangle) is logically active nutrients is selected from a library of nutrients compared with the functional genomic profile of reference of already known specific activity for this particular patho and target datasets and the comparison indicates the presence logical state. These biologically active nutrients are incubated of a pathological condition. For the square genotype, G3 was together with the cells of the test animals, the mRNA is 1 instead of 2 and for triangle genotype G4 was 2 instead of 1. 35 extracted and the expressed functional genomic profile is The selection of the biologically active nutrient is based on measured with a custom array using real time RT-PCR. The the library of the functional genomic profile contained in the custom array is designed to contain the over- or under-ex nutrient dataset. In the example, three compounds or constitu pressed genes of the test animal as compared to those of the ents are reported, namely CA, CB and CC, with the relative pool of the normal healthy animals. The biologically active functional genomic profile for the two genotypes (squares 40 nutrient is thus selected according to its specific effect on the and triangles). As can be seen, the compounds vary between test animal for that particular pathological condition. them and have a different effect on each of the two genotypes. FIG. 8 shows the data processing system of the microarray. For the square genotypes, the appropriated biologocally Spots are scanned, intensity of the colour is converted into active nutrient is CB, since it is able to increase the value of numerical value digits and then is processed with SAM (sta G3 by 1 unit, thereby restoring the value of 2 of the normal 45 tistical analysis of microarray) software. The plot of spoS healthy animals. For triangle genotypes, the appropriate biol (genes) intensity of red and yellow colours leads to the iden ogocally active nutrient is CC, since it is able to reduce the tification of the genes that significantly differ from the value of G4 by 1 unit, thereby restoring the value of 1 of the straight line. An arbitrary value of the ratio is taken as thresh normal healthy animals. old for the up (higher than 1.5) or down (lower than -1.5) FIGS. 6A and 6B describe flow diagrams showing the 50 regulated genes. method of dynamic nutrient determination using biological The system accesses biological samples by the method of samples. dynamic nutrient determination, wherein the functional FIG. 7 shows a microarray hybridised with mRNA genomic profile of a reference data set pool of a biological obtained from blood of a test animal, labelled with Cy3 dye sample for each genotype of the animals in physiological (green), and a pool of mRNA of a pool of healthy animals of 55 homeostasis is compared with the functional genomic profile the same genotype, labelled with Cy5 dye (red). of a test animal of a defined genotype. Mismatching indicates In the example, a direct comparison between the test ani an abnormal unhealthy animal, which can be diagnosed mal and the normal healthy animals of the same genotype is according a library of functional genomic profiles from a pool performed by means of competitive hybridisation of mRNA of data obtained for animals with the same genotype and on a microarray platform. A library of pools of mRNA from 60 pathology. The mismatching requires a change of food. blood or other biological fluid or tissue of healthy animals of FIG. 9, illustrates, respectively, two typical molecular different genotypes is stored and used to assess results dietary signatures of two different nutrients, namely, Cur obtained for a test animal of known genotype. The pool of cuminand Andrographolide on a set of genes for animals with mRNA from the blood plasma is selected that has the same the same genotype. genotype as the test animal and labelled with Cy3 dye (green). 65 The molecular dietary signature of the animal is the varia The mRNA extracted from the whole blood is labelled with tion of a set of genes which differ for each animal genotype or Cy5 dye (red) and the two labelled mRNAs are hybridised on phenotype or nutrient. US 7,970,552 B1 15 16 The protein signature is the variation of a set of metabolites The “group' can be defined at least in part by a physiologi which differs for each animal genotype orphenotype or nutri cal condition that is a product of interaction of the genotype ent. with the environment of an animal or a group of animals. The The metabolic signature is the variation of a set of protein term “physiological condition” refers to one or more of the which differs for each animal genotype orphenotype or nutri physical, behavioral and biochemical attributes of an animal ent. including its size, weight, age, sex, activity level, disposition, Generally, the phenotype is the genetic nature of an organ and condition of heath or disease. ism that is revealed by visible characteristics or measurable "Functional Genomic Profile' as used in this disclosure performance, in contradistinction to the genotype, which may includes DNA regions transcribed into RNA, expressed not be evident without a breeding test or genetic map. 10 genes, expressed sequence tag (EST), microRNA, translated The term “phenotype' as used herein refers to the appear proteins and their derived metabolites. A functional genomic ance of an individual resulting from the interaction of envi profile can be established using any one or more of a genomic, ronmental factor with the genotype of the individual. “Phe proteomic or metabolomic approach. A functional genomic notypic information' is the physical descriptive and health profile can result from information from DNA, RNAs, pep assessment profiles and characteristics such as the physi 15 tides, proteins, or metabolites associated with a phenotypic ological, pathological, endocrinological, hematological, epi condition of an animal in response to exposure to one or more demiological, behavioral, and immunological data from biologically active nutrients. parameters such as phenotype, breed, lifespan, health history, Information for the Functional genomic profile as used in and presence of infectious diseases and metabolic disorders. this disclosure is generated from biological samples by any The term “genotype” refers to the genetic information car technique known in the art of functional genomics. Examples ried both in and extrachromosomally. of techniques useful in generating functional genomic analy The 'genotypic information relates to genetic mapping, sis include, without limitation, the following techniques that genetic background, and genetic screening databases. This can be used individually or in combination: (a) DNA, cDNA, includes data obtained from the pedigree, family history, RNAS and protein arrays and microarrays in the existing low heritable physical characteristics, genetic screening tests, 25 and high density formats; (b) polymerase chain reaction DNA testing, genomic mapping, and related laboratory (PCR) techniques including single and multiplexed quantita assessment of the gene product for known or Suspected con tive real-time PCR techniques; (c) serial analysis of gene genital and heritable traits. In this application, the term 'gene expression (SAGE); (d) DNA and RNA sequencing; (e) product” means the specific phenotypic characteristic(s) Southern blot analysis, Northern blot analysis and Western resulting from the expression of the genotype, and may 30 blot analysis, (f) gel electrophoresis, including two-color 2D include certain specific laboratory or other test data. gel methodologies, SDS-polyacrylamide gel electrophoresis The “genotypic information” typically relates to individual (SDS-PAGE), and 2D PAGE: (g) protein sequencing, using animals, or a group or class of animals. This genotypic infor variable existing mass spectrometry techniques: (h) metabo mation, namely the physical characteristics and genetic lite analysis, using variable existing mass spectrometry tech makeup (pedigree), heritable disorder history, and related 35 niques: (i) liquid chromatography by itself or in tandem with health history of animals in the group is usually manually mass spectrometry techniques and other separative analytical recorded by breeders, owners, and researchers of companion techniques. and other valued animals. The genetic constitution of an As used in this disclosure, the functional genomic profile individual includes genes without visible effects as well as extends beyond measurements of clinical pathology analytes those revealed by the phenotype. It may refer to all the genes 40 Such as complete blood count, serum chemistry, hormone or to a single pair of . assays and analysis. "Genotyping refers to the process of determining the The functional genomic profile of an animal can be asso genotype of an individual by the use of biological assay, Such ciated with a “normal” or "abnormal” phenotype. A “normal' as polymerase chain reaction (PCR), DNA sequencing, and phenotype is one occurring in an animal exhibiting a condi DNA microarrays. The technique provides a measurement of 45 tion of health as defined herein, and generally indicative of the genetic variation between members of a species and is Such a state. A "normal” phenotype is associated with physi uses to investigate disease, productive, reproductive and ological homeostasis, i.e., a tendency to stability of optimal nutrition-associated genes. The most common type of genetic bodily functions. An "abnormal phenotype is one that is variation is the single nucleotide polymorphisms (SNP) that outside the range identified as “normal” and can be associated is a single mutation at a specific , usually 50 with a breakdown in physiological homeostasis or patho consisting of two alleles. SNPs are often found to be associ physiological condition. ated with many diseases, productive and reproductive traits of A functional genomic profile from a normal phenotype animals and are becoming of particular interest in pharmaco differs at least in one piece of data or information from the genetic, pharmacogenomic, nutrigenetic and nutrigenomic functional genomic profile of an abnormal phenotype. A pro studies. 55 gressive drift from normality can lead to the death of the A group of animals of the same specie having the same individual, requiring an intervention to restore the physi genotype includes individuals that share a minimum number ological homeostasis to a healthy, normal condition. of common SNPs or other DNA markers that are related to a A normal phenotype can present a functional genomic defined characteristic. In that sense, one animal can be profile generally associated with an abnormal phenotype, included in several genotype groups, according to the specific 60 indicating a latent non-physiological homeostasis or heredi characteristic to which that the group relates. tary predisposition. This drift from the normality requires a In humans, the use of SNPs is being extended to the hap preventive or prophylactic intervention to restore the physi lotype (HapMap project), which is attempting to provide the ological homeostasis to abnormal healthy condition. minimal set of SNPs needed to genotype the . “Biological samples' include for instance feces and urine, Similar haplotyping is being extended to animals. 65 blood, lymph, tears, cheek Swab, Saliva, amniotic fluid, SNPs can also provide a genetic fingerprint for use in serum, prostatic and vaginal secretions, hair, tissue biopsies identity testing. and necropsy specimens. US 7,970,552 B1 17 18 The “reference dataset' includes the functional genomic Soy hulls profile of biological samples and genotype information for Potato the animals with normal phenotype, typically stored in digital Oats form and organized in one to a plurality of databases. Oil, vegetable The “target group dataset contains the functional genomic Examples of micronutrients and biologocally active nutri profile of biological samples and genotype information for ents are Set Out: the animals in abnormal unhealthy conditions. Micro-Nutrients and Biologically Active Nutrients The “nutrient dataset comprises genotype information Leucine and the different effects of biologocally active nutrients on a Isoleucine functional genomic profile of animal of different genotypes. 10 The different genotypes respond differently to the same Valine nutritional components, and according to the present disclo Alanine sure, effects of biologocally active nutrients on the functional Glutamine genomic profile can be determined by controlled experiments Taurine L-Carnitine in animals having different genotypes and exposed to differ 15 ent levels of, and/or different durations of exposure to, one or Portulaca oleracea more biologocally active nutrients. Andrographis paniculata In one embodiment, an alternative testing model of biol Butea frondosa ogocally active nutrients is an ex vivo model using tissue Syllibum marianum explants obtained from an animal of the same species and the Echinacea angustifolia same genotypes, and maintained outside the body of the Curcuma longa animal. Eleutherococcus senticosus The nutrition data set can include data not only on chemical Valeriana officinalis or biological entities known as biologocally active nutrients Matricaria recutrita but on a variety of materials that have nutritional, or nutriceu 25 Conjugated linoleic acid tical or pharmacological effect. All Such materials are con Na sulphate sidered biologocally active nutrients herein if a useful effect Glucosamine HCI on expression of at least one gene, function of at least one Vaccinum nirillus protein or production of at least one metabolite is found. In Vitamin. E one embodiment, biologocally active nutrients of interest 30 Vitamin. C herein are materials having GRAS (generally regarded as Vitamin B1 safe) or equivalent status under U.S. FDA (Food and Drug Vitamin B2 Administration) regulations or counterpart regulations in Di-methylglycine other countries, or are eligible for such status. In other g-orizanol embodiments a biologocally active nutrient can be a thera 35 EPA+DHA peutically or pharmacologically effective compound, e.g. a Green tea polyphenols drug or herbal medicine. Data defines the genotype and physiological condition of Otherwise, the macronutrients required in a balanced ani the individual animal for which a diet is designed, and a mal diet (protein, carbohydrate, fat and fiber) are considered nutrition product or composition prepared. This includes the separately from biologocally active nutrients such as those 40 functional genomic profile. In order to design the nutritional listed above in designing a nutritional formula, as will be formula, the input data for an animal is compared with refer discussed below. ence data set and target data set to identify the normal or Certain biological materials, especially botanical materi abnormal unhealthy conditions of the individual animal. als, can be considered biologocally active nutrients and can, The nutrient data set contains the effects of biologocally if desired, be included in the nutrition data set. In many of 45 active nutrients on the functional genomic profile of an indi these, a bioactive chemical entity has been identified. Even vidual animal with different genotypes. The nutritional for where a bioactive component is known, other unknown, bio mula is computed to incorporate effective amounts of one or active components may be present and contribute to the bio more biologocally active nutrients according to the specific active effect or effects of the biological material. effects on the functional genomic profile in order to restore Examples of macronutrients are set out: 50 the physiological homeostasis. The nutritional formula can be Macro-Nutrients computed as a dietary or nutritional Supplement which can be Chicken meat related to, exclude, or include basic energy, protein, meta Beef meat bolic or other nutrient requirements. Lamb meat Where a nutritional formula, food or composition is gen Horse meat 55 erated, the biolologically active nutrients and other compo Turkey meat nents can be in any Suitable form. For example, components Bison meat can be expressed in terms of their content in a food compo Ostrich or Enu meat sition (e.g., in 96 or in mg/g, usually on a dry matter basis), in Rabbit meat terms of a daily dosage or allowance (e.g., in g/day), or Venison meat 60 optionally on a live weight basis (e.g., in mg/kg/day). An Fish, illustrative nutritional formula, food or nutrient composition Egg can be obtained by the present disclosure and can for instance Rice include any one or more of the exemplary macro-nutrients, Carrot micro-nutrients and/or additives set out above. The food.com Pumpkin 65 position could be one or more biologically active nutrient Peas formulas selected from the exemplary macro-nutrients, Beet, Sugar pulp micro-nutrient and/or additives Setout above and self con US 7,970,552 B1 19 20 tained and/or added as a 'sprinkle Supplement in a dry liquid -continued or semi-moist form to an existing regular or specialized or therapeutic diet. Gene symbol Gene name Animals in conditions of health or disease are identified. CYP1A1 cytochrome P450, family 1, subfamily A, polypeptide 1 Each sample is subjected to functional genomic analysis, for 5 CYP1A2 cytochrome P450, family 1, subfamily A, polypeptide 2 example using an established microarray technique, to evalu CYP1B1 cytochrome P450, family 1, subfamily B, polypeptide 1 CYP2OA1 cytochrome P450, family 20, subfamily A, polypeptide 1 ate an functional genomic profile for the animal that provided CYP24A1 cytochrome P450, family 24, subfamily A, polypeptide 1 the sample, which reflects the genotype, and physiological, FMO1 flavin containing 1 and pathophysiological or other condition of the animal at the FMO4 flavin containing monooxygenase 4 time the sample was collected. 10 FMO5 flavin containing monooxygenase 5 NOS2 2A Biologically active nutrients are tested in one or more CYP2A1. cytochrome P450, family 2, subfamily A animal having different genotypes. CYP2B cytochrome P450, family 2, subfamily B An end-product of one form of the disclosure is the nutri CYP2C cytochrome P450, family 2, subfamily C tional formula, food or composition. A nutritional formula CYP2C13 cytochrome P450, family 2, subfamily C, polypeptide 13 CYP2C7 cytochrome P450, family 2, subfamily C, polypeptide 7 can be designed to provide a therapy for a state of disease or 15 CYP2E1 cytochrome P450, family 2, subfamily E, polypeptide 1 physiological disorder. The pet food can be manufactured to CYP2F2 cytochrome P450, family 2, subfamily F, polypeptide 2 be customized to an individual animal providing the input CYP3A3 cytochrome P450, family 3, subfamily A3 data, or to an animal population represented by an animal CYP4A1 cytochrome P450, family 4, subfamily A, polypeptide 1 CYP4A22 cytochrome P450, family 4, subfamily A, polypeptide 22 providing the input data. The manufacture can be individually CYP4B1 cytochrome P450, family 4, subfamily B, polypeptide 1 prepared in a manual form or automatically composed by an CYP4F2 cytochrome P450, family 4, subfamily F, polypeptide 2 automated or computerized system. CYP7A1 cytochrome P450, family 7, subfamily A, polypeptide 1 The formulas, food or food composition is designed in a CYP7B1 cytochrome P450, family 7, subfamily B, polypeptide 1 GSTM1 glutathione S-transferase M1 dynamic manner for individual animals so as to promote GSTM3 glutathione S-transferase M3 health. This can further include (1) restoring one or more GSTMS glutathione S-transferase M5 constituents of the functional genomic profile to a healthy 25 GSTT1 glutathione S-transferase theta 1 condition, including expression of a gene, function of a pro GSTT2 glutathione S-transferase theta 2 SOD1 Superoxidodismutase 1 tein or production of a metabolite; (2) adapting or altering the SOD2 Superoxidodismutase 2 nutritional management of an animal for specific stressful life GPX1 Glutathione peroxidase 1 stages, even where no disease or disorder is present or detect GPX2 Glutathione peroxidase 2 able, and (3) improving the health in offspring of the indi 30 GSTA1 glutathione S-transferase A1 GSTA2 glutathione S-transferase A2 vidual animal by promoting the health of an individual parent. GSTA2 glutathione S-transferase A4 EXAMPLES No differences in the gene expression levels for this panel The disclosure can be further illustrated by the following 35 examples. of genes were observed between the five haplotypes (A to E). Values for gene expression of individual normal healthy Example 1 dogs are part of the functional genomic profile of the refer ence data set, in this case being identical for dogs of geno The example reports the method to build the reference data 40 types D1 or D2. The functional genomic profile is the molecu set, the target data set and the nutrient data set. In the example, lar dietary signature of normal, healthy dogs. the effect of sylimarin to treat liver disease of dogs with different genotypes is reported. Construction of the Target Data Set Construction of the Reference Data Set A second population of 30 dogs suffering liver diseases Twenty normal, healthy dogs (with genotypes D1 or D2) 45 was screened for haplotypes. Blood was sampled and total was used to build the reference data set. Blood was sampled DNA and RNA extracted. DNA was used for genotyping and and total DNA and RNA extracted. DNA was used for geno haplotype identification and RNA for the determination of typing and haplotype identification, using restriction frag gene expressions, using the pathway specific microarray. ment length polymorphism (RFLP) and gel electrophoresis, The expression profile of the dogs was clustered in two including nine known single polymorphisms (SNPs) along 50 patterns, according to the severity of clinical symptoms, a So CF15. RNA was used for the determination of called Severe (D1, 18 dogs) and Mild (D2, 12 dogs), func gene expressions, by means of a pathway specific microarray. tional genomic profile of the two population differed, The technique is based on the quantitative real time RT-PCR. severely affected dogs (D1) showing an higher increase of The functional genomic profile of a population of 20 mixed detoxifying and antioxidant enzymes than the mildly affected breed dogs, from 2 to 4 years old, in healthy clinical condition 55 dogs (D2). and considered normal, was measured using a pathway-spe cific microarray. The pathway for drug metabolizing enzymes The functional genomic profile in the target data set of the two defined genotypes is the molecular signature of liver was used, and included the genes reported in the table below. disease for the dogs of genotypes D1 or D2. 60 Values for gene expression of individual unhealthy dogs severely and mildly affected with liver disease are part of the Gene symbol Gene name functional genomic profile of the target data set, in this case Acadsb acyl-coenzyme A dehydrogenase, short branched chain being different for genotypes D1 or D2. CAT catalase CYP11A1 cytochrome P450, family 11, subfamily A, polypeptide 1 The number of known haplotypes was 5 (from A to E. 65 shown below), and an association is shown between those CYP11B2 cytochrome P450, family 11, subfamily B, polypeptide 2 dogs with haplotypes including the causal variant of SNP'A' and “B” and the severity of liver diseases. US 7,970,552 B1 21 22

SNP profiling pattern of dogs

SNPVariant HAPLOTYPE A. B C D C E A. G. E. Response

1 A. C T T T C A. C C Severe Illness 2 A. C T T T C A. A C Severe Illness 3 T G T A T C G C C Mild Illness 4 T G T T T C G C C Mild Illness 5 A. C A T A G A. C G Severe Illness 6 A. C A A A G A. C G Severe Causal Causal Causal Illness variant variant variant

Construction of the Nutrient Data Set -continued The two genotyped populations of 18 (D1) and 12 (D2) unhealthy dogs (30 overall) suffering from liver disease were GENE EXPRESSION LEVELS fed orally with a standardized extract of Svlibum marianum, a CYP2E1 1.7 O.2 1.9 O.3 2.1 O.1 dose of 1.5 mg/kg body weight of sylimarin for 15 days. At CYP2F2 2.8 O.3 2.8 O.3 2.5 O.S CYP3A3 4.3 O.9 5.7 O.9 3.8 1.O the end of the treatment, blood was collected from each of the 25 CYP4A1 2.5 4.9 4.9 1.1 4.8 O.9 individual animals of the severe (D1) and mild (D2) illness CYP4A22 1.1 O.1 1.7 0.4 O.2 O.1 groups and their total RNA was extracted. The RNA of indi CYP4B1 O.3 O.1 2.1 O.3 3.1 0.4 CYP4F2 O.2 O.1 2.2 O.3 4.6 O.9 viduals animals of the severe and mild illness groups were CYP7A1 O.8 O.2 1.4 O.3 3.8 O.1 analysed for gene expressions in duplicate using the pathway CYP7B1 O.9 O.2 1.8 O.2 1.7 O.2 specific microarray. 30 GSTM1 1.3 0.4 13.3 1.8 6.3 O.8 GSTM3 17.9 2.5 14.7 1.7 9.6 1.O The gene expression profiles of individuals from these two GSTMS 20.1 2.8 19.4 2.1 13.5 1.2 populations of unhealthy dogs after 15 days of sylimarin GSTT1 12.8 2.6 27.1 2.0 7.8 O.9 treatment (severe D1 and mild D2) showed a different pattern. GSTT2 16.1 2.9 21.1 1.7 17.0 2.1 The nutrient data set contains the molecular dietary signa SOD1 23.9 3.8 228.2 34.7 267.7 34.5 35 SOD2 16.7 1.7 115.O 14.3 1647 28.0 ture of the sylimarin for individual dogs of genotypes D1 or GPX1 8.8 1.1 25.3 2.3 S.1 0.7 D2. In the example, Sylimarin Supplementation can be used to GPX2 9.5 1.2 27.3 2.1 68.7 8.6 effectively treat affected unhealthy dogs of the D1 genotype GSTA1 11.8 1.9 18.8 2.0 16.8 2.3 but not affected unhealthy dogs of the D2 genotype. GSTA2 12.4 1.5 28.0 3.1 30.8 2.1 Expression levels of the genes encoding for individual GSTA2 14.1 2.O SO.2 S.6 39.7 3.4 normal healthy dogs of genotypes D1 or D2, and individual 40 Sylimarin Sylimarin unhealthy dogs, severely (D1) and mildly (D2) affected with D1 D2 liver disease before and after sylimarin administration (Syli marin D1 and Sylimarin D2) for 15 days. Mean S.d. Mean S.d. Acadsb O.8 O.2 1.1 O.3 45 CAT 7 O.3 1.8 O.2 CYP11A1 3.9 O.3 4.7 3.4 GENE EXPRESSION LEVELS CYP11B2 7.2 O.6 10.7 2.3 CYP1A1 2.1 O.3 3.9 O.9 Severe Mild CYP1A2 .5 O.2 3.6 O.3 Normal D1 D2 CYP1B1 3.6 1.2 10.2 0.7 50 CYP2OA1 S.1 3.4 8.1 1.O Mean S.d. Mean S.d. Mean S.d. CYP24A1 68.5 8.9 68.3 S.6 FMO1 .5 O.3 4.0 1.3 Acadsb O.9 O.1 2.3 O.3 1.O O.1 FMO4 .5 O.S 8.0 O.8 CAT 1.4 0.4 S.O O.9 1.7 O.2 FMO5 4.2 3.4 3.1 O.2 CYP11A1 1.5 O.2 12.9 1.1 6.5 1.1 NOS2 4.1 4.0 5.8 O.3 CYP11B2 2.0 0.4 24.9 2.3 16.0 3.0 55 CYP2A1. .1 O.2 1.3 O.1 CYP1A1 O.3 O.1 7.7 1.1 6.3 O.9 CYP2B 3 O.3 1.9 O.2 CYP1A2 0.4 O.1 5.3 O.9 5.7 O.8 CYP2C .5 O.S 2.3 O.9 CYP1B1 1.4 O.6 12.2 O.9 15.8 1.4 CYP2C13 2.2 O.8 3.9 O.6 CYP2OA1 2.4 O.6 16.8 3.4 11.3 1.5 CYP2C7 .4 O.3 3.2 O.2 CYP24A1 1.3 O.2 2S3.0 25.6 113.9 14.3 CYP2E1 O O.3 2.2 O.1 FMO1 2.3 0.4 3.4 O.8 4.5 1.1 FMO4 2.1 O.6 3.4 1.O 11.6 2.0 60 CYP2F2 .5 O.S 3.1 0.4 FMO5 2.5 O.3 13.3 1.9 2.8 0.7 CYP3A3 2.7 2.1 4.7 O.S NOS2 3.0 O.9 12.4 1.9 6.9 O.9 CYP4A1 2.O 2.1 4.3 O.2 CYP2A1. 1.7 O.2 2.4 O.S O.S O.1 CYP4A22 O.8 O.3 O.8 O.1 CYP2B 1.9 O.2 3.0 0.4 1.3 0.4 CYP4B1 O.6 O.2 2.0 0.4 CYP2C 1.8 O.3 3.8 O.3 2.2 O.6 CYP4F2 0.7 O.2 2.9 O.6 CYP2C13 2.4 O.6 5.8 O.9 4.1 1.O 65 CYP7A1 O.6 O.1 2.7 O.2 CYP2C7 2.5 0.4 2.8 O.2 2.9 O.1 CYP7B1 0.7 O.2 1.5 O.1 US 7,970,552 B1 23 24 -continued -continued

GENE EXPRESSION LEVELS MEAN CHANGES OF GENE EXPRESSION

GSTM1 3.9 3.8 4.5 O.2 GENE MDS D1 MDS D2 GSTM3 8.7 9.6 16.2 1.2 5 GSTMS 10.6 1.3 19.8 2.0 CYP2E1 -O.7 -1.9 GSTT1 10.7 2.O 12.2 O.9 CYP2F2 -1.5 -3.4 GSTT2 9.9 1.8 19.5 O.8 CYP3A3 -1.2 -5.2 SOD1 674 7.0 172.S. 21.3 CYP4A1 0.4 -1.9 SOD2 35.2 3.5 107.3 14-0 CYP4A22 -0.1 -1.7 GPX1 9.2 O.9 8.2 1.6 10 CYP4B1 1.2 O.8 GPX2 9.9 1.O 46.0 3.9 CYP4F2 1.3 1.5 GSTA1 8.2 O.S 16.8 3.0 CYP7A1 O.O O.3 GSTA2 10.9 O.9 25.4 2.7 CYP7B1 O.2 -O.7 GSTA2 17.2 2.1 31.8 4.5 GSTM1 8.0 0.4 GSTM3 -12.0 -24.6 15 GSTMS -11.3 -26.4 Genotype induces a different response to sylimarin, a GSTT1 3.6 -17.1 micro-nutrient, in dogs with haplotypes including the causal GSTT2 -4.9 -18.6 SOD1 136.9 71.4 variant of SNPA and B. Sylimarin is one of the main bioactive SOD2 63.1 40.7 compounds of the Sylibum marianum plant and is known to GPX1 7.3 -12.0 cleanse the liver and spare liver metabolism. GPX2 7.9 13.2 The heat map shows individual normal dogs of both the D1 GSTA1 -1.1 -11.8 or D2 genotypes, having negative (green)values for almost all GSTA2 4.7 -7.1 the genes. Individual unhealthy dogs, severely affected with GSTA2 18.9 -6.1 liver disease (D1) showed positive (red) values, indicating a gene over expression. Administration of Sylimarin restored The heat map shows the molecular dietary signature of the normal values of the genes, which were clustered together. 25 sylimarin on genotype D1 and D2. The pattern of expression of the individual unhealthy dogs Comparing the functional genomic profile of a test sample mildly affected with liver disease (D2) was different from that of dog with known genotype with the reference and target dat of individual dogs severely affected with liver disease (D1). Set indicating a different state of their liver disease. Sylimarin The diagnosis of liver disease in a dog can be performed administration to these individual D2 dogs was neitherable to 30 determining the functional genomic profile of a blood sample, restore the normal condition, nor to change the pattern of gene using the patter designed microarray. expression in comparison to the condition seen in the indi The DNA of the individual dog needs to be genotyped for vidual D2 dogs before the treatment. This is apparent from the the known SNP, enabling to identify the presence of a causal cluster analysis, since the gene expression of the individual 35 variant of SNPA and B. mildly affected dogs before and after sylimarin administra The comparison of values for gene expression of the tion remained clustered together. Molecular Dietary Signature sample of an individual test dog of a defined genotype (D1 or Effect of sylimarin administration on individual unhealthy D2) with the gene expression of the reference and target data dogs of Genotypes D1 or D2 affected by liver disease Values sets permits identification of the presence of liver disease. shown are changes of gene expression. 40 According to the genotype, Sylimarin is administered. If genotype is D1, Sylimarin is effective in treating the liver Molecular Dietary Signature (MDS): disease, if the genotype is D2 another biologically active MDS D1=(Severe D1-Normal)-Sylimarin D1 nutrient needs to be used.

MDS D2=(Mild D2-Normal)-Sylimarin D2 45 Example 2 This is the use of individual samples from normal healthy sheep with different genotypes to diagnose disease conditions MEAN CHANGES OF GENE EXPRESSION in affected unhealthy sheep, and identifies the nutrient com 50 position to add to the feed to restore the health of the GENE MDS D1 MDS D2 unhealthy sheep. Acadsb O.S -1.0 Reference Data Set CAT 1.8 -1.6 The individual blood samples were obtained from 20 nor CYP11A1 7.6 O.3 mal healthy sheep of the Sardinian breed and 20 normal CYP11B2 15.7 3.4 55 healthy sheep of the Bergamscabreed. The animals, selected CYP1A1 5.3 2.2 CYP1A2 3.4 1.8 within the flocks, were female, clinically healthy, not preg CYP1B1 7.2 4.3 nant and not lactating and in normal body condition score. CYP20A1 9.2 O.8 The age of the sheep ranged from 3 to 5 years. These animals CYP24A1 1831 44.2 represented the reference dataset for the two genotypes (G1 or FMO1 -0.4 -1.9 FMO4 -0.1 1.5 60 G2). FMO5 6.5 -2.9 Target Data Set NOS2 5.3 -1.9 A second population of individual sheep was selected from CYP2A1 -0.4 -2.4 the two breed flocks, Sardinian and Bergamasca, for having CYP2B -0.2 -2.5 CYP2C O.S -2.0 an inflammatory condition of laminitis. The number of indi CYP2C13 1.2 -2.2 65 vidual affected unhealthy sheep was 10 for each breed. The CYP2C7 -1.1 -2.7 sheep were female, not pregnant and not lactating and in normal body condition score. The age of the sheep ranged US 7,970,552 B1 25 26 from 3 to 5 years. These animals represented the target dataset -continued for the two genotypes (G1 or G2). Nutrient Data Set MEANGENE EXPRESSION LEVELS The animals were fed a maintenance ration, based on hay and concentrate, supplemented with 2 mg/kg body weight of 5 symbol Normal Normal Abnormal Abnormal Treated Treated dry extract of Echinacea angustifolia for 20 days. The IL1RAP 81.74 94.73 95.27 106.08 95.23 95.26 samples collected from each of the animals after the treatment IL1RN 341.33 231.38 275.11 552.67 589.34 228.83 showed the effect of Echinacea angustifolia (nutrient dataset) IL27RA 19446 246.16 76.93 149.70 212.99 249.06 for the two genotypes (G1 or G2). Animals after the treatment ILA 97.25 68.70 77.13 93.49 90.59 69.78 represented the nutrient data set. 10 IL6 175.83 16935 74.58 192.55 177.54 171.65 IL8RA 245.94 229.87 344.28 276.43 314.39 233.77 Blood was sampled from each sheep of the reference, tar TNC 32.32 27.63 27.94 44.40 36.9S 28.92 get and nutrient datasets and mRNA was extracted employing TNF 243.28 2SO.19 212.8O 375.71 158.65 252.77 PAXgene blood RNA kit (PreAnalitiX Qiagen). The TNFAIP2 181SO 150.84 74.51 1912S 18431, 1 SO.80 mRNA from the individual healthy and unhealthy sheep of TNFAIP3 84.48 70.67 89.36 158.22 123.87 70.15 15 TNFAIP6 29.90 52.14 36.56 21.54 S2.95 SO.84 each breed and dataset and their individual gene expressions TNFAIP8 73.25 57.66 73.02 98.36 99.72 57.19 were analysed in duplicate using a custom microarray. TNFRSF13B 96.70 91.02 1969 89.34 75.10 90.07 The probes of the microarray were designed with Oligowiz TNFRSF13C 79.68 55.92 6092 56.58 56.63 55.76 Software, from a collection of gene sequences and EST and TNFRSF1A 97.06 112.97 O7.37 102.81 99.32 11412 clustered, producing 12.194 Unigenes NCBI. For each TNFRSF6B 54.62 53.62 42.17 87.42 78.79 54.59 cluster two 35-40mer probes were designed. Quality check of all mRNA samples was performed with Agilent 2100 bio Hierarchical clustering, reported in the heat map figure, analyser. Two rounds of amplification of the target genes were further shows the different molecular dietary signature of the performed with Ambion Amino Allyl Message AmpTM II individual sheep of the two breeds, as well as the positive mRNA Amplification Kit. Labeling of target genes was therapeutic action of Echinacea angustifolia, in restoring the achieved with Cy5 fluorophore, in duplicate, hybridized to 25 individual affected unhealthy sheep to the individual normal microarray and scanned. healthy condition. This is apparent from the homogeneous Scanning and image acquisition. Raw data were normal cluster that G2-1a and G2-1b produced with G2-3a and ized using the function “Normalize Gene/Row Vectors' of G2-3b. MeV software and a two way analysis of variance (fixed The molecular dietary signatures of Echinacea angustifo factors genotype, G1 or G2; datasets, reference, target and 30 nutrient), was performed with ANOVA (MeV software lia on the two genotypes are reported in the heat map. v4.1 TIGR). Results were considered statistically signifi Comparing the functional genomic profile of a test sample cant for p-values.<0.01. of sheep with known genotype with the reference and target Hierarchical clustering analysis of differentially expressed dataset genes and heat maps were generated for genes which were 35 The diagnosis of inflammatory conditions in a sheep can be significantly different for interaction, treatment and time of performed determining the functional genomic profile of a sampling. (MeV software V 4.1—TIGR). Genes were anno blood sample, using a gene expression microarray. tated with HomoloGene system (about 50% of the genes Genetic data of the individual sheep (i.e. breed) needs to be present on the array have been annotated). recorded. The number of genes which significantly differed in the 40 The comparison of values for gene expression of the individual sheep was 20 between genotypes and 12 between sample of an individual test sheep of a defined breed (G1 or datasets. The interaction of genotype X dataset showed 20 G2) with the gene expression of the reference and target genes differently expressed. In this example, only this last set dataset permits the identification of the presence of inflam of genes is reported. matory conditions. As can be seen from the Tables, the two G1 or G2 geno 45 According to the breed, Echinacia angustifolia is admin types of the individual normal healthy sheep showed different istered. If genotype is G1, Echinacia angustifolia is ineffec basal values of expression for the 20 genes, indicating the tive in treating the inflammatory conditions, if the genotype is effect of the individuals of the two different breeds. Also the G2 Echinacia angustifolia is effective in treating the inflam individual affected unhealthy sheep—i.e. with laminitis pre matory conditions. sented a different response to their inflammatory conditions. 50 The administration of Echinacea angustifolia for 20 days was Example 3 notable to restore the normal condition in the individual G1 Sardinian sheep. Conversely, the individual sheep of G2 Ber Using the technique of Example 1, the biologically active gamasca breed responded positively to the treatment and the nutrient for kidney disease is identified. The relevant genes level of expression of the genes were similar to that of indi 55 for this identification would include: vidual normal healthy animals of the reference dataset.

Gene MEANGENE EXPRESSION LEVELS 60 symbol Description G1 G2 G1 G2 G1 G2 A2M alpha-2-macroglobulin Symbol Normal Normal Abnormal Abnormal Treated Treated ABCB7 ATP-binding cassette, sub-family B (MDR/TAP), member 7 IL12RB1 111.71 113.32 96.50 151.59 133.20 116.75 ABCC2 ATP-binding cassette, sub-family C IL12RB1 97.73 154.96 108.07 168.91 137.83 152.11 (CFTR/MRP), member 2 IL1E10 SS.26 44.39 61.52 36.OS 36.67 46.02 65 AB1 abil-interactor 1 IL1R2 2O4.99 233.22 199.82 145.04 186.25 234.70 ABL1. c-abl oncogene 1, receptor tyrosine kinase US 7,970,552 B1 27 28 -continued -continued

Gene Gene symbol Description symbol Description ABP1 amiloride binding protein 1 COL4A1 collagen, type IV, alpha 1 (amine oxidase (copper-containing)) COL4A2 collagen, type IV, alpha 2 ACAN aggrecan COL4A3 collagen, type IV, alpha 3 (Goodpasture antigen) ACE angiotensin I converting enzyme COL4A4 collagen, type IV, alpha 4 (peptidyl-dipeptidase A) 1 COL4A5 collagen, type IV, alpha 5 ACY1 aminoacylase 1 COL4A6 collagen, type IV, alpha 6 ACYP2 acylphosphatase 2, muscle type 10 CPS1 carbamoyl-phosphate synthetase 1, mitochondrial ADAM10 ADAM metallopeptidase domain 10 CREB1 cAMP responsive element binding protein 1 ADAM28 ADAM metallopeptidase domain 28 CRP C-reactive protein, pentraxin-related ADAM9 ADAM metallopeptidase domain 9 (meltringamma) CRYAB crystallin, alpha B ADAMTS13 ADAM metallopeptidase with CSDA cold shock domain protein A thrombospondin type 1 motif, 13 CSF3 colony stimulating factor 3 (granulocyte) ADAMTS4 ADAM metallopeptidase with 15 CSK c-Src tyrosine kinase thrombospondin type 1 motif, 4 CSPG4 chondroitin Sulfate proteoglycan 4 ADAMTSS ADAM metallopeptidase with CTGF connective tissue growth factor thrombospondin type 1 motif, 5 CXCL12 chemokine (C-X-C motif) ligand 12 arginine decarboxylase (stromal cell-derived factor 1) adenylate cyclase 1 (brain) CXCLS chemokine (C-X-C motif) ligand 5 acireductone dioxygenase 1 CXCL6 chemokine (C-X-C motif) ligand 6 enosine A2b receptor (granulocyte chemotactic protein 2) renergic, beta-3-, receptor chemokine (C-X-C motif) receptor 4 enyloSuccinate lyase cytochrome P450, family 1, subfamily A, polypeptide 2 vanced glycosylation end product-specific receptor cytochrome P450, family 2, subfamily B, polypeptide 6 gmatine ureohydrolase (agmatinase) cysteine-rich, angiogenic inducer, 61 angiotensinogen (Serpin peptidase epidermal growth factor receptor (erythroblastic leukemia inhibitor, clade A, member 8) 25 viral (v-erb-b) oncogene homolog, avian) AKR1C3 aldo-keto reductase family 1, member C3 (3-alpha EIF4B eukaryotic translation initiation factor 4B hydroxysteroid dehydrogenase, type II) ELAVL1 ELAV (embryonic lethal, abnormal vision, AKT1 V-akt murine thymoma viral oncogene homolog 1 Drosophila)-like 1 (Huantigen R) AKT2 V-akt murine thymoma viral oncogene homolog 2 ELF3 E74-like factor 3 (ets domain transcription ALB albumin factor, epithelial-specific) ALLC allantoicase 30 ENPP1 ectonucleotide pyrophosphatase/phosphodiesterase 1 AOC2 amine oxidase, copper containing 2 (retina-specific) EPHA2 EPH receptor A2 AOC3 amine oxidase, copper containing 3 EPHX2 epoxide hydrolase 2, cytoplasmic (vascular adhesion protein 1) EPO erythropoietin AQP1 aquaporin 1 (Colton blood group) ERBB4 v-erb-a erythroblastic leukemia viral AQP10 aquaporin 10 oncogene homolog 4 (avian) AQP11 aquaporin 11 35 ESR2 estrogen receptor 2 (ER beta) aquaporin 2 (collecting duct) ETS1 v-ets erythroblastosis virus E26 aquaporin 3 (Gill blood group) oncogene homolog 1 (avian) aquaporin 4 ETV1 ets variant gene 1 aquaporin 7 ETV4 ets variant gene 4 (E1A enhancer binding protein, E1AF) aquaporin 8 ETV5 ets variant gene 5 (ets-related molecule) aquaporin 9 ETV 7 ets variant gene 7 (TEL2 oncogene) amphiregulin (Schwannoma-derived growth factor) 40 F12 coagulation factor XII (Hageman factor) arginase, liver F13A1 coagulation factor XIII, A1 polypeptide arginase, type II F13A2 coagulation factor XIII, A2 polypeptide Rho GTPase activating protein 5 F2 coagulation factor II (thrombin) argininosuccinate lyase coagulation factor II (thrombin) receptor argininosuccinate synthetase 1 F5 coagulation factor V (proaccelerin, labile factor) beta-2-microglobulin 45 FABP2 atty acid binding protein 2, intestinal basic leucine Zipper transcription factor, ATF-like 3 FASLG Fas ligand (TNF Superfamily, member 6) carbonic anhydrase I FBLN2 bulin 2 carbonic anhydrase II FGA brinogen alpha chain carbonic anhydrase IX FGF13 broblast growth factor 13 calreticulin FGF broblast growth factor 2 (basic) 1, protein, 22 kDa 50 FGF broblast growth factor 4 (heparin Secretory transforming chemokine (C-C motif) ligand 4 protein 1, Kaposi sarcoma oncogene) chemokine (C-C motif) ligand 5 FGF5 fibroblast growth factor 5 CCNE2 cyclin E2 FH fumarate hydratase CCR3 chemokine (C-C motif) receptor 3 FKBP1A FK506 binding protein 1A, 12 kDa CCR5 chemokine (C-C motif) receptor 5 FLG filaggrin CDH1 cadherin 1, type 1, E-cadherin (epithelial) 55 FN fibronectin 1 CDHS cadherin 5, type 2 (vascular endothelium) FOLH1 olate hydrolase (prostate-specific membrane antigen) 1 CEACAMS carcinoembryonic antigen-related cell FOSB BJ murine osteosarcoma viral oncogene homolog B adhesion molecule 5 FOSL1 OS-like antigen 1 CEBPB CCAAT?enhancer binding protein (C/EBP), beta FURIN furin (paired basic amino acid cleaving enzyme) CES1 carboxylesterase 1 G6PC glucose-6-phosphatase, catalytic subunit (monocyte/macrophage serine esterase 1) GADD45B growth arrest and DNA-damage-inducible, beta CFLAR CASP8 and FADD-like apoptosis regulator 60 GATM glycine amidinotransferase CGBS chorionic gonadotropin, beta polypeptide 5 (L-arginine:glycine amidinotransferase) CLDN4 claudin 4 GBP1 guanylate binding protein 1, interferon-inducible, 67kDa CLU clusterin GC group-specific component (vitamin D binding protein) COL18A1 collagen, type XVIII, alpha 1 GCGR glucagon receptor COL1A1 collagen, type I, alpha 1 GGT1 gamma-glutamyltransferase 1 COL1A2 collagen, type I, alpha 2 65 GH1 growth hormone 1 COL3A1 collagen, type III, alpha 1 GHRH growth hormone releasing hormone US 7,970,552 B1 29 30 -continued -continued

Gene Gene symbol Description symbol Description GHRL ghrelinfobestatin prepropeptide KRT14 keratin 14 (epidermolysis bullosa simplex, GLB1 galactosidase, beta 1 Dowling-Meara, Koebner) GLO1 glyoxalase I KRT18 keratin 18 GLS glutaminase KRTS keratin 5 (epidermolysis bullosa simplex, GLS2 glutaminase 2 (liver, mitochondrial) Dowling-Meara Kobner/Weber-Cockayne types) GLUD1 glutamate dehydrogenase 1 KRT8 keratin 8 GLUL glutamate-ammonia ligase (glutamine synthetase) 10 LALBA actalbumin, alpha GNMT glycine N-methyltransferase LAMC2 aminin, gamma 2 GOT1 glutamic-oxaloacetic transaminase 1, LCN1 ipocalin 1 (tear prealbumin) Soluble (aspartate aminotransferase 1) ipocalin 2 GOT2 glutamic-oxaloacetic transaminase 2, LEF1 ymphoid enhancer-binding factor 1 mitochondrial (aspartate aminotransferase 2) LGALS7 ectin, galactoside-binding, soluble, 7 GPRC6A G protein-coupled receptor, family C, group 6, member A 15 LIMS1 LIM and Senescent cell antigen-like domains 1 GPT glutamic-pyruvate transaminase (alanine aminotransferase) LOC73241S similar to Matrix metalloproteinase-19 precursor GRLF1 glucocorticoid receptor DNA binding factor 1 (MMP-19) (Matrix metalloproteinase RASI) (MMP-18) GRN granulin LOX ySyl oxidase HBEGF heparin-binding EGF-like growth factor LPA ipoprotein, Lp(a) HELLS helicase, lymphoid-specific LRP1 ow density lipoprotein-related protein 1 HGF hepatocyte growth factor (hepapoietin A; Scatter factor) (alpha-2-macroglobulin receptor) HIF1A hypoxia-inducible factor 1, alpha Subunit LRPAP1 ow density lipoprotein receptor-related (basic helix-loop-helix transcription factor) protein associated protein 1 histone cluster 2, H3c MAOA monoamine oxidase A major histocompatibility complex, class II, DR beta 1 MAOB monoamine oxidase B major histocompatibility complex, class I, G MAP2K1 mitogen-activated protein kinase kinase 1 hepatocyte nuclear factor 4, alpha MAP2K2 mitogen-activated protein kinase kinase 2 haptoglobin 25 MAP2K3 mitogen-activated protein kinase kinase 3 heparanase MAP2KS mitogen-activated protein kinase kinase 5 hemopexin MAP2K6 mitogen-activated protein kinase kinase 6 histamine receptor H2 MAP3K1 mitogen-activated protein kinase kinase kinase 1 heat shock factor binding protein 1 MAP3K7 mitogen-activated protein kinase kinase kinase 7 heat shock protein 90 kDa alpha (cytosolic), MAPK1 mitogen-activated protein kinase 1 class A member 2 30 MAPK10 mitogen-activated protein kinase 10 HSPA1A heat shock 70 kDa protein 1A MAPK11 mitogen-activated protein kinase 11 HSPA4L heat shock 70 kDa protein 4-like MAPK14 mitogen-activated protein kinase 14 HSPA8 heat shock 70 kDa protein 8 MAPK3 mitogen-activated protein kinase 3 HSPB2 heat shock 27 kDa protein 2 MAPK7 mitogen-activated protein kinase 7 HSPD1 heat shock 60 kDa protein 1 (chaperonin) MAPK8 mitogen-activated protein kinase 8 HSPE1 heat shock 10 kDa protein 1 (chaperonin 10) 35 MAPK9 mitogen-activated protein kinase 9 BSP integrin-binding sialoprotein MAPT microtubule-associated protein tau CAM1 intercellular adhesion molecule 1 MAZ MYC-associated zinc finger protein GF1R insulin-like growth factor 1 receptor (purine-binding transcription factor) GF2 insulin-like growth factor 2 (somatomedin A) MBP myelin basic protein L10 interleukin 10 MCCC1 methylcrotonoyl-Coenzyme A carboxylase 1 (alpha) L11RA interleukin 11 receptor, alpha MCHR1 melanin-concentrating hormone receptor 1 L13 interleukin 13 40 MCRS1 microspherule protein 1 L17RA interleukin 17 receptor A MDH1 malate dehydrogenase 1, NAD (soluble) L18 interleukin 18 (interferon-gamma-inducing factor) MDH2 malate dehydrogenase 2, NAD (mitochondrial) L1B interleukin 1, beta MEP1B meprin A, beta interleukin 5 (colony-stimulating factor, eosinophil) MEPE matrix, extracellular phosphoglycoprotein interleukin 6 (interferon, beta 2) with ASARM motif (bone) interleukin 8 45 MIF macrophage migration inhibitory factor interleukin 8 receptor, beta (glycosylation-inhibiting factor) integrin-linked kinase MIP major intrinsic protein of lens fiber integrin, alpha2b (platelet glycoprotein MKI67 antigen identified by monoclonal antibody Ki-67 IIb of IIb, IIIa complex, antigen CD41) MLNR motilin receptor TGA3 integrin, alpha 3 (antigen CD49C, MMP1 matrix metallopeptidase 1 (interstitial collagenase) alpha 3 subunit of VLA-3 receptor) 50 MMP10 matrix metallopeptidase 10 (stromelysin 2) TGA4 integrin, alpha 4 (antigen CD49D, alpha 4 MMP11 matrix metallopeptidase 11 (stromelysin 3) subunit of VLA-4 receptor) MMP12 matrix metallopeptidase 12 (macrophage elastase) TGAV integrin, alpha V (vitronectin receptor, MMP13 matrix metallopeptidase 13 (collagenase 3) alpha polypeptide, antigen CD51) MMP14 matrix metallopeptidase 14 (membrane-inserted) TGB1 integrin, beta 1 (fibronectin receptor, beta polypeptide, MMP15 matrix metallopeptidase 15 (membrane-inserted) antigen CD29 includes MDF2, MSK12) 55 MMP16 matrix metallopeptidase 16 (membrane-inserted) TGB3 integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) MMP17 matrix metallopeptidase 17 (membrane-inserted) TGB4 integrin, beta 4 MMP19 matrix metallopeptidase 19 TGB6 integrin, beta 6 MMP2 matrix metallopeptidase 2 (gelatinase A, 72 kDa TGB8 integrin, beta 8 gelatinase, 72 kDa type IV collagenase) TIH4 inter-alpha (globulin) inhibitor H4 MMP2O matrix metallopeptidase 20 (plasma Kallikrein-sensitive glycoprotein) MMP21 matrix metallopeptidase 21 involucrin 60 MMP23A matrix metallopeptidase 23A (pseudogene) junctophilin 4 MMP23B matrix metallopeptidase 23B jun oncogene MMP24 matrix metallopeptidase 24 (membrane-inserted) jun B proto-oncogene MMP25 matrix metallopeptidase 25 kinase insert domain receptor MMP26 matrix metallopeptidase 26 (a type III receptor tyrosine kinase) MMP27 matrix metallopeptidase 27 KLF12 Kruppel-like factor 12 65 MMP28 matrix metallopeptidase 28 KLKB1 kallikrein B, plasma (Fletcher factor) 1 MMP3 matrix metallopeptidase 3 (stromelysin 1, progelatinase) US 7,970,552 B1 31 32 -continued -continued

Gene Gene symbol Description symbol Description MMP7 matrix metallopeptidase 7 (matrilysin, uterine) PTEN phosphatase and tensin homolog MMP8 matrix metallopeptidase 8 (neutrophil collagenase) PTGER4 prostaglandin E receptor 4 (Subtype EP4) MMP9 matrix metallopeptidase 9 (gelatinase B, PTGIR prostaglandin I2 (prostacyclin) receptor (IP) 92 kDa gelatinase, 92 kDa type IV collagenase) PTGIS prostaglandin I2 (prostacyclin) synthase MPV17 MpV17 mitochondrial inner membrane protein PTGS2 prostaglandin-endoperoxide synthase 2 MRC1 mannose receptor, C type 1 (prostaglandin GH synthase and ) MRC2 mannose receptor, C type 2 10 PTK2 PTK2 protein tyrosine kinase 2 MRGPRX1 MAS-related GPR, member X1 PTK7 PTK7 protein tyrosine kinase 7 MSBP3 minisatellite binding protein 3, 115 kDa PTN pleiotrophin MSH6 mutS homolog 6 (E. coli) PTTG1 pituitary tumor-transforming 1 MUC1 mucin 1, cell Surface associated PYGB phosphorylase, glycogen; brain MYLK myosin light chain kinase RAB8A RAB8A, member RAS oncogene family NAGLU N-acetylglucosaminidase, alpha 15 RAC1 ras-related C3 botulinum toxin substrate 1 NAGS N-acetylglutamate synthase (rho family, Small GTP binding protein Rac1) NAMPT nicotinamide phosphoribosyltransferase RADS1 RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae) NANOS1 nanos homolog 1 (Drosophila) RBP4. retinol binding protein 4, plasma NCL nucleolin RECK reversion-inducing-cysteine-rich protein with kazal motifs NCOR2 nuclear receptor co-repressor 2 RELA V-rel reticuloendotheliosis viral oncogene FAT5 nuclear factor of activated T-cells 5, tonicity-responsive homolog A (avian) N FKB1 nuclear factor of kappa light polypeptide RETN resistin gene enhancer in B-cells 1 RHOA ras homolog gene family, member A NFKB repressing factor RLN1 relaxin 1 nitric oxide synthase 1 (neuronal) RLN2 relaxin 2 nitric oxide synthase 2A (inducible, hepatocytes) RPE ribulose-5-phosphate-3-epimerase nitric oxide synthase 3 (endothelial cell) RRM2 ribonucleotide reductase M2 polypeptide aminopeptidase puromycin sensitive 25 RUNX2 runt-related transcription factor 2 neuropeptide Y receptor Y5 S OOA8 S100 calcium binding protein A8 nuclear receptor Subfamily 1, group H, member 2 S AT spermidine?spermine N1-acetyltransferase 1 nuclear receptor Subfamily 1, group I, member 3 SAT2AT spermidine?spermine N1-acetyltransferase family nuclear receptor Subfamily 4, group A, member 1 member 2 neuropilin 2 SERPINA3 Serpin peptidase inhibitor, clade A neurotrophic tyrosine kinase, receptor, type 1 30 (alpha-1 antiproteinase, antitrypsin), member 3 T ornithine aminotransferase (gyrate atrophy) SERPINAf Serpin peptidase inhibitor, clade A occludin (alpha-1 antiproteinase, antitrypsin), member 7 ornithine decarboxylase 1 SERPINB1 serpin peptidase inhibitor, clade B opticin (ovalbumin), member ornithine carbamoyltransferase SERPINB3 serpin peptidase inhibitor, clade B ovostatin 35 (ovalbumin), member 3 oxidase (cytochrome c) assembly 1-like SERPINE1 Serpin peptidase inhibitor, clade E (nexin, procollagen-proline, 2-oxoglutarate 4-dioxygenase plasminogen activator inhibitor type 1), member 1 (proline 4-hydroxylase), beta polypeptide SERPINF2 serpin peptidase inhibitor, clade F PAH phenylalanine hydroxylase (alpha-2 antiplasmin, pigment epithelium derived pyruvate carboxylase factor), member 2 PCK2 phosphoenolpyruvate carboxykinase 2 (mitochondrial) SERPINH1 Serpin peptidase inhibitor, clade H (heat shock PCNT pericentrin 40 protein 47), member 1, (collagen binding PCSK6 proprotein convertase subtilisinkexin type 6 protein 1) PCSK7 proprotein convertase subtilisin?kexin type 7 stratifin DIA2 protein disulfide isomerase family A, member 2 LAMF7 SLAM family member 7 platelet factor 4 (chemokine (C-X-C motif) ligand 4) LC14A2 Solute carrier family 14 (urea transporter), member 2 HB prohibitin LC17AS Solute carrier family 17 (anion sugar HEX phosphate regulating endopeptidase homolog, X-linked 45 transporter), member 5 3 peptidase inhibitor 3, skin-derived (SKALP) Solute carrier family 1 (glial high affinity glutamate phosphoinositide-3-kinase, class 2, alpha polypeptide transporter), member 3 phospholipase A2, group IB (pancreas) SLC25A10 solute carrier family 25 (mitochondrial carrier; plasminogen activator, urokinase dicarboxylate transporter), member 10 pleckstrin homology domain containing, family F SLC25A12 solute carrier family 25 (mitochondrial carrier, (with FYVE domain) member 1 50 Aralar), member 12 plasminogen SLC25A13 solute carrier family 25, member 13 (citrin) LXNB1 plexin B1 SLC25A15 solute carrier family 25 (mitochondrial carrier; LXNC1 plexin C1 ornithine transporter) member 15 OR P450 (cytochrome) SLC25A2 solute carrier family 25 (mitochondrial carrier; PARA peroxisome proliferator-activated receptor alpha ornithine transporter) member 2 PARG peroxisome proliferator-activated receptor gamma 55 Solute carrier family 2 (facilitated glucose PIA peptidylprolyl isomerase A (cyclophilin A) transporter), member 1 RDM2 PR domain containing 2, with ZNF domain SLC2A10 REP prolyl endopeptidase Solute carrier family 2 (facilitated glucose RKACA protein kinase, cAMP-dependent, catalytic, alpha transporter), member 10 RKCA protein kinase C, alpha Solute carrier family 2 (facilitated glucose RKG1 protein kinase, c0MP-dependent, type I transporter), member 3 RSS2 protease, serine, 2 (trypsin 2) 60 SLC2AS Solute carrier family 2 (facilitated glucosef fructose RSS7 protease, serine, 7 (enterokinase) transporter), member 5 RTN3 proteinase 3 Solute carrier family 2 (facilitated glucose PSG2 pregnancy specific beta-1-glycoprotein 2 transporter), member 6 PSMB8 proteasome (prosome, macropain) subunit, Solute carrier family 2, (facilitated glucose beta type, 8 (large multifunctional peptidase 7) transporter) member 8 PSMC6 proteasome (prosome, macropain) 26S subunit, ATPase, 6 65 Solute carrier family 2 (facilitated glucose PTAFR platelet-activating factor receptor transporter), member 9 US 7,970,552 B1 33 34 -continued

Gene Gene symbol Description symbol Description SLC37A4 Solute carrier family 37 (glucose-6-phosphate AKT1 V-akt murine thymoma viral oncogene homolog 1 transporter), member 4 AMHR2 anti-Mullerian hormone receptor, type II SLC38A1 solute carrier family 38, member 1 APAF1 apoptotic peptidase activating factor 1 SLPI Secretory leukocyte peptidase inhibitor APEX1 APEX nuclease (multifunctional DNA repair enzyme)1 SLPI Secretory leukocyte peptidase inhibitor APOBEC1 apolipoprotein B mRNA editing enzyme, SMAD1 SMAD family member 1 catalytic polypeptide 1 SMPD2 sphingomyelin phosphodiesterase 2, neutral 10 ATADS ATPase family, AAA domain containing 5 membrane (neutral sphingomyelinase) BAD BCL2-antagonist of cell death SMPD3 sphingomyelin phosphodiesterase 3, neutral BAG1 BCL2-associated athanogene membrane (neutral sphingomyelinase II) BAG2 BCL2-associated athanogene 2 SMS spermine synthase BAG3 BCL2-associated athanogene 3 SNAI1 Snail homolog 1 (Drosophila) BAG4 BCL2-associated athanogene 4 SNAP23 synaptosomal-associated protein, 23 kDa 15 BAGS BCL2-associated athanogene 5 SNAPIN SNAP-associated protein BAK1 BCL2-antagonist/killer 1 SOD3 Superoxide dismutase 3, extracellular BAX BCL2-associated X protein SPARC Secreted protein, acidic, cysteine-rich (osteonectin) BBC3 BCL2 binding component 3 SPOCK3 sparcosteonectin, cwcv and kazal-like domains BCAP29 B-cell receptor-associated protein 29 proteoglycan (testican) 3 BCAP31 B-cell receptor-associated protein 31 SPP1 Secreted phosphoprotein 1 BCL10 B-cell CLL/lymphoma 10 SRM spermidine synthase BCL2 B-cell CLL/lymphoma 2 STAR steroidogenic acute regulatory protein BCL2A1 BCL2-related protein Al STAT3 signal transducer and activator of transcription 3 BCL2L1 BCL2-like 1 (acute-phase response factor) BCL2L10 BCL2-like 10 (apoptosis facilitator) STX4 4 BCL2L11 BCL2-like 11 (apoptosis facilitator) SUMO1 SMT3 suppressor of miftwo BCL2L12 BCL2-like 12 (proline rich) 3 homolog 1 (S. cerevisiae) 25 BCL2L13 BCL2-like 13 (apoptosis facilitator) TAT tyrosine aminotransferase BCL2L14 BCL2-like 14 (apoptosis facilitator) TE TEK tyrosine kinase, endothelial BCL2L15 BCL2-like 15 TFAP2A transcription factor AP-2 alpha BCL2L2 BCL2-like 2 (activating enhancer binding protein 2 alpha) BCL2L7P1 BCL2-like 7 pseudogene 1 TFAP2B transcription factor AP-2 beta BCL2L7P2 BCL2-like 7 pseudogene 2 (activating enhancer binding protein 2 beta) 30 BID BH3 interacting domain death agonist TFAP2C transcription factor AP-2 gamma BIK BCL2-interacting killer (apoptosis-inducing) (activating enhancer binding protein 2 gamma) BIRC2 baculoviral IAP repeat-containing 2 TFPI2 tissue factor pathway inhibitor 2 BIRCS baculoviral IAP repeat-containing 5 (Survivin) TGFB1 transforming growth factor, beta 1 BMF Bcl2 modifying factor TGM2 transglutaminase 2 (C polypeptide, BNIP1 BCL2/adenovirus E1B 19 kDa interacting protein 1 protein-glutamine-gamma-glutamyltransferase) 35 BNIP2 BCL2/adenovirus E1B 19 kDa interacting protein 2 THEBS1 thrombospondin 1 BOK BCL2-related ovarian killer THEBS2 thrombospondin2 CAMKK1 calcium calmodulin-dependent TIMP1 TIMP metallopeptidase inhibitor 1 protein kinase kinase 1, alpha TIMP2 TIMP metallopeptidase inhibitor 2 CARD10 caspase recruitment domain family, member 10 TIMP3 TIMP metallopeptidase inhibitor 3 CARD8 caspase recruitment domain family, member 8 TIMP4 TIMP metallopeptidase inhibitor 4 CASP1 caspase 1, apoptosis-related cysteine TNF tumor necrosis factor (TNF Superfamily, member 2) 40 peptidase (interleukin 1, beta, convertase) TP53 tumor protein p53 CASP3 caspase 3, apoptosis-related cysteine peptidase TRH thyrotropin-releasing hormone CASP7 caspase 7, apoptosis-related cysteine peptidase TSPANT tetraspanin 7 CASP8 caspase 8, apoptosis-related cysteine peptidase TTR transthyretin CC2D1A coiled-coil and C2 domain containing 1A TUBB tubulin, beta CHUK conserved helix-loop-helix ubiquitous kinase TUSC4 tumor Suppressor candidate 4 45 CIAPIN1 cytokine induced apoptosis inhibitor 1 TYRP1 -related protein 1 CNDP2 CNDP dipeptidase 2 (metallopeptidase M20 family) UCN urocortin COP1 caspase-1 dominant-negative inhibitor pseudo-ICE UMOD uromodulin CP ceruloplasmin (ferroxidase) UTS2R urotensin 2 receptor CREBBP CREB binding protein (Rubinstein-Taybi syndrome) WAMP2 vesicle-associated membrane protein CXCL12 chemokine (C-X-C motif) ligand 12 2 ( 2) 50 (stromal cell-derived factor 1) WCAM1 vascular cell adhesion molecule 1 CXCLS chemokine (C-X-C motif) ligand 5 VCL. Vinculin CYCS cytochrome c, somatic VEGFA vascular endothelial growth factor A CYP24A1 cytochrome P450, family 24, VTN vitronectin Subfamily A, polypeptide 1 WASF3 WAS protein family, member 3 DDIT3 DNA-damage-inducible transcript 3 WEE1 WEE1 homolog (S. pombe) 55 EDN1 endothelin 1 YBX1 Y box binding protein 1 EGF epidermal growth factor (beta-urogastrone) ZEB2 Zinc finger E-box binding homeobox2 EGFR epidermal growth factor receptor ZNF148 Zinc finger protein 148 EIF4A1 eukaryotic translation initiation factor 4A, isoform 1 ZNF267 Zinc finger protein 267 EIF4A2 eukaryotic translation initiation factor 4A, isoform 2 ZNF318 Zinc finger protein 318 EP300 E1A binding protein p300 F3 coagulation factor III (thromboplastin, tissue factor) 60 FAIM3 Fas apoptotic inhibitory molecule 3 FAS Fas (TNF receptor Superfamily, member 6) FASLG Fas ligand (TNF Superfamily, member 6) Example 4 FGF1 fibroblast growth factor 1 (acidic) FGF10 fibroblast growth factor 10 FGF11 fibroblast growth factor 11 Using the technique of Example 1, the biologically active 65 FGF12 fibroblast growth factor 12 nutrient for inflammatory and immune disease is identified. FGF13 fibroblast growth factor 13 The relevant genes for this identification would include: US 7,970,552 B1 35 36 -continued -continued

Gene Gene symbol Description symbol Description

O ast grow actor 14 MAP3K7IP1 mitogen-activated protein kinase kinase O ast grow actor 16 kinase 7 interacting protein 1 O ast grow actor 17 MAP3K7IP2 mitogen-activated protein kinase kinase O ast grow actor 18 kinase 7 interacting protein 2 O ast grow actor 19 MAP3K7IP3 mitogen-activated protein kinase kinase O ast grow actor 2 (basic) kinase 7 interacting protein 3 O ast grow actor 20 10 MAPK1 mitogen-activated protein kinase 1 O ast grow actor 21 MAPK10 mitogen-activated protein kinase 10 O ast grow actor 22 MAPK14 mitogen-activated protein kinase 14 O ast grow actor 3 MAPK3 mitogen-activated protein kinase 3 O ast grow actor 4 MAPK8 mitogen-activated protein kinase 8 O ast grow actor 5 MAPK8IP2 mitogen-activated protein kinase 8 O ast grow actor 6 15 interacting protein 2 O ast grow actor 7 (keratinocyte growth factor) MAPK9 mitogen-activated protein kinase 9 O ast grow actor 8 (androgen-induced) MAPKAPK2 mitogen-activated protein kinase-activated O ast grow actor 9 (glia-activating factor) protein kinase 2 v-Ha-ras Harvey rat Sarcoma viral oncogene homolog MIRN1SA microRNA. 15a HIV-1 Rev binding protein MKI67 antigen identified by monoclonal antibody Ki-67 harakiri, BCL2 interacting protein MOAP1 modulator of apoptosis 1 (contains only BH3 domain) MRPL41 mitochondrial ribosomal protein LA-1 heat shock protein 90 kDa alpha MSH2 mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) (cytosolic), class A member 1 MSH6 mutS homolog 6 (E. coli) heat shock 70 kDa protein 4 MUL1 mitochondrial ubiquitin ligase activator of NFKB 1 heat shock 70 kDa protein 8 MYC v-myc myelocytomatosis viral oncogene homolog (avian) H ErA serine peptidase 2 MYD88 myeloid differentiation primary response gene (88) in ercellular adhesion molecule 1 25 FATC1 nuclear factor of activated T-cells, cytoplasmic, immediate early response 3 calcineurin-dependent 1 in e eron, alpha 4 FATC2 nuclear factor of activated T-cells, cytoplasmic, in e eron (alpha, beta and omega) receptor 1 calcineurin-dependent 2 in e eron (alpha, beta and omega) receptor 2 FKB nuclear factor of kappa light polypeptide gene in e eron, gamma enhancer in B-cells 1 insul in-like growth factor 1 (Somatomedin C) 30 FKB2 nuclear factor of kappa light polypeptide gene in e eukin 10 enhancer in B-cells 2 (p49, p100) in e eukin 15 receptor, alpha nuclear factor of kappa light polypeptide gene in e eukin 17B enhancer in B-cells inhibitor, alpha in e eukin 18 binding protein nuclear factor of kappa light polypeptide gene in e eukin 18 receptor 1 enhancer in B-cells inhibitor, beta in e eukin 18 receptor accessory protein 35 nuclear factor of kappa light polypeptide gene in e eukin 1, alpha enhancer in B-cells inhibitor, delta in e eukin 1, beta nuclear factor of kappa light polypeptide gene in e eukin 1 family, member 10 (theta) enhancer in B-cells inhibitor, epsilon in e eukin 1 family, member 6 (epsilon) nuclear factor of kappa light polypeptide gene in e eukin 1 family, member 8 (eta) enhancer in B-cells inhibitor, epsilon in e eukin 1 receptor, type I nuclear factor of kappa light polypeptide gene 40 in e eukin 1 receptor, type II enhancer in B-cells inhibitor, Zeta in e eukin 1 receptor accessory protein NFKB activating protein in e eukin 1 receptor accessory protein-like 1 NFKB activating protein-like in e eukin 1 receptor accessory protein-like 2 NFKB inhibitor interacting Ras-like in e eukin 1 receptor-like 1 NFKB inhibitor interacting Ras-like 2 in e eukin 1 receptor-like 2 NFKB repressing factor in e eukin 1 receptor antagonist 45 NLR family, pyrin domain containing 1 in e eukin 6 (interferon, beta 2) NO D 2 nucleotide-binding oligomerization domain containing 2 RAK1 in e eukin-1 receptor-associated kinase 1 NOS2A nitric oxide synthase 2A (inducible, hepatocytes) RAK1 in e eukin-1 receptor-associated kinase 1 NOX4 NADPH oxidase 4 RAK2 in e eukin-1 receptor-associated kinase 2 PAWR PRKC, apoptosis, WT1, regulator RAK4 in e eukin-1 receptor-associated kinase 4 PI3 peptidase inhibitor 3, skin-derived (SKALP) RF1 in e eron regulatory factor 1 50 PLCG2 phospholipase C, gamma 2 RF9 in e eron regulatory factor 9 (phosphatidylinositol-specific) TGB4 in egrin, beta 4 PLEKHGS pleckstrin homology domain containing, AG1 jagged 1 (Alagile syndrome) amily G (with RhoGef domain) member 5 AK1 Janus kinase 1 (a protein tyrosine kinase) phorbol-12-myristate-13-acetate-induced protein 1 JUN jun oncogene protein phosphatase 1, catalytic Subunit, alpha isoform JUNB jun B proto-oncogene 55 protein phosphatase 1, catalytic Subunit, beta isoform KDSR 3-ketodihydrosphingosine reductase protein phosphatase 1, catalytic Subunit, gamma isoform protein phosphatase 1, regulatory (inhibitor) subunit 13 like KGFLP1 keratinocyte growth factor-like protein 1 protein phosphatase 1, regulatory (inhibitor) subunit 1B LAG3 lymphocyte-activation gene 3 protein phosphatase 2 (formerly 2A), LAMA2 laminin, alpha 2 (merosin, congenital muscular dystrophy) catalytic subunit, alpha isoform LBR lamin B receptor PPP2R4 protein phosphatase 2A activator, regulatory Subunit 4 MAP2K1 mitogen-activated protein kinase kinase 1 60 PPP2RSA protein phosphatase 2, regulatory subunit B, alpha isoform MAP2K3 mitogen-activated protein kinase kinase 3 PRKACA protein kinase, cAMP-dependent, catalytic, alpha MAP2K4 mitogen-activated protein kinase kinase 4 PRKCA protein kinase C, alpha MAP2K6 mitogen-activated protein kinase kinase 6 PRKCZ protein kinase C, Zeta MAP3K1 mitogen-activated protein kinase kinase kinase 1 PSIP1 PC4 and SFRS1 interacting protein 1 MAP3K14 mitogen-activated protein kinase kinase kinase 14 PTGIR prostaglandin I2 (prostacyclin) receptor (IP) MAP3K3 mitogen-activated protein kinase kinase kinase 3 65 PTGS2 prostaglandin-endoperoxide synthase 2 MAP3K7 mitogen-activated protein kinase kinase kinase 7 (prostaglandin GH synthase and cyclooxygenase) US 7,970,552 B1 37 38 -continued -continued

Gene Gene symbol Description symbol Description CEBPB CCAAT?enhancer binding protein (C/EBP), beta PTGS2 prostaglandin-endoperoxide synthase 2 CES1 carboxylesterase 1 (monocyte/macrophage (prostaglandin GH synthase and cyclooxygenase) serine esterase 1) REL V-rel reticuloendotheliosis viral oncogene homolog (avian) CETP cholesteryl ester transfer protein, plasma RELA V-rel reticuloendotheliosis viral oncogene homolog A (avian) CHUK conserved helix-loop-helix ubiquitous kinase RELB V-rel reticuloendotheliosis viral oncogene homolog B CP ceruloplasmin (ferroxidase) RIPK1 receptor (TNFRSF)-interacting serine-threonine kinase 1 CXCL12 chemokine (C-X-C motif) ligand 12 RNF216 ring finger protein 216 10 (stromal cell-derived factor 1) RNF216L ring finger protein 21.6-like CXCLS chemokine (C-X-C motif) ligand 5 RNF25 ring finger protein 25 CYP1A2 cytochrome P450, family 1, subfamily A, polypeptide 2 ROS1 c-ros oncogene 1, receptor tyrosine kinase CYP2C19 cytochrome P450, family 2, subfamily C, polypeptide 19 RPL17 ribosomal protein L17 CYP2C8 cytochrome P450, family 2, subfamily C, polypeptide 8 RTN3 reticulon 3 CYP2C9 cytochrome P450, family 2, subfamily C, polypeptide 9 SATB1 SATB homeobox 1 15 CYP22 cytochrome P450, family 2, subfamily J, polypeptide 2 SCNN1B Sodium channel, nonvoltage-gated 1, beta CYP3A cytochrome P450, family 3, subfamily A SCNN1G Sodium channel, nonvoltage-gated 1, gamma CYP3A4 cytochrome P450, family 3, subfamily A, polypeptide 4 SERPINE1 Serpin peptidase inhibitor, clade E CYP3A5 cytochrome P450, family 3, subfamily A, polypeptide 5 (nexin, plasminogen activator inhibitor type 1), member 1 CYP51A1 cytochrome P450, family 51, subfamily A, polypeptide 1 SIRTS sirtuin (silent mating type information regulation 2 DDX11 DEAD H (Asp-Glu-Ala-Asp/His) box polypeptide 11 homolog) 5 DHFR dihydrofolate reductase SLC34A3 solute carrier family 34 (sodium phosphate), member 3 ECSIT ECSIT homolog (Drosophila) ST2 Suppression of tumorigenicity 2 EDN1 endothelin 1 STAT1 signal transducer and activator of transcription 1, 91 kDa EFNB3 ephrin-B3 STAT2 signal transducer and activator of transcription 2, 113 kDa FGF19 fibroblast growth factor 19 TANK TRAF family member-associated NFKB activator FMO3 flavin containing monooxygenase 3 TBK1 TANK-binding kinase 1 FOXO3 forkhead box O3 TBKBP1 TBK1 binding protein 1 25 GSTA2 glutathione S-transferase A2 TGFB1 transforming growth factor, beta 1 GSTM1 glutathione S-transferase M1 TICAM2 oll-like receptor adaptor molecule 2 GSTM3 glutathione S-transferase M3 (brain) TMED4 transmembrane emp24 protein transport GSTP1 glutathione S-transferase pi 1 domain containing 4 GSTT1 glutathione S-transferase theta 1 TNF tumor necrosis factor (TNF Superfamily, member 2) HMOX1 heme (decycling) 1 TNFAIP1 tumor necrosis factor, alpha-induced 30 HSF1 heat shock transcription factor 1 protein 1 (endothelial) HSPB2 heat shock 27 kDa protein 2 TNFRSF1OD tumor necrosis factor receptor Superfamily, GHG1 immunoglobulin heavy constant gamma 1 (Glm marker) member 10d, decoy with truncated death domain GKC immunoglobulin kappa constant TNFRSF11A tumor necrosis factor receptor Superfamily, KBKB inhibitor of kappa light polypeptide gene member 11a, NFKB activator enhancer in B-cells, kinase beta TNFRSF11B tumor necrosis factor receptor Superfamily, 35 interleukin 10 member 11b interleukin 17B TNFRSF1A tumor necrosis factor receptor Superfamily, interleukin 18 receptor 1 member 1A interleukin 18 receptor accessory protein TNFRSF4 tumor necrosis factor receptor Superfamily, interleukin 1, alpha member 4 interleukin 1, beta TNFSF10 tumor necrosis factor (ligand) Superfamily, interleukin 1 family, member 10 (theta) member 10 40 interleukin 1 family, member 6 (epsilon) TNFSF11 tumor necrosis factor (ligand) Superfamily, interleukin 1 family, member 8 (eta) member 11 interleukin 1 receptor, type I TNFSF14 tumor necrosis factor (ligand) Superfamily, interleukin 1 receptor, type II member 14 interleukin 1 receptor accessory protein TOLLIP oll interacting protein interleukin 1 receptor accessory protein-like 1 TP53 tumor protein p53 45 interleukin 1 receptor accessory protein-like 2 TP53BP2 tumor protein p53 binding protein, 2 interleukin 1 receptor-like 1 TRAF1 TNF receptor-associated factor 1 interleukin 1 receptor-like 2 TRAF2 TNF receptor-associated factor 2 interleukin 1 receptor antagonist TRAF3 TNF receptor-associated factor 3 interleukin 2 receptor, gamma (severe TRAF3IP2 TRAF3 interacting protein 2 combined immunodeficiency) TRAFS TNF receptor-associated factor 5 50 interleukin 6 (interferon, beta 2) TRAF6 TNF receptor-associated factor 6 interleukin-1 receptor-associated kinase 1 TRAF6 TNF receptor-associated factor 6 interleukin-1 receptor-associated kinase 2 TRIM38 tripartite motif-containing 38 interleukin-1 receptor-associated kinase 4 TYK2 tyrosine kinase 2 interferon regulatory factor 1 jun oncogene 55 KGFLP1 keratinocyte growth factor-like protein 1 LAG3 lymphocyte-activation gene 3 Example 5 LBR lamin B receptor MAP2K3 mitogen-activated protein kinase kinase 3 MAP2K4 mitogen-activated protein kinase kinase 4 Using the technique of Example 1, the biologically active MAP2K6 mitogen-activated protein kinase kinase 6 nutrient for gastro intestinal disease is identified. The relevant MAP3K1 mitogen-activated protein kinase kinase kinase 1 60 MAP3K14 mitogen-activated protein kinase kinase kinase 14 genes for this identification would include: MAP3K7 mitogen-activated protein kinase kinase kinase 7 MAP3K7IP1 mitogen-activated protein kinase kinase kinase 7 interacting protein 1 Gene symbol Description MAP3K7IP2 mitogen-activated protein kinase kinase kinase 7 interacting protein 2 aldehyde dehydrogenase 1 family, member A1 65 MAP3K7IP3 mitogen-activated protein kinase kinase kinase 7 ATPase, Cu++ transporting, beta polypeptide interacting protein 3 US 7,970,552 B1 39 40 -continued

Gene symbol Description Gene symbol Description MAPK10 mitogen-activated protein kinase 10 MAPK14 mitogen-activated protein kinase 14 alpha-fetoprotein MAPK8 mitogen-activated protein kinase 8 AGMAT agmatine ureohydrolase (agmatinase) MAPK8IP2 mitogen-activated protein kinase 8 interacting protein 2 AGTR1 angiotensin II receptor, type 1 MAPK9 mitogen-activated protein kinase 9 ALB albumin MT2A metallothionein 2A ALDH2 aldehyde dehydrogenase 2 family (mitochondrial) MTHFR 5,10-methylenetetrahydrofolate reductase (NADPH) ALDH3A1 aldehyde dehydrogenase 3 family, memberA1 MUC1 mucin 1, cell Surface associated 10 ALDH9A1 aldehyde dehydrogenase 9 family, member A1 MYC v-myc myelocytomatosis viral oncogene homolog (avian) ALDOB aldolase B, fructose-bisphosphate MYCN v-myc myelocytomatosis viral related AL CC) alkaline phosphatase, placental (Regan isozyme) oncogene, neuroblastoma derived (avian) ANGPT2 angiopoietin 2 MYD88 myeloid differentiation primary response gene (88) ANKH ankylosis, progressive homolog (mouse) NFKB1 nuclear factor of kappa light polypeptide ANPE alanyl (membrane) aminopeptidase gene enhancer in B-cells 1 15 ASL argininosuccinate lyase NFKB2 nuclear factor of kappa light polypeptide ASS argininosuccinate synthetase 1 gene enhancer in B-cells 2 (p49, p100) BMP6 bone morphogenetic protein 6 NFKBLA nuclear factor of kappa light polypeptide BMP8B bone morphogenetic protein 8b gene enhancer in B-cells inhibitor, alpha BTBD1 BTB (POZ) domain containing 1 NFKBIB nuclear factor of kappa light polypeptide BTD biotinidase gene enhancer in B-cells inhibitor, beta CA2 carbonic anhydrase II NFKBIE nuclear factor of kappa light polypeptide CA3 carbonic anhydrase III, muscle specific gene enhancer in B-cells inhibitor, epsilon CABIN1. calcineurin binding protein 1 NFYA nuclear transcription factor Y, alpha CALR calreticulin NR12 nuclear receptor Subfamily 1, group I, member 2 CASP3 caspase 3, apoptosis-related cysteine peptidase NR13 nuclear receptor Subfamily 1, group I, member 3 CASP4 caspase 4, apoptosis-related cysteine peptidase peptidase inhibitor 3, skin-derived (SKALP) CASPS caspase 5, apoptosis-related cysteine peptidase POR P450 (cytochrome) oxidoreductase 25 CASP8 caspase 8, apoptosis-related cysteine peptidase PPARA peroxisome proliferator-activated receptor alpha CASP9 caspase 9, apoptosis-related cysteine peptidase AT catalase PPARD peroxisome proliferator-activated receptor delta PPARG peroxisome proliferator-activated receptor gamma cadherin 1, type 1, E-cadherin (epithelial) cadherin 5, type 2 (vascular endothelium) PPARGC1A peroxisome proliferator-activated receptor cyclin-dependent kinase 7 gamma, coactivator 1 alpha 30 cyclin-dependent kinase inhibitor 1A (p21, Cip1) PPARGC1B peroxisome proliferator-activated receptor cyclin-dependent kinase inhibitor 2A gamma, coactivator 1 beta (melanoma, p16, inhibits CDK4) PRIC285 peroxisomal proliferator-activated receptor A C cyclin-dependent kinase inhibitor 3 interacting complex (CDK2-associated dual specificity PRKCZ protein kinase C, Zeta phosphatase) PTGS2 prostaglandin-endoperoxide synthase 2 35 LIC1 chloride intracellular channel 1 RARA retinoic acid receptor, alpha ceruloplasmin (ferroxidase) RIPK1 receptor (TNFRSF)-interacting serine-threonine PB1 carboxypeptidase B1 (tissue) kinase 1 PD carboxypeptidase D RNF216 ring finger protein 216 PE carboxypeptidase E RNF216L ring finger protein 21.6-like PS1 carbamoyl-phosphate synthetase 1, mitochondrial PT1A carnitine palmitoyltransferase 1A (liver) RXRA retinoid X receptor, alpha 40 complement component (3b/4b) receptor 1 RXRB retinoid X receptor, beta (Knops blood group) SCNN1B Sodium channel, nonvoltage-gated 1, beta cellular retinoic acid binding protein 1 SCNN1G Sodium channel, nonvoltage-gated 1, gamma carnitine acetyltransferase SERPINE1 Serpin peptidase inhibitor, clade E cAMP responsive element binding protein 3 SIGIRR single immunoglobulin and toll-interleukin 1 CREB binding protein (Rubinstein-Taybi syndrome) receptor (TIR) domain 45 cAMP responsive element binding protein-like 1 S RT1 sirtuin (silent mating type information regulation cytochrome P450, family 17, subfamily A, polypeptide 1 2 homolog) 1 (S. cerevisiae) cytochrome P450, family 1, subfamily A, polypeptide 1 solute carrier family 34 (sodium phosphate), member 3 cytochrome P450, family 1, subfamily A, polypeptide 2 Suppression of tumorigenicity 2 cytochrome P450, family 2, subfamily A, polypeptide 6 transforming growth factor, beta 1 cytochrome P450, family 2, subfamily C, polypeptide 19 toll-like receptor adaptor molecule 2 50 cytochrome P450, family 2, subfamily C, polypeptide 9 tumor necrosis factor (TNF Superfamily, member 2) cytochrome P450, family 2, subfamily D, polypeptide 6 1 tumor necrosis factor, alpha-induced protein 1 (endothelial) cytochrome P450, family 2, subfamily E, polypeptide 1 S. R toll interacting protein cytochrome P450, family 3, subfamily A, polypeptide 4 tumor protein p53 cytochrome P450, family 3, subfamily A, polypeptide 5 tumor protein p73 decorin 55 dCMP deaminase RAF6 TNF receptor-associated factor 6 dipeptidyl-peptidase 4 (CD26, adenosine deaminase RIM38 tripartite motif-containing 38 complexing protein 2) BE2N ubiquitin-conjugating enzyme E2N dual oxidase 1 (UBC13 homolog, yeast) E2F transcription factor 1 U ubiquitin-conjugating enzyme E.2 variant 1 E4F transcription factor 1 epidermal growth factor (beta-urogastrone) 60 epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) Example 6 EGR2 early growth response 2 (Krox-20 homolog, Drosophila) EGR3 early growth response 3 ENG endoglin (Osler-Rendu-Weber syndrome 1) Using the technique of Example 1, the biologically active 65 ENO1 enolase 1, (alpha) nutrient for liver situations is identified. The relevant genes ESR1 estrogen receptor 1 for this identification would include: US 7,970,552 B1 41 42 -continued -continued

Gene Gene symbol Description symbol Description ESR2 estrogen receptor 2 (ER beta) interleukin 1 receptor accessory protein-like 2 ESRRB estrogen-related receptor beta interleukin 1 receptor-like 1 F10 coagulation factor X interleukin 1 receptor antagonist F8 coagulation factor VIII, procoagulant component interleukin 2 FADD Fas (TNFRSF6)-associated via death domain interleukin 20 FAH umarylacetoacetate hydrolase (fumarylacetoacetase) interleukin 22 FAP fibroblast activation protein, alpha 10 interleukin 28A (interferon, lambda 2) FAS Fas (TNF receptor Superfamily, member 6) interleukin 2 receptor, alpha FASLG Fas ligand (TNF Superfamily, member 6) interleukin 2 receptor, beta FASN atty acid synthase interleukin 4 FASTK Fas-activated serine/threonine kinase LAR interleukin 4 receptor FBP2 ructose-1,6-bisphosphatase 2 interleukin 6 (interferon, beta 2) FBXL2 F-box and leucine-rich repeat protein 2 15 interleukin 6 receptor FGL1 fibrinogen-like 1 interleukin 7 FGL2 fibrinogen-like 2 interleukin 7 receptor FIGF c-fos induced growth factor (vascular interleukin 8 endothelial growth factor D) interleukin 8 receptor, alpha FRY urry homolog (Drosophila) interleukin 8 receptor, beta FTH1 erritin, heavy polypeptide 1 interleukin enhancer binding factor 2, 45 kDa G3BP1 GTPase activating protein (SH3 domain) interleukin enhancer binding factor 3, 90 kDa binding protein 1 integrin, alpha 4 (antigen CD49D, glucose-6-phosphate dehydrogenase alpha 4subunit of VLA-4 receptor) UDP-galactose-4-epimerase TGAL integrin, alpha L (antigen CD11A (p180), lymphocyte glyceraldehyde-3-phosphate dehydrogenase function-associated antigen 1: alpha polypeptide) ap junction protein, beta 1, 32 kDa TGB1 integrin, beta 1 (fibronectin receptor, beta polypeptide, utamate dehydrogenase 1 25 antigen CD29 includes MDF2, MSK12) utamate-ammonia ligase (glutamine synthetase) TIEH4 inter-alpha (globulin) inhibitor H4 (plasma olgi autoantigen, golgin subfamily a 4 Kallikrein-sensitive glycoprotein) olgi membrane protein 1 inositol 14,5-triphosphate receptor, type 2 ycoprotein 2 (zymogen granule membrane) jagged 1 (Alagile syndrome) ycoprotein VI (platelet) anus kinase 1 (a protein tyrosine kinase) ycoprotein A33 (transmembrane) 30 ketohexokinase (fructokinase) ypican 3 aminin, beta 2 (laminin S) utamic-pyruvate transaminase ike-glycosyltransferase 8. lanine aminotransferase) ecithin-cholesterol acyltransferase PT2 l amic pyruvate transaminase ymphocyte-specific protein tyrosine kinase lanine aminotransferase) 2 ipocalin 1 (tear prealbumin) athione peroxidase 1 ymphocyte cytosolic protein 1 (L-plastin) athione peroxidase 2 (gastrointestinal) 35 ow density lipoprotein receptor athione peroxidase 5 (familial hypercholesterolemia) ididymal androgen-related protein) eukocyte cell-derived chemotaxin 2 GSR i athione reductase ymphoid enhancer-binding factor 1 GSTM1 athione S-transferase M1 eptin GSTP1 athione S-transferase pi 1 eptin receptor GSTT1 athione S-transferase theta 1 40 Leptin, serum levels of GUSB curonidase, beta LTA ymphotoxin alpha (TNF Superfamily, member 1) HBXIP e atitis B virus X interacting protein LTB ymphotoxin beta (TNF Superfamily, member 3) HDAC1 S one deacetylase 1 LTBP2 atent transforming growth factor beta binding protein 2 HDDC2 D domain containing 2 LTBR ymphotoxin beta receptor (TNFRSuperfamily, HIC1 hypermethylated in cancer 1 member 3) HNF4A hepatocyte nuclear factor 4, alpha 45 LTF actotransferrin HNRNPA1 heterogeneous nuclear ribonucleoprotein A1 MAGEA1 melanoma antigen family A, 1 NRNPA2B1 heterogeneous nuclear ribonucleoprotein A2, B1 (directs expression of antigen MZ2-E) HNRNPC heterogeneous nuclear ribonucleoprotein C (C1/C2) MAGEA4 melanoma antigen family A, 4 CAM1 intercellular adhesion molecule 1 MAP2K4 mitogen-activated protein kinase kinase 4 CAM3 intercellular adhesion molecule 3 MAP2K6 mitogen-activated protein kinase kinase 6 KBKB inhibitor of kappa light polypeptide gene 50 MAP2K7 mitogen-activated protein kinase kinase 7 enhancer in B-cells, kinase beta MAP3K14 mitogen-activated protein kinase kinase kinase 14 inhibitor of kappa light polypeptide gene MAP4K4 mitogen-activated protein kinase kinase kinase kinase 4 enhancer in B-cells, kinase epsilon MAPK1 mitogen-activated protein kinase 1 interleukin 10 MAPK10 mitogen-activated protein kinase 10 interleukin 10 receptor, alpha MAPK14 mitogen-activated protein kinase 14 interleukin 10 receptor, beta 55 MAPK8 mitogen-activated protein kinase 8 interleukin 12A (natural killer cell stimulatory factor 1, MARCKS myristoylated alanine-rich protein kinase C Substrate cytotoxic lymphocyte maturation factor 1, p35) MARCKSL1 MARCKS-like 1 interleukin 12B (natural killer cell stimulatory factor 2, MAT1A methionine adenosyltransferase I, alpha cytotoxic lymphocyte maturation factor 2, p.40) MAZ MYC-associated zinc finger protein interleukin 12 receptor, beta 1 (purine-binding transcription factor) interleukin 15 MBL1P1 mannose-binding lectin (protein A) 1, pseudogene 1 interleukin 18 (interferon-gamma-inducing factor) 60 MBL2 mannose-binding lectin (protein C) 2, interleukin 18 binding protein soluble (opsonic defect) interleukin 18 receptor 1 MBP myelin basic protein interleukin 19 MCM2 minichromosome maintenance complex component 2 interleukin 1, alpha MCM7 minichromosome maintenance complex component 7 interleukin 1, beta MCRS1 microspherule protein 1 interleukin 1 receptor, type I 65 MDM2 Mdm2 p53 binding protein homolog (mouse) interleukin 1 receptor accessory protein MEMO1 mediator of cell motility 1 US 7,970,552 B1 43 44 -continued -continued

Gene Gene symbol Description symbol Description MET met proto-oncogene (hepatocyte growth factor receptor) 5 RSF1 remodeling and spacing factor 1 MGAT3 mannosyl (beta-1,4-)-glycoprotein beta-1,4-N- RXRA retinoid X receptor, alpha acetylglucosaminyltransferase S1 OOB S100 calcium binding protein B MGATS mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl SCARB1 Scavenger receptor class B, member 1 glucosaminyltransferase SCARB2 Scavenger receptor class B, member 2 MGMT O-6-methylguanine-DNA methyltransferase SCLY Selenocysteine lyase NAGLU N-acetylglucosaminidase, alpha 10 SULT1A3 Sulfotransferase family, cytosolic, 1A, NAT1 N-acetyltransferase 1 (arylamine N-acetyltransferase) phenol-preferring, member 3 NAT2 N-acetyltransferase 2 (arylamine N-acetyltransferase) SULT2B1 Sulfotransferase family, cytosolic, 2B, member 1 NCL nucleolin SUOX sulfite oxidase NCOA6 nuclear receptor coactivator 6 TBK1 TANK-binding kinase 1 NDRG1 N-myc downstream regulated gene 1 TBP TATA box binding protein NFIL3 nuclear factor, interleukin 3 regulated 15 TBX21 T-box 21 NFKB1 nuclear factor of kappa light polypeptide gene TCEA1 transcription elongation factor A (SII), 1 enhancer in B-cells 1 TCF3 transcription factor 3 (E2A immunoglobulin nuclear factor of kappa light polypeptide gene enhancer binding factors E12/E47) enhancer in B-cells inhibitor, beta TDF tumor differentiation factor nuclear factor of kappa light polypeptide TERT elomerase reverse transcriptase gene enhancer in B-cells inhibitor-like 1 TG hyroglobulin NINJ1 ninjurin 1 TGFA transforming growth factor, alpha NNMT nicotinamide N-methyltransferase TGFB1 transforming growth factor, beta 1 NOS2A nitric oxide synthase 2A (inducible, hepatocytes) TGFB3 transforming growth factor, beta 3 NOSTRIN nitric oxide synthase trafficker TGFBR1 transforming growth factor, beta receptor 1 PAX5 paired box 5 TGFBRAP1 transforming growth factor, beta receptor PDCD1 programmed cell death 1 associated protein 1 PDGFB platelet-derived growth factor beta polypeptide 25 TGIF1 TGFB-induced factor homeobox 1 (simian sarcoma viral (v-sis) oncogene homolog) THEBS2 hrombospondin2 PDLIM3 PDZ and LIM domain 3 THEBS4 hrombospondin 4 PDXP pyridoxal (pyridoxine, vitamin B6) phosphatase THOC1 THO complex 1 PI4KA phosphatidylinositol 4-kinase, catalytic, alpha THPO hrombopoietin PIAS1 protein inhibitor of activated STAT, 1 THY1 Thy-1 cell Surface antigen PIAS3 protein inhibitor of activated STAT, 3 30 TK hymidine kinase 1, Soluble PIK3R1 phosphoinositide-3-kinase, regulatory Subunit 1 (alpha) TK2 hymidine kinase 2, mitochondrial PIN1 peptidylprolyl cis/trans isomerase, NIMA-interacting 1 TKT transketolase PITX1 paired-like homeodomain 1 TOP1 opoisomerase (DNA) I PNKD paroxysmal nonkinesigenic dyskinesia hioredoxin C PARA peroxisome proliferator-activated receptor alpha TXNIP hioredoxin interacting protein C PAT phosphoribosyl pyrophosphate amidotransferase 35 ubiquitin D C PIA peptidylprolyl isomerase A (cyclophilin A) ubiquitin-conjugating enzyme E2B (RAD6 homolog) C PIB peptidylprolyl isomerase B (cyclophilin B) ubiquitin-conjugating enzyme E2E 3 C PIG peptidylprolyl isomerase G (cyclophilin G) UBC4/5 homolog, yeast) PPM2C protein phosphatase 2C, magnesium-dependent, ubiquitin-conjugating enzyme E2K catalytic Subunit UBC1 homolog, yeast) PPP2R4 protein phosphatase 2A activator, regulatory subunit 4 ubiquitin-conjugating enzyme E2L 3 PRDX2 peroxiredoxin 2 40 ubiquitin protein ligase E3A PRF1 perforin 1 (pore forming protein) ubiquilin 1 PRKACA protein kinase, cAMP-dependent, catalytic, alpha uridine-cytidine kinase 1 PRKCB1 protein kinase C, beta 1 vitamin D (1,25-dihydroxyvitamin D3) receptor PRKCZ protein kinase C, Zeta vascular endothelial growth factor A PRKG1 protein kinase, c0MP-dependent, type I von Willebrand factor C and EGF domains PRL prolactin 45 Wolf-Hirschhorn syndrome candidate 2 PRM1 protamine 1 X-ray repair complementing defective repair in PRMT1 protein arginine methyltransferase 1 Chinese hamster cells 1 PRSM2 protease, metallo, 2 PRSS1 protease, serine, 1 (trypsin 1) PRTN3 proteinase 3 PTBP1 polypyrimidine tract binding protein 1 50 PTBP2 polypyrimidine tract binding protein 2 Example 7 PTEN phosphatase and tensin homolog PTGS1 prostaglandin-endoperoxide synthase 1 Using the technique of Example 1, the biologically active (prostaglandin GH synthase and cyclooxygenase) PTGS2 prostaglandin-endoperoxide synthase 2 nutrient for anxiety syndromes is identified. The relevant (prostaglandin GH synthase and cyclooxygenase) 55 genes for this identification would include: PTMA prothymosin, alpha PTPLAD1 protein tyrosine phosphatase-like A domain containing 1 PTPN11 protein tyrosine phosphatase, non-receptor type 11 Gene PTPN3 protein tyrosine phosphatase, non-receptor type 3 symbol Description PTPRC protein tyrosine phosphatase, receptor type, C PTPRCAP protein tyrosine phosphatase, receptor type, 60 ABCA1 ATP-binding cassette, sub-family A (ABC1), member 1 C-associated protein ABCC9 ATP-binding cassette, sub-family C PVR poliovirus receptor (CFTR/MRP), member 9 RELA V-rel reticuloendotheliosis viral oncogene ADARB1 adenosine deaminase, RNA-specific, homolog A (avian) B1 (RED1 homolog rat) REX1, RNA exonuclease 1 homolog ADAT1 adenosine deaminase, tRNA-specific 1 (S. cerevisiae)-like 1 65 ADCY1O adenylate cyclase 10 (soluble) RSAD2 radical S-adenosyl methionine domain containing 2 ADCYAP1 adenylate cyclase activating polypeptide 1 (pituitary) US 7,970,552 B1 45 46 -continued -continued

Gene Gene symbol Description symbol Description ADM adrenomedullin DNM1 1 ADORA1 adenosine A1 receptor DRDS dopamine receptor D5 ADORA2A adenosine A2a receptor EGR1 early growth response 1 ADORA3 adenosine A3 receptor EMP2 epithelial membrane protein 2 ADRA1B adrenergic, alpha-1B-, receptor ERN1 endoplasmic reticulum to nucleus signaling 1 ADRBK1 adrenergic, beta, receptor kinase 1 ERN2 endoplasmic reticulum to nucleus signaling 2 AGT angiotensinogen (serpin peptidase inhibitor, 10 F2R coagulation factor II (thrombin) receptor clade A, member 8) F2RL1 coagulation factor II (thrombin) receptor-like 1 AGTR1 angiotensin II receptor, type 1 F2RL2 coagulation factor II (thrombin) receptor-like 2 ANK2 ankyrin 2, neurona FAS Fas (TNF receptor Superfamily, member 6) ANXA3 annexin A3 FBP1 fructose-1,6-bisphosphatase 1 ANXA4 annexin A4 FBP2 fructose-1,6-bisphosphatase 2 P1 G1 adaptor-related protein complex 1, gamma 1 subunit 15 FIG4 FIG4 homolog (S. cerevisiae) P1 G1 adaptor-related protein complex 1, gamma 1 subunit FLNA filamin A, alpha (actin binding protein 280) PBA1 amyloid beta (A4) precursor protein-binding, FLNB filamin B, beta (actin binding protein 278) family A, member FLNC filamin C, gamma (actin binding protein 280) amyloid beta (A4) precursor protein-binding, FLOT1 flotill in 1 family A, member 2 FLOT1 flotill in 1 PB B1 IP amyloid beta (A4) precursor protein-binding, GABBR1 gamma-aminobutyric acid (GABA) B receptor, 1 family B, member 1 interacting protein GABBR2 gamma-aminobutyric acid (GABA) B receptor, 2 archain 1 GAL galanin prepropeptide arrestin 3, retinal (X-arrestin) GAP43 growth associated protein 43 ATPase, Ca++ transporting, cardiac GGA1 golgi associated, gamma adaptin ear containing, muscle, fast twitch 1 ARF binding protein 1 ATP2A2 ATPase, Ca++ transporting, cardiac GHRL ghrelinfobestatin prepropeptide muscle, slow twitch 2 25 GJA8 gap junction protein, alpha 8, 50 kDa ATPase, Ca++ transporting, ubiquitous GLP1R glucagon-like peptide 1 receptor ATPase, H+/K+ exchanging, beta polypeptide GNRHR gonadotropin-releasing hormone receptor ATPase, H+ transporting, lysosomal 56/58 kDa, GRM1 glutamate receptor, metabotropic 1 V1 Subunit B1 GRMS glutamate receptor, metabotropic 5 ATP8B1 ATPase, class I, type 8B, member 1 GRM7 glutamate receptor, metabotropic 7 ATR ataxiatelangiectasia and Rad3 related 30 GRP gastrin-releasing peptide VPRIA arginine vasopressin receptor 1A HCRTR1 hypocretin (orexin) receptor 1 VPR1B arginine vasopressin receptor 1B HDACS histone deacetylase 5 DKRB2 bradykinin receptor B2 HRH1 histamine receptor H1 DNF brain-derived neurotrophic factor HRH2 histamine receptor H2 ECN1 beclin 1, autophagy relate HTR2A 5-hydroxytryptamine (serotonin) receptor 2A ET1 blocked early in transport 1 homolog 35 HTR2B 5-hydroxytryptamine (serotonin) receptor 2B (S. cerevisiae) HTR2C 5-hydroxytryptamine (serotonin) receptor 2C blocked early in transport 1 homolog HTT huntingtin (S. cerevisiae)-like HTT huntingtin CACNA1A calcium channel, Voltage-dependent, P/O type, GF1R insulin-like growth factor 1 receptor alpha 1A Subuni HPK1 inositol hexaphosphate kinase 1 CACNA1A calcium channel, Voltage-dependent, P/O type, HPK2 inositol hexaphosphate kinase 2 alpha 1A Subuni 40 HPK3 inositol hexaphosphate kinase 3 CACNA1B calcium channel, Voltage-dependent, N type, L2 interleukin 2 alpha 1B subuni L6 interleukin 6 (interferon, beta 2) CACNA1C calcium channel, Voltage-dependent, L type, MPA2 inositol(myo)-1 (or 4)-monophosphatase 2 alpha 1C Subuni MPAD1 inositol monophosphatase domain containing 1 CACNA1D calcium channel, Voltage-dependent, L type, NPP1 inositol polyphosphate-1-phosphatase alpha 1D Subuni 45 NPP3 inositol polyphosphate-3-phosphatase CALB1 calbindin 1, 28 kDa NPP4A inositol polyphosphate-4-phosphatase, type I, 107 kDa CALCR calcitonin receptor NPP4B inositol polyphosphate-4-phosphatase, type II, 105 kDa CALM1 calmodulin 1 (phosphorylase kinase, delta) NPP5A inositol polyphosphate-5-phosphatase, 40 kDa CALM3 calmodulin 3 (phosphorylase kinase, delta) NPP5E inositol polyphosphate-5-phosphatase, 75 kDa CALR calreticulin NPP5C inositol polyphosphate-5-phosphatase, 120 kDa CAMK2A calcium calmodulin-dependent protein kinase 50 NPP5D inositol polyphosphate-5-phosphatase, 145 kDa (CaM kinase) II alpha NPP5E inositol polyphosphate-5-phosphatase, 72 kDa CAMK2B calcium calmodulin-dependent protein kinase NPPSF inositol polyphosphate-5-phosphatase F (CaM kinase) II beta NPPL1 inositol polyphosphate phosphatase-like 1 CAMK2G calcium calmodulin-dependent protein kinase NSR insulin receptor (CaM kinase) II gamma SYNA1 myo-inositol 1-phosphate synthase A1 CAMK2G calcium calmodulin-dependent protein kinase 55 TPK1 inositol 1,3,4-triphosphate 5/6 kinase (CaM kinase) II gamma TPKA inositol 14,5-trisphosphate 3-kinase A CANT1 calcium activated nucleotidase 1 TPKB inositol 1,4,5-trisphosphate 3-kinase B CANX calnexin TPKC inositol 1,4,5-trisphosphate 3-kinase C CAPN1 calpain 1, (mul) large subunit TPR1 inositol 14,5-triphosphate receptor, type 1 CASK calcium calmodulin-dependent serine TPR2 inositol 14,5-triphosphate receptor, type 2 protein kinase (MAGUK family) TPR3 inositol 14,5-triphosphate receptor, type 3 CASR calcium-sensing receptor (hypocalciuric 60 AK1 Janus kinase 1 (a protein tyrosine kinase) hypercalcemia 1, severe neonatal hyperparathyroidism) AK2 Janus kinase 2 (a protein tyrosine kinase) CAV1 , caveolae protein, 22 kDa KCNA2 potassium voltage-gated channel, CAV1 caveolin 1, caveolae protein, 22 kDa shaker-related subfamily, member 2 CAV2 caveolin 2 KCNB1 potassium voltage-gated channel, CCNB1 cyclin B1 Shab-related subfamily, member 1 CRH corticotropin releasing hormone 65 KCND3 potassium voltage-gated channel, CRHR1 corticotropin releasing hormone receptor 1 Shal-related subfamily, member 3 US 7,970,552 B1 47 48 -continued -continued

Gene Gene symbol Description symbo Description KCNJ6 potassium inwardly-rectifying channel, hosphatidylinositol-5-phosphate Subfamily J, member 6 -kinase, type II, gamma KCNMA1 potassium large conductance calcium-activated hosphatidylinositol-4-phosphate channel, Subfamily M, alpha member 1 ase, type I, alpha KIFSB kinesin family member 5B hosphatidylinositol-4-phosphate LHB luteinizing hormone beta polypeptide inase, type I, beta LHCGR. luteinizing hormonefchoriogonadotropin receptor 10 hosphatidylinositol-4-phosphate ARCH2 membrane-associated ring finger (C3HC4)2 ll ase, type I, gamma NPP1 multiple inositol polyphosphate histidine phosphatase, 1 PSK3 hosphatidylinositol-3-phosphatef OX myo-inositol oxygenase hosphatidylinositol 5-kinase, type III MP14 matrix metallopeptidase 14 (membrane-inserted) PSKL1 hosphatidylinositol-4-phosphate 5-kinase-like 1 MP17 matrix metallopeptidase 17 (membrane-inserted) TPNA hosphatidylinositol transfer protein, alpha MP25 matrix metallopeptidase 25 15 TPNM1 hosphatidylinositol transfer protein, PP2 membrane protein, palmitoylated 2 membrane-associated 1 (MAGUK p55 subfamily member 2) hospholipase C, beta 1 (phosphoinositide-specific) membrane protein, palmitoylated 7 hospholipasei C, beta 2 (MAGUK p55 subfamily member 7) hospholipasei C, beta 3 (phosphatidylinositol-specific) mannose receptor, C type 1 hospholipasei C, beta 4 mitochondrial ribosomal protein S6 hospholipasei C, delta 1 murine retrovirus integration site 1 homolog hospholipasei C, delta 3 membrane-spanning 4-domains, Subfamily A, member 14 hospholipasei C, delta 4 methylenetetrahydrofolate dehydrogenase hospholipasei C, epsilon 1 (NADP+ dependent) 2-like hospholipasei C, gamma 1 NAGA N-acetylgalactosaminidase, alpha hospholipasei C, gamma 2 (phosphatidylinositol-specific) NAPA N-ethylmaleimide-sensitive factor attachment protein, alpha hospholipasei C, eta 1 NAPB N-ethylmaleimide-sensitive factor attachment protein, beta 25 hospholipasei C, eta 2 NAPG N-ethylmaleimide-sensitive factor attachment protein, gamma hospholipasei C-like 1 NFAT5 nuclear factor of activa ed T-cells 5, tonicity-responsive hospholipasei C, Zeta 1 NFATC1 nuclear factor of activa ed T-cells; cytoplasmic, hospholipasei D1, phosphatidylcholine-specific calcineurin-dependent hospholipid scramblase 1 nuclear factor of kappa light polypeptide gene hosphomannomutase 1 enhancer in B-cells 1 30 presenilin 1 NMBR neuromedin B receptor presenilin 1 NMUR1 neuromedin U receptor 1 presenilin 2 (Alzheimer disease 4) NMUR2 neuromedin U receptor 2 prostaglandin D2 receptor (DP) NOLC1 nucleolar and coiled-body phosphoprotein 1 prostaglandin F receptor (FP) NPY neuropeptide Y parathyroid hormone NTS neurotensin 35 parathyroid hormone-like hormone NTSR1 neurotensin receptor 1 (high affinity) parathyroid hormone receptor 1 OR4D2 factory receptor, family 4, Subfamily D, member 2 PTK2B protein tyrosine kinase 2 beta OXT xytocin, prepropeptide Sodium channel, nonvoltage-gated 1 alpha OXTR xytocin receptor Sodium channel, nonvoltage-gated 1, beta PDE2A hosphodiesterase 2A, coMP-stimulated Sodium channel, nonvoltage-gated 1, gamma PDE3B hosphodiesterase 3B, c0MP-inhibited Syndecan 1 PDE4D hosphodiesterase 4D, cAMP-specific 40 Syndecan 2 (phosphodiesterase E3 dunce homolog, Drosophila) Syndecan 4 osphoinositide dependent protein kinase-1 alpha(1.2) fucosyltransferase pseudogene OS phatidylinositol 4-kinase type 2 alpha septin 2 OS phatidylinositol 4-kinase type 2 beta septin 5 OS phatidylinositol 4-kinase, catalytic, alpha SFT2 domain containing 3 OS phatidylinositol 4-kinase, catalytic, alpha pseudogene 2 45 serum glucocorticoid regulated kinase family, member 3 OS phatidylinositol 4-kinase, catalytic, beta Sedoheptulokinase OS phatidylinositol (4,5) bisphosphate 5-phosphatase, A Sucrase-isomaltase (alpha-glucosidase) OS phatidylinositol glycan anchor biosynthesis, class A Solute carrier family 1 (neuronal epithelial high OS phatidylinositol glycan anchor biosynthesis, class C affinity glutamate transporter, system Xag), OS phatidylinositol glycan anchor biosynthesis, class H member 1 OS phatidylinositol glycan anchor biosynthesis, class L 50 Solute carrier family 2 (facilitated glucose OS phatidylinositol glycan anchor biosynthesis, class P transporter), member 13 hosphatidylinositol glycan anchor biosynthesis, class Q Solute carrier family 2 (facilitated glucose hosphatidylinositol glycan anchor biosynthesis, class T transporter), member 4 hosphatidylinositol glycan anchor biosynthesis, class W Solute carrier family 2 (facilitated glucose hosphatidylinositol glycan anchor biosynthesis, class Z transporter), member 6 hosphoinositide-3-kinase, class 2, alpha polypeptide hosphoinositide-3-kinase, class 2, beta polypeptide 55 Solute carrier family 4, Sodium bicarbonate hosphoinositide-3-kinase, class 2, gamma polypeptide cotransporter, member 4 hosphoinositide-3-kinase, class 3 Solute carrier family 5 (sodium glucose hosphoinositide-3-kinase, catalytic, alpha polypeptide cotransporter), member 11 hosphoinositide-3-kinase, catalytic, beta polypeptide LCSA3 Solute carrier family 5 (inositol transporters), hosphoinositide-3-kinase, catalytic, delta polypeptide member 3 hosphoinositide-3-kinase, catalytic, gamma polypeptide 60 solute carrier family 5 (sodium-dependent hosphoinositide-3-kinase, regulatory Subunit 1 (alpha) vitamin transporter), member 6 hosphoinositide-3-kinase, regulatory Subunit 2 (beta) solute carrier family 6 (neurotransmitter hosphoinositide-3-kinase, regulatory Subunit 3 (gamma) transporter, GABA), member 1 hatidylinositol-5-phosphate solute carrier family 6 (neurotransmitter -kinase, type II, alpha transporter, noradrenalin), member 2 hosphatidylinositol-5-phosphate 65 solute carrier family 6 (neurotransmitter 4-kinase, type II, beta transporter, dopamine), member 3 US 7,970,552 B1 49 50 -continued -continued

Gene Gene symbol Description symbol Description solute carrier family 6 (neurotransmitter VEGFA vascular endothelial growth factor A transporter, serotonin), member 4 VIM vimentin solute carrier family 6 (neurotransmitter VNN1 vanin 1 transporter, glycine), member 5 VNN2 vanin 2 solute carrier family 6 (neurotransmitter WNT2 wingless-type MMTV integration site family member 2 transporter, glycine), member 9 Solute carrier family 8 (sodium calcium 10 exchanger), member 1 SMG1 PI-3-kinase-related kinase SMG-1 SMPD1 sphingomyelin phosphodiesterase 1, acid lysosomal Example 8 SMPD2 sphingomyelin phosphodiesterase 2, neutral membrane (neutral sphingomyelinase) Using the technique of Example 1, the biologically active SORD Sorbitol dehydrogenase 15 STX10 Syntaxin 10 nutrient for obesity is identified. The relevant genes for this STX11 Syntaxin 11 identification would include: STX12 Syntaxin 12 STX16 Syntaxin 16 STX17 Syntaxin 17 STX18 Syntaxin 18 Gene STX19 Syntaxin 19 symbol Description STXIA Syntaxin 1A (brain) STX1B Syntaxin 1B ACACA acetyl-Coenzyme A carboxylase alpha STX2 Syntaxin 2 ACACB acetyl-Coenzyme A carboxylase beta STX3 ACTG1 actin, gamma 1 STX4 Syntaxin 4 ADIPOQ adiponectin, C1O and collagen domain containing STX5 Syntaxin 5 25 ADIPOR1 adiponectin receptor 1 STX6 Syntaxin 6 ADIPOR2 adiponectin receptor 2 STX7 Syntaxin 7 ADRB2 adrenergic, beta-2-, receptor, Surface STX8 Syntaxin 8 ADRB3 adrenergic, beta-3-, receptor STXBP1 Syntaxin binding protein 1 AGRP agouti related protein homolog (mouse) STXBP2 Syntaxin binding protein 2 AKT V-akt murine thymoma viral oncogene homolog 1 STXBP3 Syntaxin binding protein 3 30 ANGPTL4 angiopoietin-like 4 STXBP4 Syntaxin binding protein 4 APLN apelin STXBP5 Syntaxin binding protein 5 (tomosyn) APOA4 apolipoprotein A-IV STXBPSL Syntaxin binding protein 5-like APOD apolipoprotein D STXBP6 Syntaxin binding protein 6 (amisyn) APOM apolipoprotein M SV2B glycoprotein 2B AR androgen receptor (dihydrotestosterone receptor; testicular SYCN Syncollin 35 feminization; spinal and bulbar muscular atrophy; SYN1 synapsin I Kennedy disease) SYP BDNF brain-derived neurotrophic factor SYT1 I BIRCS baculoviral IAP repeat-containing 5 (survivin) SYT1 synaptotagmin I C1OTNF3 C1q and tumor necrosis factor related protein 3 SYT2 synaptotagmin II CAQ5 Circulating adiponectin QTL on SYT3 synaptotagmin III CARTPT CART prepropeptide SYTL4 synaptotagmin-like 4 40 CCK cholecystokinin TAC1 achykinin, precursor 1 CCND1 cyclin D1 TACR1 achykinin receptor 1 CEBPA CCAAT?enhancer binding protein (C/EBP), alpha TACR2 achykinin receptor 2 CGBS chorionic gonadotropin, beta polypeptide 5 TACR3 achykinin receptor 3 CLU clusterin TPTE transmembrane phosphatase with tensin homology CNTF ciliary neurotrophic factor TPTE2 transmembrane phosphoinositide 3-phosphatase 45 CNTFR ciliary neurotrophic factor receptor and tensin homolog 2 COL1A1 collagen, type I, alpha 1 TRAF2 TNF receptor-associated factor 2 CPT1B carnitine palmitoyltransferase 1B (muscle) TRAF6 TNF receptor-associated factor 6 CPT2 carnitine palmitoyltransferase II TRH hyrotropin-releasing hormone CREB1 cAMP responsive element binding protein 1 TRHR hyrotropin-releasing hormone receptor CRHR2 corticotropin releasing hormone receptor 2 TSHR hyroid stimulating hormone receptor 50 CRP C-reactive protein, pentraxin-related TSPAN4 etraspanin 4 CTGF connective tissue growth factor TXK CYP19A1 cytochrome P450, family 19, subfamily A, polypeptide 1 TXLNA DGAT1 diacylglycerol O-acyltransferase homolog 1 (mouse) TXLNB DGKZ diacylglycerol kinase, Zeta 104 kDa TXNDC4 hioredoxin domain containing 4 DRD2 dopamine receptor D2 (endoplasmic reticulum) 55 EDN endothelin 1 TYK2 tyrosine kinase 2 EPHA3 EPH receptor A3 TYRP1 tyrosinase-related protein 1 ESR1 estrogen receptor 1 WAMP1 vesicle-associated membrane protein 1 (synaptobrevin 1) FAAH fatty acid amide hydrolase WAMP2 vesicle-associated membrane protein 2 (Synaptobrevin 2) FABP7 fatty acid binding protein 7, brain WAMP3 vesicle-associated membrane protein 3 (cellubrevin) FASN fatty acid synthase VAMP7 vesicle-associated membrane protein 7 FFAR3 free fatty acid receptor 3 WAMP8 vesicle-associated membrane protein 8 (endobrevin) 60 FTO fat mass and obesity associated WAPA VAMP (vesicle-associated membrane GALP galanin-like peptide protein)-associated protein A, 33 kDa GCG glucagon WAPB VAMP (vesicle-associated membrane GCKR glucokinase (hexokinase 4) regulator protein)-associated protein B and C GH1 growth hormone 1 VCL. Vinculin GHR growth hormone receptor VCP valosin-containing protein 65 GHRL ghrelinfobestatin prepropeptide WDAC1 voltage-dependent anion channel 1 GHSR growth hormone secretagogue receptor US 7,970,552 B1 51 52 -continued -continued

Gene Gene symbol Description symbol Description gonadotropin-releasing hormone 1 PSMC6 proteasome (prosome, macropain) 26S Subunit, ATPase, 6 (luteinizing-releasing hormone) PTGDS prostaglandin D2 synthase 21 kDa (brain) GNRH2 gonadotropin-releasing hormone 2 PYY peptide YY GPLD1 glycosylphosphatidylinositol specific phospholipase D1 RETN resistin GRB2 growth factor receptor-bound protein 2 RNYS RNA, Ro-associated Y5 GRIP1 glutamate receptor interacting protein 1 SCD stearoyl-CoA desaturase (delta-9-desaturase) GRLF1 glucocorticoid receptor DNA binding factor 1 10 SCD5 stearoyl-CoA desaturase 5 GRP gastrin-releasing peptide SELE selectin E H6PD hexose-6-phosphate dehydrogenase SLEP1 Serum leptin concentration QTL 1 (glucose 1-dehydrogenase) SLEP2 Serum leptin concentration QTL2 HAMP hepcidin antimicrobial peptide SLEP3 Serum leptin concentration QTL3 HCRT hypocretin (orexin) neuropeptide precursor SMAD2 SMAD family member 2 HCRTR1 hypocretin (orexin) receptor 1 15 SMAD3 SMAD family member 3 HCRTR2 hypocretin (orexin) receptor 2 SNCG Synuclein, gamma (breast cancer-specific protein 1) HK2 hexokinase 2 SNX4 4 HSD11B1 hydroxysteroid (11-beta) dehydrogenase 1 SNX6 Sorting nexin 6 HTR2C 5-hydroxytryptamine (serotonin) receptor 2C SOAT1 sterol O-acyltransferase (acyl-Coenzyme A: cholesterol APP islet amyloid polypeptide acyltransferase) 1 GF1 insulin-like growth factor 1 (Somatomedin C) SRA1 steroid receptor RNA activator 1 GF1R insulin-like growth factor 1 receptor SREBF1 sterol regulatory element binding transcription factor 1 GFBP1 insulin-like growth factor binding protein 1 STAT3 signal transducer and activator of transcription 3 GFBP2 insulin-like growth factor binding protein 2, 36 kDa STATSA signal transducer and activator of transcription 5A GFBP3 insulin-like growth factor binding protein 3 STATSB signal transducer and activator of transcription 5B GFBP4. insulin-like growth factor binding protein 4 TRH thyrotropin-releasing hormone GH1 immunoglobulin heavy joining 1 TTF2 transcription termination factor, RNA polymerase II NS insulin 25 UBC ubiquitin C NSR insulin receptor UCN urocortin RS1 insulin receptor Substrate 1 UCP1 uncoupling protein 1 (mitochondrial, proton carrier) RS2 insulin receptor Substrate 2 UCP2 uncoupling protein 2 (mitochondrial, proton carrier) RS4 insulin receptor Substrate 4 UCP3 uncoupling protein 3 (mitochondrial, proton carrier) TLN1 intelectin 1 (galactofuranose binding) WDR vitamin D (1,25-dihydroxyvitamin D3) receptor AK1 anus kinase 1 (a protein tyrosine kinase) 30 VEGFA vascular endothelial growth factor A AK2 anus kinase 2 (a protein tyrosine kinase) VLDLR very low density lipoprotein receptor KATS K(lysine) acetyltransferase 5 ZBTB17 Zinc finger and BTB domain containing 17 KISS1 KiSS-1 metastasis-suppressor ZNF318 Zinc finger protein 318 KISS1R KISS1 receptor LEP eptin LEPR eptin receptor 35 LEPROT eptin receptor overlapping transcript Example 9 LEPROTL1 eptin receptor overlapping transcript-like 1 LMNA aminAC LPA ipoprotein, Lp(a) The method to assess the biologically active nutrient to LPIN3 ipin 3 include in the diet based on the differential gene expressions LRP2 ow density lipoprotein-related protein 2 ow density lipoprotein receptor-related protein 8, 40 of samples from healthy and unhealthy animals of different apolipoproteine receptor genotypes is reported. Leptin, serum levels of In the example, the effect of curcuminor andrographolide melanocortin 2 receptor (adrenocorticotropic hormone) administrations on arthrosis of German Shepherd dogs is MC4R, melanocortin 4 receptor described. In the example, the differential gene expressions MCSR melanocortin 5 receptor 45 profiles between healthy and affected dogs is evaluated by MCHR1 melanin-concentrating hormone receptor 1 means of microarray. The exposure of cells of affected dogs to MDK midkine (neurite growth-promoting factor 2) MICE MHC class I polypeptide-related sequence E biologically active nutrients with known ant inflammatory MUC3A mucin 3A, cell Surface associated activity allows the identification of the more appropriate bio MUC4 mucin 4, cell Surface associated logically active nutrients to include in the diet. NAMPT nicotinamide phosphoribosyltransferase 50 NGF nerve growth factor (beta polypeptide) Synovial fluid from the knee of 10 dogs affected by arthro NMB neuromedin B sis (age 4-6 years) and 10 healthy dogs (age 5-7 years) were NOS3 nitric oxide synthase 3 (endothelial cell) sampled. Synovial fluid was centrifuged at 10000 rpm for 30 NOX4 NADPH oxidase 4 NPY neuropeptide Y minutes and cell pellets recovered and store at -80 degrees C. NPY1R neuropeptide Y receptor Y1 55 until analysis. NPYSR neuropeptide Y receptor Y5 Total RNA of cells was extracted using phenol/guanidine NR3C1 nuclear receptor Subfamily 3, group C, member 1 NTS neurotensin HCI reagents (Trizol, Invitrogen). RNA quality integrity was PDE3A phosphodiesterase 3A, c0MP-inhibited analysed using the Agilent 2100 Bioanalyser (Agilent Tech PDE3B phosphodiesterase 3B, c0MP-inhibited nologies) of the sample. The samples determined to have no. PELP1 proline, glutamate and leucine rich protein 1 PMCH pro-melanin-concentrating hormone 60 or minimal, loss of integrity and thus were considered Suit POMC proopiomelanocortin able for use in experiments. mRNA was amplified for each PPARG peroxisome proliferator-activated receptor gamma sample, starting with 500 ng total RNA using a commercially PPYR1 pancreatic polypeptide receptor 1 available kit (Ambion T7 MEGAScript high yield transcrip PRL prolactin PRLR prolactin receptor tion kit, Ambion). The mRNA was quantified using a spec PSMB8 proteasome (prosome, macropain) subunit, 65 trophotometer. mRNA was directly reverse transcribed to beta type, 8 (large multifunctional peptidase 7) cDNA from 25ug of total RNA using the Superscript indirect cDNA labeling Core kit (Invitrogen, Milan, Italy). US 7,970,552 B1 53 54 Two micrograms of cDNA were labelled with Cyanine -continued 3dCTP (Cy3) or Cyanine-5dCTP (Cy5) fluorochromes using Fold change the cDNA labeling purification module kit (Invitrogen, Individual Milan, Italy). Samples were hybridised to a canine specific, Unhealthy Dogs whole genome 44k spot 60mer oligonucleotide (Agilent 5 Technologies). The labelled cDNA was appropriately Gene symbol Gene name Mean S.e. coupled and used for competitive hybridization on the same IL2 Interlukin 2 3.0 0.7 microarray at 42°C. for 16h. Fluorescence incorporation was MMP2 matrix metallopeptidase 2 1O.O 2.1 determined using a spectrophotometer. The relative intensity 10 N 2 O R 2A de Swnth : 8. of labelled cDNA in was acquired with Scan Array LITE SPARC E.OS898ll-elOOCOXOle p SWill3St.y 6.3 1.1 scanner (PerkinElmer Life Sciences, Inc). STMN1 Stathmin 1 6.2 O.6 TIMP1 TIMP metallopeptidase inhibitor 1 6.2 0.4 Expression data were then exported into Excel 2007 and TIMP2 TIMP metallopeptidase inhibitor 2 2.O O.1 processed with SAM Software; comparison between groups TNF-a tumor necrosis factor alpha 1O.O 1.1 was achieved using paired student's t tests. Comparisons of 15 TE Alpha-tubulin 4.7 0.7 the number of genes up- or down-regulated in both the normal TUBB Beta-tublin 4.8 O.S and affected cells were made using Chi squared analysis. Correction for multiple hypothesis testing was performed The comparison between the healthy and affected dogs using the false discovery rate (FDR). indicated that each of the affected unhealthy dogs underwent For each of the 10 individual- healthy and 10 individual- 20 degenerative"S and- inflammatorv0 ryp processes. - nhealthy d ffected with arthrosis, th t of 21 Searching within the nutrient data set for biologically unnealuny dogs allected win artnrosis, une same set o active nutrients with anti-arthritic and anti-inflammatory genes was determined to be differently expressed between the properties identified curcumin and androgropholide as the cartilage cells of both the healthy and unhealthy dogs. Two appropriated compositions. The cartilage cells of individual were down-regulated and 19 were up-regulated (Table 1). affected unhealthy dogs were cultured in vitro with 0, or 60 25 mg/l of curcumin or androgropholide for 6 hours. At the end of the incubation, these cartilage cells from individual affected dogs were washed and used for RNA extraction. For Fold change the microarray analysis, co-hybridisation of the RNA from 0 Individual and 60 mg/l was conducted, using the material and the meth Unhealthy Dogs 30 ods described previously. s 9. Gene symbol Gene name Mean S.C. Table 2 ACTB Beta actin 3.8 O.1 The table below reports the mean fold change in gene ACTR3 Actin-related protein 3 O.3 O.1 expression of two biologically active nutrients, namely cur ADK Adenosine kinase, transcript variant 3 3.6 O.2 cumun and andrographolide. As can be seen, the androgra ANKRD10 Ankyrin repeat domain 10 5.2 0.9 35 pholide possesses an anti-inflammatory activity, but does not CAV1 Caveolin 1 4.8 O.6 exactly match the regulation of all the genes up- or down CDH11 Cadherin 11, type 2, OB-cadherin 6.9 O.9 regulated in the individual affected unhealthy cartilage cells. COX1COL3A1 Cycloxygenase-21Collagen 3, alpha 1 9.5O.8 O.21.7 Instead, curcumin completely satisfies these requirements, so COX2 Cycloxygenase-2 23.0 2.8 that the food composition is designed to include curcumn. IGFBP7 IGFBP7 Insulin-like growth 3.7 0.8 . The dose to be added to the food or the nutrient composition factor binding protein 7 is computed using literature data, which indicates a dose of 4 mg/kg body weight for either curcumin or andrographolide.

FOLD CHANGE OF GENE NET EFFECT OF THENBC ON FOLD EXPRESSION CHANGE Gene Curcumin Androgrpholide Curcumin Androgrpholide

Name le:8 S.d. (8. S.d. (8. S.d. (8. S.d.

ACTB -5.O. O.1 -1.0 O.1 -1.2 O.1 2.8 O.1 ACTR3 -1.0 0.2 1.O 1 -4.0 O.2 1.3 0.7 ADK -4.3 0.1 O.O O.1 -1.4 O.2 3.6 O.2 ANKRD10 - 7.O 1.1 O.O O.8 -1.8 1.O 5.2 O.9 CAV1 -3.O O.3 O.O O.3 1.8 O.S 4.8 O.S CDH11 -6.0 1.1 -1.0 O.1 O.9 1.O 5.9 O.6 COL3A1 -6.0 0.6 -2.0 O.2 3.5 1.3 7.5 1.2 COX1 -1.0 (0.3 O.O O.1 -0.2 O.3 O.8 O.2 COX2 -15.O 2 O.O O.2 8.0 2.4 23.0 2.O IGFBP7 -1.5 0.4 -2.0 O.2 2.2 O6 1.7 O.6 IL2 -4.0 0.9 -6.0 O.9 -1.0 O.8 -3.0 O.8 MMP2 -6.5 1.1 -50 O.9 3.5 1.7 S.O 1.6 NOS2 -0.5 O1 -6.0 1.1 -0.3 O.1 -58 O.8 PTGS-2 -3.0 0.2 -50 O.6 -1.8 O.3 -3.8 O.S SPARC -4.0 0.9 O.O O.2 2.3 1.O 6.3 O.8 STMN1 -5.O O.6 O.O O.3 1.2 O6 6.2 O.S TIMP1 -5.O O.6 O.O O.1 1.2 O.S 6.2 O.3 TIMP2 -2.0 0.2 O.O O.2 O.O O.2 2.0 O.2 TNF-a -14.5 1.3 -8.0 1.8 -4.5 1.2 2.0 1.5 US 7,970,552 B1 55 56 -continued

FOLD CHANGE OF GENE NET EFFECT OF THENBC ON FOLD EXPRESSION CHANGE Gene Curcumin Androgrpholide Curcumin Androgrpholide

Name le:8 S.d. le:8 S.d. le:8 S.d. le:8 S.d.

TUBA -2.0 0.4 O.O O.2 2.7 O6 4.7 O.S TUBB -5.O O.2 O.O O.1 -0.2 0.4 4.8 0.4

For complete provision of anti-arthritic and anti-inflamma As used in this disclosure a “pharmaceutical product’ tory properties, the food or nutrient composition is thus refers to one or more therapeutic drugs, compounds or com designed to include curcumin and not andrographolide at the positions including one or more nutrients or other Supple dose indicated above. 15 ments or constituents having properties which prevent, treat, The disclosure also concerns a method of analyzing the control or modulate a state of physiological homeostasis or diagnostic genetic profile of a non-human animal comprising: pathophysiological condition. (a) providing a genotypic database for the species of the The Examples above, and Examples 1-9 above identify non-human animal Subject or a selected group of the particular genes related to specific healthy or unhealthy con non-human species; ditions in an animal. These genes are identified as relating to (b) obtaining animal phenotypic data; biologically active nutrients. Similarly, these genes are (c) correlating the database of (a) with the data of (b) to related to pharmaceutical products and compositions and the determine a relationship between the database of (a) and disclosure is also applicable to the relevant pharmacokinetic the data of (b). 25 conditions. The diagnostic genetic profile of the animal or selected Genetic variations in response to a pharmaceutical product group of animals is determined based on the correlating step have involved the muscle relaxant Suxamethonium chloride, (c), and selecting the biologically active nutrient from an and pharmaceutical products metabolized by N-acetyltrans obtained molecular dietary signature, the molecular dietary ferase. About one in 3500 Caucasian people has a less effi signature being a variation of expression of a set of genes 30 cient variant of the enzyme (butyrylcholinesterase) that which may differ for the genotype of each animal or selected metabolizes suxamethonium chloride. Consequently, the group of animals. pharmaceutical product’s effect is prolonged, with slower The data of the animal is one or more data items related to recovery from Surgical paralysis. An enzyme system known genotype, selected from the group consisting of breed, breed as the cytochrome P450 oxidases provides the body with an (s) of parents, pedigree, sex, coat type, and evident hereditary 35 inborn System for clearing Xenobiotics (chemicals not nor conditions and disorders and the phenotypic data are selected mally produced by or expected to be present in the body). The from the group consisting of age, weight, veterinary medical cytochrome P450 oxidases are involved in pharmaceutical history, reproductive history, present wellness or disease product metabolism, and genetic variations in their pathways state, appetite, physical activity level, mental acuity, behav 40 should affect large populations of animals. ioral abnormalities and disposition. The thiopurines and thiopurine methyltransferase enzyme Another aspect of the disclosure is a method of identifying system has been involved in one test for a genetic variation in a pharmacological product for an individual animal having a drug metabolism that had a clinically important consequence. genotype, comprising: This system metabolizes 6-mercaptopurine and azathioprine, (a) using a “reference' dataset containing functional 45 two thiopurine drugs used in a range of clinical indications, genomic profiles of biological samples of the genotypes from leukemia to autoimmune diseases. In humans with thi of different animals of the species, the different animals opurine methyltransferase deficiency, thiopurine metabolism being healthy animals; proceeds by other pathways, one of which leads to production (b) selecting a “target' dataset containing the functional of an active thiopurine metabolite that is toxic to the bone genomic profile of biological samples of the genotypes 50 marrow. The frequency of this mutation is one in 300 people. of different animals, the different animals being These individuals need about 6-10% of the standard dose of unhealthy animals; the drug. If treated inadvertently with the full dose of the (c) using a “pharmacological product dataset comprising pharmaceutical product, these individuals are at risk for different effects of pharmacological product on func severe Suppression. For these humans, geno tional genomic profiles of the different animals of dif 55 type predicts clinical outcome, which is now considered a ferent genotypes from those of the target group (b), the prerequisite for an effective pharmacogenetic test. When different genotypes being differently responsive to the applied to animals the genetic variations in Such pathways same pharmacological product; and should affect large populations of animals. (d) having the reference dataset or target dataset include an The disclosure is directed to a method, apparatus and sys individual animal for which the biologocally active 60 tem of obtaining, analyzing and reporting laboratory test data nutrient is to be identified. in relation to the nutrition assessment data of an animal At least one of the “reference' or “target group' datasets together with the genetic data related to that same animal. are related with the pharmacological product dataset to iden These data include tests related to at least one of the func tify pharmacological product is for the selected animal geno tion of and nutritional analysis, paternity, DNA fingerprint type to prevent, treat, control, or modulate a state of physi 65 ing, and the functional genomic profile. These data are rel ological homeostasis or pathophysiological condition of the evant to the likely morbidity, likely longevity, and/or the individual animal in the reference dataset or target group. potential risk for disease or disorder for the animal. US 7,970,552 B1 57 58 According to one aspect of the disclosure, nutrition profil type and genetic data. The disclosure includes relating the ing of an animal is affected to determine characteristics phenotypic data to either one or both types of the genotypic related to the temperament of the animal which impacts on its data. longevity. Biological and genetic laboratory test data from a According to the disclosure there is an analysis of the bodily fluid or tissue of an animal are analyzed. profile of a non-human animal. The analysis comprises: a) More particularly the disclosure comprises analyzing providing a genotypic database to the species of the non genetic data of animals, analyzing nutrition assessment data human animal Subject or a selected group of the species; b) of animals, combining this as necessary, and permitting an obtaining animal data; c) correlating the database of a) with analysis predicting nutrition, disease and disorder probabili the data ofb) to determine a relationship between the database ties and longevity of selected animals. The analysis and diag 10 of a) and the data of b); c) determining the profile of the nosis is made, and a report is provided to a remote user based animal based on the correlating step; and d) determining a on the analysis the nutrition assessment data of the animal genetic profile based on the molecular signature, the molecu and/or the genetic data. lar signature being a variation of expression of a set of genes In light of the above, there is provided by this disclosure a which may differ for the genotype of each animal or a group system for managing animal comprehensive nutrition assess 15 of animals. ments of animals and genetic diagnosis, including the perfor The nutritional aspects and regimen is at least related to the mance of specific tests. nutrient or caloric composition, or the food allergies and food The disclosure also provides a bioinformatics system for intolerances. The therapeutic intervention or maintenance is inputting, controlling, analyzing and outputting of a broad at least one of drugs, nutraceuticals, or holistic treatments, range of criteria related to the nutrition, genetic background exercise or liquid intake. The diagnostic genetic and labora and longevity of animals. This includes a system concerning tory test data is a comprehensive general nutrition profile and phenotype data and genetic data relating to animals. Further, selectively at least one selected diagnostic profile for a there is provided a system for screening of genetic data and selected subject. Preferably the genetic and laboratory data genomic mapping, and integrating the phenotype nutrition for the subject is obtained overtime from the same laboratory. assessment data and genetic identifier and assessment data in 25 This is likely to enhance the uniformity of the data, and render a central database processing resource (“CDPR). Moreover, the determinations more accurate, and predictive of nutrition, there is provided a system for analyzing the nutrition assess nutritional requirements, temperament, and longevity. ment or phenotypic data with the interrelated genetic or geno The interrelationship is affected by a computer which is at typic data. Thereafter, those data and analyses are communi least one of an expert system or interrelationship program or cated from the CDPR in a broad range and in a manner that 30 network for determining data base and data relationships. has not previously been possible. This can be a system such as a neural network, or other The present disclosure offers a unique solution to above statistical sampling systems and networks. described problems by providing an apparatus, method and The database of at least one of the species or the group is system, in relation to animals, for performing data analyses of periodically updated thereby to obtain cumulative data of the genetic and biological specimens from specific Subject ani 35 species or group. Preferably both these databases are used, mals or animal groups in relation to specific Subject animal or and preferably both are updated to obtain the cumulative data. animal groups of genetic data. The apparatus, method and The data of the subject is periodically updated thereby to system comprises a controller for obtaining, inputting, and obtain cumulative data. Preferably, both the databases are analyzing genetic, biological, physiological, and pathologi periodically updated. The updating picks up data drift in cal test data together with genomic mapping and genetic 40 different populations of the Subjects, groups and species over screening data into the CDPR. time, and thereby allows for the regulation of the database so The biological, physiological, and pathological data of the as to be substantially or essentially current. Subject animal or animal group and the genetic data of the By having this feature there is obtained a method and Subject animal or animal group are communicated to a remote system which provides for enhances nutrition care and well user as raw data or as related, analyzed biological, physiologi 45 being management of the Subject. Thus the data of the Subject cal, and pathological data and genetic data. The remote user is compared to Substantially or essentially current data. Simi can also appropriately access the CDPR to input data to, or larly by retaining a history of the Subject data and relating this obtain data from, the CDPR. to the updated databases, the accuracy with which the nutri The CDPR includes at least two databases, one of the tion care and well-being is managed is significantly databases contains genetic information in relation to animals 50 enhanced. and the other is a phenotypic database. The disclosure also includes the step of reporting the deter The genetic database is either a specific file of a selected mination of the care, well-being, nutrition or other therapeu animal or a generalized animal database relating to group tic requirements and Suggestions for nutrition on a commu characteristics, and is cross-relatable with the phenotypic nications network including the Internet. Preferably, there is a database of particular selected Subject animals. 55 payment procedure for the report which is achieved through Additionally other databases can be used and cross-related the Internet. This communication network and structure is to these databases. The genetic database includes data from described here in further detail. selected animals, animal families, animal breeds and/or data There is provided means for inputting data into the genetic related to selected animal diseases and/or disorders. Other database and phenotypic database, and other databases, Stor databases include those related to genetic markers or maps of 60 ing the data in these databases, analyzing the data in a rela animals, databases related to epidemiology, purebred animal tional sense from the different databases, and retrieving the ownership, identification registries, and studbook registries. data from these databases, namely the databases which are The phenotype, nutrition profile, or nutrition assessment part of the CDPR. database contains data which is mostly phenotypic. The geno A further aspect of the disclosure is the accessibility of the type database includes data which is in the category of mostly 65 nutrition assessment database and/or genetic database or genotype or genetic and which may include a second category other databases of the CDPR by the remote user selected on of some phenotype data which predicts or manifests the geno the basis of password, security control, and financial payment US 7,970,552 B1 59 60 such that the data can be transmitted into and from the CDPR The diagnostic laboratory test data is a comprehensive by a computer network. Use of selected passwords, encryp general nutrition profile and selectively at least one selected tion systems, and payment systems are employed to facilitate diagnostic profiles for a selected subject. The laboratory data and restrict the flow of data in and/or out of the databases. for the subject is ideally obtained over time from the same Alerts can be set up to advise of attempts at unauthorized 5 laboratory. This is likely to enhance the uniformity of the access to the CDPR. The computer network may conve data, and render the determinations more accurate, and pre niently include the Internet. dictive of nutrition, nutritional requirements, temperament, As required, the data in the CDPR can also be distributed to and longevity. multiple authorized remote parties, namely third parties for The database of at least one of the species or the group is research or other analysis. The disclosure also includes a 10 method and system for achieving this. periodically updated thereby to obtain cumulative data of the Further aspects of the present disclosure will become dog species or group within the dog species. Both of these apparent in the course of the following description and by data bases generally should be used, and both should be reference to the attached drawings. updated to obtain the cumulative data. In some cases, only The present disclosure will now be described in detail with 15 one of the databases is used and/or one of them is periodically reference to a few preferred embodiments thereof, as illus updated. trated in the accompanying drawings. In the following The data of the dog Subject is also periodically updated. description, numerous specific details are set forth in order to Overall there is obtained cumulative data of the dog subject, provide a thorough understanding of the present disclosure. It species or group. The updating picks up data drift or data will be apparent, however, to one skilled in the art, that the trends within different populations of the particular dog sub present disclosure may be practiced without some or all of ject, the groups (for instance, breed) and the species (for these specific details. In other instances, well known process instance, the dog generally as a species) over time. This steps have not been described in detail in order to not unnec allows for the review and oversight of the database so as to be essarily obscure the present disclosure. Substantially or essentially current. C. Genotypic Markers of Disease 25 Enhanced nutrition care and well-being management of the Recent advances in molecular genetics have focused on dog Subject is obtained. Thus the data of the dog Subject is mapping the human genome, and this has stimulated interest compared to Substantially or essentially current data. Simi in developing parallel genetic maps for animals. For example, larly, by retaining a historical record of the dog Subject data it is estimated that a minimum often years and several million and relating this to the updated databases, the accuracy with dollars will be needed to map the canine genome. Once devel 30 which the management of the nutrition care and well-being, oped, a genetic map provides information about the relative and the nutrition design of nutritional requirements or thera order and placement of genes or specific DNA markers on peutic and maintenance interventions is significantly specific chromosomes. This allows one to locate specific enhanced. In this manner, for instance the food, Supplements, regions on chromosomes where genes of interest are likely to nutraceuticals and the like, can be modified by additions be found. Once a molecular marker is identified close to a 35 and/or subtractions of components based on the determined specific gene of interest, Screening tests for this particular relationship, since these cumulative and dynamic databases marker can be used to identify individuals carrying or and data analyte changes over time, whereby the determined expressing the trait. relationship is significantly enhanced. Management of the One or more of a panel of tests relate to at least one the dog subject in one or all of these respects is dealt with a high function of and nutritional analysis, DNA fingerprinting, and 40 level of precision and predictability. the functional genomic profile. These data are relevant to the The computer is at least one of an expert System or inter likely morbidity, likely longevity, and/or the potential risk for relationship program or network for determining data base disease or disorder for the animal. and data relationships. This can be a system Such as a neural There is a dynamic method and system of managing the network, or other statistical sampling systems and networks, nutrition care, well-being and nutritional requirements of 45 and is discussed in more detail. dogs or cats. An example is set out for a dog. It can be equally The determination of the nutrition care, well-being, nutri applicable to a cat. tional or other therapeutic requirements and Suggestions for A database relating to the dog species generally, and a data promoting and maintaining nutrition of the dog is reported on base relating to a selected group, for instance, the breed, of the a communications network including the Internet. There is a dog is used. Data is obtained relating to the particular dog 50 payment procedure for the report which is achieved through Subject, and this data includes laboratory test data, and ideally the Internet. This is discussed in more detail. diagnostic laboratory data relating to that dog. The database A more detailed explanation of the features is now of the dog and the breed, for example, is related to the data of described. the subject data of the dog by a computer. There is then An initial database, from a recent temporal period made on determined, based on this relationship, a regimen for the 55 a group of nutrition dogs. will use physical characteristics, management and are of the dog Subject. nutrition history, and comprehensive laboratory data of these The nutrition care and well-being could include the nutri dogs for a specific geographic area Such as the U.S.A. and tional management or the lifestyle management. The data Canada, but also other geographic areas could be used. Addi base of the selected group of the species is at least one of tional databases could be developed for other countries. Gen breed, age, sex, size, weight, performance use, or geographi 60 erally the same laboratory is used to generate the database. cal location. The temporal retrospective database will be augmented in an The nutritional regimen is at least related to the nutrient or on-going fashion with prospective data that continues to caloric composition needed for the dog subject, or the food accumulate over the future testing years. Results will be ana allergies and food intolerances of the dog Subject. The thera lyzed from the temporal retrospective database and then will peutic intervention or maintenance needs of the dog are at 65 be periodically reanalyzed every 6-12 months depending on least one of drugs, nutraceuticals, liquid intake, holistic treat the size of the database to search for any trends or drift in the ments or exercise. values of specific analytes over time. US 7,970,552 B1 61 62 This is an important database to accumulate because the One of the most effective and least invasive or harmful presence of drift over time means that Subsequent studies of ways to intervene in promoting animal nutrition and longev the same or other animals, whether they be nutrition or have ity would be to utilize dietary management. Specifically, diseases, disorders or changes in lifestyle, diet or other wholesome foods are the key to a balanced functioning parameters including reproduction, or performance use need immune system and the resistance to disease. Given the tight to take such drift in the database into account in order to database that is developed by this approach, extraneous noise accurately interpret the values obtained. Animals of specific in the results of comprehensive laboratory analyses is mini breed or type characteristics, size, age, weight, performance mized. One can take the findings then for individual animals level, lifestyle, geographic location will then have their labo or groups of animals having cumulative laboratory evidence ratory profiles and physical characteristics and nutrition his 10 of trends or drift from the normal ranges and design specific tory entered into a database that will start from entry into the dietary interventions that will rebalance the system and pro system and continue on a regular basis over time, preferably mote immunological function and resistance to disease. This at least annually. Also entered into this database will be pup method of identifying what changes could be made in dietary pies that will be tested for the first time at about six months of 15 components or Supplements does not depend on single point age, pre-puberty, and then before puberty, preferably in in time individual pet or other animal data, but in fact the key anestrus females, and then annually thereafter in a compre is developing a cumulative comprehensive database overtime hensive manner to establish a cumulative laboratory database for normal animals in a like-group location or activity level, as for the individual animal. well as specific animals within the group in order to determine These data will also be put into a group-specific database what trends are evidenced over time and thereby use the trend for the breed characteristics or the activity characteristics or to give a more solid determination of what these changes in any other parameter that is useful to group together for ana nutritional requirements or nutritional Supplements or other lytical purposes. This method of gathering comprehensive intervention should be. and cumulative data will permit not only analysis of indi An example of the comprehensive diagnostic testing used vidual animals, whether they be nutrition or expressing some 25 in this disclosure are selected examples for diagnostic genetic stage of disease or disorder, but also will allow analysis of panels, screens and microarray analysis or other High their membership in a group. When the group is analyzed it Through Put systems (“HTS”). will provide a database for predictive laboratory value expec In a practical application on a large scale, the comprehen tation for similar members of the group. By developing these sive individual and group databases that relate to thyroid databases in a cumulative manner the trends for particular 30 function and animal behavior are important. As the key to analytes or groups of analytes predictive of organ function, having an individual animal become a successful companion for example, can then be compared within individual animals, animal or member of a household, the social interaction of the nutrition or diseased, with that of the retrospective and pro animal with the caregivers is crucial. If the animal has an spective nutrition animal database to look for differences in undesirable behavior or social bad habits, very likely the trends. Those differences in trends, as well as differences in 35 animal will be isolated, stressed, and may be treated unkindly individuals or groups of animals, can then be used as a pre and even ostracized by some family members, so that the dictor of nutrition, disease and longevity. animal may eventually be given up or even sent to a pound and Once trends or changes are identified within individual sheltered and be euthanized. If kept by the family, the animal animals or within the related groups of animals or within will likely undergo significant stress which would contribute specific analytes or groups of analytes from a database, this 40 to immune Suppression and lack of well-being and thus fur will permit intervention in a management and treatment per ther promote the abnormal behavior. By using this database spective. The intervention can be nutritional, can include the and identifying animals that have very early Subtle changes in use of dietary Supplements, use of specific nutraceuticals, and laboratory analytes shown by their individual or cumulative can include, of course, other conventional and alternative data drift from the expected normal parameters, one can inter treatments and management of nutrition care. The database so 45 vene before the abnormal behavior becomes unbearable for gathered, while primarily phenotypic in its laboratory ana the family caregivers. lytical sense and its patient descriptive sense, will also be The databases of the functional genomic profile and the predictive for the most part of the genotype of the individual nutritional database can be used to assist in resolution of the animals or groups of animals in the population, because the disease state or condition. canine genome has changed very little over the last hundred 50 As a relationship for has been recognized between thyroid years, and so the majority of the canine genome is identical function and behavior, early detection of thyroid imbalance between dogs, breeds and individuals. Differences in pheno allows one to intervene, specifically with nutritional Support, type (physical appearance and size and weight, for example) and managing individual foodstuffs and Supplements that within dog breeds constitutes a very Small genetic variation, would optimize thyroid function before the disease less than 1%, within the overall genome. Predicting genotype 55 progresses to the stage where thyroid hormone Supplement and phenotype with these comprehensive and cumulative becomes an essential component of the management and laboratory test panels permits a novel approach to intervening treatment. For instance, food Supplements containing kelp, in the management and treatment of canine disease and dis iodine and the minerals from green leafy vegetables could be orders and also in the maintenance of canine nutrition and very helpful in enhancing thyroid glandular output and func longevity. 60 tion. Soybean-derived products by contrast tend to inhibit The comprehensive cumulative database developed with thyroid function, as can the quality and content of protein in this disclosure allows one to look at very early Subtle changes the diet. Because the thyroid is a major master gland that that are consistent within individuals or groups of related controls the metabolism of So many functions in the body, individuals, animals within a related group so that one can being able to balance it with optimum nutrition would be predict disease Sooner, make interventions that are less 65 extremely important because thyroid disorder is the most expensive, less invasive, and more effective, and thereby common endocrine dysfunction amongst companion animals reverse the process before it becomes more serious clinically. today. US 7,970,552 B1 63 64 As it is well known that specific breeds that are used for Overall System performance events can have quite different basal thyroid FIG. 14 is an overview of the web-based system to provide metabolism, for example sighthounds and other coursing access to the invented database management system. With breeds vs. toy breeds or working breeds, it is important in the this system multiple users, for instance, remote users 8, cumulative database to determine these characteristics by access the web site 4 using the Internet 6. Each of the users 8 comprehensive profiling expected of this group as a whole, so has a computer terminal with the appropriate Software for that the data for individual animals could be compared to the accessing Internet. The users 8 may be unknown to the web group. Values for this specific functional group by breed server computers 10 and 12. Each user 8 is allowed to browse would then be compared to the entire database for the canine the web site and explore how the system functions. as a species and specific trends over time would be developed 10 There are several aspects to maintain security of informa relating to age and to environmental influences. Once the tion maintained in the database server 22 and a banking specific determinants of the individuals and the group that system 28. A firewall 20 prevents any user 8 from accessing they belong to have been made, the trends that have been any of the components behind the firewall 20. In this way the identified would be used to modify and intervene to promote users 8 have access to the web server computers 10 and 12, but nutrition and longevity, specifically again with modifications 15 only have access to the database server 22 through the firewall to dietary components or Supplements as well as other 20. The database server 22 maintains, among other things, changes in lifestyle, including exercise, group housing, indi various database fields with respect to each of the nutrition vidual housing and parameters that would promote wellness profiles of Subjects and the genetic information of a subject and longevity. and groups. The database 22 maintains the services with a The term “group” here has many different characteristics. designation associated to determine what nutrition assess It could include, for example, a specific breed of canine, a ment data and genetic data can be browsed by the users 8. specific purpose for which these canines are used, such as Each of the web server computers 10 and 12 allow users 8 to those who are purely companion pets in a home situation, view Subject and group categories and actual services and performance animals for show conformation, for obedience, data products which are available from the database. working trials, coursing trials, and for sheep herding and 25 The web server computers 10 and 12 can be identical and other herding purposes. It could also involve groups of ani can be duplicated as additional load or growth on the system mals depending on where they live—in a temperate climate, occurs. The web server computers 10 and 12 share the respon a warm or tropical climate, an arid desert climate, or a cold sibility for servicing the users of the site. This arrangement northern climate. It will include, of course, animals that live in provides for expandability of the system by merely adding urban and rural areas, animals that live near water, animals of 30 additional web server computers as necessary. variousages, intact or neutered, and for reproduction. In other Preferably, the system includes an appropriate computer words, the term “group' is used in avery broad sense here and terminal 24 for interfacing with independent financial insti can apply to any group that the user wishes to inquire of the tutions which are connected on-line via the serial connection database. Thus, the group is any selected Subset of the nutri 26 to the financial institution computers 28. This allows auto tion or diseased or disordered animals within the entire data 35 matic real time confirmation of the access of nutrition profile base. and genetic data services and products. Once a user requires The determination of the interrelationships between indi access to a product or service, the user goes through identifi viduals or groups of individuals in the database can use any cation or registration process and the exchange of financial one of a number of computerized or other methods of analy information to allow for credit or debit card payment of the sis, simple or complex, including Such things as neural net 40 purchase. This is verified, confirmed and authorized by the working or other kinds of relational technology evaluative appropriate bank system institution 28. Confirmation of the databases. purchase or deposit of data, or a service is made by a mail The system and procedure for carrying out the genetic test server 34 which sends an E-mail to the user 8 confirming the is, for instance a microarray analysis of DNA or RNA. There purchase or deposit. The mail server 34 allows for mail to be after there will be an analysis with a nutritional database. This 45 received and sent out. Security of the various databases is is be done by a comparison of the functional genomic profiles maintained. Alert messages are generated when an unautho as necessary with one or more of the nutritional databases. rized access is attempted. Verification messages, authoriza This disclosure utilizes comprehensive and cumulative tion messages and confirmation messages are generated as data profiling in a novel way over time to allow one to predict appropriate. the specific nutritional management interventions that will 50 The database server 22 is also designed to interact with an assist in the care and management of the very earliest stages input computer 32 operated by a CDPR. A firewall 30 serves of specific abnormalities or trends that have been identified in to prevent unauthorized access to the database server 22 or to the nutrition profile of animals, thereby extending and the input computer 32. The input computer 32 can input improving their nutrition and longevity. This is a unique nutrition profile data and genetic data to the database, after approach to Scientifically and medically determining by com 55 appropriate access and/or passwords are entered into the sys prehensive and cumulative laboratory profiling of individual tem. Similarly, users 8 through their own computers can use animals and animals within specified defined groups to per appropriate access codes and passwords to access input data mit intervention in preventive and management and treatment to the database server 22. This is tightly controlled for secu of general and veterinary medical nutrition care. Specifically, rity reasons. The data may only be added to an independent this disclosure directs the outcome of the laboratory profiling 60 sub-database of the data server 22, and only after scrutiny by to nutritional and nutritional Supplement management of the the CDPR operator of the database through input computer specific identified abnormalities and trends over time to 32, will this data from users 8 be subsequently added to the accomplish this goal. This is not only important but also main database server 22. practical because nutritional intervention and management is FIG. 15 is an illustration of the Internet and its use in the relatively inexpensive, non-invasive and easily accepted by 65 system of the disclosure. The Internet 6 is a network of mil the pet owner and the Veterinary professional making these lions of interconnected computers 40 including systems recommendations. owned by Internet providers 42 and information systems 44. US 7,970,552 B1 65 66 Individual or corporate users may establish connections to the software implemented by microprocessor 116 can be used to Internet in several ways. A user on a home PC 46 may pur connect computer system 100 to an existing network and chase an account through the Internet provider 42. Using a transfer data according to standard protocols. As such the modem 48, the PC user can dial up the Internet provider to computer system is connectable through an interface device connect to a high speed modem 50 which, in turn, provides a with the Internet 6. full service connection to the Internet. A user 52 may also Keyboard 114 is used by a user to input commands and make a somewhat limited connection to the Internet through other instructions to computer system 100. Other types of a system 20 that provides an Internet gateway connection 54 user input devices can also be used in conjunction with the and 56 to its customers. The database 22 is also connected into present disclosure. For example, pointing devices such as a the Internet 6 through an appropriate modem or high speed or 10 computer mouse, a track ball, a stylus, or a tablet can be used direct interface 58. The database 22 is operable and main to manipulate a pointer on a screen of a general-purpose tained by the CDPR operator computer 60. Users of the computer. databases of the disclosure would access the Internet in an The present disclosure in relation to the animal database appropriately selected manner. management of data can also be embodied as computer read FIG. 16 is a block diagram of an exemplary computer 15 able code on a computer readable medium. The computer system 100 for practicing various aspects of the disclosure. readable medium is any data storage device that can store data The computer system 100 includes a display screen or moni which can be thereafter read by a computer system. Examples tor 104, a printer 106, a disk drive 108, a hard disk drive 100, of the computer readable medium include read-only memory, a network interface 112, and a keyboard 114. The computer random-access memory, magnetic data storage devices Such system 100 includes a microprocessor 116, a memory bus as diskettes, and optical data storage devices such as CD 118, random access memory (RAM) 129, read only memory ROMs. The computer readable medium can also be distrib (ROM) 122, a peripheral bus 124, and a keyboard controller uted over network coupled computer systems so that the 126. The computer system 100 can be a personal computer. computer readable code is stored and executed in a distributed Microprocessor 116 is a general purpose digital processor fashion. which controls the operation of computer system 100. Micro 25 Specific System processor 116 can be a single-chip processor or can be imple FIG. 17 illustrates a browser system for use with the data mented with multiple components. Using instructions base system of the disclosure. A browser goes through a retrieve from memory, the microprocessor 116 controls the number of preliminary Screens and logic steps, and reaches a reception and manipulation of input data and the output and screen 60 entitled “Next Entry'. This screen provides data display of data on output devices. 30 details or information generally indicated as 62. Clicking on Memory bus 188 is used by the microprocessor 116 to any of these categories allows the user to review database access RAM 120 and ROM 122. RAM 129 is used by micro details 64, data specific details as generally indicated by 66. In processor 116 as a general storage area and as Scratch-pad this way, the user can index through a number of screens to get memory, and can also be used to store input data and pro information regarding the different databases of the system. cessed data. ROM 122 can be used to store instructions or 35 In addition, clicking on any of the triggers 70, 72, 74 and 76 program code followed by microprocessor 116 as well as is possible. These correspond to HOW IT WORKS, SECU other data. RITY, EXTENDED DATA and PRE-REGISTRATION. Peripheral bus 124 is used to access the input, output, and Clicking on trigger 70 provides the user with information on storage devices used by computer system 10. These devices how the process works, explains the system, and provides include the display screen 104, printer device 106, disk drive 40 details on how the user can participate in the database and 108, hard disk drive 110, and network interface 112. The obtain data or input data. Clicking on trigger 72 provides keyboard controller 126 is used to receive input from the details regarding security of the system and automatic pay keyboard 114 and send decoded symbols for each pressed key ment. In some cases, products and services are offered with to microprocessor 116 over bus 128. extended data and clicking on trigger 74 which can provide The display screen or monitor 104 is an output device that 45 details of the extended data and explains that this may only be displays images of data provided by microprocessor 116 via available on certain services or products. peripheral bus 124 or provided by other components in com Trigger 76 allows a user to pre-register and obtain userID puter system 100. The printer device 106 when operating as a number. This ID number is combined with financial informa printer provides an image on a sheet of paper or a similar tion retained in the database in an encrypted form. The pre Surface. Other output devices such as a plotter, typesetter, etc. 50 registration trigger 76 follows with step 78 which is to gather can be used in place of, or in addition to the printer device 106. personal information Such as credit card number and expiry The disk drive 108 and hard disk drive 110 can be used to date to allow for automatic payment. Step 80 is to validate a store various types of data. The disk drive 108 facilitates current existence in the database, if this occurs. With a nega transporting Such data to other computer systems, and hard tive answer, the user is directed into a registration process disk drive 110 permits fast access to large amounts of stored 55 indicate as 82. A user ID is assigned and a password is data. entered. This information is maintained in a portion of the Microprocessor 116 together with an operating system database 22. At 84 the user is provided a screen identifying the operate to execute computer code and produce and use data. user ID at screen 86. If the user already exists, the registration The computer code and data may reside on RAM 120, ROM process is rejected at 88 and the user is advised of the infor 122, or hard disk drive 120. The computer code and data could 60 mation at the display 86. The screen at 86 would also repre also reside on a removable program medium and loaded or sent the information which is available in the database 22. installed onto computer system 100 when needed. Remov In FIG. 18 there is shown a basic block diagram of the able program mediums include, for example, CD-ROM, PC components making up the CDPR. There is the phenotype CARD, floppy disk and magnetic tape. database or physical nutrition database 200 and a genotype The network interface circuit 112 is used to send and 65 database or genetic information database 201. These are con receive data over a network connected to other computer tained in part of the overall CDPR database 202. User input systems. An interface card or similar device and appropriate 203 can be obtained from a remote user such as a veterinarian, US 7,970,552 B1 67 68 owner, breeder, or the operator of the database, an agent or In an alternative approach, the reader can request an analy researcher. The output from the database 204 could be to the sis 215 from the genotype database as indicated by line 216. Veterinarian, owner, breeder, operator, agent or researcher. This analysis can receive data along line 217 from the analy FIG. 19 shows a relationship for retrieving data from the sis information of the physical nutrition assessment. Interpre database 202. The user 8 is represented here as a veterinarian, tation of the PT and GT can be obtained as indicated by 218, owner, breeder, operator, or researcher 203 who accesses the and this can then be outputted as indicated along line 219. The CDPR 202 accesses a first screen through a computer network interpretation of PT and GT 218 can be performed by an 6 which inquires about information about the user. An access algorithm relating to the coefficients and predictability of request message is sent, and an appropriate access enabling information relating to disorders, disease and longevity when message is transmitted. The user 203 can obtain partial or full 10 considering the data from the two databases PT 200 and GT access to the CDPR 202 according to the scale of authority 201. This can be done automatically and outputted along line given to the user 203 to access data. There is a computer 219, or there can be an expert interface 220 using skilled program system 205 to ensure that payment is made as appro personnel to interpret the data of block 218, and this can, in priate before access to the CDPR 202 is granted. In some turn, be outputted along line 221 to the output 212. situations, the appropriate access code 204 can permit 15 Database 207 can be a genetic marker database, and the bypassing the payment requirement 205 as indicated by line information from that database can be directly input into the 206. Payments 205 through the computer program can be output through a read-out 222 and 223 to the output 212. effected by a credit card entry and automatic transfer to a Alternatively, the data from database 207 can be added to the financial institution on behalf of the operator of the CDPR interpretation section 218 of the physical nutrition and 202. Such payment for access to the database is effected by a genetic information by directing the data along line 224. This system which is well known in the art. The financial institu data can then be made the Subject of the output along the line tion will appropriately credit the operator of the CDPR 202 in 219 and 221 as required. a financial manner as established between the operator and Similarly other databases 207 208,209, respectively, have the financial institution. read-outs 225 and 226 which can be directly coupled along Within the CDPR 202 there is the ability to access the 25 lines 227 and 228 to the output, or can be directed optionally physical nutrition phenotype database 200, the genotype along lines 229 and 230 to the interpretation module 218. It database 201, and other databases 207, 208 and 209, respec can then be the subject of interpretation for an expert interface tively. The phenotypic and genotypic information together 220 reviews which is, in turn, made the subject of the output with other database information can be presented on a single 219 and 221. screen or monitor or other viewing means, for instance, hard 30 In each of the outputlines 219,221, 222,223,227,228, and copy format. The access therefore can be to multiple data 214 there is also provided an encryption program 231 which bases contained within the CDPR 202. After accessing the can be optionally used in the system. The output 212 can physical nutrition database 200, the user obtains an analysis include paper, electronic, or Voice read-out as is required. report from module 210. The user is then able to read the In this manner, the output 212 provides a compilation analysis as indicated by 211 and output the analysis from the 35 which combines the physical nutrition and genetic informa read-out 211 as indicated by output 212. The output 212 can tion relating to a subject, the breed, disease, disorder and be a computer screen read-out, fax or voice information. lifespan, thereby enabling the receiver of the output 212 to use The other databases 207 are respectively a pharmacokinet the compiled information in a manner to facilitate breeding ics database to determine the fate of substances administered criteria which can be important in relation to animals which externally to a living organism. This is applied to drug Sub 40 are usually inbred or line bred. The information can also be stances, compounds ingested or otherwise delivered exter used to facilitate on-going monitoring of particular subject nally to an organism, such as nutrients, metabolites, hor animals. The data from this system can be used to manipulate mones, toxins. The pharmacokinetics database permits and regulate breeding, nutrition, and longevity effectively exploration of what a drug does to the body, whereas phar among animals. macokinetics explores what the body does to the drug. The 45 The system of the disclosure is further described with other database 208 includes the biologically active nutrient regard to FIG. 20 which is a system for inputting data to the database. The database 210 is for the target group. CDPR 202. Here multiple users 203, which can be a remote The physical nutrition orphenotype database 200 is subject user Such as a laboratory, a breeder, an owner, hospital, agent, or group specific. In other words, the data obtained in that or an operator of the CDPR 202 accesses the system through database is specific to a particular animal or animal group 50 module 204 which, in turn, accesses the CDPR 202. Appro (breed, family, species, etc.) which has been the subject of a priate access request and access enable messages are sent. laboratory or research biological examination Such that fluid Within the CDPR 202 there is a physical nutrition or pheno or tissue samples have been Subject to analysis in one or more type module 200, a genetic or genotype data module 201, and laboratory or research environments. These biological reports other database modules 207, etc. After accessing the CDPR can include those from specimens of blood, urine, other body 55 202, additional data can be added to the modules 200, 201, fluids, skin, eyes, skeletal and other tissues. The PT database 207, etc. through any of the users 203, if authorized. Depos 200 has the ability to store the subject specific information as iting data into each of the modules 200, 201 and 207 can required within the CDPR 202. optionally require the payment to the operator of the CDPR The genotype specific or genetic disorder or disease data is 202 as is indicated by block 205. This system can function in retained in the database 201 within the CDPR database 202. 60 the same manner as the retrieval of data from CDPR 202. This data is either subject specific, family specific, breed The stored data in each of the blocks 200, 201, and 207 can specific, species specific, disorder specific, or disease spe be set up as indicated by block 232 in a manner which is cific, and is group or Subject specific. The user can access the restricted or unrestricted to selected users 203. This may be genotype database 201 and obtain a read-out 213 which can necessary according to the protocols governing the inputted then be transmitted along line 214 to an output 212 in the 65 data to the different databases. In some cases, the waiving of same manner that the physical nutrition assessment is deposit fees is made in the interest of freedom of the database obtained as an output. to subsequent users who wish to retrieve data from the data US 7,970,552 B1 69 70 base. After storage of the data as indicated by block 234, the be disposed of a waste material. The test results, or output is user 203 exits CDPR 202 as indicated by block 233. provided as part of a nutrition profile database 200 of the As is apparent, the physical nutrition or phenotype profile CDPR 202 and is available to the remote user 8. of Subject animals is dynamic and grows as more data is If desired, the remote user 8 can arrange to have the data added into the system. Likewise, the genetic genotype data stored in the CDPR 202, made available to other remote users base also grows as increasing research of particular subjects, 8. The remote user 8 can also request the laboratory to per breeds, and the like is obtained. The deposit of new informa form analysis on the nutrition profile data 200 generated. tion into the CDPR 202 is regulated in a manner that the data In one embodiment, the communications link is a computer cannot distort the databases 202 in an in appropriate manner. network and the message transfer modality is, for instance, Likewise, users 203 cannot access the secured databases 10 the Internet 6, and/or an Intranet and/or an Extranet. The within CDPR 202 in an inappropriate manner. network systems are particularly Suited to the application Different algorithms regulate the relationship between the described herein since it offers global or widespread accessi nutrition profile, the genetic data, and other data relating to bility and high speed data transfer of large amounts of infor animals. These algorithms determine the probabilities, pos mation. sibilities, and likelihood of disorders and disease in subject 15 A security unit allows remote users to designate who has animals and offspring animals. They are used as predictors of permission to view or use their data. Feasible options for these the future evolvement of nutrition of the animal. information management requirements include: access by the Analyzing the data from the CDPR 102 in the manner of Submitting remote users only, access by certain designated the present disclosure permits for genetic screening, nutrition researchers and collaborators, time-embargoed data followed assessment profiling, and the diagnostic, prophylactic, and by wider access, and unrestricted access by all. therapeutic management of animals. A commerce unit can implement functions related to the An exemplary server performs all the operations of a con business aspects of the CDPR facility, including billing, ventional database system and performs additional opera inventory management of Support materials. tions in accordance with the present disclosure as has been A multimedia unit comprises means to store, manipulate, discussed. The server includes a central processing unit 25 and present audio, graphical, video information. This infor (CPU) together with associated memory for processing infor mation may include a video explaining how the CDPR is mation about different animal species and history. The inquir used, a visual depiction of the data, methodology, or a com ies concern animal species and history and inquiries and ment regarding the background of the data. The multimedia requests for nutrition profiling and genetic information, and unit may also implement Subscription functions, so that providing nutrition profiles and genetic information. The 30 updated data automatically provided to remote users or other CPU is coupled to the database and to users via a communi interested parties. cations port. The CPU is also coupled to an electronic mail The operations performed by the present disclosure begins processor for processing and storing (in a storage device) when the controller receives an access request message from e-mail messages transmitted between the CPU and various the remote user via a communication link. Using information agents, users and the like. The CPU is further coupled to a data 35 in the access request message and any other available infor storage device. A data storage device may include a variety of mation, the controller determines if the remote user is autho the databases. The system permits for the requesting, storing rized to access the CDPR 202. If so, an access enabling and providing of data with respect to animal phenotypic infor message is transmitted from the controller to the remote user mation and genetic information. The format and content of 8. The access enabling message can comprise a set of com the databases have been discussed in detail. 40 puter instructions transmitted over the Internet 6 which is FIG. 21 presents an overview of the laboratory instruments downloaded into the remote user memory for execution by apparatus, System, and method operable with the present the remote user processor. These instructions may be disclosure in relation to a CDPR 202. The present disclosure enabling, that is, they may allow direct communication allows access by remote users with computers or processors between the remote user 8 and the CDPR 202 with no further 100 to receive and access data on specimens. Using the Inter 45 need for the controller. In another embodiment, the access net 6 or other computer network or communication link capa enabling message may simply comprise a password or other bility, the remote user 8 sends a message to request access to enabling message which allows the remote user 8 to proceed. the services provided by the laboratory or operator which has The remote user 8 can access or submit data to the CDPR 202 a CDPR 202. If access to the CDPR 202 is granted, a message according to different protocols and regimes and security is sent to the remote user computers 100. This message 50 arrangements. includes instructions enabling the remote user 8 to define and Different forms of expert System computing and Software access data stored in the CDPR 202. programming can be used to determine the relationship of the In one form of the disclosure, the desired data is based on databases and data. Parallel distributed processing, and neu the Submission of test specimens of a specific animal to the romorphic systems, such as neural networks can be used. laboratory. In some other cases nutrition profile test data 200 55 They are good pattern recognition engines and robust classi can be inputted into the CDPR 202 having the genetic data fiers, with the ability to generalize in making decisions about base 201. The CDPR 202 can performan analysis and corre imprecise input data. There are multitudes of different types lation between the nutrition profile database 200 and the of networks such as a multilayer perception which is gener genetic database 201. ally trained with the back propagation of error algorithm, Using the communications link, the remote user 8 commu 60 learning vector quantization, radial basis function, Hopfield, nicates with the laboratory or the CDPR 202. Specimens can and Kohonen. Some are feed forward while others are recur be packaged and physically transported to the laboratory site rent (i.e., implement feedback) depending on how data is via commercially available common carriers, such as the processed through the network. Some may require training postal service or courier services. When the packages arrive, while others are unsupervised or self-organizing. This can be the laboratory places them in Storage, or the tests are per 65 implemented in Software or in specialized hardware. formed. Instruments 300 perform the tests to obtain data as Alternatively or additionally fuzzy logic can be used due to specified by the remote user 8. The biohazardous samples can the dynamic nature of the data applications, rules and func US 7,970,552 B1 71 72 tions. Such logic is adaptive to the changing environment. The data storage devices of the disclosure include a variety This logic and the neural networks can be integrated in the of databases including a database relating to the phenotypic system. data of a particular species, a database relating to nutrition Adaptive Logic Networks technology is an effective alter assessment or other phenotypic data of particular animals in a native or additional technology. The Adaptive Logic Network particular species, and genetic characteristics of different spe is neuro-computing capable of modeling complex non-linear cies and different family trees relating to different species. systems by using piece-wise linear data. The inputs to an The family trees would contain information including the Adaptive Logic Network may be the data from large data origin, genomic map, and parental lines of a species and bases as described, observations recorded by a scientist, vet records of nutrition and performance of a species. These 10 databases are interrelated in an analytical manner and in erinarian or owner. The outputs of an Adaptive Logic Net accordance with different algorithms of permutations and work can be used for analysis, prediction, or real-time probabilities to facilitate useful output information based on management. the combination of data in the genotypic and the phenotypic Conclusion databases, and the selected databases. As is clear, the tests above relate to at least one of endocrine 15 Many other examples of the disclosure exist, each differing function, immunologic function, gastrointestinal function from others in matters of detail only. The disclosure is to be and nutritional analysis, metabolism, paternity, DNA finger determined solely by the following claims. printing, and the functional genomic profile. These data are In the specification, there have been disclosed typical pre relevant to the likely morbidity, likely longevity, and/or the ferred embodiments of the disclosure and, although specific potential risk for disease or disorder for the animal provide terms are employed, they are used in a generic and descriptive useful information. This is in a manner previously not sense only and not for purposes of limitation, the scope of the obtained. disclosure being set forth in the claims. Many modifications As the above demonstrates, there is a need for providing and variations of the disclosure are possible in light of the data analysis and dissemination services to a wide variety of above teachings. It is therefore to be understood that within globally-distributed remote users. There is a need for provid 25 the scope of the appended claims the disclosure may be prac ing a system for inputting, storing and retrieving data related ticed otherwise than as specifically described. to animal nutrition assessment and genetics in a manner Some typical embodiments of the disclosure have been which permits for the effective use of this information. described. Many more examples, modifications and varia The system also permits for the access to the genetic and/or tions of the disclosure are possible in light of the above phenotype data through a password and a system whereby 30 teachings. For instance, although the disclosure and the access to the data generates a fee. This system permits for the claims indicate specific steps to perform the invention, the access or to provide data with regard to credit cards or the like steps described are not limited to a particular sequence of to ensure that the fee is transmitted automatically to a banking performance and in some circumstances two or more of these system for the account of the database when such data is steps could be undertaken simultaneously. It is therefore to be accessed. 35 understood that within the scope of the appended claims the This system also provides for a situation wherein payments disclosure may be practiced otherwise than as specifically can be made by credit card for requests to perform nutrition described, and the scope of the disclosure is set out in the assessment profiles and secure genomic mapping and genetic claims. screening information. Such bioinformatics system can also We claim: permit for the automatic payment for Such services and prod 40 1. A method for determining the nutrition of a non-human ucts to the banking system of the database or laboratory. As animal comprising the steps of Such, the database may require that the payments be guaran (a) providing a genotypic database for the species of the teed, for instance by Supplying a credit card number with a non-human animal Subject or a selected group of the request for performance of services and a product, and for the non-human species; retrieval of such data. 45 (b) obtaining animal phenotypic data; A user can Submit a request to the database in any number (c) correlating the database of (a) with the data of (b) to of ways. For example, the request can be submitted via on determine a relationship between the database of (a) and line direct connection, namely through a computer network the data of (b); and Such as the Internet. An intermediate researcher Such as a (d) determining a diagnostic profile of the animal or veterinarian or scientist other than the owner could also sub 50 Selected group of animals based on the correlating step mit the request on behalf of the owner using the e-mail capa (c), and selecting the biologically active nutrient from an bilities of the central database system. Alternatively, the user obtained molecular dietary signature, the molecular can Submit the data via an interactive Voice response unit dietary signature being the functional genomic profile coupled to the database system of the Supplier. In some situ which is a variation of expression of a set of genes which ations, the database Supplier can decide whether to Supply the 55 may differ for the genotype of each animal or selected nutrition assessment information and/or genomic mapping group of animals. and genetic screening information based on the criteria of the 2. A method of claim 1 comprising: user or its intermediary agent. Such user or intermediary using a “reference' dataset containing the functional agent can be notified of the decision via the interactive genomic profiles of biological samples for the multiple response unit or a live operator. 60 genotypes of different animals or groups of animals in The user or agent can log into the database system and varying physiological conditions; obtain the necessary records relating to an animal physical using a “target group' dataset containing the functional nutrition and/or genetic ancestry or offspring. The database genomic profiles of biological samples for the multiple system can transmit in real time or on a periodic basis as genotypes of different animals or groups of animals in determined, thereby, providing information regarding the 65 varying pathophysiological conditions; nutrition assessment or the genetic background and forward using a “nutrient dataset comprising the variable effects of this information to the user and/or its intermediary agent. nutritional components on a functional genomic profile US 7,970,552 B1 73 74 of an animal or a group of animals of different geno 14. A method of determining nutrition for an animal com types, the different genotypes that are responsive differ prising: ently to the same nutritional components; and (a) using a “reference' dataset containing functional relating the “reference' or “target group' datasets with the genomic profiles of biological samples of the genotypes “nutrient datasets, to derive a nutrient for the selected 5 of different animals of the species, the different animals animal genotypes to prevent, or treat, or control, or being healthy animals; modulate a state of physiological homeostasis or patho (b) selecting a “target group' dataset containing the func physiological condition of the animal or a group of ani tional genomic profile of biological samples of the geno mals. types of different animals, the different animals being 10 unhealthy animals; 3. The method of claim 1 including the step of reporting the (c) using a “biologically active nutrient dataset compris determination on a communications network including the ing different effects of biologically active nutritional Internet. components on functional genomic profiles of the dif 4. The method of claim 1 wherein the nutrition is a food, a ferent animals of different genotypes from those of the Supplement, a botanical, a nutraceutical selected to promote 15 target group (b), the different genotypes being differ wellness by enhancing an aspect of health of one or more ently responsive to the same biologically active nutri animals and wherein wellness is promoted by preventing, tional components; attenuating or eliminating at least one disease state in one or (d) having the reference group or target group include the more animals. individual animal; and 5. A method of formulating nutritional regime for a non (e) relating at least one of the “reference' or “target group' human animal comprising the steps of claim 1 to determine a datasets with the “biologically active nutrient dataset to relationship between the databases; and determining the identify a biologically active nutrient for the selected nutritional regimen for the animal based on the correlating animal genotype to prevent, treat, control, or modulate a step. state of physiological homeostasis or pathophysiologi 6. A method of determining a nutritional regime for a 25 cal condition of the individual animal in the reference non-human animal comprising the steps of claim 2 to deter group or target group. mine a relationship between the databases and determining 15. A method as claimed in claim 14 wherein this diagnosis the nutritional regimen for the animal based on the correlating results in prescribing a treatment or prophylaxis for the Sub step. ject based on the diagnosis. 30 16. A method of determining nutrition for an animal, com 7. A method as claimed in claim 1 wherein the animal is prising: either a canine or a feline; the canine or feline is from the (a) using a “reference' dataset containing functional group consisting of one or more of breed type, specific breed, genomic profiles of biological samples of the genotypes chronological age, physiological age, activity level, state of of different animals of the species, the different animals wellness, and State of disease. 35 being healthy animals; 8. A method as claimed in claim 2 wherein the condition is (b) selecting a “target group' dataset containing the func autoimmunity, anxiety, arthritis, depression, variable body tional genomic profile of biological samples of the geno condition score, immune Suppression, or inflammation. types of different animals, the animals being unhealthy 9. A method as claimed in claim 1, wherein the data of the animals; animal is one or more data items related to genotype, selected 40 (c) using a “biologically active nutrient dataset compris from the group consisting of breed, breed(s) of parents, pedi ing different effects of biologically active nutritional gree, sex, coat type, and evident hereditary conditions and components on functional genomic profiles of the dif disorders and the phenotypic data are selected from the group ferent animals of different genotypes from those of the consisting of age, weight, veterinary medical history, repro target group (b), the different genotypes being differ ductive history, present wellness or disease state, appetite, 45 ently responsive to the same biologically active nutri physical activity level, mental acuity, behavioral abnormali tional components; ties and disposition. (d) having the reference group or target group include the 10. A method as claimed in claim 2 wherein the reference animals; and data is data selected from group of animals with different (e) relating at least one of the “reference' or “target group' genotypes in physiological homeostasis and includes at least 50 datasets with the “biologically active nutrient dataset to one of DNA, RNA, proteins, metabolites and biomarkers. identify a biologically active nutrient for the selected 11. A method as claimed in claim 2, wherein the target animal genotypes to prevent, treat, control, or modulate group data is data selected from groups of animals with dif a state of physiological homeostasis or pathophysiologi ferent genotypes in non-physiological homeostasis and cal condition of the animal in the reference group or includes at least one of DNA, RNA, proteins, metabolites and 55 target group, and the relating being by using an analysis biomarkers. of gene or protein expression or the metabolytes in the 12. A method as claimed in claim 2, wherein the nutrient biological sample of the target group. data is data selected from groups of animals with different 17. A method as claimed in claim 16 wherein this diagnosis genotypes, the different genotypes being responsive differ results in prescribing a treatment or prophylaxis for the Sub ently to the same nutritional components, and includes at least 60 ject based on the diagnosis. one of DNA, RNA, proteins, metabolites and biomarkers. 18. A method of identifying a pharmacological product for 13. A method as claimed in claim 2, wherein the identified animals, the animals being selectively companion animals nutrient is a food, a Supplement, a botanical, a nutraceutical and selectively being a canine comprising: selected to promote wellness by enhancing an aspect of health (a) using a “reference' dataset containing functional of one or more animals and wherein wellness is promoted by 65 genomic profiles of biological samples of the genotypes preventing, attenuating or eliminating at least one disease of different animals of the species, the different animals state in one or more animals. being healthy animals; US 7,970,552 B1 75 76 (b) selecting a “target' dataset containing the functional 22. A method as claimed in claim 18, wherein the target genomic profile of biological samples of the genotypes dataset includes data selected from groups of animals with of different animals, the different animals being different genotypes in non physiological homeostasis and unhealthy animals; includes at least one of DNA, RNA, proteins, metabolites and (c) using a “pharmacological product dataset comprising biomarkers. 23. A method as claimed in claim 19, wherein the target different effects of pharmacological product on func dataset includes data selected from groups of animals with tional genomic profiles of the different animals of dif different genotypes in non physiological homeostasis and ferent genotypes from those of the target group (b), the includes at least one of DNA, RNA, proteins, metabolites and different genotypes being differently responsive to the biomarkers. same pharmacological product; 10 24. A method of identifying a pharmacological product for (d) having the reference dataset or target dataset include an animals, the animals being selectively companion animals individual animal; and and selectively being a canine comprising: (e) relating at least one of the “reference' or “target group' (a) using a “reference' dataset containing functional datasets with the pharmacological product dataset to genomic profiles of biological samples of the genotypes identify a pharmacological product for the selected ani 15 of different animals of the species, the different animals mal genotype to prevent, treat, control, or modulate a being healthy animals; state of physiological homeostasis or pathophysiologi (b) selecting a “target group' dataset containing the func cal condition of the individual animal in the reference tional genomic profile of biological samples of the geno dataset or target group. types of different animals, the animals being unhealthy animals; 19. A method as claimed in claim 18 wherein the identifi (c) using a “pharmacological product' dataset comprising cation is based on a molecular dietary signature being the different effects of pharmacological product on func expression of a gene or a set of genes which may differ for the tional genomic profiles of the different animals of dif genotypes of different animals of the same species, and the ferent genotypes from those of the target group (b), the nutrient identification includes the molecular dietary signa 25 different genotypes being differently responsive to the ture that the biologically active nutrient induces in the indi same pharmacological product; vidual animal. (d) having the reference group or target group include the 20. A method as claimed in claim 18, wherein the reference animals; and dataset includes data selected from group of animals with (e) relating at least one of the “reference' or “target group' different genotypes in physiological homeostasis and 30 datasets with the pharmacological product dataset to includes at least one of DNA, RNA, proteins, metabolites and select a biologically active formulation for the selected biomarkers. animal genotypes to prevent, treat, control, or modulate 21. A method as claimed in claim 19, wherein the reference a state of physiological homeostasis or pathophysiologi dataset includes data selected from group of animals with cal condition of the animal in the reference dataset or different genotypes in physiological homeostasis and 35 includes at least one of DNA, RNA, proteins, metabolites and target group. biomarkers.