US20060178837A1 - Computer-assisted means for assessing lifestyle risk factors - Google Patents

Computer-assisted means for assessing lifestyle risk factors Download PDF

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US20060178837A1
US20060178837A1 US11/277,525 US27752506A US2006178837A1 US 20060178837 A1 US20060178837 A1 US 20060178837A1 US 27752506 A US27752506 A US 27752506A US 2006178837 A1 US2006178837 A1 US 2006178837A1
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genes
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gene
polymorphisms
cancer
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Rosalynn Gill-Garrison
Christopher Martin
Manuel Sanchez-Felix
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Priority to US12/627,554 priority patent/US20100286923A1/en
Priority to US13/073,601 priority patent/US20110264375A1/en
Priority to US13/590,919 priority patent/US20120322036A1/en
Priority to US14/069,749 priority patent/US20160210880A1/en
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    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16HHEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H10/00ICT specially adapted for the handling or processing of patient-related medical or healthcare data
    • G16H10/40ICT specially adapted for the handling or processing of patient-related medical or healthcare data for data related to laboratory analysis, e.g. patient specimen analysis
    • GPHYSICS
    • G09EDUCATION; CRYPTOGRAPHY; DISPLAY; ADVERTISING; SEALS
    • G09BEDUCATIONAL OR DEMONSTRATION APPLIANCES; APPLIANCES FOR TEACHING, OR COMMUNICATING WITH, THE BLIND, DEAF OR MUTE; MODELS; PLANETARIA; GLOBES; MAPS; DIAGRAMS
    • G09B19/00Teaching not covered by other main groups of this subclass
    • G09B19/0092Nutrition
    • GPHYSICS
    • G09EDUCATION; CRYPTOGRAPHY; DISPLAY; ADVERTISING; SEALS
    • G09BEDUCATIONAL OR DEMONSTRATION APPLIANCES; APPLIANCES FOR TEACHING, OR COMMUNICATING WITH, THE BLIND, DEAF OR MUTE; MODELS; PLANETARIA; GLOBES; MAPS; DIAGRAMS
    • G09B5/00Electrically-operated educational appliances
    • G09B5/02Electrically-operated educational appliances with visual presentation of the material to be studied, e.g. using film strip
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/10Signal processing, e.g. from mass spectrometry [MS] or from PCR
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16HHEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H10/00ICT specially adapted for the handling or processing of patient-related medical or healthcare data
    • G16H10/60ICT specially adapted for the handling or processing of patient-related medical or healthcare data for patient-specific data, e.g. for electronic patient records
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16HHEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H20/00ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance
    • G16H20/70ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance relating to mental therapies, e.g. psychological therapy or autogenous training
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16HHEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/30ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for calculating health indices; for individual health risk assessment
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Definitions

  • the present invention relates to methods of assessing disease susceptibility.
  • it relates to methods of assessing disease susceptibility associated with dietary and lifestyle risk factors.
  • Cancer is a disease influenced primarily by external factors. Up to 80% of human cancers arise from exposure to environmental agents. The majority of cancer is believed to be preventable because exposure to these external factors should be manageable (Giovannucci, 1999; Perera, 2000).
  • Phase I Metabolism involves the addition of an oxygen atom or a nitrogen atom to lipophilic (fat soluble) compounds such as steroids, fatty acids, xenobiotics (from external sources like diet, smoke, etc.) so that they can be conjugated to glutathione or N-acetylated by the Phase II enzymes (thus made water-soluble) and excreted from the body.
  • lipophilic (fat soluble) compounds such as steroids, fatty acids, xenobiotics (from external sources like diet, smoke, etc.) so that they can be conjugated to glutathione or N-acetylated by the Phase II enzymes (thus made water-soluble) and excreted from the body.
  • xenobiotic-metabolising enzymes There are superfamilies of xenobiotic-metabolising enzymes: cytochrome P450's (Phase I), GSTs (Phase II) and NATs (Phase I and II) which are thought to have evolved as an adaptive response to environmental insult. Alterations in the activity of these enzymes are predicted to result in an altered susceptibility to cancer (Hirvonen, 1999).
  • Enzymatic activation of xenobiotics is not, however, the only route to cancer development.
  • Epidemiological studies suggest that nutritional factors may also play a causative role in more than 30% of human cancers.
  • defining the precise roles of specific dietary factors in the development of cancer is difficult due to the multitude of variables involved (Perera, 2000). Specific dietary factors are not easily measured as a single quantifiable variable, such as number of cigarettes smoked per day. Further complications arise due to differences in methodology, control populations, types of carcinogens, and amounts of exposure to carcinogens.
  • Priorities for studies relating to the interrelationship of dietary factors and cancer susceptibility include identification of genetic factors that contribute to individual cancer risk, identification of cancer-preventative chemicals in fruits and vegetables, better understanding of carcinogenic role of polycyclic aromatic hydrocarbons and heterocyclic amines generated by cooking meats at high temperature, and better understanding of the role of increased caloric intake with increased cancer risk (Perera, 2000).
  • colon cancer colon cancer
  • WHO colorectal cancers are the second most common cause of cancer death in Germany (WHO, 1997).
  • Worldwide colon cancer represents 8.5% of new cancer cases reported, with the highest rates seen in the developed world and the lowest rates in India.
  • Colon cancer occurs with approximately equal frequency in men and women, and the occurrence appears to be highly sensitive to changes in the environment. Immigrant populations assume the incidence rates of the host country very rapidly, often within the generation of the initial immigrant (Potter, 1999).
  • Risk factors for colon cancer include a positive family history, meat consumption, smoking and alcohol consumption (Giovannuci, 1999). There is an inverse relationship, i.e. lower risk, associated with consumption of vegetables, high folate intakes, use of non-steroidal anti-inflammatory drugs, hormone replacement therapy and physical activity. Meat and tobacco smoke are sources of carcinogens, while vegetables are a source of folate, antioxidants, and have Phase II (detoxifying) enzyme-inducing ability (Taningher, 1999).
  • Diets rich in raw vegetables, green vegetables, and cruciferous vegetables have a decreased risk of colon cancer Diets high in fibre, from vegetables and cereals, have been associated with a greater than two-fold decrease in risk of colorectal adenomas in men.
  • the data on fruit in the diet is not as consistent to date (WCRF, 1997), but a recent report (Eberhart, 2000) measured potent anti-oxidant activity of phytochemicals in apple skins with the ability to inhibit growth of tumour cell lines in vitro, so it is possible that more clearly defined links will emerge in the future.
  • Lower risk of colon cancer is associated with high folate intakes, but actual consumption of vegetables, rather than specific micronutrient preparations or vitamin supplements, has the most consistent low risk (Potter, 1999).
  • prostate and breast Other cancers that have been correlated with nutrition include prostate and breast. These malignancies are largely influenced by a combination of factors related to diet and nutrition. Prostate cancer is associated with high consumption of milk, dairy products and meats. These products decrease levels of 1,25(OH)2 vitamin D, which is a cell differentiator. Low levels of 1,25(OH)2 vitamin D may enhance prostate carcinogenesis by preventing cells from undergoing terminal differentiation and continuing to proliferate (Giovannucci, 1999). Breast, colon, and prostate cancers are relatively rare in less economically developed countries, where malignancies of the upper gastrointestinal tract are quite common. The cancers of the upper gastrointestinal tract have been related to various food practices or preservation methods other than refrigeration.
  • cancer of the mouth and pharynx is the sixth most common cancer world-wide and has been linked to alcohol consumption, tobacco, salt-preserved meat and fish, smoked foods and charcoal-grilled meat, as well as ingestion of beverages drunk very hot.
  • diet can be a direct supply of genotoxic compounds or may cause chronic irritation or inflammation (Giovannucci, 1999).
  • the invention provides a computer assisted method of providing a personalized lifestyle advice plan for a human subject comprising:
  • lifestyle risk factors it is meant risk factors associated with dietary factors, exposure to environmental factors, such as smoking, environmental chemicals or sunlight.
  • lifestyle recommendations should be interpreted as relating to recommendations relating to dietary factors and exposure to environmental factors, such as smoking, environmental chemicals or sunlight.
  • Disease susceptibility should be interpreted to include susceptibility to conditions such as allergies.
  • the method allows individualised advice to be generated based on the unique genetic profile of an individual and the susceptibility to disease associated with the profile.
  • specific risk factors can be identified and dietary and other health advice tailored to the individual's needs.
  • the lifestyle advice will include recommended minimum or maximum amounts of foodtypes. (Note that an amount may be 0).
  • Information concerning the sex and health of the individual and/or of the individual's family may also provide indications that a particular polymorphism or group of polymorphisms associated with a particular condition should be investigated. Such information may therefore be used in selection of polymorphisms to be screened for in the method of the invention.
  • Such factors may also be used in the determination of appropriate lifestyle recommendations in step (v) of the method. For example, recommendations relating to reducing susceptibility to prostate cancer would not be given to women and recommendations relating to susceptibility to ovarian cancer would not be given to men. Other factors, such as information regarding the age, alcohol consumption, and existing diet of the client may be incorporated into the determination of appropriate lifestyle recommendations in step (v).
  • the report comprising the personalised dietary advice may be delivered to the client by any suitable means, for example by letter, facsimile or electronic means, such as e-mail.
  • the report may be posted on a secure Web-page of the service provider with access limited to the client by the use of a unique identifier notified to the client either by conventional or electronic mail.
  • the report can therefore comprise one or more hyperlinks to other documents of the report provider's Web-site or to other Web-sites giving relevant information on the particular polymorphisms identified, disease prevention and/or dietary advice.
  • the method will involve assessing a variety of loci in order to give a broad view of susceptibility and possible means of minimising disease risk.
  • individual polymorphisms may be considered biomarkers for individual cancer risk, the different biomarkers, when considered together, may also reveal a significant cancer risk.
  • the correlation between CYP1A1 activity and cancer susceptibility varies, dependent on the presence of specific types of CYP1A1 polymorphism as well as the presence of GSTM1 polymorphisms.
  • An individual with an extremely active CYP1A1 gene, leading to high Phase I P450 activity in combination with a null GSTM1 genotype that lacks the detoxifying Phase II activities has a very high risk of developing cancer (Taningher, 1999).
  • the presence of a particular polymorphism may be indicative of increased susceptibilty to one disease while being indicative of decreased susceptibility to another disease.
  • one allele of the gene encoding epoxide hydrolase which catalyses the conversion of toxic PAH metabolites formed by CYP1A1 and CYP1A2 into less toxic and more water-soluble trans-dihydrodiols, has recently been found to be associated with increased risk of aflatoxin-induced liver cancer, but also with decreased risk of ovarian cancer (Pluth, 200; Taningher, 1999).
  • Preferred genes for which polymorphisms are identified include genes that encode Phase I metabolism enzymes responsible for detoxification of xenobiotics, genes that encode Phase II metabolism enzymes responsible for further detoxification and excretion of xenobiotics, genes that encode enzymes that combat oxidative stress, genes associated with micronutrient deficiency (for example, deficiency of folate, B12 or B6), genes that encode enzymes responsible for metabolism of alcohol, genes that encode enzymes involved in lipid and/or cholesterol metabolism, genes that encode enzymes involved in clotting, genes that encode trypsin inhibitors, genes that encode enzymes related to susceptibility to metal toxicity, genes which encode proteins required for normal cellular metabolism and growth and genes which encoded HLA Class 2 molecules.
  • the method of the invention may include the step of determining the presence of individual alleles at one or more genetic loci of the DNA in a DNA sample of the subject, and constructing the dataset used in step (iii) using results of that determination.
  • Techniques for determining the presence or absence of individual alleles are known to the skilled person. They may include techniques such as hybridization with allele-specific oligonucleotides (ASO)(Wallace, 1981; Ikuta, 1987; Nickerson, 1990, Varlaan-de Vries, 1986, Saiki, 1989 and Zhang, 1991) allele specific PCR (Newton 1989, Gibbs, 1989), solid-phase minisequencing (Syvanen, 1993), oligonucleotide ligation assay (OLA) (Wu, 1989, Barany, 1991; Abravaya, 1995), 5′ fluorogenic nuclease assay (Holland, 1991 & 1992, Lee, 1998) U.S. Pat. Nos. 4,683,202, 4,683,195, 5,723,591 and 5,801,155, or Restriction fragment length polymorphism (RFLP) (Donis-Keller, 1987).
  • ASO allele-specific oligonucleotides
  • RFLP Rest
  • the genetic loci are assessed via a specialised type of PCR used to detect polymorphisms, commonly referred to as the Taqman® assay, in which hybridisation of a probe comprising a fluorescent reporter molecule, a fluorescent quencher molecule and a minor groove binding chemical to a region of interest is detected by removal of quenching of the fluorescent molecule and detection of resultant fluorescence. Details are given below.
  • the genetic loci are assessed via hybridisation with allele-specific oligonucleotides, the allele specific oligonucleotides being preferably arranged as an array of oligonucleotide spots stably associated with the surface of a solid support.
  • the arrays suitable for use in the method of the invention form a further aspect of the present invention.
  • the fragments of DNA comprising the gene(s) of interest may be amplified to produce a sufficient amount of material to be tested.
  • the present inventors have designed a number of specific primer sets for amplification of gene regions of interest. Such primers may be used in pairs to isolate a particular region of interest in isolation. Therefore in a further aspect of the invention, there is provided a primer having a sequence selected from SEQ ID NO: 86-99, 104-163. In another aspect, there is provided a primer pair comprising primers having SEQ ID NO:n, where n is an even number from 86-98 or 104-162 in conjunction with a primer having SEQ ID NO:(n+1).
  • primer sets will be used together with other primer sets to provide multiplexed amplification of a number of regions to allow determination of a number of polymorphisms from the same sample. Therefore in a further aspect of the invention, there is provided a primer set comprising at least 5, more preferably 10, 15 primer pairs selected from SEQ ID NO: 86-121.
  • FIG. 1 shows examples of databases 1 and 2 which may be used in an embodiment of the present invention.
  • FIG. 2 is a flow chart illustrating an embodiment of the invention.
  • markers for polymorphisms of one or more of the following classes of genes are used:
  • the first dataset of the method of the invention may comprise information relating to two or more alleles of one or more genetic loci of genes selected from the group comprising:
  • the dataset will preferably comprise information relating to two or more alleles of at least two genetic loci of genes selected from the group comprising categories a-k as described above, for example, a+b, a+c, a ⁇ d, a+e, a+f, a+g, a+h, a+i, a+j, a+k, b+c, b+d, b+e etc., c ⁇ d, c+e etc, d+e, d+f etc, e+f, e+g etc, f+g, f+h etc., g+h, g+i, g+k, h+i, h+k.
  • the dataset comprises information relating to two or more alleles of at least two genetic loci
  • at least one of the genetic loci is of category d, due to the central role of micronutrients in the maintenance of proper cellular growth and DNA repair, and due to the association of micronutrient metabolism or utilisation disorders with several different types of diseases (Ames 1999; Perera, 2000; Potter, 2000).
  • the dataset will preferably comprise information relating to two or more alleles of at least three genetic loci selected from the group comprising categories a-k as described above.
  • the dataset comprises information relating to alleles of at least three genetic loci, it is preferred that at least two of the genetic loci are of categories d and e.
  • Information relating to polymorphisms present in both of these categories is particularly useful due to the effects of alcohol consumption and metabolism on the efficiency of enzymes related to micronutrient metabolism and utilisation (Ulrich, 1999).
  • the dataset comprises information relating to alleles of at least three genetic loci
  • it is preferred that at least two of the genetic loci are of categories a and b due to the close interaction of Phase I and Phase II enzymes in the metabolism of xenobiotics.
  • the dataset will comprise information relating to two or more alleles of at least four genetic loci of genes selected from the group comprising categories a-k as defined above, for example, a+b+c+d, a+b+c+e, a+b+d+e, a+c+d+e, b+c+d+e etc.
  • the dataset comprises information relating to alleles of at least four genetic loci
  • at least three of the genetic loci are of categories d and e and f
  • Information relating to polymorphisms present in these three categories is particularly useful due to the strong correlation of polymorphisms of these alleles with coronary artery disease due to the combined effects of altered micronutrient utilisation, affected adversely by alcohol metabolism, together with imbalances in fat and cholesterol metabolism.
  • the dataset comprises information relating to alleles of at least five genetic loci
  • at least four of the genetic loci are of categories a, b, d and e.
  • the dataset may comprise information relating to two or more alleles of at least five, for example a, b, d, e and f, six, seven, eight, nine or ten genetic loci of genes selected from the group comprising categories a-k as defined above.
  • the dataset will comprise information relating to two or more alleles of one or more genetic loci of genes selected from each member of the group comprising categories a-k as described above.
  • the first dataset comprises information relating to two or more alleles of the genetic loci of genes encoding each of the cytochrome P450 monooxygenase, N-acetyltransferase 1, N-acetyltransferase 2, glutathione-S-transferase, manganese superoxide dismutase, 5,10-methylenetetrahydrofolatereductase and alcohol dehydrogenase 2 enzymes.
  • the first dataset further comprises information relating to two or more alleles of the genetic loci of genes encoding one or more, preferably each of epoxide hydrolase (EH), NADPH-quinone reductase (NQ01), paraxonaoase (PON1), myeloperoxidase (MPO), alcohol dehydrogenase 1, alcohol dehydrogenase 3, cholesteryl ester transfer protein, apolipoprotein A IV, apolipoprotein E, apolipoprotein C III, angiotensin, factor VII, prothrombin 20210, ⁇ -fibrinogen, heme-oxygenase-1, ⁇ -antitrypsin, SPINK1, ⁇ -aminolevulinacid dehydratase, interleukin 1, interleukin 1, vitamin D receptor, B1 kinin receptor, cystathionine-beta-synthase, methionine synthase (B
  • Xenobiotics are potentially toxic compounds found in, for example, char-grilled red meat. Meat consumption is associated with increased risk of cancer, especially well-done meat cooked at high temperatures (Sinha, 1999). Cooking meat in this fashion leads to the production of heterocyclic amines (HCA), nitrosamines (NA), and polycyclic aromatic hydrocarbons (PAH), which have known carcinogenic activity in animals (Hirvonen, 1999; Layton, 1995).
  • HCA heterocyclic amines
  • NA nitrosamines
  • PAH polycyclic aromatic hydrocarbons
  • Phase I metabolism involves the addition of an oxygen atom or a nitrogen atom to lipophilic (fat soluble) compounds, such as steroids, fatty acids, xenobiotics (from external sources like diet, smoke, etc.) so that they can be conjugated by the Phase II enzymes (thus made water-soluble) and excreted from the body (Hirvonen, 1999).
  • Phase II enzymes thus made water-soluble
  • Individuals with genetic polymorphisms correlated with cancer risk in these genes should avoid consumption of char-grilled foods, smoked fish, well-done red meat whether grilled or pan-fried (Sinha, 1999). They should also increase consumption of food products known to increase Phase II metabolism so the products of Phase I metabolism may be cleared more efficiently.
  • genes of category a for which information relating to polymorphisms may be used in the present invention include genes encoding cytochrome P450 monooxygenase (CYP) e.g. CYP1A1, CYA1A2, CYP2C, CYP2D6, CYP2E1, CYP3A4, CYP11B2, genes encoding N-acetyltransferase 1 e.g. NAT1, genes encoding N-acetyltransferase 2 e.g.
  • CYP cytochrome P450 monooxygenase
  • genes encoding N-acetyltransferase 1 e.g. NAT1 genes encoding N-acetyltransferase 2 e.g.
  • NAT2 genes encoding epoxide hydrolase (EH), genes encoding NADPH-quinone reductase (NQ01, genes encoding paraxonaoase (PON1), genes encoding myeloperoxidase (MPO).
  • EH epoxide hydrolase
  • NQ01 genes encoding NADPH-quinone reductase
  • PON1 genes encoding paraxonaoase (PON1)
  • MPO myeloperoxidase
  • CYP is also referred to as cytochromome P450 monooxygenase (gene is called CYP, enzyme is called P450).
  • P450 enzymes belong to a super-family with wide substrate activity that catalyses the insertion of an oxygen atom into a substrate. The reaction can convert a molecule (procarcinogen) into a DNA-reactive electrophilic carcinogen (Hirvonen, 1999; Smith, 1995). Polymorphisms in genes encoding cytochrome P450 (CYP family of genes) are associated with altered susceptibility to cancer, CAD and altered metabolisim of various pharmaceutical agents (Poolsup, 2000; Miki, 1999; Cramer, 2000; Marchand, 1999; Sinha, 1997).
  • CYP1A1 codes for a P450 enzyme that metabolises polycyclic aromatic hydrocarbons (PAH).
  • PAH polycyclic aromatic hydrocarbons
  • the CYP1A1 gene is polymorphic and is inducible by PAH, which means that expression of the enzyme is increased upon exposure to PAH (MacLeod, 1997).
  • CYP1A1 is located on chromosome 15q 22 -q24 (Smith, 1995). This gene has been linked to colorectal, urinary bladder, breast, oral cavity, stomach, and lung cancers (Perera, 2000; Garte, 1998).
  • the gene product, the P450 enzyme is inducible by exposure to the agents that it metabolises, so the consumption of high levels of a potential source of carcinogens, such as well-done red meat, would increase the production of the enzyme and thus the creation of carcinogenic substances (Mooney, 1996; Perera, 2000; Alexandrie, A. K., 2000).
  • Studies of polymorphisms of the CYP1A1 gene have revealed considerable differences in enzyme activity, with corresponding differences in cancer risk after exposure to known substrates of the enzyme (Alexandrie, 2000; Rojas, 2000; Garte, 2000). Both the Ile-Val polymorphism I, which comprises an A4889G substitution (i.e.
  • the adenine residue at position 4889 of the 5′-3′ strand is substituted by a guanine residue
  • the CYP1A1*C polymorphism, which comprises an T6235C substitution are induced to a greater extent than the wild type gene after exposure to PAH, and have been associated with a significant increase in cancer risk (Taningher, 1999; Garte, 1998; Kawajiri, 1996; MacLeod, S., 1997; Smith, 1995).
  • Approximately 10 percent of the Caucasian population carries polymorphisms linked to cancer risk, according to a recent American review paper (Shields, 2000).
  • CYP1A2, CYP2C, CYP2D6, CYP2E1, CYP3A4, CYP11B2 are associated with altered susceptibility to cancer and drug sensitivity. (Poolsup, 2000; Miki, 1999; Cramer,2000; Marchand, 1999; Sinha, 1997).
  • NAT1 N-acetyltransferase 1
  • NAT2 N-acetyltransferase 2
  • PAH and heterocyclic amines The enzymes catalyse N-acetylation, O-acetylation, and N,O-acetylathon.
  • the O-acetylation reaction is considered the most risky, with the potential for forming chemical carcinogens that can bind to DNA.
  • the N-acetylation reaction can occur on a compound after a P450 has inserted an oxygen, thus increasing the water solubility of the compound so it may be excreted. Due to this activity, the NAT genes are often considered as both Phase I and Phase II type enzymes.
  • N-acetyltransferase genes There are 3 separate N-acetyltransferase genes in humans, two are active genes: NAT1 and NAT2, and a pseudogene, NATP. Pseudogenes have the same sequence, but lack apparent function and promoter elements and are not expressed in cells (i.e. the gene is not transcribed into RNA then translated into amino acids to make a protein/enzyme) (Perera, 2000).
  • NAT1 and NAT2 genes are located on chromosome 8 at 8p21.3-21.1, both genes are 870 bp long and both code for a protein 290 amino acids in length.
  • the genes are highly polymorphic and epidemiological studies have sometimes given conflicting information regarding links with cancer.
  • the genes show geographical and ethnic variation and the enzyme activity varies considerably within different tissues or organs.
  • the current list of nomenclature and polymorphisms is kept at a web site: http://www.louisville.edu/medschool/pharmacology/NAT.html.
  • NAT1 is expressed to a higher degree than NAT2 in the colon, so NAT1 may be associated with localised activity of activated HAA or PAH in the colon (Brockton, 2000; Perera, 2000).
  • the polymorphism NAT1*10 which comprises T1088A and C1095A substitutions, and which has a fast phenotype, has been consistently linked with an increased risk of colon cancer and higher DNA adduct levels (i.e. DNA damage that can lead to cancer) in colon tissue (Perera, 2000; Ilett, 1987).
  • the NAT1*11 polymorphism has been linked to risk of breast cancer in women who smoke or consume well-done red meat (Zheng, 1999).
  • NAT1*11A polymorphisms which comprises C( ⁇ 344)T, A( ⁇ 40)T, G445A, G459A, T640G, C1095A substitutions and a ⁇ 9:1065-1090 deletion
  • NAT1*113 polymorphism which comprises C( ⁇ 344)T, A( ⁇ 40)T, G445A, G459A, T640G substitutions and a A9:1065-1090 deletion.
  • NAT1*11 polymorphisms should be understood to include reference to NAT1*11A or NAT1*11B polymorphisms.
  • NAT1*14 on the other hand has little or no enzyme activity (Brockton, 2000) and has been associated with increased lung cancer risk (Bouchardy, C., 1998).
  • Two alleles of the NAT1*14 polymorphism are known: the NAT1*14A polymorphism, which comprises G560A, T1088A and C1095A substitutions; and the NAT1*14B polymorphism, which comprises a G560A substitution.
  • NAT1*14 polymorphisms should, except where the context dictates otherwise, be understood to include reference to NAT1*14A or NAT1*14B polymorphisms.
  • the NAT1*14 polymorphism shares a restriction enzyme site with the NAT1*11polymorphism, and some of the conflicting results reported in the literature are believed to be due to the inability of the assay used (restriction fragment length polymorphism assay (RFLP)) to distinguish the polymorphisms (Hein, 2000a).
  • RFLP tion fragment length polymorphism assay
  • the oligonucleotide array suitable for use in the present invention can distinguish all polymorphisms and therefore will be more precise than the RFLP procedure.
  • NAT2 is expressed primarily in the liver, but has been linked with cancer incidence in other organs (Hein, 2000b).
  • NAT2*5A which comprises T481C and T341C substitutions
  • NAT2*6A which comprises C282T and G590A substitutions
  • NAT2*7A which comprises a G857A substitution
  • NAT2*4 is considered the normal, or wild type, sequence.
  • NAT2*4 has fast acetylator activity and has been linked to increased cancer risk in several studies (reviewed in Hein, 2000b; Gil, 1998), but especially in conjunction with the NAT1*10 polymorphism (Bell, 1995).
  • NAT2 rapid/intermediate acetylators with at least one NAT2*4 allele have been linked to breast cancer in women who consumed well-done red meat (Dietz, 1999).
  • Approximately 55% of the Caucasian population carry NAT1 polymorphisms linked to cancer. (Shields, 2000).
  • Polymorphisms in genes encoding epoxide hydrolase are associated with cancer and chronic obstructive pulmonary disease (Pluth, 200; Miki,1999). Polymorphisms in genes encoding NADPH-quinone reductase are associated with altered susceptibility to cancer (Nakajima, 2000). Polymorphisms in genes encoding paraxonoase are associated with altered susceptibility to cancer and to CAD (MacKness, 2000). Polymorphisms in genes encoding mycloperoxidase are associated with altered susceptibility to CAD (Schabath, 2000).
  • genes of category b for which information relating to polymorphisms may be used in the present invention include genes encoding glutathione-S-transferase e.g GSTM1, GSTP1, GSTT1.
  • Glutathione-S-transferases catalyse the reaction of electrophilic compounds with glutathione so the compounds may be excreted from the body.
  • the enzymes belong to a super-family with broad and overlapping substrate specificities.
  • Glutatione-S-transferases provide a major pathway of protection against chemical toxins and carcinogens and are thought to have evolved as an adaptive response to environmental insult, thus accounting for their wide substrate specificity (Hirvonen, 1999).
  • GSTmu has 3 alleles: null, a, which is considered to be the wild type, and b, which comprises a C534G substitution, with no functional difference between the a and b alleles.
  • the GSTmu sub-type has the highest activity of the 4 types and is predominately located in the liver (Hirvonen, 1999). Approximately half of the population has a 10 complete deletion of this gene with a corresponding risk of lung, bladder, breast, liver, and oral cavity cancer (Shields, 2000; Perera, 2000). It has been estimated that 17% of all lung and bladder cancers may be attributable to GSTM1 null genotypes (Hirvonen, 1999).
  • GSTM1 null genotype together with a highly active CYP1A1 polymorphism has been linked to a very high cancer risk in several studies (Rojas, 2000; Shields, 2000).
  • the GSTM1 gene is located on chromosome 1p13.3 (Cotton, 2000).
  • GSTpi gene is located on chromosome 11q13. This sub-type is known to metabolise many carcinogenic compounds and is the most abundant sub-type in the lungs (Hirvonen, 1999).
  • Two single nucleotide polymorphisms have been linked to cancer to date GSTP1*B, which comprises an A313G substitution, and GSTP1*C, which comprises a C341T substitution.
  • the enzymes of these polymorphic genes have decreased activity compared to the wild type and a corresponding increased risk of bladder, testicular, larynx and lung cancer (Harries, 1997; Matthias, 1998; Ryberg, 1997).
  • GSTtheta gene is on chromosome 22q11.2 and is deleted in approximately 20% of the Caucasian population.
  • the enzyme is found in a variety of tissues, including red blood cells, liver, and lung (Potter, 1999).
  • the deletion is associated with an increased risk of lung, larynx and bladder cancers (Hirvonen, 1999). Links with GSTM1 null genotypes are currently being searched, as it is believed that individuals that have both GSTM1 and GSTT1 alleles deleted will have a greatly increased risk of developing cancer (Potter, 1999).
  • genes of category c for which information relating to polymorphisms may be used in the present invention include genes encoding manganese superoxide dismutase (MnSOD or SOD2 gene).
  • Manganese superoxide dismutase is an enzyme that destroys free radicals or a free-radical scavenger.
  • the gene is located on chromosome 6q25.3, but the enzyme is found within the mitochondria of cells.
  • Ile 58Thr allele which comprises an T175C substitution
  • Val( ⁇ 9)Ala allele which comprises a T( ⁇ 28)C substitution.
  • a study of premenopausal women found a four-fold increased risk of breast cancer in individuals with the Val( ⁇ 9)Ala polymorphism and the highest risk within this group is found in women who consumed low amounts of fruits and vegetables (Ambrosone, 1999). This polymorphism occurs in the signal sequence of the amino acid chain.
  • the signal sequence ensures transport of the enzyme into the mitochondria of the cell, and so the polymorphism is believed to reduce the amount of enzyme delivered to the mitochondria (Ambrosone, 1999).
  • the mitochondria is commonly referred to as the workhorse of the cell, where the energy-yielding reactions take place. This is the site of many oxidative reactions, so many free radicals are generated here. Individuals with low activity of this enzyme should be advised to take antioxidant supplements and increase consumption of fruits and vegetables (Giovannucci, 1999; Perera, 2000).
  • Micronutrient Deficiency e.g. of Folate, Vitamin B12 or Vitamin B6
  • genes of category d for which information relating to polymorphisms may be used in the present invention include the gene encoding 5,10-methylenetetrahydrofolatereductase (MTHFR) activity.
  • MTHFR 5,10-methylenetetrahydrofolatereductase
  • 5,10-methylenetetrahydrofolate reductase is active in the Iolate-dependent methylation of DNA precursors. Low activity of this enzyme leads to an increase of uracil incorporation into DNA (instead of thymine) (Ames, 1999).
  • the MTHFR gene is polymorphic and has been linked to colon cancel, adult acute lymphocytic leukaemia and infant leukaemia (Ames, 1999; Perera, 2000; Potter, 2000). Both the wt and polymorphic alleles have been linked to disease, each being dependent on levels of folate in the diet. Approximately 35% of the Caucasian population has genetic polymorphisms at this locus with corresponding risk of colon cancer (Shields, 2000).
  • Polymorphisms at this locus include those with a C677T or A1298C substitution. Dietary recommendations for individuals lacking in MTHFR activity include taking supplements with folate and increasing consumption of fruit and vegetables (Ames, 1999). Low levels of vitamins B12 and B6 have been associated with low MTHFP activity and increased cancer risk, so individuals should increase intake of these vitamins; B12 is found primarily in meat and B6 is found in whole grains, cereals, bananas, and liver (Ames, 1999). Alcohol has a deleterious effect on folate metabolism, affecting individuals with the A1298C polymorphism most severely (Ulrich, 1999). These individuals should be advised to avoid alcohol.
  • genes of category e for which information relating to polymorphisms may be used in the present invention include genes encoding alcohol dehydrogenase e.g. the ALDH2 gene, ALDH1 gene and ALDH3 gene.
  • Alcohol dehydrogenase 2 (ALDH2) is involved in the second step of ethanol utilisation. Reduced activity of this enzyme leads to accumulation of acetaldehyde, a potent DNA adduct former (Bosron, 1986).
  • ALDH2*2 polymorphism which comprises a G1156A substitution, and which has links with oesophageal/throat cancer, stomach, lung, and colon cancer (IARC, 1998; Yokoyama, 1998).
  • the advice to individuals with the polymorphism would be to avoid alcohol.
  • Polymorphisms in ALDH1 and 3 are associated with increased susceptibility to cancers and Parkinson's disease.
  • genes of category f for which information relating to polymorphisms may be used in the present invention include genes encoding cholesteryl ester transfer protein e.g. the CETP gene, polymorphisms of which genes are associated with altered susceptibility to coronary artery disease (CAD)((Raknew, 2000; Ordovas, 2000); genes encoding apolipoprotein A, IV (ApoA-IV), polymorphisms of which genes are associated with altered susceptibility to coronary artery disease (CAD) (Wallace,2000; Heilbronn, 2000); apolipoprotein E(ApoE), polymorphisms of which genes are associated with altered susceptibility to CAD and Alzheimer's disease (Corbo,1999; Bullido, 2000); or apolipoprotein C, III (ApoC-III), polymorphisms of which genes are associated with altered susceptibility to CAD, hypertension and insulin resistance (Salas, 1998).
  • CAD coronary artery disease
  • genes of category g for which information relating to polymorphisms may be used in the present invention include genes encoding angiotensin (AGT-1) and angiotensin converting enzyme (ACE), polymorphisms of which genes are associated with altered susceptibility to hypertension (Brand 2000;de Padua Mansur, 2000), factor VII, polymorphisms of which genes are associated with altered susceptibility to CAD (Donati, 2000; Di Castelnuovo, 2000); prothrombin 20210, polymorphisms of which genes are associated with altered susceptibility to venous thrombosis (Vicente, 1999); ⁇ -fibrinogen, polymorphisms of which genes are associated with altered susceptibility to CAD (Humphries, 1999); or heme-oxygenase-1, polymorphisms of which genes are associated with altered susceptibility to emphysema (Yamada, 2000).
  • AGT-1 angiotensin
  • ACE angiotensin
  • genes of category h for which information relating to polymorphisms may be used in the present invention include genes encoding ⁇ -antitrypsin, polymorphisms of which genes are associated with altered susceptibility to chronic obstructive pulmonary disease (COED) (Miki, 1999); or serine protease inhibitor, Kazal type 1(SPINK), polymorphisms of which genes are associated with altered susceptibility to pancreatitis (Pfutzer, 2000).
  • genes of category i for which information relating to polymorphisms may be used in the present invention include genes encoding ⁇ -aminolevulinacid dehydratase, polymorphisms of which genes are associated with altered susceptibility to lead toxicity (Costa, 2000).
  • genes of category j for which information relating to polymorphisms may be used in the present invention include genes encoding the vitamin D receptor, polymorphisms of which genes are associated with altered susceptibility to osteoporosis, tuberculosis, Graves disease, COPD, and early periodontal disease (Ban, 2000; Wilkinson, 2000; Gelder, 2000; Miki, 1999; Hennig, 1999); the B1 kinin receptor (B1R), polymorphisms of which genes are associated with altered susceptibility to kidney disease (Zychma, 1999); cystathionine-beta-synthase, polymorphisms of which genes are associated with altered susceptibility to CAD (Tsai, 1999); methionine synthase (B12 MS), polymorphisms of which genes are associated with altered susceptibility to CAD (Tsai, 1999); the 5-HT transporter, polymorphisms of which genes are associated with altered susceptibility to neurological disorders, Alzheimer'
  • genes of category k for which information relating to polymorphisms may be used in the present invention include genes encoding HLA Class 2 molecules, polymorphisms of which genes are associated with altered susceptibility to cervical cancer and human papilloma virus (HPV) infection (Maciag, 2000); T-lymphocyte associated antigen 4 (CTLA-4), polymorphisms of which genes are associated with altered susceptibility to liver disease (Argawal, 2000); interleukin 1 (IL-1), polymorphisms of which are associated with cardiovascular disease and periodontal disease (macaiag, 2000; Nakajima, 2000); IL-4, polymorphisms of which genes are associated with altered susceptibility to atopy and asthma (Rosa-Rosa, 1999); IL-3, polymorphisms of which genes are associated with altered susceptibility to atopy and asthma (Rosa-Rosa, 1999); IL-6, polymorphisms of which genes are associated with altered susceptibility to
  • the method of the invention may include the step of analysing a DNA sample of a human subject in order to construct the dataset to be used in the method of the invention.
  • DNA for analysis using the method or arrays of the invention can be isolated from any suitable client or patient cell sample.
  • the DNA is isolated from cheek (buccal) cells. This enables easy and painless collection of cells by the client, with the convenience of being able to post the sample to the provider of the genetic test without the problems associated with posting a liquid sample.
  • Cells may be isolated from the inside of the mouth using a disposable scraping device with a plastic or paper matrix “brush”, for example, the C.E.P. SwabTM (Life Technologies Ltd., UK). Cells are deposited onto the matrix upon gentle abrasion of the inner cheek, resulting in the collection of approximately 2000 cells (Aron, 1994). The paper brush can then be left to dry completely, ejected from the handle placed into a microcentrifuge tube and posted by the client or patient to the provider of the genetic test.
  • a plastic or paper matrix “brush” for example, the C.E.P. SwabTM (Life Technologies Ltd., UK). Cells are deposited onto the matrix upon gentle abrasion of the inner cheek, resulting in the collection of approximately 2000 cells (Aron, 1994).
  • the paper brush can then be left to dry completely, ejected from the handle placed into a microcentrifuge tube and posted by the client or patient to the provider of the genetic test.
  • DNA from the cell samples can be isolated using conventional procedures.
  • DNA may be immobilised onto filters, column matrices, or magnetic beads.
  • Numerous commercial kits such as the Qiagen QIAamp kit (Quiagen, Crawley, UK) may be used. Briefly, the cell sample may be placed in a microcentrifuge tube and combined with Proteinase K, mixed, and allowed to incubate to lyse the cells. Ethanol is then added and the lysate is transferred to a QIAamp spin column from which DNA is eluted after several washings.
  • the amount of DNA isolated by the particular method used may be quantified to ensure that sufficient DNA is available for the assay and to determine the dilution required to achieve the desired concentration of DNA for PCR amplification.
  • the desired target DNA concentration may be in the range 10 ng and 50 nq. DNA concentrations outside this range may impact the FCR amplification of the individual alleles and thus impact the sensitivity and selectivity of the polymorphism determination step.
  • the quantity of DNA obtained from a sample may be determined using any suitable technique.
  • suitable techniques are well known to persons skilled in the art and include UV (Maniatis, 1982) or fluorescence based methods.
  • UV methods may suffer from the interfering absorbance caused by contaminating molecules such as nucleotides, RNA, EDTA and phenol and the dynamic range and sensitivity of this technique is not as great as that of fluorescent methods, fluorescence methods are preferred.
  • Commercially available fluorescence based kits such as the PicoGreen dsDNA Quantification (Molecular Probes, Eugene, Oreg., USA).
  • the nucleic acids in the sample may be selectively amplified, for example using Polymerase Chain Reaction (PCR) amplification. as described in U.S. Pat. Nos. 4,683,202 AND 4,683,195.
  • PCR Polymerase Chain Reaction
  • Preferred primers for use in the present invention are from 18 to 23 nucleotides in length, without internal homology or primer-primer homology.
  • the two primers of a pair are preferably selected to hybridise to either side of the region of interest so that about 150 bases in length are amplified, although amplification of shorter and longer fragments may also be used.
  • the site of polymorphism should be at or near the centre of the region amplified.
  • Table 1 provides preferred examples of primer pairs which may be used in the invention, particularly when the Taqman® assay is used in the method of the invention.
  • the primers are shown together with the gene targets and preferred examples of the wt probes and polymorphism probes used in the Taqman® assay for each gene target.
  • Table 2 provides preferred examples of the primer pairs which may be used in the invention together with the gene targets and the size of the fragment isolated using the primers, which they amplify.
  • the primers and primer pairs form a further aspect of the invention. Therefore the invention provides a primer having a sequence selected from SEQ ID NO: 86-99, 104-163.
  • a primer pair comprising primers having SEQ ID NO:n, where n is an even number from 86-98 or 104-162 in conjunction with a primer having SEQ ID NO:(n+1).
  • primer pairs to be used in the same reaction are preferably selected by position, similarity of melting temperature, internal stability, absence of internal homology or homology to each other to prevent self-hybridisation or hybridisation with other primers and lack of propensity of each primer to form a stable hairpin loop structure.
  • the sets of primer pairs to be coamplified together preferably have approximately the same thermal profile, so that they can be effectively coamplified together. This may be achieved by having groups of primer pairs with approximately the same length and the same G/C content.
  • a primer set comprising at least 5, more preferably 10, 15 primer pairs selected from SEQ ID NO: 86-121.
  • TABLE 1 Forward Reverse Polymorphism Gene primer primer WT probe probe 1. CYP1A1 A4889G CATGGGCAAGCGGAAG CAGGATAGCCAGGAA CGGTGAGACCaTTG CGGTGAGACCgTTG TG GAGAAAGAC (SEQ ID NO:164) (SEQ ID NO:165) (SEQ ID NO:122) (SEQ ID NO:123) T6235C AGACACGGTCCCCAGG CAGAGGCTGAGGTGG CTCCACCTCCtGGG CTCCACCTCCcGGG TCAT GAGAA (SEQ ID NO:166) (SEQ ID NO:167) (SEQ ID NO:124) (SEQ ID NO:125) 2.
  • GSTP1 A313G CCTGGTGGACATGGTG GCAGATGCTCACATA GCAAATACaTCTCCCT GCAAATACgTCTCCC AATG GTTGGTGTAG (SEQ ID NO:188) T (SEQ ID NO:148) (SEQ ID NO:149) (SEQ ID NO:189) C341T CGGATGAGACTAGGAT GGGTCTCAAAAGGCT CCTTGCCCgCCTC CTTGCCCaCCTCC GATACATGGT TCAGTTG (SEQ ID NO:190) (SEQ ID NO:191) (SEQ ID NO:150) (SEQ ID NO:151) 6.
  • polymorphisms may be identified. Identification of the markers for the polymorphisms involves the discriminative detection of allelic forms of the same gene that differ by nucleotide substitution, or in the case of some genes, for example the GSTM1 and GSTT1 genes, deletion of the entire gene. Methods for the detection of known nucleotide differences are well known to the skilled person. These may include, but are not limited to:
  • the genetic loci are assessed via a specialised type of PCR used to detect polymorphisms, commonly referred to as the Taqman® assay and performed using an AB7700 instrument (Applied Blosystems, Warrington, UK).
  • a probe is synthesised which hybridises to a region of interest containing the polymorphism.
  • the probe contains three modifications: a fluorescent reporter molecule, a fluorescent quencher molecule and a minor groove binding chemical to enhance binding to the genomic DNA strand.
  • the probe may be bound to either strand of DNA.
  • the polymerase will encounter the probe and begin to remove bases from the probe one at a time using a 5′-3′ exonuclease activity.
  • the fluorescent molecule is no longer quenched by the quencher molecule and the molecule will begin to fluoresce.
  • This type of reaction can only take place if the probe has hybridised perfectly to the matched genomic sequence. As successive cycles of amplification take place, i.e. more probes and primers are bound to the DNA present in the reaction mixture, the amount of fluorescence will increase and a positive result will be detected. If the genomic DNA does not have a sequence that matches the probe perfectly, no fluorescent signal is detected.
  • oligonucleotide probes which may be used in the invention, particularly when the Taqman® assay is used in the method of the invention together with primers which may be used. These oligonucleotide probes form another aspect of the present invention.
  • an oligonucleotide having a sequence selected from SEQ ID NO: 164-202.
  • the invention further provides a set of oligonucleotides comprising at least 5, 10, 20, 30, 40, 50, 60 or 70 oligonucleotides selected from the group comprising SEQ ID NO:164-202.
  • hybridisation with allele specific oligonucleotides is conveniently carried out using oligonucleotide arrays, preferably microarrays, to determine the presence of particular polymorphisms.
  • microarrays allow miniaturisation of assays, e.g. making use of binding agents (such as nucleic acid sequences) immobilised in small, discrete locations (microspots) and/or as arrays on solid supports or on diagnostic chips.
  • binding agents such as nucleic acid sequences
  • microspots discrete locations
  • diagnostic chips diagnostic chips.
  • These approaches can be particularly valuable as they can provide great sensitivity (particularly through the use of fluorescent labelled reagents), require only very small amounts of biological sample from individuals being tested and allow a variety of separate assays to be carried out simultaneously. This latter advantage can be useful as it provides an assay for different a number of polymorphisms of one or more genes to be carried out using a single sample.
  • DNA microarrays have been shown to provide appropriate discrimination for polymorphism detection. Yershov, 1996; Cheung, 1999 and Schena 1999 have described the principles of the technique.
  • the DNA microarray may be generated using oligonucleotides that have been selected to hybridise with the specific target polymorphism. These oligonucleotides may be applied by a robot onto a predetermined location of a glass slide, e.g. at predetermined X,Y Cartesian coordinates, and immobilised.
  • the FCR product e.g.
  • RNA or DNA fluorescently labelled RNA or DNA
  • a hybridisation reaction conducted so that sample RNA or DNA hinds to complementary sequences of oligonucleotides in a sequence-specific manner, and allow unbound material to be washed away.
  • Gene target polymorphisms can thus be detected by their ability to bind to complementary oligonucleotides on the array and produce a signal.
  • the absence of a fluorescent signal for a specific oligonucleotide probe indicates that the client does not have the corresponding polymorphism.
  • the method is not limited to the use of fluorescence labelling but may use other suitable labels known in the art.
  • the fluorescence at each coordinate can be read using a suitable automated detector in order to correlate each fluorescence signal with a particular oligonucleotide.
  • Oligonucleotides for use in the array may be selected to span the site of the polymorphism, each oligonucleotide comprising one of the following at a central location within the sequence:
  • arrays used in the present method form another independent aspect of the present invention.
  • Arrays of the invention comprise a set of two or more oligonucleotides, each oligonucleotide being specific to a sequence comprising one or more polymorphisms of a gene selected from the group comprising categories a-k as defined above.
  • the array will comprise oligonucleotides each being specific to a sequence comprising one or more polymorphisms of an individual gene of at least two different categories a-k as defined above, for example a+b (i.e. at least one oligonucleotide specific for a sequence comprising one or more polymorphisms of a first gene, the first gene being of category a and at least one oligonucleotide specific for a sequence comprising one or more polymorphisms of a second gene, the second gene being of category b), a+c, a+d, a+e, a+f, a+g, a+h, a+i, a+j, a+k, b+c, b+d, b+e etc., c+d, c+e etc, d+e, d+f etc, e+f, e+g etc, f+g, f+h etc., g+h
  • the array comprises two or more oligonucleotides
  • at least one of the oligonucleotides is an oligonucleotide specific for a sequence of a polymorphism of a gene of category d, due to the central role of micronutrients in the maintenance of proper cellular growth and DNA repair, and due to the association of micronutrient metabolism or utilisation disorders with several different types of diseases (Ames 1999; Perera, 2000; Potter, 2000).
  • the array will comprise oligonucleotides each being specific to a sequence comprising one or more polymorphisms of an individual gene of at least three different categories a-k as defined above, for example, a+b+c, a+b+d, a+b+e, a+b+f, a+b+g, a+b+h, a+b+i, a+b+j, a+b+k a+c+d, a+c+e etc, a+d+e, etc, b+c+d, etc, c+d+e etc, d+e+f etc, and all other combinations of three categories.
  • the array comprises three or more oligonucleotides
  • at least two of the oligonucleotides are oligonucleotides specific for a sequence of a polymorphism of a gene of categories d and e.
  • Information relating to polymorphisms present in both of these categories is particularly useful due to the effects of alcohol consumption and metabolism on the efficiency of enzymes related to micronutrient metabolism and utilisation. (Ulrich, 1999).
  • the array comprises three or more oligonucleotides
  • at least two of the oligonucleotides are oligonucleotides specific for a sequence of a polymorphism of a gene of c categories a and b due to the close interaction of Phase I and Phase II enzymes in the metabolism of xenobiotics.
  • the array will comprise oligonucleotides each being specific to a sequence comprising one or more polymorphisms of an individual gene of at least four different categories a-k as defined above, for example, a+b+c+d, a+b+c+e, a+b+d+e, a+c+d+e, b+c+d+e etc.
  • the array comprises four or more oligonucleotides
  • at least three of the oligonucleotides are oligonucleotides specific for a sequence of a polymorphism of a gene of categories d and e and f Information relating to polymorphisms present in these three categories is particularly useful due to the strong correlation of polymorphisms of these alleles with coronary artery disease due to the combined effects of altered micronutrient utilisation, affected adversely by alcohol metabolism, together with imbalances in fat and cholesterol metabolism.
  • the array comprises five or more oligonucleotides
  • at least four of the oligonucleotides are oligonucleotides specific for a sequence of a polymorphism of a gene of categories a, b, d and e.
  • Information relating to polymorphisms present in these four categories is particularly useful due to the combined effects of micronutrients utilisation, alcohol metabolism, Phase 1 metabolism of xenobiotics and Phase II metabolism on the further metabolism and excretion of potentially harmful metabolites produced in the body (Taningher, 1999; Ulrich, 1999).
  • the array may comprise oligonucleotides each being specific to a sequence comprising one or more polymorphisms of an individual gene of at least five, for example a, b, d, e and f, six, seven, eight, nine or ten different categories a-k as defined above.
  • the array will comprise oligonucleotides each being specific to a sequence comprising one or more polymorphisms of an individual gene of each of categories a-k as defined above.
  • the array comprises oligonucleotides each being specific to a sequence comprising one or more polymorphisms of individual genes, the individual genes comprising each member of the group comprising genes encoding cytochrome P45C monooxygenase, N-acetyltransferase 1, N-acetyltransferase 2, glutathione-S-transferase, manganese superoxide dismutase, 5,10-methylenetetrahydrofolatereductase and alcohol dehydrogenase 2 enzymes.
  • the array further comprises oligonucleotides specific for one or more alleles of the genetic loci of genes encoding one or more, preferably each of epoxide hydrolase (EH), NADPH-quinone reductase (NQ01), paraxonaoase (PON1), myeloperoxidase (MPO), alcohol dehydrogenase 1, alcohol dehydrogenase 3, cholesteryl ester transfer protein, apolipoprotein A IV, apolipoprotein E, apolipoprotein C III, angiotensin, factor VII, prothrombin 20210, ⁇ -fibrinogen, heme-oxygenaso-1, ⁇ -antitrypsin, SPINK1, ⁇ -aminolevulinacid dehydratase, interleukin 1, interleukin 1, vitamin D receptor, B1 kinin receptor, cystathionine-beta-synthase, methionine synthe
  • the oligonucleotides in the array comprise at least 5, 10, 20, 30, 40, 50, 60 or 70 oligonucleotides selected from the group comprising SEQ ID NO:1-SEQ ID NO: 85 illustrated in TABLE 3 which shows preferred oligonucleotides listed in the right column with the primer set used to amplify the appropriate fragments of sample DNA listed in the left column.
  • the array will comprise all of the oligonucleotides SEQ ID NO:1-85.
  • TABLE 3 Gene Target 25 nt sequence 1.
  • CYP1A1 Primer set1 A4889G wt-lead 5′ atc ggt gag acc Att gcc cgc tgg g (SEQ ID NO: 1)
  • SEQ ID NO: 2 Primer set1 A4889G polymorph- 5′ atc ggt gag acc Gtt gcc cgc tgg g lead (SEQ ID NO: 3)
  • Primer set2 T6235P wt-
  • GSTM1 Primer set1 wt-lead 5′ gct aca ttg ccc gca agc aca acc t (SEQ ID NO: 52) Primer set1 wt-lag 5′ agg ttg tgc ttg cgg gca atg tag c (SEQ ID NO: 53) 5.
  • the results of genetic polymorphism analysis may be used to correlate the genetic profile of the donor of the sample with disease susceptibility using the first dataset, which provides details of the relative disease susceptibility associated with particular polymorphisms and their interactions.
  • the risk factors identified using dataset 1 can then be matched with dietary and other lifestyle recommendations from dataset 2 to produce a lifestyle advice plan individualised to the genetic profile of the donor of the sample. Examples of datasets 1 and 2 which may be used to generate such advice is illustrated in FIG. 1 .
  • a ranking system is preferably used to provide an indication of the degree of susceptibility of a specific polymorph to risk of cancer(s) and/or other conditions.
  • the ranking system may be designed to take into account of homozygous or heterozygous alleles in the client's sample, i.e. the same or different alleles being present in diploid nucleus. Five categories which may be used are summarised below:
  • a susceptibility may be assigned to each polymorphism identified and, from dataset 2 , a lifestyle recommendation corresponding to each susceptibility identified may be assigned. For example, if an individual is found to have the NAT1*10 polymorphism, the decision tree may indicate that the there is an enhanced susceptibility of colonic cancer. Recommendations appropriate to minimising the risk of colonic cancer are then generated. For example, the recommendations may he to avoid particular foods associated with increased risk and to increase consumption of other foods associated with a protective effect against such cancers. The totality of recommendations may be combined to generate a lifestyle advice plan individualised to the donor of the sample.
  • the decision tree is preferably arranged to recognise particular combinations of polymorphisms and/or susceptibilities which interact either positively to produce a susceptibility greater than would be expected from the risk factors associated with each individually, and/or, which interact negatively to reduce the susceptibility associated with each individually. Where such combinations are identified, the advice generated can be tailored accordingly. For example, the combination of NAT2*4 and NAT1*10 polymorphisms have been linked to increased cancer risk (Bell, 1995). Therefore, when such a combination of polymorphisms is identified from a subject's DNA, the associated very high susceptibility to cancer is assigned and the advice tailored to emphasise the need to reduce consumption of xenobiotics, e.g. by reducing or eliminating consumption of char-grilled foodstuffs.
  • DNA is prepared from a buccal cell sample on a brush using a Qiagen QIAamp kit according to the manufacturer's instructions (Qiagen, Crawley, UK). Briefly, the brush is cut in half and one half stored at room temperature in a sealed tube in case retesting is required. The other half of the brush is placed in a microcentrifuge tube. 400 ⁇ l PBS is added and the brush allowed to rehydrate for 45 minutes at room temperature. Quiagen lysis buffer and Proteinase K is then added, the contents are mixed, and allowed to incubate at 56 C for 15 minutes to lyse the cells. Ethanol is added and the lysate transferred to a QIAamp spin column from which DNA is eluted after several washings.
  • Qiagen QIAamp kit according to the manufacturer's instructions (Qiagen, Crawley, UK). Briefly, the brush is cut in half and one half stored at room temperature in a sealed tube in case retesting is required. The other half
  • a quantification step is carried out using the PicoGreen dsDNA Quantification kit (Molecular Probes, Eugene, Oreg., USA).
  • client DNA samples are prepared by transferring a 10 ⁇ l aliquot into a microcentrifuge tube with 90 ⁇ l TE. 100 ⁇ l of the working PicoGreen dsDNA quantification reagent is added, mixed well, and transferred into a black 96 well plate with flat well bottoms. The plate is then incubated for 5 minutes in the dark before a fluorescent reading is taken. The quantity of DNA present in the clients' samples is determined by extrapolating from a calibration plot prepared using DNA standards.
  • a quantity of DNA in the range of 5-50 ng total is used in the subsequent PCR step. Remaining client DNA sample is stored at ⁇ 20° C. for retesting if required.
  • the modified reaction mixture contains Taq polymerase (1.25 units/ ⁇ l), optimised PCR buffer, dNTP (200 ⁇ M each), 2 mM MgCl 2 and primer pairs SEQ ID NO: 160 and 161 and polymorphism probe SEQ ID NO: 200.
  • the reaction mixture is initially incubated for 10 minutes at 50° C., then 5 minutes at 95° C., followed by 40 cycles of 1 minute of annealing at between 55° C. and 60° C. and 30 seconds of denaturation at 95° C. Both during the cycles and at the end of the run, fluorescence of the released reporter molecules of the probe is measured by an integral CCD detection system of the AB7700 thermocycler. The presence of a fluorescent signal which increases in magnitude through the course of the run indicates a positive result.
  • the assay is then repeated with the same primer pair and wt probe SEQ ID NO: 199. If the sample is homozygous for the polymorphism, no fluorescence signal is seen with the wt probe. However, if the sample is heterozygous for the polymorphism, a fluorescence signal is also seen with the wt probe. If single reporter results from homozygous wt, homozygous polymorphic and heterozygous polymorphic samples are plotted are plotted on an X/Y axis, the homozygous alleles will cluster at opposite ends of the axes relative to each reporter, and the heterozygous alleles will cluster at a midway region.
  • the PCR reaction mix contains Taq polymerase (1.25 units/reaction), optimised PCR buffer, dNTP's (200 ⁇ M each) and MgCl 2 at an appropriate concentration of between 1 and 4 mM, and 40 pmol of each primer(SEQ ID NOS: 1-8, 17-63) for amplification of seven fragments and the sample DNA.
  • reaction mixture is initially incubated at 95° C. for 1 minute, and then subjected to 45 cycles of PCR in a MWG TC9600 thermocycler (MWG-Biotech-AG Ltd., Milton Keynes, UK) as follows:
  • thermocyclers such as the Applied Biosystems 9700 thermocycler (Applied Biosystems, Warrington, UK), may be used.
  • the PCR mplification products are then purified using the Qiagen QIAquick PCR Purification Kit (Qiagen, Crawley, UK) to remove dNTPs, primers, and enzyme from the PCR product.
  • the PCR product is layered onto a QIAquick spin column, a vacuum applied to separate the PCR product from the other reaction products and the DNA eluted in buffer.
  • the DNA is then transcribed into RNA using T3 and T7 RNA polymerases together with fluorescently labelled UTP for incorporation into the growing chain of RNA.
  • the reaction mixture comprises:
  • the mixture is incubated at 37° C. for 1 hour.
  • the mixture is then treated with DNAse to remove DNA so that only newly synthesised fluorescent RNA is left.
  • the RNA is then precipitated, microcentrifuged and resuspended in buffer for hybridisation on the array.
  • sample amplified fragments are then tested using a DNA microarray
  • the DNA microarray used comprises oligonucleotides SEQ ID NOs: 1-85. These oligonucleotides are applied by a robot onto a glass slide and immobilised. The fluorescently labelled amplified DNA is introduced onto the DNA microarray and a hybridisation reaction conducted to bind any complementary sequences in the sample, allowing unbound material to be washed away. The presence of bound samples is detected using a scanner. The absence of a fluorescent signal for a specific oligonucleotide probe indicates that the client does not have the corresponding polymorphism.
  • the PCR reaction mix contains Taq polymerase (1.25 units/reaction), optimised PCR buffer, dNTP's (200 ⁇ M each) and MgCl 2 at an appropriate concentration of between 1 and 4 mM, and 40 pmol of each primer (SEQ ID NOs: 88, 89) for amplification of the fragment.
  • the methods used is the same as detailed in Example 4, with the array comprising oligonucleotides SEQ ID NO: 17, 18, 19 and 20.
  • results of the microarray or Taqman® analysis are input into a computer comprising a first dataset correlating the presence of individual alleles with a risk factor and a second dataset correlating risk factors with lifestyle advice.
  • a report is generated identifying the presence of particular polymorphisms and providing lifestyle recommendations based on the identified polymorphisms. An example of such a decision process is shown in FIG. 2 .
  • a sample of DNA is screened and the alleles identified input to a dataprocessor as Dataset 3 .
  • Each allele is matched to lifestyle risk factor from dataset 1 , e.g. high susceptibility to colon cancer due to the presence of the NAT1*10 allele and the absence of the GSTM1 allele.
  • the identified risk factor is then matched with one or more lifestyle recommendations from dataset 2 , for example “avoid red meat, chargrilled food, smoked meats and fish; stop smoking immediately” (in order to avoid production of potentially toxic byproducts by Phase 1 enzymes with increased activity) and “increase consumption of vegetables of the allium family e.g. onions and garlic, and the brassaicae family e.g.
  • broccoli in order to increase the activity of Phase 11 enzymes present, such as GSTP1 and GSTT1 and others, in order to increase the excretion of toxic byproducts of Phase 1 metabolism.
  • Phase 11 enzymes present such as GSTP1 and GSTT1 and others, in order to increase the excretion of toxic byproducts of Phase 1 metabolism.
  • the lifestyle recommendations are then assembled to generate a comprehensive personalised lifestyle advice plan.

Abstract

The present invention relates to methods of assessing disease susceptibility associated with dietary and lifestyle risk factors. The invention provides for analysis of alleles at loci of genes associated with lifestyle risk factors, and the disease susceptibility profile of an individual is determined by reference to datasets which further match the risk factor with lifestyle recommendations in order to produce a personalized lifestyle advice plan.

Description

  • This application is a divisional of Ser. No. 09/771,933, filed Jan. 30, 2001 (allowed), the entire contents of which is incorporated herein by reference.
  • FIELD OF THE INVENTION
  • The present invention relates to methods of assessing disease susceptibility. In particular, it relates to methods of assessing disease susceptibility associated with dietary and lifestyle risk factors.
  • BACKGROUND TO THE INVENTION
  • Cancer is a disease influenced primarily by external factors. Up to 80% of human cancers arise from exposure to environmental agents. The majority of cancer is believed to be preventable because exposure to these external factors should be manageable (Giovannucci, 1999; Perera, 2000).
  • Human tumours result from a series of mutational events, leading to the loss of the regulatory mechanisms that govern normal cell behaviour and ultimately resulting in the formation of a tumour with full metastatic (or invasive) potential (Smith, 1995). All higher organisms have developed a complex variety of mechanisms to protect themselves from environmental insult, for example from ingested plant toxins. One of the most important protection measures involves the metabolism of toxins (or xenobiotics) leading to detoxification and ultimately excretion of the toxin (Smith, 1995). Unfortunately, the metabolic pathways do not always lead to detoxification of the toxin. Indeed many chemical carcinogens are activated by these same metabolic pathways to react with cellular macromolecules.
  • Improvements in genetic analysis and the availability of human genetic sequence information arising from the Human Genome Project has added another facet to the analysis of cancer susceptibility, that of inter-individual variation at the genome level. Molecular epidemiology has already begun to clarify some of the gene-environment interactions that may lead to disease. The ultimate goal of molecular epidemiology is to develop risk assessment models for individuals, and already the field has provided insight into inter-individual variation in human cancer risk (Shields, 2000). Molecular epidemiology focuses on three major determinants of human cancer risk: inherited host susceptibility factors, molecular dosimetry of carcinogen exposure, and biomarkers of early effects of carcinogenic exposure. The variability in metabolic activity, detoxification and DNA repair of the US population could be as high as 85-500-fold with correspondingly high variability in cancer risk (Hattis, 1986). Considering the latency of cancer, the importance of correlating individual risk with biomarkers at an early stage becomes apparent. These biomarkers can help to identify populations or individuals at risk of cancer resulting from specific environment-gene interactions.
  • Defining the factors that contribute to inter-individual variations in cancer susceptibility has been a major focus of research for many years. Given the suggested role of environmental factors in carcinogenesis, some of the candidate genes are those that encode the xenobiotic-metabolising enzymes that activate or inactivate carcinogens. Variable levels of expression of these enzymes could result in increased or decreased carcinogen activation. Other genetic factors that could contribute to cancer susceptibility include genes involved in DNA repair, proto-oncogenes, tumour suppressor genes, cell-cycle genes, as well as genes involved in aspects of nutrition, hormonal status, and immunological responses. Emerging data from the Human Genome Project has led to studies that show combinations of metabolic polymorphisms are increasingly being linked to a greater risk of cancer (Perera, 1997). Studies which have measured the formation of DNA adducts as a marker of enzyme activity have found that the levels of DNA damage or protein adducts vary considerably between persons with apparently similar exposure (Bryant, 1987; Perera, 1992; Mooney, 1995). The observed variability reflects a combination of true biologic factors, unaccounted for by differences in exposure or laboratory variation (Dickey, 1997). In fact, lower exposures to carcinogens can result in proportionately higher adduct levels because of a person's genetic predisposition for increased carcinogen metabolic activation (Kato, 1995; Vineis, 1997)
  • The existence of multiple alleles at loci that encode xenobiotic-metabolising enzymes can result in differential susceptibilities of individuals to the carcinogenic effects of various chemicals. Metabolism in humans occurs in two distinct phases: Phase I Metabolism involves the addition of an oxygen atom or a nitrogen atom to lipophilic (fat soluble) compounds such as steroids, fatty acids, xenobiotics (from external sources like diet, smoke, etc.) so that they can be conjugated to glutathione or N-acetylated by the Phase II enzymes (thus made water-soluble) and excreted from the body. There are superfamilies of xenobiotic-metabolising enzymes: cytochrome P450's (Phase I), GSTs (Phase II) and NATs (Phase I and II) which are thought to have evolved as an adaptive response to environmental insult. Alterations in the activity of these enzymes are predicted to result in an altered susceptibility to cancer (Hirvonen, 1999).
  • Enzymatic activation of xenobiotics is not, however, the only route to cancer development. Epidemiological studies suggest that nutritional factors may also play a causative role in more than 30% of human cancers. However, defining the precise roles of specific dietary factors in the development of cancer is difficult due to the multitude of variables involved (Perera, 2000). Specific dietary factors are not easily measured as a single quantifiable variable, such as number of cigarettes smoked per day. Further complications arise due to differences in methodology, control populations, types of carcinogens, and amounts of exposure to carcinogens.
  • Priorities for studies relating to the interrelationship of dietary factors and cancer susceptibility include identification of genetic factors that contribute to individual cancer risk, identification of cancer-preventative chemicals in fruits and vegetables, better understanding of carcinogenic role of polycyclic aromatic hydrocarbons and heterocyclic amines generated by cooking meats at high temperature, and better understanding of the role of increased caloric intake with increased cancer risk (Perera, 2000).
  • Increased consumption of vegetables and fruits is correlated with a decreased risk of cancer, and studies of this aspect of nutritional effects on cancer has led to the identification of other enzymes and micronutrients involved in the maintenance of a normal cellular phenotype (Giovannuci, 1999).
  • One quarter of the US population with low intake of fruits and vegetables has roughly twice the cancer rate for most types of cancer (lung, larynx, oral cavity, oesophagus, stomach, colon and rectum, bladder, pancreas, cervix, and ovary) when compared with the quarter with the highest intake (Ames, 1999). Fruit and vegetables are high in folate and antioxidants. Low intake can lead to micronutrient deficiency, which has been shown to cause DNA damage in a way that mimics radiation damage by causing single and double-stranded breaks, oxidative lesions or both. The micronutrients correlated with DNA-damaging activity include folate (or folic acid), iron, zinc, and vitamins B12, B6, C and E (Ames, 1999).
  • Of the cancers that are correlated with nutritional effects, colon cancer (colorectal neoplasia) has among the strongest links to diet. In the US, colon cancer is the fourth most common incident cancer and second most common cause of cancer death in the US, with 130,000 new cases and 55,000 deaths per year (Potter, 1999). According to the WHO, colorectal cancers are the second most common cause of cancer death in Britain (WHO, 1997). Worldwide colon cancer represents 8.5% of new cancer cases reported, with the highest rates seen in the developed world and the lowest rates in India. Colon cancer occurs with approximately equal frequency in men and women, and the occurrence appears to be highly sensitive to changes in the environment. Immigrant populations assume the incidence rates of the host country very rapidly, often within the generation of the initial immigrant (Potter, 1999).
  • Risk factors for colon cancer include a positive family history, meat consumption, smoking and alcohol consumption (Giovannuci, 1999). There is an inverse relationship, i.e. lower risk, associated with consumption of vegetables, high folate intakes, use of non-steroidal anti-inflammatory drugs, hormone replacement therapy and physical activity. Meat and tobacco smoke are sources of carcinogens, while vegetables are a source of folate, antioxidants, and have Phase II (detoxifying) enzyme-inducing ability (Taningher, 1999).
  • Diets rich in raw vegetables, green vegetables, and cruciferous vegetables have a decreased risk of colon cancer Diets high in fibre, from vegetables and cereals, have been associated with a greater than two-fold decrease in risk of colorectal adenomas in men. The data on fruit in the diet is not as consistent to date (WCRF, 1997), but a recent report (Eberhart, 2000) measured potent anti-oxidant activity of phytochemicals in apple skins with the ability to inhibit growth of tumour cell lines in vitro, so it is possible that more clearly defined links will emerge in the future. Lower risk of colon cancer is associated with high folate intakes, but actual consumption of vegetables, rather than specific micronutrient preparations or vitamin supplements, has the most consistent low risk (Potter, 1999).
  • Other cancers that have been correlated with nutrition include prostate and breast. These malignancies are largely influenced by a combination of factors related to diet and nutrition. Prostate cancer is associated with high consumption of milk, dairy products and meats. These products decrease levels of 1,25(OH)2 vitamin D, which is a cell differentiator. Low levels of 1,25(OH)2 vitamin D may enhance prostate carcinogenesis by preventing cells from undergoing terminal differentiation and continuing to proliferate (Giovannucci, 1999). Breast, colon, and prostate cancers are relatively rare in less economically developed countries, where malignancies of the upper gastrointestinal tract are quite common. The cancers of the upper gastrointestinal tract have been related to various food practices or preservation methods other than refrigeration. For example, cancer of the mouth and pharynx is the sixth most common cancer world-wide and has been linked to alcohol consumption, tobacco, salt-preserved meat and fish, smoked foods and charcoal-grilled meat, as well as ingestion of beverages drunk very hot. Thus, diet can be a direct supply of genotoxic compounds or may cause chronic irritation or inflammation (Giovannucci, 1999).
  • In recent years, many genes involved in the processes described above and other areas of metabolism have been found to exist in allelic form. Therefore, certain populations, subpopulations, races etc have greater or lesser susceptibility to particular diseases linked with variation in alleles of some genes. For many decades, health advice, for example relating to diet, exercise, smoking, sunbathing has been issued by Governments, charities and health advisory bodies, such advice has been directed only at the population as a whole, or, at best, to groups such as the elderly, children and pregnant women. Such advice can therefore only be very general and cannot, by its very nature, take account of the particular genotype of an individual. Moreover, in recent years, there has been much media publicity of research findings on links between particular foods, drugs etc and medical conditions, often causing health scares. As the factors that contribute to disease susceptibility, for example cancer, or cardiovascular disease susceptibility vary between populations and between individuals of populations, it is often impossible for an individuals to derive useful advice appropriate to his or her particular circumstances from such reports.
  • SUMMARY OF THE INVENTION
  • In order to enable individuals to protect and manage their own health, there is a need for individuals to have personally-tailored information about risk factors which may be important to that individual's well-being and personally-tailored advice on reducing the risk of disease.
  • Accordingly, the invention provides a computer assisted method of providing a personalized lifestyle advice plan for a human subject comprising:
  • (i) providing a first dataset on a data processing means, said first dataset comprising information correlating the presence of individual alleles at genetic loci with a lifestyle risk factor, wherein at least one allele of each genetic locus is known to be associated with increased or decreased disease susceptibility;
  • (ii) providing a second dataset on a data processing means, said second dataset comprising information matching each said risk factor with at least one lifestyle recommendation;
  • (iii) inputting a third dataset identifying alleles at one or more of the genetic loci of said first dataset of said human subject;
  • (iv) determining the risk factors associated with said alleles of said human subject using said first dataset;
  • (v) determining at least one appropriate lifestyle recommendation based on each identified risk factor from step (iv) using said second dataset; and
  • (vi) generating a personalized lifestyle advice plan based on said lifestyle recommendations.
  • By lifestyle risk factors, it is meant risk factors associated with dietary factors, exposure to environmental factors, such as smoking, environmental chemicals or sunlight. Similarly lifestyle recommendations should be interpreted as relating to recommendations relating to dietary factors and exposure to environmental factors, such as smoking, environmental chemicals or sunlight. Disease susceptibility should be interpreted to include susceptibility to conditions such as allergies.
  • Thus, the method allows individualised advice to be generated based on the unique genetic profile of an individual and the susceptibility to disease associated with the profile. By individually assessing the genetic make-up of the client, specific risk factors can be identified and dietary and other health advice tailored to the individual's needs. In a preferred embodiment, the lifestyle advice will include recommended minimum or maximum amounts of foodtypes. (Note that an amount may be 0).
  • Information concerning the sex and health of the individual and/or of the individual's family may also provide indications that a particular polymorphism or group of polymorphisms associated with a particular condition should be investigated. Such information may therefore be used in selection of polymorphisms to be screened for in the method of the invention.
  • Such factors may also be used in the determination of appropriate lifestyle recommendations in step (v) of the method. For example, recommendations relating to reducing susceptibility to prostate cancer would not be given to women and recommendations relating to susceptibility to ovarian cancer would not be given to men. Other factors, such as information regarding the age, alcohol consumption, and existing diet of the client may be incorporated into the determination of appropriate lifestyle recommendations in step (v).
  • The report comprising the personalised dietary advice may be delivered to the client by any suitable means, for example by letter, facsimile or electronic means, such as e-mail. Alternatively, the report may be posted on a secure Web-page of the service provider with access limited to the client by the use of a unique identifier notified to the client either by conventional or electronic mail. The report can therefore comprise one or more hyperlinks to other documents of the report provider's Web-site or to other Web-sites giving relevant information on the particular polymorphisms identified, disease prevention and/or dietary advice.
  • As such sites would be able to be updated and new hyperlinks added to the report after the report is initially delivered to the client, the information and advice would be able to be updated at any time, thereby allowing the client to access up-to-date yet personalised health and dietary advice over a prolonged period, without the need for requesting another report.
  • Preferably, the method will involve assessing a variety of loci in order to give a broad view of susceptibility and possible means of minimising disease risk. Although individual polymorphisms may be considered biomarkers for individual cancer risk, the different biomarkers, when considered together, may also reveal a significant cancer risk. For example, the correlation between CYP1A1 activity and cancer susceptibility varies, dependent on the presence of specific types of CYP1A1 polymorphism as well as the presence of GSTM1 polymorphisms. An individual with an extremely active CYP1A1 gene, leading to high Phase I P450 activity in combination with a null GSTM1 genotype that lacks the detoxifying Phase II activities has a very high risk of developing cancer (Taningher, 1999).
  • The presence of a particular polymorphism may be indicative of increased susceptibilty to one disease while being indicative of decreased susceptibility to another disease. For example, one allele of the gene encoding epoxide hydrolase, which catalyses the conversion of toxic PAH metabolites formed by CYP1A1 and CYP1A2 into less toxic and more water-soluble trans-dihydrodiols, has recently been found to be associated with increased risk of aflatoxin-induced liver cancer, but also with decreased risk of ovarian cancer (Pluth, 200; Taningher, 1999).
  • Therefore, it will be important to assess the risk factors associated with other polymorphisms to give meaningful advice on maintaining optimal health.
  • Preferred genes for which polymorphisms are identified include genes that encode Phase I metabolism enzymes responsible for detoxification of xenobiotics, genes that encode Phase II metabolism enzymes responsible for further detoxification and excretion of xenobiotics, genes that encode enzymes that combat oxidative stress, genes associated with micronutrient deficiency (for example, deficiency of folate, B12 or B6), genes that encode enzymes responsible for metabolism of alcohol, genes that encode enzymes involved in lipid and/or cholesterol metabolism, genes that encode enzymes involved in clotting, genes that encode trypsin inhibitors, genes that encode enzymes related to susceptibility to metal toxicity, genes which encode proteins required for normal cellular metabolism and growth and genes which encoded HLA Class 2 molecules.
  • The method of the invention may include the step of determining the presence of individual alleles at one or more genetic loci of the DNA in a DNA sample of the subject, and constructing the dataset used in step (iii) using results of that determination.
  • Techniques for determining the presence or absence of individual alleles are known to the skilled person. They may include techniques such as hybridization with allele-specific oligonucleotides (ASO)(Wallace, 1981; Ikuta, 1987; Nickerson, 1990, Varlaan-de Vries, 1986, Saiki, 1989 and Zhang, 1991) allele specific PCR (Newton 1989, Gibbs, 1989), solid-phase minisequencing (Syvanen, 1993), oligonucleotide ligation assay (OLA) (Wu, 1989, Barany, 1991; Abravaya, 1995), 5′ fluorogenic nuclease assay (Holland, 1991 & 1992, Lee, 1998) U.S. Pat. Nos. 4,683,202, 4,683,195, 5,723,591 and 5,801,155, or Restriction fragment length polymorphism (RFLP) (Donis-Keller, 1987).
  • In a preferred embodiment, the genetic loci are assessed via a specialised type of PCR used to detect polymorphisms, commonly referred to as the Taqman® assay, in which hybridisation of a probe comprising a fluorescent reporter molecule, a fluorescent quencher molecule and a minor groove binding chemical to a region of interest is detected by removal of quenching of the fluorescent molecule and detection of resultant fluorescence. Details are given below.
  • In another embodiment, the genetic loci are assessed via hybridisation with allele-specific oligonucleotides, the allele specific oligonucleotides being preferably arranged as an array of oligonucleotide spots stably associated with the surface of a solid support.
  • The arrays suitable for use in the method of the invention form a further aspect of the present invention.
  • In order to assay the sample for the alleles to be identified the fragments of DNA comprising the gene(s) of interest may be amplified to produce a sufficient amount of material to be tested.
  • The present inventors have designed a number of specific primer sets for amplification of gene regions of interest. Such primers may be used in pairs to isolate a particular region of interest in isolation. Therefore in a further aspect of the invention, there is provided a primer having a sequence selected from SEQ ID NO: 86-99, 104-163. In another aspect, there is provided a primer pair comprising primers having SEQ ID NO:n, where n is an even number from 86-98 or 104-162 in conjunction with a primer having SEQ ID NO:(n+1).
  • Preferably, however, the primer sets will be used together with other primer sets to provide multiplexed amplification of a number of regions to allow determination of a number of polymorphisms from the same sample. Therefore in a further aspect of the invention, there is provided a primer set comprising at least 5, more preferably 10, 15 primer pairs selected from SEQ ID NO: 86-121.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows examples of databases 1 and 2 which may be used in an embodiment of the present invention.
  • FIG. 2 is a flow chart illustrating an embodiment of the invention.
  • DETAILED DESCRIPTION OF THE INVENTION
  • Selection of Genetic Polymorphisms for Datasets
  • The correct selection of genetic polymorphisms is important to the provision of accurate and meaningful advice Although not limited to such classes of polymorphisms, in a preferred embodiment of the present invention, markers for polymorphisms of one or more of the following classes of genes are used:
  • The first dataset of the method of the invention may comprise information relating to two or more alleles of one or more genetic loci of genes selected from the group comprising:
  • (a) genes that encode enzymes responsible for detoxification of xenobiotics in Phase I metabolism;
  • (b) genes that encode enzymes responsible for conjugation reactions in Phase II metabolism;
  • (c) genes that encode enzymes that help cells to combat oxidative stress;
  • (d) genes associated with micronutrient deficiency;
  • (e) genes that encode enzymes responsible for metabolism of alcohol.
  • (f) genes that encode enzymes involved in lipid and/or cholesterol metabolism;
  • (g) genes that encode enzymes involved in clotting;
  • (h) genes that encode trypsin inhibitors;
  • (i) genes that encode enzymes related to susceptibility to metal toxicity;
  • (j) genes which encode proteins required for normal cellular metabolism and growth;
  • (k) genes which encoded HLA Class 2 molecules.
  • The dataset will preferably comprise information relating to two or more alleles of at least two genetic loci of genes selected from the group comprising categories a-k as described above, for example, a+b, a+c, a−d, a+e, a+f, a+g, a+h, a+i, a+j, a+k, b+c, b+d, b+e etc., c−d, c+e etc, d+e, d+f etc, e+f, e+g etc, f+g, f+h etc., g+h, g+i, g+k, h+i, h+k. Where the dataset comprises information relating to two or more alleles of at least two genetic loci, it is preferred that at least one of the genetic loci is of category d, due to the central role of micronutrients in the maintenance of proper cellular growth and DNA repair, and due to the association of micronutrient metabolism or utilisation disorders with several different types of diseases (Ames 1999; Perera, 2000; Potter, 2000). More preferably, the dataset will preferably comprise information relating to two or more alleles of at least three genetic loci selected from the group comprising categories a-k as described above. Where the dataset comprises information relating to alleles of at least three genetic loci, it is preferred that at least two of the genetic loci are of categories d and e. Information relating to polymorphisms present in both of these categories is particularly useful due to the effects of alcohol consumption and metabolism on the efficiency of enzymes related to micronutrient metabolism and utilisation (Ulrich, 1999). In a further preferred embodiment, where the dataset comprises information relating to alleles of at least three genetic loci, it is preferred that at least two of the genetic loci are of categories a and b due to the close interaction of Phase I and Phase II enzymes in the metabolism of xenobiotics. Even more preferably, the dataset will comprise information relating to two or more alleles of at least four genetic loci of genes selected from the group comprising categories a-k as defined above, for example, a+b+c+d, a+b+c+e, a+b+d+e, a+c+d+e, b+c+d+e etc. Where the dataset comprises information relating to alleles of at least four genetic loci, it is preferred that at least three of the genetic loci are of categories d and e and f Information relating to polymorphisms present in these three categories is particularly useful due to the strong correlation of polymorphisms of these alleles with coronary artery disease due to the combined effects of altered micronutrient utilisation, affected adversely by alcohol metabolism, together with imbalances in fat and cholesterol metabolism. Further, where the dataset comprises information relating to alleles of at least five genetic loci, it is preferred that at least four of the genetic loci are of categories a, b, d and e. Information relating to polymorphisms present in these four categories is particularly useful due to the combined effects of micronutrients utilisation, alcohol metabolism, Phase 1 metabolism of xenobiotics and Phase II metabolism on the further metabolism and excretion of potentially harmful metabolites produced in the body (Taningher, 1999; Ulrich, 1999). Similarly, the dataset may comprise information relating to two or more alleles of at least five, for example a, b, d, e and f, six, seven, eight, nine or ten genetic loci of genes selected from the group comprising categories a-k as defined above.
  • Preferably, the dataset will comprise information relating to two or more alleles of one or more genetic loci of genes selected from each member of the group comprising categories a-k as described above. In a preferred embodiment, the first dataset comprises information relating to two or more alleles of the genetic loci of genes encoding each of the cytochrome P450 monooxygenase, N-acetyltransferase 1, N-acetyltransferase 2, glutathione-S-transferase, manganese superoxide dismutase, 5,10-methylenetetrahydrofolatereductase and alcohol dehydrogenase 2 enzymes. In a more preferred embodiment the first dataset further comprises information relating to two or more alleles of the genetic loci of genes encoding one or more, preferably each of epoxide hydrolase (EH), NADPH-quinone reductase (NQ01), paraxonaoase (PON1), myeloperoxidase (MPO), alcohol dehydrogenase 1, alcohol dehydrogenase 3, cholesteryl ester transfer protein, apolipoprotein A IV, apolipoprotein E, apolipoprotein C III, angiotensin, factor VII, prothrombin 20210, β-fibrinogen, heme-oxygenase-1, α-antitrypsin, SPINK1, Δ-aminolevulinacid dehydratase, interleukin 1, interleukin 1, vitamin D receptor, B1 kinin receptor, cystathionine-beta-synthase, methionine synthase (B12 MS), 5-HT transporter, transforming growth factor beta 1 (TGFβ1), L-myc, HLA Class 2 molecules, T-lymphocyte associated antigen 4 (CTLA-4), interleukin 4, interleukin 3, interleukin 6, IgA, and/or galactose metabolism gene GALT.
  • Genes that Encode Enzymes Responsible for (a) Detoxification of Xenobiotics in Phase I Metabolism; and (b) Conjugation Reactions in Phase II Metabolism
  • Xenobiotics are potentially toxic compounds found in, for example, char-grilled red meat. Meat consumption is associated with increased risk of cancer, especially well-done meat cooked at high temperatures (Sinha, 1999). Cooking meat in this fashion leads to the production of heterocyclic amines (HCA), nitrosamines (NA), and polycyclic aromatic hydrocarbons (PAH), which have known carcinogenic activity in animals (Hirvonen, 1999; Layton, 1995).
  • Detoxification of xenobiotics occurs in 2 phases in humans: Phase I metabolism involves the addition of an oxygen atom or a nitrogen atom to lipophilic (fat soluble) compounds, such as steroids, fatty acids, xenobiotics (from external sources like diet, smoke, etc.) so that they can be conjugated by the Phase II enzymes (thus made water-soluble) and excreted from the body (Hirvonen, 1999). Individuals with genetic polymorphisms correlated with cancer risk in these genes should avoid consumption of char-grilled foods, smoked fish, well-done red meat whether grilled or pan-fried (Sinha, 1999). They should also increase consumption of food products known to increase Phase II metabolism so the products of Phase I metabolism may be cleared more efficiently.
  • Specific examples of genes of category a for which information relating to polymorphisms may be used in the present invention include genes encoding cytochrome P450 monooxygenase (CYP) e.g. CYP1A1, CYA1A2, CYP2C, CYP2D6, CYP2E1, CYP3A4, CYP11B2, genes encoding N-acetyltransferase 1 e.g. NAT1, genes encoding N-acetyltransferase 2 e.g. NAT2, genes encoding epoxide hydrolase (EH), genes encoding NADPH-quinone reductase (NQ01, genes encoding paraxonaoase (PON1), genes encoding myeloperoxidase (MPO).
  • CYP is also referred to as cytochromome P450 monooxygenase (gene is called CYP, enzyme is called P450). P450 enzymes belong to a super-family with wide substrate activity that catalyses the insertion of an oxygen atom into a substrate. The reaction can convert a molecule (procarcinogen) into a DNA-reactive electrophilic carcinogen (Hirvonen, 1999; Smith, 1995). Polymorphisms in genes encoding cytochrome P450 (CYP family of genes) are associated with altered susceptibility to cancer, CAD and altered metabolisim of various pharmaceutical agents (Poolsup, 2000; Miki, 1999; Cramer, 2000; Marchand, 1999; Sinha, 1997).
  • CYP1A1 codes for a P450 enzyme that metabolises polycyclic aromatic hydrocarbons (PAH). The CYP1A1 gene is polymorphic and is inducible by PAH, which means that expression of the enzyme is increased upon exposure to PAH (MacLeod, 1997). CYP1A1 is located on chromosome 15q22-q24 (Smith, 1995). This gene has been linked to colorectal, urinary bladder, breast, oral cavity, stomach, and lung cancers (Perera, 2000; Garte, 1998). The gene product, the P450 enzyme, is inducible by exposure to the agents that it metabolises, so the consumption of high levels of a potential source of carcinogens, such as well-done red meat, would increase the production of the enzyme and thus the creation of carcinogenic substances (Mooney, 1996; Perera, 2000; Alexandrie, A. K., 2000). Studies of polymorphisms of the CYP1A1 gene have revealed considerable differences in enzyme activity, with corresponding differences in cancer risk after exposure to known substrates of the enzyme (Alexandrie, 2000; Rojas, 2000; Garte, 2000). Both the Ile-Val polymorphism I, which comprises an A4889G substitution (i.e. the adenine residue at position 4889 of the 5′-3′ strand is substituted by a guanine residue) and the CYP1A1*C polymorphism, which comprises an T6235C substitution, are induced to a greater extent than the wild type gene after exposure to PAH, and have been associated with a significant increase in cancer risk (Taningher, 1999; Garte, 1998; Kawajiri, 1996; MacLeod, S., 1997; Smith, 1995). Approximately 10 percent of the Caucasian population carries polymorphisms linked to cancer risk, according to a recent American review paper (Shields, 2000). Polymorphisms in genes encoding CYP1A2, CYP2C, CYP2D6, CYP2E1, CYP3A4, CYP11B2 are associated with altered susceptibility to cancer and drug sensitivity. (Poolsup, 2000; Miki, 1999; Cramer,2000; Marchand, 1999; Sinha, 1997).
  • NAT1 (N-acetyltransferase 1) and NAT2 (N-acetyltransferase 2) also activate PAH and heterocyclic amines (HAA). The enzymes catalyse N-acetylation, O-acetylation, and N,O-acetylathon. The O-acetylation reaction is considered the most risky, with the potential for forming chemical carcinogens that can bind to DNA. The N-acetylation reaction can occur on a compound after a P450 has inserted an oxygen, thus increasing the water solubility of the compound so it may be excreted. Due to this activity, the NAT genes are often considered as both Phase I and Phase II type enzymes. The literature describing a cancer link focuses on the activation activity of the enzymes, so they will be listed in the Phase I section only. There are 3 separate N-acetyltransferase genes in humans, two are active genes: NAT1 and NAT2, and a pseudogene, NATP. Pseudogenes have the same sequence, but lack apparent function and promoter elements and are not expressed in cells (i.e. the gene is not transcribed into RNA then translated into amino acids to make a protein/enzyme) (Perera, 2000). NAT1 and NAT2 genes are located on chromosome 8 at 8p21.3-21.1, both genes are 870 bp long and both code for a protein 290 amino acids in length. The genes are highly polymorphic and epidemiological studies have sometimes given conflicting information regarding links with cancer. The genes show geographical and ethnic variation and the enzyme activity varies considerably within different tissues or organs. There are approximately 20 polymorphisms for NAT1 known to date, out the list below only includes the polymorphisms that have shown a link to cancer (Hein, 2000a). The current list of nomenclature and polymorphisms is kept at a web site: http://www.louisville.edu/medschool/pharmacology/NAT.html.
  • Many of the epidemiological studies of both NAT1 and NAT2 used phenotyping assays, which measured enzyme activity, and found fast and slow acetylator types, with the fast phenotype carrying an increased risk for cancer in the colon (Perera, 2000). However, later analysis of the results found that the fast/slow phenotype could vary considerably depending on the substrate chosen for acetylation (Hein, 2000a). Recent studies have used genetic sequence data to more precisely match acetylator activity and cancer risk with polymorphism (Hein, 2000b). Although the genes are the same size, they do act on different substrates. For example, caffeine is a substrate for NAT2 but not for NAT1.
  • NAT1 is expressed to a higher degree than NAT2 in the colon, so NAT1 may be associated with localised activity of activated HAA or PAH in the colon (Brockton, 2000; Perera, 2000). The polymorphism NAT1*10, which comprises T1088A and C1095A substitutions, and which has a fast phenotype, has been consistently linked with an increased risk of colon cancer and higher DNA adduct levels (i.e. DNA damage that can lead to cancer) in colon tissue (Perera, 2000; Ilett, 1987). The NAT1*11 polymorphism has been linked to risk of breast cancer in women who smoke or consume well-done red meat (Zheng, 1999). However, the phenotype is not well understood, so this marker cannot be categorized as a fast or slow acetylator (Doll, 1997). Two alleles of the NAT1*11 polymorphism are known: the NAT1*11A polymorphisms which comprises C(−344)T, A(−40)T, G445A, G459A, T640G, C1095A substitutions and a Δ9:1065-1090 deletion; and the NAT1*113 polymorphism, which comprises C(−344)T, A(−40)T, G445A, G459A, T640G substitutions and a A9:1065-1090 deletion. References to NAT1*11 polymorphisms should be understood to include reference to NAT1*11A or NAT1*11B polymorphisms. NAT1*14 on the other hand has little or no enzyme activity (Brockton, 2000) and has been associated with increased lung cancer risk (Bouchardy, C., 1998). Two alleles of the NAT1*14 polymorphism are known: the NAT1*14A polymorphism, which comprises G560A, T1088A and C1095A substitutions; and the NAT1*14B polymorphism, which comprises a G560A substitution. References to NAT1*14 polymorphisms should, except where the context dictates otherwise, be understood to include reference to NAT1*14A or NAT1*14B polymorphisms. The NAT1*14 polymorphism shares a restriction enzyme site with the NAT1*11polymorphism, and some of the conflicting results reported in the literature are believed to be due to the inability of the assay used (restriction fragment length polymorphism assay (RFLP)) to distinguish the polymorphisms (Hein, 2000a). The oligonucleotide array suitable for use in the present invention can distinguish all polymorphisms and therefore will be more precise than the RFLP procedure.
  • NAT2 is expressed primarily in the liver, but has been linked with cancer incidence in other organs (Hein, 2000b). NAT2*5A, which comprises T481C and T341C substitutions, NAT2*6A, which comprises C282T and G590A substitutions, NAT2*7A, which comprises a G857A substitution, have reduced acetylation activity (Hein, 2000b) and have been linked to risk of bladder cancer (Taningher, 1999; Lee, 1998). NAT2*4, is considered the normal, or wild type, sequence. NAT2*4 has fast acetylator activity and has been linked to increased cancer risk in several studies (reviewed in Hein, 2000b; Gil, 1998), but especially in conjunction with the NAT1*10 polymorphism (Bell, 1995). NAT2 rapid/intermediate acetylators with at least one NAT2*4 allele have been linked to breast cancer in women who consumed well-done red meat (Dietz, 1999). Approximately 55% of the Caucasian population carry NAT1 polymorphisms linked to cancer. (Shields, 2000).
  • Polymorphisms in genes encoding epoxide hydrolase are associated with cancer and chronic obstructive pulmonary disease (Pluth, 200; Miki,1999). Polymorphisms in genes encoding NADPH-quinone reductase are associated with altered susceptibility to cancer (Nakajima, 2000). Polymorphisms in genes encoding paraxonoase are associated with altered susceptibility to cancer and to CAD (MacKness, 2000). Polymorphisms in genes encoding mycloperoxidase are associated with altered susceptibility to CAD (Schabath, 2000).
  • Specific examples of genes of category b for which information relating to polymorphisms may be used in the present invention include genes encoding glutathione-S-transferase e.g GSTM1, GSTP1, GSTT1.
  • Glutathione-S-transferases catalyse the reaction of electrophilic compounds with glutathione so the compounds may be excreted from the body. The enzymes belong to a super-family with broad and overlapping substrate specificities. Glutatione-S-transferases provide a major pathway of protection against chemical toxins and carcinogens and are thought to have evolved as an adaptive response to environmental insult, thus accounting for their wide substrate specificity (Hirvonen, 1999). There are 4 family members: alpha, mu, theta, and pi, also designated as A, M, T and P. Polymorphisms have been identified in each family (Perera, 2000). Individuals with low glutathione-S-transferase activity should avoid meats cooked at higher temperatures as above, and increase fruit and vegetable consumption. Cruciferous vegetables such as broccoli and members of the allium family such as garlic and onion have been shown to be potent inducers of these enzymes, which would be expected to increase clearance of toxic substances from the body (Cotton, 2000; Giovannucci, 1999).
  • GSTmu, has 3 alleles: null, a, which is considered to be the wild type, and b, which comprises a C534G substitution, with no functional difference between the a and b alleles. The GSTmu sub-type has the highest activity of the 4 types and is predominately located in the liver (Hirvonen, 1999). Approximately half of the population has a 10 complete deletion of this gene with a corresponding risk of lung, bladder, breast, liver, and oral cavity cancer (Shields, 2000; Perera, 2000). It has been estimated that 17% of all lung and bladder cancers may be attributable to GSTM1 null genotypes (Hirvonen, 1999). GSTM1 null genotype together with a highly active CYP1A1 polymorphism has been linked to a very high cancer risk in several studies (Rojas, 2000; Shields, 2000). The GSTM1 gene is located on chromosome 1p13.3 (Cotton, 2000).
  • GSTpi gene is located on chromosome 11q13. This sub-type is known to metabolise many carcinogenic compounds and is the most abundant sub-type in the lungs (Hirvonen, 1999). Two single nucleotide polymorphisms have been linked to cancer to date GSTP1*B, which comprises an A313G substitution, and GSTP1*C, which comprises a C341T substitution. The enzymes of these polymorphic genes have decreased activity compared to the wild type and a corresponding increased risk of bladder, testicular, larynx and lung cancer (Harries, 1997; Matthias, 1998; Ryberg, 1997).
  • GSTtheta gene is on chromosome 22q11.2 and is deleted in approximately 20% of the Caucasian population. The enzyme is found in a variety of tissues, including red blood cells, liver, and lung (Potter, 1999). The deletion is associated with an increased risk of lung, larynx and bladder cancers (Hirvonen, 1999). Links with GSTM1 null genotypes are currently being searched, as it is believed that individuals that have both GSTM1 and GSTT1 alleles deleted will have a greatly increased risk of developing cancer (Potter, 1999).
  • Genes that Code for Enzymes that Help Cells to Combat Oxidative Stress
  • Specific examples of genes of category c for which information relating to polymorphisms may be used in the present invention include genes encoding manganese superoxide dismutase (MnSOD or SOD2 gene).
  • Manganese superoxide dismutase is an enzyme that destroys free radicals or a free-radical scavenger. The gene is located on chromosome 6q25.3, but the enzyme is found within the mitochondria of cells. There are 2 polymorphisms linked to cancer to date, an Ile 58Thr allele, which comprises an T175C substitution, and a Val(−9)Ala allele, which comprises a T(−28)C substitution. A study of premenopausal women found a four-fold increased risk of breast cancer in individuals with the Val(−9)Ala polymorphism and the highest risk within this group is found in women who consumed low amounts of fruits and vegetables (Ambrosone, 1999). This polymorphism occurs in the signal sequence of the amino acid chain. The signal sequence ensures transport of the enzyme into the mitochondria of the cell, and so the polymorphism is believed to reduce the amount of enzyme delivered to the mitochondria (Ambrosone, 1999). The mitochondria is commonly referred to as the workhorse of the cell, where the energy-yielding reactions take place. This is the site of many oxidative reactions, so many free radicals are generated here. Individuals with low activity of this enzyme should be advised to take antioxidant supplements and increase consumption of fruits and vegetables (Giovannucci, 1999; Perera, 2000).
  • Genes Associated with Micronutrient Deficiency e.g. of Folate, Vitamin B12 or Vitamin B6
  • Specific examples of genes of category d for which information relating to polymorphisms may be used in the present invention include the gene encoding 5,10-methylenetetrahydrofolatereductase (MTHFR) activity.
  • 5,10-methylenetetrahydrofolate reductase is active in the Iolate-dependent methylation of DNA precursors. Low activity of this enzyme leads to an increase of uracil incorporation into DNA (instead of thymine) (Ames, 1999). The MTHFR gene is polymorphic and has been linked to colon cancel, adult acute lymphocytic leukaemia and infant leukaemia (Ames, 1999; Perera, 2000; Potter, 2000). Both the wt and polymorphic alleles have been linked to disease, each being dependent on levels of folate in the diet. Approximately 35% of the Caucasian population has genetic polymorphisms at this locus with corresponding risk of colon cancer (Shields, 2000). Polymorphisms at this locus include those with a C677T or A1298C substitution. Dietary recommendations for individuals lacking in MTHFR activity include taking supplements with folate and increasing consumption of fruit and vegetables (Ames, 1999). Low levels of vitamins B12 and B6 have been associated with low MTHFP activity and increased cancer risk, so individuals should increase intake of these vitamins; B12 is found primarily in meat and B6 is found in whole grains, cereals, bananas, and liver (Ames, 1999). Alcohol has a deleterious effect on folate metabolism, affecting individuals with the A1298C polymorphism most severely (Ulrich, 1999). These individuals should be advised to avoid alcohol.
  • Genes that code for Enzymes Responsible for Metabolism of Alcohol
  • Specific examples of genes of category e for which information relating to polymorphisms may be used in the present invention include genes encoding alcohol dehydrogenase e.g. the ALDH2 gene, ALDH1 gene and ALDH3 gene.
  • Alcohol dehydrogenase 2 (ALDH2) is involved in the second step of ethanol utilisation. Reduced activity of this enzyme leads to accumulation of acetaldehyde, a potent DNA adduct former (Bosron, 1986). There has been one polymorphism identified to date, the ALDH2*2 polymorphism, which comprises a G1156A substitution, and which has links with oesophageal/throat cancer, stomach, lung, and colon cancer (IARC, 1998; Yokoyama, 1998). The advice to individuals with the polymorphism would be to avoid alcohol. Polymorphisms in ALDH1 and 3 are associated with increased susceptibility to cancers and Parkinson's disease.
  • Genes that Encode Enzymes Involved in Lipid and/or Cholesterol Metabolism
  • Specific examples of genes of category f for which information relating to polymorphisms may be used in the present invention include genes encoding cholesteryl ester transfer protein e.g. the CETP gene, polymorphisms of which genes are associated with altered susceptibility to coronary artery disease (CAD)((Raknew, 2000; Ordovas, 2000); genes encoding apolipoprotein A, IV (ApoA-IV), polymorphisms of which genes are associated with altered susceptibility to coronary artery disease (CAD) (Wallace,2000; Heilbronn, 2000); apolipoprotein E(ApoE), polymorphisms of which genes are associated with altered susceptibility to CAD and Alzheimer's disease (Corbo,1999; Bullido, 2000); or apolipoprotein C, III (ApoC-III), polymorphisms of which genes are associated with altered susceptibility to CAD, hypertension and insulin resistance (Salas, 1998).
  • Genes that Encode Enzymes Involved in Clotting Mechanisms
  • Specific examples of genes of category g for which information relating to polymorphisms may be used in the present invention include genes encoding angiotensin (AGT-1) and angiotensin converting enzyme (ACE), polymorphisms of which genes are associated with altered susceptibility to hypertension (Brand 2000;de Padua Mansur, 2000), factor VII, polymorphisms of which genes are associated with altered susceptibility to CAD (Donati, 2000; Di Castelnuovo, 2000); prothrombin 20210, polymorphisms of which genes are associated with altered susceptibility to venous thrombosis (Vicente, 1999); β-fibrinogen, polymorphisms of which genes are associated with altered susceptibility to CAD (Humphries, 1999); or heme-oxygenase-1, polymorphisms of which genes are associated with altered susceptibility to emphysema (Yamada, 2000).
  • Genes that Encode Trypsin Inhibitors
  • Specific examples of genes of category h for which information relating to polymorphisms may be used in the present invention include genes encoding α-antitrypsin, polymorphisms of which genes are associated with altered susceptibility to chronic obstructive pulmonary disease (COED) (Miki, 1999); or serine protease inhibitor, Kazal type 1(SPINK), polymorphisms of which genes are associated with altered susceptibility to pancreatitis (Pfutzer, 2000).
  • Genes that Encode Enzymes Related to Susceptibility to Metal Toxicity
  • Specific examples of genes of category i for which information relating to polymorphisms may be used in the present invention include genes encoding Δ-aminolevulinacid dehydratase, polymorphisms of which genes are associated with altered susceptibility to lead toxicity (Costa, 2000).
  • Genes which Encode Proteins Required for Normal Cellular Metabolism and Growth
  • Specific examples of genes of category j for which information relating to polymorphisms may be used in the present invention include genes encoding the vitamin D receptor, polymorphisms of which genes are associated with altered susceptibility to osteoporosis, tuberculosis, Graves disease, COPD, and early periodontal disease (Ban, 2000; Wilkinson, 2000; Gelder, 2000; Miki, 1999; Hennig, 1999); the B1 kinin receptor (B1R), polymorphisms of which genes are associated with altered susceptibility to kidney disease (Zychma, 1999); cystathionine-beta-synthase, polymorphisms of which genes are associated with altered susceptibility to CAD (Tsai, 1999); methionine synthase (B12 MS), polymorphisms of which genes are associated with altered susceptibility to CAD (Tsai, 1999); the 5-HT transporter, polymorphisms of which genes are associated with altered susceptibility to neurological disorders, Alzheimer's disease, schizophrenia, other disorders of the serotonin pathway (Oliveira, 1999); tumour necrosis factor receptor 2 (TNFR2), polymorphisms of which genes are associated with altered susceptibility to CAD (Fernandez-Real, 2000); galactose metabolism gene GALT, polymorphisms of which genes are associated with altered susceptibility to ovarian cancer (Cramer, 2000); transforming growth factor beta 1 (TGFβ1), polymorphisms of which genes are associated with altered susceptibility to CAD and cancers (Yokota, 2000); and L-myc, polymorphisms of which genes are associated with altered susceptibility to CAD (especially in relation to tolerance to smoking) and cancers (Togo, 2000).
  • Genes which Encoded Proteins Associate with Immunological Susceptibility
  • Specific examples of genes of category k for which information relating to polymorphisms may be used in the present invention include genes encoding HLA Class 2 molecules, polymorphisms of which genes are associated with altered susceptibility to cervical cancer and human papilloma virus (HPV) infection (Maciag, 2000); T-lymphocyte associated antigen 4 (CTLA-4), polymorphisms of which genes are associated with altered susceptibility to liver disease (Argawal, 2000); interleukin 1 (IL-1), polymorphisms of which are associated with cardiovascular disease and periodontal disease (macaiag, 2000; Nakajima, 2000); IL-4, polymorphisms of which genes are associated with altered susceptibility to atopy and asthma (Rosa-Rosa, 1999); IL-3, polymorphisms of which genes are associated with altered susceptibility to atopy and asthma (Rosa-Rosa, 1999); IL-6, polymorphisms of which genes are associated with altered susceptibility to osteoporosis; and IgA, polymorphisms of which genes are associated with altered susceptibility to COPD (Miki, 1999).
  • Detection of Polymorphisms
  • As described above, the method of the invention may include the step of analysing a DNA sample of a human subject in order to construct the dataset to be used in the method of the invention.
  • Testing of Samples
  • Collection of Tissue Samples
  • DNA for analysis using the method or arrays of the invention can be isolated from any suitable client or patient cell sample. For convenience, it is preferred that the DNA is isolated from cheek (buccal) cells. This enables easy and painless collection of cells by the client, with the convenience of being able to post the sample to the provider of the genetic test without the problems associated with posting a liquid sample.
  • Cells may be isolated from the inside of the mouth using a disposable scraping device with a plastic or paper matrix “brush”, for example, the C.E.P. Swab™ (Life Technologies Ltd., UK). Cells are deposited onto the matrix upon gentle abrasion of the inner cheek, resulting in the collection of approximately 2000 cells (Aron, 1994). The paper brush can then be left to dry completely, ejected from the handle placed into a microcentrifuge tube and posted by the client or patient to the provider of the genetic test.
  • Isolation of DNA from Samples
  • DNA from the cell samples can be isolated using conventional procedures. For example DNA may be immobilised onto filters, column matrices, or magnetic beads. Numerous commercial kits, such as the Qiagen QIAamp kit (Quiagen, Crawley, UK) may be used. Briefly, the cell sample may be placed in a microcentrifuge tube and combined with Proteinase K, mixed, and allowed to incubate to lyse the cells. Ethanol is then added and the lysate is transferred to a QIAamp spin column from which DNA is eluted after several washings.
  • The amount of DNA isolated by the particular method used may be quantified to ensure that sufficient DNA is available for the assay and to determine the dilution required to achieve the desired concentration of DNA for PCR amplification. For example, the desired target DNA concentration may be in the range 10 ng and 50 nq. DNA concentrations outside this range may impact the FCR amplification of the individual alleles and thus impact the sensitivity and selectivity of the polymorphism determination step.
  • The quantity of DNA obtained from a sample may be determined using any suitable technique. Such techniques are well known to persons skilled in the art and include UV (Maniatis, 1982) or fluorescence based methods. As UV methods may suffer from the interfering absorbance caused by contaminating molecules such as nucleotides, RNA, EDTA and phenol and the dynamic range and sensitivity of this technique is not as great as that of fluorescent methods, fluorescence methods are preferred. Commercially available fluorescence based kits such as the PicoGreen dsDNA Quantification (Molecular Probes, Eugene, Oreg., USA).
  • Primers
  • Prior to the testing of a sample, the nucleic acids in the sample may be selectively amplified, for example using Polymerase Chain Reaction (PCR) amplification. as described in U.S. Pat. Nos. 4,683,202 AND 4,683,195.
  • Preferred primers for use in the present invention are from 18 to 23 nucleotides in length, without internal homology or primer-primer homology.
  • Furthermore, to ensure amplification of the region of interest and specificity, the two primers of a pair are preferably selected to hybridise to either side of the region of interest so that about 150 bases in length are amplified, although amplification of shorter and longer fragments may also be used. Ideally, the site of polymorphism should be at or near the centre of the region amplified.
  • Table 1 provides preferred examples of primer pairs which may be used in the invention, particularly when the Taqman® assay is used in the method of the invention. The primers are shown together with the gene targets and preferred examples of the wt probes and polymorphism probes used in the Taqman® assay for each gene target.
  • Table 2 provides preferred examples of the primer pairs which may be used in the invention together with the gene targets and the size of the fragment isolated using the primers, which they amplify.
  • The primers and primer pairs form a further aspect of the invention. Therefore the invention provides a primer having a sequence selected from SEQ ID NO: 86-99, 104-163. In another aspect, there is provided a primer pair comprising primers having SEQ ID NO:n, where n is an even number from 86-98 or 104-162 in conjunction with a primer having SEQ ID NO:(n+1).
  • In a preferred embodiment of the invention, multiplexed amplification of a number of sequences are envisioned in order to allow determination of the presence of a plurality of polymorphisms using, for example the DNA array method. Therefore, primer pairs to be used in the same reaction are preferably selected by position, similarity of melting temperature, internal stability, absence of internal homology or homology to each other to prevent self-hybridisation or hybridisation with other primers and lack of propensity of each primer to form a stable hairpin loop structure. Thus, the sets of primer pairs to be coamplified together preferably have approximately the same thermal profile, so that they can be effectively coamplified together. This may be achieved by having groups of primer pairs with approximately the same length and the same G/C content.
  • Therefore in a further aspect of the invention, there is provided a primer set comprising at least 5, more preferably 10, 15 primer pairs selected from SEQ ID NO: 86-121.
    TABLE 1
    Forward Reverse Polymorphism
    Gene primer primer WT probe probe
    1. CYP1A1
    A4889G CATGGGCAAGCGGAAG CAGGATAGCCAGGAA CGGTGAGACCaTTG CGGTGAGACCgTTG
    TG GAGAAAGAC (SEQ ID NO:164) (SEQ ID NO:165)
    (SEQ ID NO:122) (SEQ ID NO:123)
    T6235C AGACACGGTCCCCAGG CAGAGGCTGAGGTGG CTCCACCTCCtGGG CTCCACCTCCcGGG
    TCAT GAGAA (SEQ ID NO:166) (SEQ ID NO:167)
    (SEQ ID NO:124) (SEQ ID NO:125)
    2. NAT1
    G445A CGAGTTAATTTCTGGG TGGTCTAGATACCAG GCCTTGTgTCTTC TGCCTTGTaTCTTC
    AAGGATCAG AATCCATTCTCTT (SEQ ID NO:168) (SEQ ID NO:169)
    (SEQ ID NO:126) (SEQ ID NO:127)
    G459A GGCAGCCTCTGGAGTT TTCCCTTCTCATTTG CGTTTGACgGAAGAG CGTTTGACaGAAGAG
    AATTTCT GTCTAGATACC (SEQ ID NO:170) (SEQ ID NO:171)
    (SEQ ID NO:128) (SEQ ID NO:129)
    G560A GGGAACAGTACATTCC TGTTCGAGGCTTAAG AATACCgAAAAATC CAAATACCaAAAAAT
    AAATGAAGA AGTAAAGGAGT (SEQ ID NO:172) (SEQ ID NO:173)
    (SEQ ID NO:130) (SEQ ID NO:131)
    T640G AACAATTGAAGATTTT TCTGCAAGGAACAAA CATCTCCAtCATCTG ACATCTCCAgCATCT
    GAGTCTATGAATACA ATGATTTACTAGT (SEQ ID NO:174) (SEQ ID NO:175)
    (SEQ ID NO:132) (SEQ ID NO:133)
    T1088A GAAACATAACCACAAA AAATCACCAATTTCC CCATCTTTAAAATACA CATCTTTAAAATACA
    CCTTTTCAAA AAGATAACCA TTTaTTA TTTtTTA
    (SEQ ID NO:134) (SEQ ID NO:135) (SEQ ID NO:203) (SEQ ID NO:204)
    C1095A AAACAAACCACAAAC AAATCACCAATTTCC GCCATCTTTAAAAgAC GCCATCTTTAAAAtA
    CTTTTCAAATAAT AAGATAACCA AT CATT
    (SEQ ID NO:136) (SEQ ID NO:137) (SEQ ID NO:176) (SEQ ID NO:177)
    3. NAT2
    C >T AATCAACTTCTGTACT CCATGCCAGTGCTGT AGGGTATTTTTAcATC AGGGTATTTTTAtAT
    GGGCTCTGA ATTTGTT CCT CCCTC
    (SEQ ID NO:139) (SEQ ID NO:139) (SEQ ID NO:178) (SEQ ID ND:179)
    C >T2 TGCATTTTCTGCTTGA TTTGTTTGTAATATA TCTGGTACCTGGACCA AATCTGGTACtTGGA
    CAGAAGA CTGCTCTCTCCTGAT A CCAA
    (SEQ ID NO:140) (SEQ ID NO:141) (SEQ ID ND:180) (SEQ ID NO:181)
    G >A GCCAAAGAAGAAACAC AAATGATCTGGTTAT TGAACCTCgAACAAT TTGAACCTCaAACAA
    CAAAAAAT AAATGAAGATGTTG (SEQ ID NO:182) TT
    (SEQ ID NO:142) (SEQ ID NO:143) (SEQ ID NO:183)
    G >A2 AAGAGGTTGAAGAAGT ATACATACACAAGGG CTGGTGATGgATCC CTGGTCATGaATCC
    GCTGAAAAATAT TTTATTTTGTTCCT (SEQ ID NO:184) (SEQ ID NO:185)
    (SEQ ID NO:144) (SEQ ID NO:145)
    4. GSTM1
    C534G GTTCCAGCCCACACAT CGGGAGATGAAGTCC CAAGCAqTGGGGC CAAGCAcTTGGGC
    TCTTG TTCAGATT (SEQ ID NO:186) (SEQ ID NO:187)
    (SEQ ID NO:146) (SEQ ID NO:147)
    5. GSTP1
    A313G CCTGGTGGACATGGTG GCAGATGCTCACATA GCAAATACaTCTCCCT GCAAATACgTCTCCC
    AATG GTTGGTGTAG (SEQ ID NO:188) T
    (SEQ ID NO:148) (SEQ ID NO:149) (SEQ ID NO:189)
    C341T CGGATGAGACTAGGAT GGGTCTCAAAAGGCT CCTTGCCCgCCTC CTTGCCCaCCTCC
    GATACATGGT TCAGTTG (SEQ ID NO:190) (SEQ ID NO:191)
    (SEQ ID NO:150) (SEQ ID NO:151)
    6. GSTT1
    TCATTCTGAAGGCCAA CAGGGCATCAGCTTC CCTGCAGACCCC N/A
    GGACTT TGCTT (SEQ ID NO:192)
    (SEQ ID NO:152) (SEQ ID NO:153)
    7. MnSOD
    T-28C GGCTGTGCTTTCTCGT TTCTGCCTGGAGCCC ACCCCAAAaCCGGA ACCCCAAAgCCGGA
    CTTCA AGAT (SEQ ID NO:193) (SEQ ID NO:194)
    (SEQ ID NO:154) (SEQ ID NO:155)
    T175C GTGTTGCATTTACTTC TCCAGAAAATGCTAT AGCCCAGAtAGCT AGCCCAGAcAGCT
    AGGAGATGTT GATTGATATGAC (SEQ ID NO:195) (SEQ ID NO:196)
    (SEQ ID NO:156) (SEQ ID NO:157)
    8. MTHFR
    C677T GACCTGAAGCACTTGA TCAAAGAAAAGCTGC AAATCGgCTCCCGC AAATCGaCTCCCGCA
    AGGAGAA GTGATGA (SEQ ID NO:197) GA
    (SEQ ID NO:158) (SEQ ID NO:159) (SEQ ID NO:198)
    A1298C AAGAGCAAGTCCCCCA CTTTGTGACCATTCC CAGTGAAGaAAGTGTC AGTGAAGcAAGTGTC
    AGGA GGTTTG (SEQ ID NO:199) (SEQ ID NO:200)
    (SEQ ID NO:160) (SEQ ID NO:161)
    9. ALDH2
    G1156A CCCTTTGGTGGCTACA AGACCCTCAAGCCCC TCACAGTTTTCACTTc TCACAGTTTTCACTT
    AGATGT AACA AGTGT tAGTGT
    (SEQ ID NO:162) (SEQ ID NO:163) (SEQ ID NO:201) (SEQ ID NO:202)
  • TABLE 2
    Examples of Primer pairs
    Primer
    Gene Set Forward Reverse Size
    NAT1 1 N/A same genotype as
    set 3
    2 N/A same genotype as
    set 3
    3 5′ggg ttt gga cgc tca 5′aat gta ctg ttc cct tct 141 bp
    tac c (SEQ ID NO: 86) gat ttg g (SEQ ID NO: 87)
    4b 5′tcc gtt tga cgg aag 5′ggg tct gca agg aac aaa 234 bp
    aga at (SEQ ID NO: 88) at (SEQ ID NO: 89)
    5 5′gaa aca taa cca caa 5′caa caa taa acc aac att 241 bp
    acc (SEQ TO NO: 90) aaa agc (SEQ ID NO: 91)
    NAT2 1 5′act tct gta ctg ggc 5′gca tcg aca atg taa ttc 150 bp
    tct gac a (SEQ ID NO: ctg c (SEQ ID NO: 93)
    92)
    2 5′aat aca gca ctg gca 5′caa gga aca aaa tga tgt 380 bp
    tgg (SEQ ID NO: 94) gg (SEQ ID NO: 95)
    3 5′gtg ggc ttc atc ctc 5′ggg tga tac ata cac aag 259 bp
    acc ta (SEQ ID NO: 96) ggt tt (SEQ ID NO: 97)
    GSTM1 1 5′cag ccc aca cat tct 5′aag cgg gag atg aag tcc 196 bp
    tgg (SEQ ID NO: 98) (SEQ ID NO: 99)
    MTHFR 1 5′agg tta ccc caa agg 5′gca agt gat gcc cat gtc 166 bp
    cca cc (SEQ ID NO: 100) g (SEQ ID NO: 101)
    2 5′tct tct acc tga aga 5′caa gtc act ttg tga cca 142 bp
    gca agt cc (SEQ ID NO: ttc c (SEQ ID NO: 103)
    102)
    CYP1A1 1b 5′cct gaa ctg cca ctt 5′cca gga aga gaa aga cct 199 bp
    cag c (SEQ ID NO: 104) cc (SEQ ID NO: 105)
    2 5′ccc att ctg tgt ttg 5′aga ggc tga ggt ggg aga 213 bp
    ggt ttt t (SEQ ID NO: at (SEQ ID NO: 107)
    106)
    GSTT1 1 5′gag gtc att ccg aag 5′ttt gtg gac tgc tga gga 133 bp
    gcc aag g (SEQ ID NO: cg (SEQ ID NO: 109)
    108)
    β- 1b 5′tcc tca gat cat tgc 5′taa cgc aac taa gtc ata 175 bp
    actin tcc (SEQ ID NO: 110) gtc c (SEQ ID NO: 111)
    MnSOD 1 5′ggc tgt gct ttc tcg 5′ggt gac gtt cag gtt gtt 194 bp
    tct tc (SEQ ID NO: 112) ca (SEQ ID NO: 113)
    2 5′aca gtg gtt gaa aaa 5′caa aat gta gat aag ggt 205 bp
    gta gg (SEQ ID NO: 114) gc (SEQ ID NO: 115)
    ALDH2 1 5′ttg gtg gct aca aga 5′agg tcc tga act tcc agc 345 bp
    tgt cg (SEQ ID NO: 116) ag (SEQ ID NO: 117)
    GSTP1 1 5′gct cta tgg gaa gga 5′aag cca cct gag ggg taa 192 bp
    cca gc (SEQ ID NO: 118) gg (SEQ ID NO: 119)
    2 5′cag cag ggt ctc aaa 5′gat gga cag gca gaa tgg 250 bp
    agg (SEQ ID NO: 120) (SEQ ID NO: 121)
  • Having obtained a sample of DNA, preferably with amplified regions of interest, individual polymorphisms may be identified. Identification of the markers for the polymorphisms involves the discriminative detection of allelic forms of the same gene that differ by nucleotide substitution, or in the case of some genes, for example the GSTM1 and GSTT1 genes, deletion of the entire gene. Methods for the detection of known nucleotide differences are well known to the skilled person. These may include, but are not limited to:
      • a. Hybridization with allele-specific oligonucleotides (ASO), (Wallace, 1981; Ikuta, 1987; Nickerson, 1990, Varlaan, 1986, Saiki, 1989 and Zhang, 1991).
      • b. Allele specific PCR, (Newton 1989, Gibbs, 1989).
      • c. Solid-phase minisequencing (Syvanen, 1993).
      • d. Oligonucleotide ligation assay (OLA) (Wu, 1989, Barany, 1991; Abravaya, 1995).
      • e. The 5′ fluorogenic nuclease assay (Holland, 1991 & 1992, Lee, 1998, U.S. Pat. Nos. 4,683,202, 4,683,195, 5,723,591 and 5,801,155).
      • f. Restriction fragment length polymorphism (RFLP), (Donis-Keller, 1987).
  • In a preferred embodiment, the genetic loci are assessed via a specialised type of PCR used to detect polymorphisms, commonly referred to as the Taqman® assay and performed using an AB7700 instrument (Applied Blosystems, Warrington, UK). In this method, a probe is synthesised which hybridises to a region of interest containing the polymorphism. The probe contains three modifications: a fluorescent reporter molecule, a fluorescent quencher molecule and a minor groove binding chemical to enhance binding to the genomic DNA strand. The probe may be bound to either strand of DNA. For example, in the case of binding to the coding strand, when the Taq polymerase enzyme begins to synthesise DNA from the 5′ upstream primer, the polymerase will encounter the probe and begin to remove bases from the probe one at a time using a 5′-3′ exonuclease activity. When the base bound to the fluorescent reporter molecule is removed, the fluorescent molecule is no longer quenched by the quencher molecule and the molecule will begin to fluoresce. This type of reaction can only take place if the probe has hybridised perfectly to the matched genomic sequence. As successive cycles of amplification take place, i.e. more probes and primers are bound to the DNA present in the reaction mixture, the amount of fluorescence will increase and a positive result will be detected. If the genomic DNA does not have a sequence that matches the probe perfectly, no fluorescent signal is detected.
  • Examples of oligonucleotide probes which may be used in the invention, particularly when the Taqman® assay is used in the method of the invention together with primers which may be used. These oligonucleotide probes form another aspect of the present invention.
  • Therefore in a further aspect of the invention, there is provided an oligonucleotide having a sequence selected from SEQ ID NO: 164-202. The invention further provides a set of oligonucleotides comprising at least 5, 10, 20, 30, 40, 50, 60 or 70 oligonucleotides selected from the group comprising SEQ ID NO:164-202.
  • Arrays
  • In a preferred embodiment of the invention, hybridisation with allele specific oligonucleotides is conveniently carried out using oligonucleotide arrays, preferably microarrays, to determine the presence of particular polymorphisms.
  • Such microarrays allow miniaturisation of assays, e.g. making use of binding agents (such as nucleic acid sequences) immobilised in small, discrete locations (microspots) and/or as arrays on solid supports or on diagnostic chips. These approaches can be particularly valuable as they can provide great sensitivity (particularly through the use of fluorescent labelled reagents), require only very small amounts of biological sample from individuals being tested and allow a variety of separate assays to be carried out simultaneously. This latter advantage can be useful as it provides an assay for different a number of polymorphisms of one or more genes to be carried out using a single sample. Examples of techniques enabling this miniaturised technology are provided in WO84/01031, WO88/1058, WO89/01157, WO93/8472, WO95/18376/WO95/18377, WO95/24649 and EP-A-0373203, the subject matter of which are herein incorporated by reference.
  • DNA microarrays have been shown to provide appropriate discrimination for polymorphism detection. Yershov, 1996; Cheung, 1999 and Schena 1999 have described the principles of the technique. In brief, the DNA microarray may be generated using oligonucleotides that have been selected to hybridise with the specific target polymorphism. These oligonucleotides may be applied by a robot onto a predetermined location of a glass slide, e.g. at predetermined X,Y Cartesian coordinates, and immobilised. The FCR product (e.g. fluorescently labelled RNA or DNA) is introduced on to the DNA microarray and a hybridisation reaction conducted so that sample RNA or DNA hinds to complementary sequences of oligonucleotides in a sequence-specific manner, and allow unbound material to be washed away. Gene target polymorphisms can thus be detected by their ability to bind to complementary oligonucleotides on the array and produce a signal. The absence of a fluorescent signal for a specific oligonucleotide probe indicates that the client does not have the corresponding polymorphism. Of course, the method is not limited to the use of fluorescence labelling but may use other suitable labels known in the art. the fluorescence at each coordinate can be read using a suitable automated detector in order to correlate each fluorescence signal with a particular oligonucleotide.
  • Oligonucleotides for use in the array may be selected to span the site of the polymorphism, each oligonucleotide comprising one of the following at a central location within the sequence:
      • a. wild-type or normal base at the position of interest in the leading strand
      • b. wild-type or normal base at the position of interest in the lag (non-coding) strand
      • c. altered base at the position of interest in the leading strand
      • d. altered complementary base at the position of interest in the lag strand
  • The arrays used in the present method form another independent aspect of the present invention. Arrays of the invention comprise a set of two or more oligonucleotides, each oligonucleotide being specific to a sequence comprising one or more polymorphisms of a gene selected from the group comprising categories a-k as defined above.
  • Preferably, the array will comprise oligonucleotides each being specific to a sequence comprising one or more polymorphisms of an individual gene of at least two different categories a-k as defined above, for example a+b (i.e. at least one oligonucleotide specific for a sequence comprising one or more polymorphisms of a first gene, the first gene being of category a and at least one oligonucleotide specific for a sequence comprising one or more polymorphisms of a second gene, the second gene being of category b), a+c, a+d, a+e, a+f, a+g, a+h, a+i, a+j, a+k, b+c, b+d, b+e etc., c+d, c+e etc, d+e, d+f etc, e+f, e+g etc, f+g, f+h etc., g+h, g+i, g+k, h+i, h+k. Where the array comprises two or more oligonucleotides, it is preferred that at least one of the oligonucleotides is an oligonucleotide specific for a sequence of a polymorphism of a gene of category d, due to the central role of micronutrients in the maintenance of proper cellular growth and DNA repair, and due to the association of micronutrient metabolism or utilisation disorders with several different types of diseases (Ames 1999; Perera, 2000; Potter, 2000). More preferably, the array will comprise oligonucleotides each being specific to a sequence comprising one or more polymorphisms of an individual gene of at least three different categories a-k as defined above, for example, a+b+c, a+b+d, a+b+e, a+b+f, a+b+g, a+b+h, a+b+i, a+b+j, a+b+k a+c+d, a+c+e etc, a+d+e, etc, b+c+d, etc, c+d+e etc, d+e+f etc, and all other combinations of three categories. Where the array comprises three or more oligonucleotides, it is preferred that at least two of the oligonucleotides are oligonucleotides specific for a sequence of a polymorphism of a gene of categories d and e. Information relating to polymorphisms present in both of these categories is particularly useful due to the effects of alcohol consumption and metabolism on the efficiency of enzymes related to micronutrient metabolism and utilisation. (Ulrich, 1999). In a further preferred embodiment where the array comprises three or more oligonucleotides, it is preferred that at least two of the oligonucleotides are oligonucleotides specific for a sequence of a polymorphism of a gene of c categories a and b due to the close interaction of Phase I and Phase II enzymes in the metabolism of xenobiotics. Even more preferably, the array will comprise oligonucleotides each being specific to a sequence comprising one or more polymorphisms of an individual gene of at least four different categories a-k as defined above, for example, a+b+c+d, a+b+c+e, a+b+d+e, a+c+d+e, b+c+d+e etc. Where the array comprises four or more oligonucleotides, it is preferred that at least three of the oligonucleotides are oligonucleotides specific for a sequence of a polymorphism of a gene of categories d and e and f Information relating to polymorphisms present in these three categories is particularly useful due to the strong correlation of polymorphisms of these alleles with coronary artery disease due to the combined effects of altered micronutrient utilisation, affected adversely by alcohol metabolism, together with imbalances in fat and cholesterol metabolism. Where the array comprises five or more oligonucleotides, it is preferred that at least four of the oligonucleotides are oligonucleotides specific for a sequence of a polymorphism of a gene of categories a, b, d and e. Information relating to polymorphisms present in these four categories is particularly useful due to the combined effects of micronutrients utilisation, alcohol metabolism, Phase 1 metabolism of xenobiotics and Phase II metabolism on the further metabolism and excretion of potentially harmful metabolites produced in the body (Taningher, 1999; Ulrich, 1999). Similarly, the array may comprise oligonucleotides each being specific to a sequence comprising one or more polymorphisms of an individual gene of at least five, for example a, b, d, e and f, six, seven, eight, nine or ten different categories a-k as defined above.
  • Most preferably, the array will comprise oligonucleotides each being specific to a sequence comprising one or more polymorphisms of an individual gene of each of categories a-k as defined above.
  • In one preferred embodiment, the array comprises oligonucleotides each being specific to a sequence comprising one or more polymorphisms of individual genes, the individual genes comprising each member of the group comprising genes encoding cytochrome P45C monooxygenase, N-acetyltransferase 1, N-acetyltransferase 2, glutathione-S-transferase, manganese superoxide dismutase, 5,10-methylenetetrahydrofolatereductase and alcohol dehydrogenase 2 enzymes. genetic loci of genes encoding each of the cytochrome P450 monooxygenase, N-acetyltransferase 1, N-acetyltransferase 2, glutathione-S-transferase, manganese superoxide dismutase, 5,10-methylenetetrahydrofolatereductase and alcohol dehydrogenase 2 enzymes. In a more preferred embodiment the array further comprises oligonucleotides specific for one or more alleles of the genetic loci of genes encoding one or more, preferably each of epoxide hydrolase (EH), NADPH-quinone reductase (NQ01), paraxonaoase (PON1), myeloperoxidase (MPO), alcohol dehydrogenase 1, alcohol dehydrogenase 3, cholesteryl ester transfer protein, apolipoprotein A IV, apolipoprotein E, apolipoprotein C III, angiotensin, factor VII, prothrombin 20210, β-fibrinogen, heme-oxygenaso-1, α-antitrypsin, SPINK1, Δ-aminolevulinacid dehydratase, interleukin 1, interleukin 1, vitamin D receptor, B1 kinin receptor, cystathionine-beta-synthase, methionine synthase (B12 MS), 5-HT transporter, transforming growth factor beta 1 (TGFβ1), L-myc, HLA Class 2 molecules, T-lymphocyte associated antigen 4 (CTLA-4), interleukin 4, interleukin 3, interleukin 6, IgA, and/or galactose metabolism gene GALT.
  • In preferred arrays, the oligonucleotides in the array comprise at least 5, 10, 20, 30, 40, 50, 60 or 70 oligonucleotides selected from the group comprising SEQ ID NO:1-SEQ ID NO: 85 illustrated in TABLE 3 which shows preferred oligonucleotides listed in the right column with the primer set used to amplify the appropriate fragments of sample DNA listed in the left column.
  • In a preferred embodiment the array will comprise all of the oligonucleotides SEQ ID NO:1-85.
    TABLE 3
    Gene Target 25 nt sequence
    1. CYP1A1
    Primer set1 A4889G wt-lead 5′ atc ggt gag acc Att gcc cgc tgg g
    (SEQ ID NO: 1)
    Primer set1 A4889G wt-lag 5′ ccc agc ggg caa Tgg tct cac cga t
    (SEQ ID NO: 2)
    Primer set1 A4889G polymorph- 5′ atc ggt gag acc Gtt gcc cgc tgg g
    lead (SEQ ID NO: 3)
    Primer set1 A4889G polymorph- 5′ ccc agc ggg caa Cgg tct cac cga t
    lag (SEQ ID NO: 4)
    Primer set2 T6235P wt-lead 5′ acc tcc acc tcc Tgg gct cac acg a
    (SEQ ID NO: 5)
    Primer set2 T6235C wt-lag 5′ tcg tgt gag ccc Agg agg tgg agg t
    (SEQ ID NO: 6)
    Primer set2 T6235C polymorph- 5′ acc tcc acc tcc Cgg gct cac acg a
    lead (SEQ ID NO: 7)
    Primer set2 T6235C polymorph- 5′ tcg tgt gag ccc Ggg agg tgg agg t
    lag (SEQ ID NO: 8)
    2. NAT1
    Primer set1 N/A
    Primer set2 N/A
    Primer set3 G445A wt-lead 5′ cag gtg cct tgt Gtc ttc cgt ttg a
    (SEQ ID NO: 9)
    Primer set3 G445A wt-lag 5′ tca aac gga aga Cac aag gca cct g
    (SEQ ID NO: 10)
    Primer set3 G445A polymorph- 5′ cag gtg cct tgt Atc ttc cgt ttg a
    lead (SEQ ID NO: 11)
    Primer set3 G445A polymorph- 5′ tca aac gga aga Tac aag gca cct g
    lag (SEQ ID NO: 12)
    Primer set3 G459A wt-lead 5′ ctt ccg ttt gac Gga aga gaa tgg a
    (SEQ ID NO: 13)
    Primer set3 G459A wt-lag 5′ tcc att ctc ttc Cgt caa acg gaa g
    (SEQ ID NO: 14)
    Primer set3 G459A polymorph- 5′ ctt ccg ttt gac Aga aga gaa tgg a
    lead (SEQ ID NO: 15)
    Primer set3 G459A polymorph- 5′ tcc att ctc ttc Tgt caa acg gaa g
    lag (SEQ ID NO: 16)
    Primer set4 G560A wt-lead 5′ aca gca aat acc Gaa aaa tct act c
    (SEQ ID NO: 17)
    Primer set4 G560A wt-lag 5′ gag tag att ttt Cgg tat ttg ctg t
    (SEQ ID NO: 18)
    Primer set4 G560A polymorph- 5′ aca gca aat acc Aaa aaa tct act c
    lead (SEQ ID NO: 19)
    Primer set4 G560A polymorph- 5′ gag tag att ttt Tcc tat ttg ctg t
    lag (SEQ ID NO: 20)
    Primer set5 T1088A wt-lead*a 5′ taa taa taa taa Taa atg tct ttt a
    (SEQ ID NO: 21)
    Primer set5 T1088A wt-lag*a 5′ taa aag aca ttt Att att att att a
    (SEQ ID NO: 22)
    Primer set5 T1088A wt-lead*b 5′ taa taa taa taa Taa atg tat ttt a
    (SEQ ID NO: 23)
    Primer set5 T1088A wt-lag*b 5′ taa aat aca ttt Att att tta att a
    (SEQ ID NO: 24)
    Primer set5 T1088A polymorph- 5′ taa taa taa taa Aaa atg tct ttt a
    lead*a (SEQ ID NO: 25)
    Primer set5 T1088A polymorph- 5′ taa aag aca ttt Ttt att tta att a
    lag*a (SEQ ID NO: 26)
    Primer set5 T1088A polymorph- 5′ taa taa taa taa Aaa atg tat ttt a
    lead*b
    Primer set5 T1088A polymorph- 5′ taa aat aca ttt Ttt att tta att a
    lag*b (SEQ ID NO: 27)
    *redundancy due to adjacent
    polymorphisms
    Primer set5 C1095A wt-lead*a 5′ aat aat aaa tgt Ctt tta aag atg g
    (SEQ ID NO: 28)
    Primer set5 C1095A wt-lag*a 5′ cca tct tta aaa Gac att tat tat t
    (SEQ ID NO: 29)
    Primer set5 C1095A wt-lead*b 5′ aat aaa aaa tgt Ctt tta aag atg g
    (SEQ ID NO: 30)
    Primer set5 C1095A wt-lag*b 5′ cca tct cta aaa Gac att ttt tat t
    (SEQ ID NO: 31)
    Primer set5 C1095A polymorph- 5′ aat aat aaa tgt Att tta aag atg g
    lead*a (SEQ ID NO: 32)
    Primer set5 C1095A polymorph- 5′ cca tct tta aaa Tac att tat tat t
    lag*a (SEQ ID NO: 33)
    Primer set5 C1095A polymorph- 5′ aat aaa aaa tgt Att tta aag atg g
    lead*b (SEQ ID NO: 34)
    Primer set5 C1095A polymorph- 5′ cca tct tta aaa Tac att ttt tat t
    lag*b (SEQ ID NO: 35)
    *redundancy due to adjacent
    polymorphisms
    3. NAT2
    Primer set1 C282T wt-lead 5′ agg gta ttt tta Cat ccc tcc agt t
    (SEQ ID NO: 36)
    Primer set1 C282T wt-lag 5′ aac tgg agg gat Gta aaa ata ccc t
    (SEQ ID NO: 37)
    Primer set1 C282T polymorph- 5′ agg gta ttt tta Tat ccc tcc agt t
    lead (SEQ ID NO: 38)
    Primer set1 C282T polymorph- 5′ aac tgg agg gat Ata aaa ata ccc t
    lag (SEQ ID NO: 39)
    Primer set2 C481T wt-lead 5′ gga atc tgg tac Ctg gac caa atc a
    (SEQ ID NO: 40)
    Primer set2 C481T wt-lag 5′ tga ttt ggt cca Ggt acc aga ttc c
    (SEQ ID NO: 41)
    Primer set2 C481T polymorph- 5′ gga atc tgg tac Ttg gac caa atc a
    lead (SEQ ID NO: 42)
    Primer set2 C481T polymorph- 5′ tga ttt ggt cca Agt acc aga ttc c
    lag (SEQ ID NO: 43)
    Primer set2 G590A wt-lead 5′ cgc ttg aac ctc Gaa caa ttg aag a
    (SEQ ID NO: 44)
    Primer set2 G590A wt-lag 5′ tct tca att gtt Cga ggt tca agc g
    (SEQ ID NO: 45)
    Primer set2 G590A polymorph- 5′ cgc ttg aac ctc Aaa caa ttg aag a
    lead (SEQ ID NO: 46)
    Primer set2 G590A polymorph- 5′ tct tca att gtt Tga ggt tca agc g
    lag (SEQ ID NO: 47)
    Primer set3 G857A wt-lead 5′ aac ctg gtg atg Gat ccc tta cta t
    (SEQ ID NO: 48)
    Primer set3 G857A wt-lag 5′ ata gta agg gat Cca tca cca ggt t
    (SEQ ID NO: 49)
    Primer set3 G857A polymorph- 5′ aac ctg gtg atg Aat ccc tta cta t
    lead (SEQ ID NO: 50)
    Primer set3 G857A polymorph- 5′ ata gta agg gat Tca tca cca ggt t
    lead (SEQ ID NO: 51)
    4. GSTM1
    Primer set1 wt-lead 5′ gct aca ttg ccc gca agc aca acc t
    (SEQ ID NO: 52)
    Primer set1 wt-lag 5′ agg ttg tgc ttg cgg gca atg tag c
    (SEQ ID NO: 53)
    5. GSTP1
    Primer set1 A313G wt-lead 5′ cgc tgc aaa tac Atc tcc ctc atc t
    (SEQ ID NO: 54)
    Primer set1 A313G wt-leg 5′ aga tga ggg aga Tgt att tgc agc g
    (SEQ ID NO: 55)
    Primer set1 A313G polymorph- 5′ cgc tgc aaa tac Gtc tcc ctc atc t
    lead (SEQ ID NO: 56)
    Primer set1 A313G polymorph- 5′ aga tga ggg aga Cgt att tgc agc g
    lag (SEQ ID NO: 57)
    Primer set2 C341T wt-lead 5′ tct ggc agg agg Cgg gca agg atg a
    (SEQ ID NO: 58)
    Primer set2 C341T wt-lag 5′ tca tcc ttg ccc Gcc tcc tgc cag a
    (SEQ ID NO: 59)
    Primer set2 C341T polymorph- 5′ tct ggc agg agg Tgg gca agg atg a
    lead (SEQ ID NO: 60)
    Primer set2 C341T polymorph- 5′ tca tcc ttg ccc Acc tcc tgc cag a
    lag (SEQ ID NO: 61)
    6. GSTT1
    Primer set1 wt-lead 5′ acc ata aag aag cag aag ctg ccc t
    (SEQ ID NO: 62)
    Primer set2 wt-lag 5′ agg gca tca gct tct gct tta tgg t
    (SEQ ID NO: 63)
    7. MnSOD
    Primer set1 T-26C wt-lead 5′ agc tgg ctc cgg Ttt tgg ggt atc t
    (SEQ ID NO: 64)
    Primer set1 T-26C wt-lag 5′ aga tac ccc aaa Acc gga gcc agc t
    (SEQ ID NO: 65)
    Primer set1 T-26C polymorph- 5′ agc tgg ctc cgg Ctt tgg ggt atc t
    lead (SEQ ID NO: 66)
    Primer set1 T-26C polymorph- 5′ aga tac ccc aaa Gcc gga gcc agc t
    lag (SEQ ID NO: 67)
    Primer set2 T175C wt-lead 5′ tta cag ccc aga Tag ctc ttc agc c
    (SEQ ID NO: 68)
    Primer set2 T175C wt-lag 5′ ggc tga aga gct Atc tgg gct gta a
    (SEQ ID NO: 69)
    Primer set2 T175C polymorph- 5′ tta cag ccc aga Cag ctc ttc agc c
    lead (SEQ ID NO: 70)
    Primer set2 T175C polymorph- 5′ ggc tga aga gct Gtc tgg gct gta a
    lag (SEQ ID NO: 71)
    8. MTHFR
    Primer set1 C677T wt-lead 5′ tgt ctg cgg gag Ccg att tca tca t
    (SEQ ID NO: 72)
    Primer set1 C677T wt-lag 5′ atg atg aaa tcg Gct ccc gca gac a
    (SEQ ID NO: 73)
    Primer set1 C677T polymorph- 5′ tgt ctg cgg gag Tcg att tca tca t
    lead (SEQ ID NO: 74)
    Primer set1 C677T polymorph- 5′ atg atg aaa tcg Act ccc gca gac a
    lag (SEQ ID NO: 75)
    Primer set2 A1298C wt-lead 5′ tga cca gtg aag Aaa gtg tct ttg a
    (SEQ ID NO: 76)
    Primer set2 A1298C wt-lag 5′ tca aag aca ctt Tct tca ctg gtc a
    (SEQ ID NO: 77)
    Primer set2 A1298C polymorph- 5′ tga cca gtg aag Caa gtg tct ttg a
    lead (SEQ ID NO: 78)
    Primer set2 A1298C polymorph- 5′ tca aag aca ctt Gct tca ctg gtc a
    lag (SEQ ID NO: 79)
    9. ALDH2
    Primer set1 wt-lead 5′ cag gca tac act Gaa gtg aaa act g
    (SEQ ID NO: 80)
    Primer set1 wt-lag 5′ cag ttt cca ctt Cag tgt atg cct g
    (SEQ ID NO: 81)
    Primer set1 polymorph-lead 5′ cag gca tac act Aaa gtg aaa act g
    (SEQ ID NO: 82)
    Primer set1 polymorph-lag 5′ cag ttt tca ctt Tag tgt atg cct g
    (SEQ ID NO: 83)
    10. beta-Actin
    Primer set1-lead 5′ tgc atc tct gcc tta cag atc atg t
    (SEQ ID NO: 84)
    Primer set1-lag 5′ aga tga tct gta agg cag aga tgc a
    (SEQ ID NO: 85)
  • Advice Decision Tree
  • The results of genetic polymorphism analysis may be used to correlate the genetic profile of the donor of the sample with disease susceptibility using the first dataset, which provides details of the relative disease susceptibility associated with particular polymorphisms and their interactions. The risk factors identified using dataset 1 can then be matched with dietary and other lifestyle recommendations from dataset 2 to produce a lifestyle advice plan individualised to the genetic profile of the donor of the sample. Examples of datasets 1 and 2 which may be used to generate such advice is illustrated in FIG. 1.
  • To enable appropriate advice to be tailored to particular susceptibilities, a ranking system is preferably used to provide an indication of the degree of susceptibility of a specific polymorph to risk of cancer(s) and/or other conditions. The ranking system may be designed to take into account of homozygous or heterozygous alleles in the client's sample, i.e. the same or different alleles being present in diploid nucleus. Five categories which may be used are summarised below:
      • (i) Reduced susceptibility: where an allele has been shown to reduce susceptibility.
      • (ii) Normal susceptibility: where allele has been shown to have a normal susceptibility of risk to cancer(s) or disease. This is generally the homozygous wild type allele or a polymorphism that has been shown to have similar function.
      • (iii) Moderate susceptibility: where a heterozygous genotype is present that contains the wild type of the allele (i.e. normal susceptibility) and an allele of the polymorphism known to give rise to higher susceptibility to specific cancer(s) or disease.
      • (iv) High susceptibility: where a homozygous genotype that contains the polymorphism is present with a higher risk of cancer susceptibility.
      • (v) Higher susceptibility: where a higher susceptibility has been observed for specific cancer(s) or disease due to the combined effects of two or more different gene targets.
  • Using dataset 1, a susceptibility may be assigned to each polymorphism identified and, from dataset 2, a lifestyle recommendation corresponding to each susceptibility identified may be assigned. For example, if an individual is found to have the NAT1*10 polymorphism, the decision tree may indicate that the there is an enhanced susceptibility of colonic cancer. Recommendations appropriate to minimising the risk of colonic cancer are then generated. For example, the recommendations may he to avoid particular foods associated with increased risk and to increase consumption of other foods associated with a protective effect against such cancers. The totality of recommendations may be combined to generate a lifestyle advice plan individualised to the donor of the sample. The decision tree is preferably arranged to recognise particular combinations of polymorphisms and/or susceptibilities which interact either positively to produce a susceptibility greater than would be expected from the risk factors associated with each individually, and/or, which interact negatively to reduce the susceptibility associated with each individually. Where such combinations are identified, the advice generated can be tailored accordingly. For example, the combination of NAT2*4 and NAT1*10 polymorphisms have been linked to increased cancer risk (Bell, 1995). Therefore, when such a combination of polymorphisms is identified from a subject's DNA, the associated very high susceptibility to cancer is assigned and the advice tailored to emphasise the need to reduce consumption of xenobiotics, e.g. by reducing or eliminating consumption of char-grilled foodstuffs.
  • In generating the advice, other factors such as information concerning the sex and health of the individual and/or of the individual's family, age, alcohol consumption, and existing diet may be used in the determination of appropriate lifestyle recommendations.
  • Experimental
  • EXAMPLE 1 Preparation of DNA Sample
  • DNA is prepared from a buccal cell sample on a brush using a Qiagen QIAamp kit according to the manufacturer's instructions (Qiagen, Crawley, UK). Briefly, the brush is cut in half and one half stored at room temperature in a sealed tube in case retesting is required. The other half of the brush is placed in a microcentrifuge tube. 400 μl PBS is added and the brush allowed to rehydrate for 45 minutes at room temperature. Quiagen lysis buffer and Proteinase K is then added, the contents are mixed, and allowed to incubate at 56 C for 15 minutes to lyse the cells. Ethanol is added and the lysate transferred to a QIAamp spin column from which DNA is eluted after several washings.
  • EXAMPLE 2 Quantification of DNA
  • In order to check that sufficient DNA has been isolated, a quantification step is carried out using the PicoGreen dsDNA Quantification kit (Molecular Probes, Eugene, Oreg., USA).
  • Briefly, client DNA samples are prepared by transferring a 10 μl aliquot into a microcentrifuge tube with 90 μl TE. 100 μl of the working PicoGreen dsDNA quantification reagent is added, mixed well, and transferred into a black 96 well plate with flat well bottoms. The plate is then incubated for 5 minutes in the dark before a fluorescent reading is taken. The quantity of DNA present in the clients' samples is determined by extrapolating from a calibration plot prepared using DNA standards.
  • A quantity of DNA in the range of 5-50 ng total is used in the subsequent PCR step. Remaining client DNA sample is stored at −20° C. for retesting if required.
  • EXAMPLE 3 Taqman® Assay to Identify the MTHFR A1298C Polymorphism
  • The modified reaction mixture contains Taq polymerase (1.25 units/μl), optimised PCR buffer, dNTP (200 μM each), 2 mM MgCl2 and primer pairs SEQ ID NO: 160 and 161 and polymorphism probe SEQ ID NO: 200.
  • The reaction mixture is initially incubated for 10 minutes at 50° C., then 5 minutes at 95° C., followed by 40 cycles of 1 minute of annealing at between 55° C. and 60° C. and 30 seconds of denaturation at 95° C. Both during the cycles and at the end of the run, fluorescence of the released reporter molecules of the probe is measured by an integral CCD detection system of the AB7700 thermocycler. The presence of a fluorescent signal which increases in magnitude through the course of the run indicates a positive result.
  • The assay is then repeated with the same primer pair and wt probe SEQ ID NO: 199. If the sample is homozygous for the polymorphism, no fluorescence signal is seen with the wt probe. However, if the sample is heterozygous for the polymorphism, a fluorescence signal is also seen with the wt probe. If single reporter results from homozygous wt, homozygous polymorphic and heterozygous polymorphic samples are plotted are plotted on an X/Y axis, the homozygous alleles will cluster at opposite ends of the axes relative to each reporter, and the heterozygous alleles will cluster at a midway region.
  • EXAMPLE 4 DNA Array Method for Identifying Polymorphisms for Identifying Multiple Polymorphisms
  • a) PCR Amplification
  • The PCR reaction mix contains Taq polymerase (1.25 units/reaction), optimised PCR buffer, dNTP's (200 μM each) and MgCl2 at an appropriate concentration of between 1 and 4 mM, and 40 pmol of each primer(SEQ ID NOS: 1-8, 17-63) for amplification of seven fragments and the sample DNA.
  • The reaction mixture is initially incubated at 95° C. for 1 minute, and then subjected to 45 cycles of PCR in a MWG TC9600 thermocycler (MWG-Biotech-AG Ltd., Milton Keynes, UK) as follows:
      • annealing 50° C., 1 minute
      • polymerisation 73° C., 1 minute
      • denaturation 95° C., 30 seconds.
  • After a further annealing step at 50° C., 1 minute, there is a final polymerisation step at 73° C. for 7 minutes.
  • (Instead of the MWG TC9600 thermocycler, other thermocyclers, such as the Applied Biosystems 9700 thermocycler (Applied Biosystems, Warrington, UK), may be used.
  • After amplification of the target genes, generation of product is checked by electrophoresis separation using 2% agarose gel, or a 3.5% NuSieve agarose gel.
  • The PCR mplification products are then purified using the Qiagen QIAquick PCR Purification Kit (Qiagen, Crawley, UK) to remove dNTPs, primers, and enzyme from the PCR product. The PCR product is layered onto a QIAquick spin column, a vacuum applied to separate the PCR product from the other reaction products and the DNA eluted in buffer.
  • b) RNA Transcription and Fluorescent Labelling of PCR Products
  • The DNA is then transcribed into RNA using T3 and T7 RNA polymerases together with fluorescently labelled UTP for incorporation into the growing chain of RNA. The reaction mixture comprises:
  • 20 μl 5× reaction buffer; 500 μM ATP, CTP, GTP, fluorescent UTP (Amersham Ltd, UK); DEPC treated dH2O; 1 unit T3 RNA polymerase or 1 unit T7 RNA polymerase (Promega Ltd., Southampton, UK); 1 unit Rnasin ribonuclaese inhibitor and DNA from PCR (⅓ of total, 10 μl in dH2O).
  • The mixture is incubated at 37° C. for 1 hour. The mixture is then treated with DNAse to remove DNA so that only newly synthesised fluorescent RNA is left. The RNA is then precipitated, microcentrifuged and resuspended in buffer for hybridisation on the array.
  • c) Polymorphism Analysis
  • The sample amplified fragments are then tested using a DNA microarray
  • The DNA microarray used comprises oligonucleotides SEQ ID NOs: 1-85. These oligonucleotides are applied by a robot onto a glass slide and immobilised. The fluorescently labelled amplified DNA is introduced onto the DNA microarray and a hybridisation reaction conducted to bind any complementary sequences in the sample, allowing unbound material to be washed away. The presence of bound samples is detected using a scanner. The absence of a fluorescent signal for a specific oligonucleotide probe indicates that the client does not have the corresponding polymorphism.
  • EXAMPLE 5 DNA Array Method for Identifying G560A Polymorphism
  • The PCR reaction mix contains Taq polymerase (1.25 units/reaction), optimised PCR buffer, dNTP's (200 μM each) and MgCl2 at an appropriate concentration of between 1 and 4 mM, and 40 pmol of each primer (SEQ ID NOs: 88, 89) for amplification of the fragment. The methods used is the same as detailed in Example 4, with the array comprising oligonucleotides SEQ ID NO: 17, 18, 19 and 20.
  • The presence of bound samples is detected using a scanner as described above. A highly fluorescent spot is detected at the positions corresponding to the oligonucleotides SEQ ID NO: 19 and 20. No signal is seen at the spots corresponding to SEQ ID NO: 17 and 18, demonstrating that the sample is not heterozygous for the wt allele.
  • EXAMPLE 6 Generation of Report
  • The results of the microarray or Taqman® analysis are input into a computer comprising a first dataset correlating the presence of individual alleles with a risk factor and a second dataset correlating risk factors with lifestyle advice. A report is generated identifying the presence of particular polymorphisms and providing lifestyle recommendations based on the identified polymorphisms. An example of such a decision process is shown in FIG. 2.
  • A sample of DNA is screened and the alleles identified input to a dataprocessor as Dataset 3. Each allele is matched to lifestyle risk factor from dataset 1, e.g. high susceptibility to colon cancer due to the presence of the NAT1*10 allele and the absence of the GSTM1 allele. The identified risk factor is then matched with one or more lifestyle recommendations from dataset 2, for example “avoid red meat, chargrilled food, smoked meats and fish; stop smoking immediately” (in order to avoid production of potentially toxic byproducts by Phase 1 enzymes with increased activity) and “increase consumption of vegetables of the allium family e.g. onions and garlic, and the brassaicae family e.g. broccoli” (in order to increase the activity of Phase 11 enzymes present, such as GSTP1 and GSTT1 and others, in order to increase the excretion of toxic byproducts of Phase 1 metabolism). This is then checked against other factors input into the dataprocessor, e.g. age, sex and existing diet to modify the recommendation accordingly before generating the final recommendation appropriate to the allele. The lifestyle recommendations are then assembled to generate a comprehensive personalised lifestyle advice plan.
  • REFERENCES
    • Abravaya, K., Carrino, J. J., Muldoon, S., and Lee, H. H. 1995. Detection of point mutation with a modified ligase chain reaction (Gap-LCR). Nucleic Acids Research. 23:675-682.
    • Agarwal, K., Jones, D. E., Daly, A. K., James, O. F., Vaidya, B., Pearce, S. & Bassendine, M. F., 2030, CTLA-4 gene polymorphism confers susceptibility to primary biliary cirrhosis, J Hepatol, 32, 4, p. 538-541.
    • Alexandrie, A.-K., Warholm, M., Carstensen, U., Axmon, A., Hagmar, L., Levin, J. O., Ostman, C., and Rannug, A. CYP1A1 and GSTM1 polymorphisms affect urinary 1-hydroyprene levels after PAH exposure. Carcinogenesis 21(4)669-676, 2000.
    • Ambrosone, C. B., Freudenheim, J. L., Thomopson, P. A., Bowman, E., Vena, J. E., Marshall, J. R., Graham, S., Laughlin, R., Nemoto, T., and Shields P. G. Manganese Superoxide Dismutase (MnSOD) Genetic Polymorphisms, Dietary Antioxidants, and Risk of Breast Cancer. Cancer Research 59: 602-606, 1999.
    • Ames, B. N. Cancer prevontion and diet: Help from single nucleotide polymorphisms. Proceedings of the National Academy of Science USA 96(22): 12216-12218, 1999.
    • Aron, Y., Swierczewski, E., Lockhart, A., 1994. A simple and rapid micromethod for genomic DNA extraction from jugal epithelial cells. Application to human lymphocyte antigen typing in one large family of atopic/asthmatic probands. Allergy 49 (9): 788-90.
    • Ban, Y. & Taniyama, M., 2000, Vitamin D Receptor Gene Polymorphism Is Associated with Graves' Disease in the Japanese Population, J Clin Endocrine Metab, 85, 12, p. 4639-4643.
    • Barany, F. 1991. Genetic disease detection and DNA amplification and DNA amplification using cloned thermostable ligase. Proceedings of the National Academy of Science. USA 88:189-193.
    • Bell, D. A, Stephens, F., Castranio, T., Umback, D. M., Watson, M., Deakin, M., Elder, J., Duncan, H., Hendrickse, C., Strange, R. C. Polyadenylation polymorphism in the N-actetyltransferase gene 1 (NAT1) increases risk of colorectal cancer. Cancer Research 55: 3537-3542, 1995.
    • Bosron, W. F. and Li, T. K. Genetic polymorphism of human liver alcohol and aldehyde dehydrogenases and their relationship to alcohol metabolism and alcoholism. Hepatology 6: 502-510, 1986.
    • Brand, E., Ringel, J. & Sharma, A. M., 2000, Role of the angiotensinogen gene for essential hypertension, Herz, 25, 1, p. 15-25.
    • Breslauer, et al., “Predicting DNA duplex stability from base sequence”, Proc. Nat'l Acad. Sci. USA, 83: 3746-3750 (1986)
    • Brockton, N., Little, J., Sharp, L, and Cotton, S. C. N-Acetyltransferase Polymorphisms and Colorectal Cancer: A HuGE Review. American Journal of Epidemiology 151(9): 846-861, 2000.
    • Bryant, M. S., Skipper, P. L., Tannenbaum, S. R., and Niure, M. Haemoglobin adducts of 4-aminobiphenyl in smokers and non-smokers. Cancer Research 47: 612-618, 1987.
    • Buervenich, S., Sydow, O., Carmine, A., Zhang, Z., Anvret, M. & Olson, L., 2000, Alcohol dehydrogenase alleles in Parkinson's disease, Mov Disord, 15, 5, p. 813-818.
    • Pullido, M. J. & Valdivieso, F., 2000, Apolipoproteln E gene promoter polymorphisms in Alzheimer's disease, Microsc Res Tech, 50, 4, p. 261-267.
    • Cheung, V. G., et. al., 1999, Nature, Genetics, vol. 21, 15-19.
    • Corbo, R. M. & Scacchi, R., 1999, Apolipoprotein E (APOE) allele distribution in the world. Is APOE*4 a ‘thrifty’ allele?, Ann Hum Genet, 63, PT1, p. 301-310.
    • Costa, L. G., 2000, The emerging field of ecogenetics, Neurotoxicology, 21, 1-2, p. 85-89.
    • Cotton, S. C., Sharp, L., Little, J., and Brockton, N. Glutathione S-Transferase Polymorphisms and Colorectal Cancer (A HuGE review). American Journal of Epidemiology 151(1)7-32, 2000.
    • Cramer, D. W., Greenberg, E. R., Titus-Ernstoff, L., Liberman, R. F., Welch, W. R., Li, E. & Ng, W. G., 2000, A case-control study of galactose consumption and metabolism in relation to ovarian cancer, Cancer Epidemiol Biomarkers Prev, 9, 1, p. 95-101.
    • Cramer, D. W., Greenberg, E. R., Titus-Ernstoff, L., Liberman, R. F., Welch, W. R., Li, E. & Ng, W. G., 2000, A case-control study of galactose consumption and metabolism in relation to ovarian cancer, Cancer Epidemiol Biomarkers Prev, 9, 1, p. 95-101.
    • de Padua Mansur, A.; Annicchino-Bizzacchi, J.; Favarato, D.; Avakian, S. D.; Machado Cesar, L. A.; Franchini Ramires, J. A., 2000. Angiotensin-converting enzyme end apolipoprotein B polymorphisms in coronary artery disease. Am J Cardiol 85 (9): 1089-93.
    • Di Castelnuovo, A., D'Orazio, A., Amore, C., Falanga, A., Donati, M. B. & Iacoviello, L., 2000, The decanucleotide insertion/deletior polymorphism in the promoter region of the coagulation factor VII gene and the risk of familial myocardial infarction, Thromb Res, 98, 1, p. 9-17.
    • Dickey, C., Snatella, R., Hattis, D., Tang, D., Hsu, Y., Cooper, T., Young, T. and Perera F., Variability in PAH-DNA adduct measurements in peripheral mononuclear cells: implications for quantitative cancer risk assessment. Risk Analysis 17: 649-655, 1997.
    • Dietz, A. C., Zheng, W., Leff, M. A., Gross, M., Xiao, G.-F., Doll, M. A., Wen, W.-Q., Folsom, A. R., Hein, D. W. N-acetyltransferase-2 (NAT2) acetylation polymorphism, well-done meat intake and breast cancer risk among post-menopausal women. Proceedings of the American Association for Cancer Research, 40: 148, 1999.
    • Doll, M. A., Jiang, W., Deitz, A. C., Rustan, T. D., and Hein, D. W. Identification of a novel allele at the human NAT1 acetyltransferase locus. Biochem. Buiophys. Res. Commun. 233: 584-591, 1997.
    • Donati, M. B., Zito, F., Castelnuovo, A. D. & Iacoviello, L., 2000, Genes, coagulation and cardiovascular risk, J Hum Hypertens, 14, 6, p. 369-372.
    • Donis-Keller H., Green P, Helms C., et. al. (1987), A genetic map of the human genome. Cell, 51, 319-337
    • Eberhart, M. V., Lee, C. Y., Liu, R. H. Antioxidant activity of fresh apples. Nature 405: 903-904, 2000.
    • Fernandez-Real, J. M., Vendrell, J., Ricart, W., Broch, M., Gutierrez, C.,
    • Casamitjana, R., Oriola, J. & Richart, C., 2000, Polymorphism of the tumor necrosis factor-alpha receptor 2 gene is associated with obesity, leptin levels, and insulin resistance in young subjects and diet-treated type 2 diabetic patients, Diabetes Care, 23, 6, p. 831-837.
    • Garte, S. The role of ethoricity in cancer susceptibility gene polymorphisms: the example of CYP1A1. Carcinogenesis 19(8) 1329-1332, 1998.
    • Gelder, C. M., Hart, K. W., Williams, O. M., Lyons, E., Welsh, K. I., Campbell, I. A., Marshall, S. E., 2000, Vitamin D receptor gene polymorphisms and susceptibility to Mycobacterium malmoense pulmonary disease, J Infect Dis, 181, 6, p. 2099-2102.
    • Gibbs, R. A., Nguyen, P. N., and Caskey, C. T. 1989. Detection of single DNA base differences by competitive oligonucleolides priming. Nucleic Acids Research. 17:2437-2448.
    • Gil, J. P., Lechner, M. C. Increased frequency of wild type arylamine-Nactyltransferase allel NAT2*4 homozygotes in Portuguese patients with colorectal cancer. Carcinogenesis 19(1) 37-41, 1998.
    • Giovannucci, E. Nutritional factors in human cancers. Advances in Experimental Medicine and Biology 472:29-42, 1999.
    • Grossman, P. D., Bloch, W., Brinson, E., Chang, C. C., Eggerding, F. A.,
    • Fung, S., lovannisci, D. A., Woo, S., and Winn-Deen, E. S. 1994. High-density multiplex detection of nucleic acid sequences: oligonucleotides ligation assay and sequence-coded separation. Nucleic Acid Research. 22:4527-4534.
    • Harries, L. W., Stubbins, M. J., Forman, D., Howard, G. c. W, Wolf R. Identification of genetic polymorphisms at the glutathione S-transferase pi locus and association with susceptibility to bladder, testicular, and prostate cancer. Carcinogenesis 18:641-644, 1997.
    • Hattis D., Erdreich, L, and DiMauro, T. Human Variability in Parameters that are Potentially Related to Susceptibility to Carcinogenesis-I. Preliminary Observations. Center for Technology, Policy and Industrial Development, MIT, Cambridge, Mass., 1986.
    • Heilbronn, I. K., Noakes, M., Morris, A. M., Kind, K. L., Clifton, P. M., 2000, 360His polymorphism of the apolipoproteinA-IV gene and plasma lipid response to energy restricted diets in overweight subjects, Atherosclerosis, 150, 1, p. 187-192.
    • Hein, D., Doll, M. A., Fretland, A. J., Leff, M. A., Webb, S. J., Xiao, U.-S. D., Nangju, N., Feng, Y., Molecular Genetics and Epidemiology of the NAT1 and NAT2 Acerylation Polymorphisms. Cancer Epidemiology, Biomarkers & Prevention 9: 29-42, 2000 (a).
    • H ein, D., N-Acetyltransferase genetics and their role in predisposition to aromatic and heterocyclic amine-induced carcinogenesis. Toxicology Letters 112-113: 349-356, 2000 (b).
    • Hennig, B. J., Parkhill, J. M., Chapple, I. L., Heasman, P. A. & Taylor, J. J., 1999, Association of a vitamin D receptor gene polymorphism with localized early-onset periodontal diseases, J Periodontol, 70, 9, p. 1032-1038.
    • Hirvoner, A. Polymorphisms of Xeno-biotic-Metaboilzing Enzymes and Susceptibility to Cancer. Environ Health Perspect 107 Supplement 1: 37-47, 1999.
    • Humphries, S. E., Henry, J. A. & Montgomery, H. E., 1999, Gene-environment interaction in the determination of levels of haemostatic variables involved in thrombosis and fibrinolysis, Blood Coagul Fibrinolysis, 10 Suppl 1, p. S17-S21.
    • Ikuta, S., Takagi K., Wallace, R. B., and Itakura, K. 1987. Dissociation Kinetics of 19 base paired oligonucleotides-DNA Duplexes containing different single mismatched base pairs. Nucleic Acids Research. 15:797-811.
    • Ilett, K. F., David, B. M., Dethen, P., Castlden, W. M, and Kwa, R. Acetylation pehotype in colorectoal carcinonoma. Cancer Research 47: 1466-1469, 1987.
    • International Agency for Research on Cancer (IARC). Alcohol Drinking. IARC monographs on the evaluation of the carcinogenic risks to humans, IARC, Lyon. 44: 153-246, 1998.
    • Kate, S., Bowman, F. D., Harrington, A. M., et al Human lung carcinogen DNA adduct levels mediated by genetic polymorphisms in vivo. Journal of the National Cancer Institute 87:902-907, 1995.
    • Kawajiri, K., Equchi, H., Nakachi, K., Sekiya., T., Yamamoto, M. Association of CYP1A1 germ line polymorphisms with mutations of the p53 gene in lung cancer. Cancer Research 56:72-76, 1996.
    • Landegren, U., Kaiser, R., Sanders, J., and Hood, L, 1988. A ligand-mediated gene detection technique. Science. 241:1077-1080.
    • Laplaud, P. M., Dantoine, T. & Chapman, M. J., 1998, Paraoxonase as a risk marker for cardiovascular disease: facts and hypotheses, Clin Chem Lab Med, 36, 7, p. 431-441.
    • Layton, D. W., Bogen, K. T., Knize, M-G., Hatch, F. T., Johnson, V. M., and Felton, J. S. Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research. Carcinogenesis 16: 39-52, 1995.
    • Lee, E., Huang, Y., Zhao, B. et al Genetic polymorphism of conjugating enzymes and cancer risk: GSTM1, GSTT1, NAT1 and NAT2. Journal of the Toxicological Society 23: 140-142, 1998.
    • Maciag, P. C., Schlecht, N. F., Souza, P. S., Franco, E. L., Villa, L. L. & Petzl-Erler, M. L., 2000, Major histocompatibility complex class II polymorphisms and risk of cervical cancer and human papillomavirus infection in Brazilian women, Cancer Epidemiol Piomarkers Prev, 9, 11, p. 1183-1191.
    • MacKness, B., Mackness, M. I., Durrington, P. N., Arrol, S., Evans, A. E., McMaster, D., Ferrieres, J., Ruidavets, J. B., Williams, N. R. & Howard, A. N., 2000, Paraoxonase activity in two healthy populations with differing rates of coronary heart disease, Eur J Clin Invest, 30, 1, p. 4-10.
    • MacLeod, S., Sinha, R., Kadlubar, F. F., Lang, N. P. Polymorphisms of CYP1A1 and GSTM1 influence the in vivo function of CYP1A2. Mutation Research 376(1-2): 135-142, 1997.
    • Maniatis T., Fritsch E. F., and Sambrock J., (1982) Molecular Cloning A Laboratory Manual, Cold Sorint Harbor Laboratory, Cold Springs Harbor, N.Y.
    • Marchand, L. L., Wilkinson, G. R. & Wilkons, L. R., 1999, Genetic and dietary predictors of CYP2E1 activity: a phenotyping study in Hawaii Japanese using chlorzoxazone, Cancer Epidemiol Biomarkers Prev, 8, 5, p. 495-500.
    • Matthias, C., Bockmuhl, U., Jahnke, V., Harries, L., Wolf, C. R., Jones, P. W., Alidersea, J. Worrall, S. F., Hand, P., Fryer, A. A. et al, The glutathione-S-transferase GSTP1 polymorphism: effects on susceptibility to oral/phryngeal and laryngeal carcinomas. Pharmacogenetics 8: 1-6, 1997.
    • Miki, M. & Satoli, K., 1999, Genetic risk factors for chronic obstructive pulmonary disease (COED), Nippon Rinsho, 57, 9, p. 1954-1958.
    • Mooney, L. A., Perera, F. P. Application of molecular epidemiology to lung cancer chemoprevention. Journal of Cellular Biochemistry Supplement 25:63-8, 1996.
    • Mooney, L. A., Santella, R. M., Covey, L., Jeffrey, A. M., Bigbee, W., Randall, M. C., Cooper, T. B., Ottman, R., Tsai, W.-Y., Wazneh, L. et al. Decline in DNA damage and other biomarkers in peripheral blood following smoking cessation. Cancer Epidemiolocical Biomarkers Prevention 4: 627-634, 1995.
    • Nakajima, T. & Aoyama, T., 2000, Polymorphism of drug-metabolizing enzymes in relation to individual susceptibility to industrial chemicals, Ind Health, 38, 2, p. 143-152.
    • Newton, C. R., Graham, A., Heptinstall, L. E., Powell, S. J., Summers, C., Kalsheker, N., Smith, J. C., and Markham, A. F. 1989. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Research. 17:2503-2516.
    • Nickerson, D. A., Kaiser, R., Lappin, S., Stewart, J., Hood, L., and Landegran, U. 1990. Automated DNA diagnostics using an ELISA-based oligonucleotides ligation assay. Proceedings of the National Academy of Science. USA 87:8923-8927.
    • Oliveira, J. R. & Zatz, M., 1999, The study of genetic polymorphisms related to serotonin in Alzheimer's disease: a new perspective in a heterogenic disorder, Braz J Med Biol Res, 32, 4, p. 463-467.
    • Ordovas, J. Y., Cupples, L. A., Corella, D., Otvos, J. D., Osgood, D., Martinez, A., Lahoz, C., Coitell, O., Wilson, P. W., Schaefer, E. J., 2000, Association of cholesteryl ester transfer protein-TaqIB polymorphism with variations in lipoprotein subclasses and coronary heart disease risk: the Framingham study, Arterioscler Thromb Vase Biol, 20, 5, p. 1323-1329.
    • Ota, N., Hunt, S. C., Nakajima, T., Suzuki, T., Hosoi, T., Orimo, H., Shirai, Y. & Emi, M., 1999, Linkage of interleukin 6 locus to human osteopenia by sibling pair analysis, Hum Genet, 105, 3, p. 253-257. P
    • Perera, F. P. Molecular epidemiology and prevention of cancer. Environmental Health Perspectives 103 Suppl 8: 233-6, 1995.
    • Perera, F. P. Biomarkers and Molecular Epidemiology of Cancer. Proceedings of the 9th International Symposium in Epidemiology in Occupational Health. National Institute for Occupational Safety and Health, Cincinnati, Ohio. PP 54-66, 1992.
    • Perera, F. P. Environment and cancer: Who are susceptible? Science 278: 1068-1073, 1997.
    • Perera, F. P. and Weinstein I. B. Molecular epidemiology: recent advances and future directions. carcinogenesis 21 (3): 517-524, 2000.
    • Pfutzer, R. H., Barmada, M. M., Brunskill, A. P., Finch, R., Hart, P. S., Neoptolemos, J., Furey, W. F. & Whitcomb, D. C., 2000, SPINK1/PSTI polymorphisms act as disease modifiers in familial and idiopathic chronic pancreatitis, Gastroenterology, 119, 3, p. 615-623.
    • PicoGreen dsDNA Quantitation Reagent and Kit Instruction, (1996) Molecular Probes, Eugene, Oreg.
    • Pluth, J. M., Nelson, D. O., Ramsey, M. J. & Tucker, J. D., 2000, The relationship between genotype and chromosome aberration frequencies in a normal adult population, Pharmacogenetics, 10, 4, p. 311-319.
    • Poolsup, N., Li Wan Po, A. & Knight, T. a., 2000, Pharmacogenetics and psychopharmacotherapy, J Clin Pharm Ther, 25, 3, p. 197-220.
    • Potter, J. D. Colorectal cancer: Molecules and Populations. Journal of the National Cancer Institute 91(11): 916-932, 1999.
    • Raknes, G., Fernandes Filho, J. A., Pandey, J. P., Myhr, K. M., Ulvestad, E., Nyland, H., Vedeler, C. A., 2000, IgG allotypes and subclasses in Norwegian patients with multiple sclerosis, J Neurol Sci, 175, 2, p. 111-115.
    • Rojas, M., Cascorbi, I., Alexandrov, K., Kried, E., Auburtin, G., Mayer, L., Kopp-Schnieder, A., Roots, I., and Bartsch, H. Modulation of benzo[a]pyrene diolepoxide-DNA adduct levels in human white blood cells by CYP1A1 GSTM1 and GSTT1 polymorphism, Carcinogenesis 21(1): 35-41, 2000.
    • Rosa-Rosa, L., Zimmermann, N., Bernstein, J. A., Rothenberg, M. E. & Khurana Hershey, G. K., 1999, The R576 IL-4 receptor alpha allele correlates with asthma severity, J Allergy Clin Immunol, 104, 5, p. 1008-1014.
    • Ryberg, D., Skaug, V., Hewer, A., Phillips, D. H., Harries, L. W., Wolf, C. R., Ogreid, D., Ulvik, A., Vu, P. Haugen, A. Genotypes of glutathione transferase M1 and P1 and their significance for lung DNA adduct levels and cancer risk. Carcinogenesis 18:1285-1289, 1997.
    • Rylchik, W., “Selection of Primers for Polymerase Chain Reaction”, Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, pp 31-40 (1993) Humana Press.
    • Saiki, R. K., Walsh, P. S., Levenson, C. H., and Erlich, H. A. 1989. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotides probes. Proceedings of the National Academy of Science. USA 86:6230-6234.
    • Salas, J., Jansen, S., Lopez-Miranda, J., Crdovas, J. M., Castro, P., Marin, C., Ostos, M. A., Bravo, M. D., Jimenez Pereperez, J., Blanco, A., Lopez Segura, F., Perez-Jimenez, P., 1998, The SstI polymorphism of the apolipoprotein C-III gene determines the insulin response to an oral-glucose tolerance test after consumption of a diet rich in saturated fats, An J Clin Nutr, 68, 2, p. 396-401.
    • Schabath, M. B., Spitz, M. R., Zhang, X., Delclos, G. L. & Wu, X., 2000, Genetic variants of myeloperoxidase and lung cancer risk, Carcinogenesis, 21, 6, p. 1163 1166.
    • Schena, M., 1999, DNA Microarrays “a practical approach”, ISBN, 0-19-963777-6, Oxford press, editor B. D. Hames
    • Shields, P. G., Harris, C. C. Cancer Risk and low-Penetrance Susceptibility Genes in Gene-Environment Interactions. Journal of Clinical Oncology 18(11) 2309-2315, 2000.
    • Sinha, R. & Caporaso, N., 1997, Heterocyclic amines, cytochrome P4501A2, and N-acetyltransferase: issues involved in incorporating putative genetic susceptibility markers into epidemiological studies, Ann Epidemios, 7, 5, p. 350-356.
    • Sinha, R., Chow, W. H., Kulldorff, M., Denobile, J., Butler, J., Garcia-Closas, M., Weil, R., Hoover, R. N., and Rothman, N. Well-done, Grilled Red Meat Increases the Risk of Colorectal Adenomas. Cancer Research 59: 4320-4324, 1999.
    • Smith, G., Stanley, L. A., Sim, E., Strange, R., and Wolf, C. R. Metabolic Polymorphisms and Cancer Susceptibility. Cancer Surveys 25: 27-65, 1995.
    • Syvanen, A. C., Sayantile, A., and Lukka, M. 1993. Identification of individuals by analysis of biallelic DNA markers, Using PCR and solid-phase minisequencing. American Journal of Human Genetics. 52:46-59.
    • Taningher, M., Malacarne, D., Izzotti, A., Ugolini, D. Parodi, S. Drug metabolism polymorphisms as modulators of cancer susceptibility. Mutation Research 436: 227-261, 1999.
    • Togo, A. V., Suspitsin, E. N., Grigoriev, M. Y., Ilyushik, E. S., Karpova, M. B., Hanson, K. P. & Imyanitov, E. N., 2000, L-myc polymorphism in cancer patients, healthy blood donors and elderly, tumor-free individuals in Russia, Int J Cancer, 85, 6, p. 747-750.
    • Tsai, M. Y., Welge, B. G., Hanson, N. Q., Bignell, M. K., Vessey, J., Schwichtenberg, K., Yang, F., Bullomer, E. E., Rasmussen, R. & Graham, K. J., 1999, Genetic causes of mild hyperhomocysteinemia in patients with premature occlusive coronary artery diseases, Atherosclerosis, 143, 1, p. 163-170.
    • Ulrich, C. M., Kampman, F., Bigler, J., Schwartz, S. M., Chen, C., Bostick, R., Fosdick, L., Bereford, S. A. A., Yasui, Y., and Potter, J. D. Colorectal adenomas and the C677T MTHFR polymorphism: evidence for gene-environment interaction? Cancer Epidemiological Biomarkers Prevention 8: 659-668, 1999.
    • Verlaan-de Vries, M., Bogaard, M. E., van den Elst, H., van Boom, J. H., van der Eb, A. J., and Bos, J. L. 1986. A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides. Gene. 50:313-320.
    • Vicente, V., Gonzalez-Conejero, R., Rivera, J. & Corral, J., 1999, The prothrombin gene variant 20210A in venous and arterial thromboembolism, Haematologica, 84, 4, p 356-362.
    • Vineis, P. Molecular Epidemiology: Low-dose Carcinogens and genetic susceptibility. International Journal of Cancer 71: 1-3, 1997.
    • Wallace, A. J., Humphries, S. E., Fisher, R. M., Mann, J. I , Chisholm, A., Sutherland, W. H, 2000, Genetic factors associated with response of LDL subfractions to change in the nature of dietary fat, Atherosclerosis, 149, 2, p 387-394.
    • Wallace, R. B., Johnson, M. J., Hirose, T., Miyake, T., Kawashima, E. H. and Itakura, K., 1981. The use of synthetic oligonucleotides as hybridisation probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA. Nucleic Acids Research. 9:879-894.
    • Wilkinson, R. J., Llewelyn, M., Toossi, Z., Patel, P., Pasvol, G., Lalvani, A., Wright, D., Latif, M. & Davidson, R. N., 2000, influence of vitamin D deficiency and vitamin D receptor polymorphisms on tuberculosis among Gujarati Asians in west London: a case-control study, Lancet, 355, 9204, p. 618-621.
    • World Cancer Research Fund (WCRF) Panel. (Potter, J. D. Chair) Diet, nutrition, and the prevention of cancer: a global perspective. Washington, D.C.: WCRF/American Institute of Cancer Research, 1997.
    • Wu, D. Y., and Wallace, R. B. 1989. The ligation amplification reaction (LAR)-amplification of specific DNA sequences using sequential rounds of template-dependent ligation. Genomics. 4:560-569.
    • Yamada, N., Yamaya, M., Okinaga, S., Nakayama, K., Sekizawa, K., Shibahara, S. & Sasaki, H., 2000, Microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with susceptibility to emphysema, Am J Hum Genet, 66, 1, p. 187-195.
    • Yershov, G., Barsky, V., et. al., 1996, Proc. Natl. Acad. Sci. USA, Genetics, Vol. 93, 4913-4918.
    • Yokota, M., Ichihara, S., Lin, T. L., Nakashima, N. & Yamada, Y., 2000, Association of a T29→C polymorphism of the transforming growth factor-beta1 gene with genetic susceptibility to myocardial infarction in Japanese, Circulation, 101, 24, p. 2783-2787.
    • Yokoyama, A., Muramatsu, T., Ohmori, T., Yokoyama, T., Okuyama, K., Takahashi, H., Hasegawa, Y., Higuchi, S., Maruyama, K., Shirakura, K., Ishii, H. Alcohol-related cancers and aldehyde dehydrogenase-2 in Japanese alcoholics. Carcinogenesis 19(8)1383-1387, 1998.
    • Zhang, Y., Coyne, M. Y., Will, S. G., Levenson, C. H., and Kawasaki, E. S. 1991. Single-base mutational analysis of cancer and genetic disease using membrane bound modified oligonucleotides. Nucleic Acids Research. 19: 3929-3933.
    • Zheng, W., Deitz, A. C., Campbell, D. R., Wen, W-Q., Cerhan, J. R., Sellers, T. A., Folsom, A. R., and Hein, D. W. N-acetyltransferase I genetic polymorphism, cigarette smoking, well-done meat intake, and breast cancer risk. Cancer Epidemiological Biomarkers Prevention 8: 233-239, 1999.
    • Zychma, M. J., Gumprecht, J., Zukowska-Szczechowska, E. & Grzeszczak, W., 1999, Polymorphisms in the genes encoding for human kinin receptors and the risk of end-stage renal failure: results of transmission/disequilibrium test. The End-Stage Renal Disease Study Group, J Am Soc Nephrol, 13, 10, p. 2120-2124.

Claims (12)

1. A computer assisted method of providing a personalized lifestyle advice plan for a human subject comprising:
(i) providing a first dataset on a data processing means, said first dataset comprising information correlating the presence of individual alleles at genetic loci with a lifestyle risk factor, wherein at least one allele of each genetic locus is known to be associated with increased or decreased disease susceptibility;
(ii) providing a second dataset on a data processing means, said second dataset comprising information matching each said risk factor with at least one lifestyle recommendation;
(iii) inputting a third dataset identifying alleles at one or more of the genetic loci of said first dataset of said human subject;
(iv) determining the risk factors associated with said alleles of said human subject using said first dataset;
(v) determining at least one appropriate lifestyle recommendation based on each identified risk factor from step (iv) using said second dataset; and
(vi) generating a personalized lifestyle advice plan based on said lifestyle recommendations.
2. The method according to the method of claim 1 wherein the personalised lifestyle advice plan includes recommended minimum and/or maximum amounts of food subtypes.
3. The method according to claim 1 wherein the method comprises the step of delivering the report to the client.
4. The method according to claim 3 wherein the plan is delivered via the Internet and accessible via a unique identifier code.
5. The method according to claim 4 wherein the plan comprises hyperlinks to one or more Web pages.
6. The method according to claim 1 wherein said first dataset comprises information relating to two or more alleles of one or more genetic loci of genes selected from the group comprising:
(a) genes that encode enzymes responsible for detoxification of xenobiotics in Phase I metabolism;
(b) genes that encode enzymes responsible for conjugation reactions in Phase II metabolism;
(c) genes that encode enzymes that help cells to combat oxidative stress;
(d) genes associated with micronutrient deficiency; and
(e) genes that encode enzymes responsible for metabolism of alcohol.
(f) genes that encode enzymes involved in lipid and/or cholesterol metabolism;
(g) genes that encode enzymes involved in clotting;
(h) genes that encode trypsin inhibitors;
(i) genes that encode enzymes related to susceptibility to metal toxicity;
(j) genes which encode proteins required for normal cellular metabolism and growth;
(k) genes which encoded HLA Class 2 molecules.
7. The method according to claim 6 wherein said first dataset comprises information relating to two or more alleles of one or more genetic loci of genes selected from each member of the group comprising:
(a) genes that encode enzymes responsible for detoxification of xenobiotics in Phase I metabolism;
(b) genes that encode enzymes responsible for conjugation reactions in Phase II metabolism;
(c) genes that encode enzymes that help cells to combat oxidative stress;
(d) genes associated with micronutrient deficiency; and
(e) genes that encode enzymes responsible for metabolism of alcohol.
(f) genes that encode enzymes involved in lipid and/or cholesterol metabolism;
(g) genes that encode enzymes involved in clotting;
(h) genes that encode trypsin inhibitors;
(i) genes that encode enzymes related to susceptibility to metal toxicity;
(j) genes which encode proteins required for normal cellular metabolism and growth;
(k) genes which encoded HLA Class 2 molecules.
8. The method according to claim 6 wherein said first dataset comprises information relating to two or more alleles of one or more genetic loci of genes encoding an enzyme selected from the group comprising: cytochrome P450 monooxygenase, N-acetyltransferase 1 , N-acetyltransferase 2, glutathione-S-transferase, manganese superoxide dismutase, 5,10-methylenetetrahydrofolatereductase and alcohol dehydrogenase 2.
9. The method according to claim 8 wherein said first dataset comprises information relating to two or more alleles of one or more genetic loci of each of the genes encoding cytochrome P450 monooxygenase, N-acetyltransferase 1, N-acetyltransferase 2, glutathione-S-transferase, manganese superoxide dismutase, 5,10-methylene-tetrahydrofolatereductase and alcohol dehydrogenase 2.
10. The method according to claim 1 including the step of determining the presence of individual alleles at one or more genetic loci of the DNA in a DNA sample of said human subject, and constructing the dataset used in step (iii) using results of said determination.
11. The method according to claim 10 wherein said presence of said individual alleles is determined by hybridisation with allele-specific oligonucleotides.
12. The method according to claim 11 wherein said allele specific oligonucleotides are selected from oligonucleotides each specific for one of the genes selected from the group comprising the CYP1A1 gene, the GSTμ gene, the GSTπ gene, the GSTθ gene, the NAT1 gene, the NAT2 gene, the MnSOD gene, the MTHFR gene and the ALDH2 gene.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090055217A1 (en) * 2007-08-23 2009-02-26 Grichnik Anthony J Method and system for identifying and communicating a health risk
US20090062621A1 (en) * 2007-08-31 2009-03-05 Grichnik Anthony J Method and system for prioritizing communication of a health risk
US20100169022A1 (en) * 2008-12-30 2010-07-01 Searete Llc, A Limited Liability Corporation Of The State Of Delaware Computational methods and systems for treatment in relation to modulation of CYP450 enzyme activity
US8315815B2 (en) 2008-12-30 2012-11-20 The Invention Science Fund I, Llc Computational methods and systems for suggesting modulators of CYP450 as treatment options
US8321151B2 (en) 2008-12-30 2012-11-27 The Invention Science Fund I, Llc Computational methods and systems for treatment in relation to modulation of CYP450 enzyme activity
US8676608B2 (en) 2007-02-14 2014-03-18 Genelex Corporation Genetic data analysis and database tools
US10210312B2 (en) 2013-02-03 2019-02-19 Youscript Inc. Systems and methods for quantification and presentation of medical risk arising from unknown factors
CN109430731A (en) * 2018-10-31 2019-03-08 河北农业大学 A kind of formula and processing technology of high-quality meat mincing recombination mutton dried meat

Families Citing this family (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1255867A2 (en) 2000-02-18 2002-11-13 Science Applications International Corporation Method for detecting a biological entity in a sample
US7455966B1 (en) 2000-05-01 2008-11-25 Science Applications International Corporation System and method for detecting a biological entity in a water sample
US7054758B2 (en) * 2001-01-30 2006-05-30 Sciona Limited Computer-assisted means for assessing lifestyle risk factors
US7874841B1 (en) * 2001-08-08 2011-01-25 Lycas Geoffrey S Method and apparatus for personal awareness and growth
WO2004072887A2 (en) * 2003-02-11 2004-08-26 Sciona Limited Computer-assisted means for assessing lifestyle risk factors of an individual
WO2005008555A1 (en) * 2003-07-11 2005-01-27 Sciona Limited Computer-assisted means for providing personalised healthcare products
US7769626B2 (en) 2003-08-25 2010-08-03 Tom Reynolds Determining strategies for increasing loyalty of a population to an entity
US8301482B2 (en) * 2003-08-25 2012-10-30 Tom Reynolds Determining strategies for increasing loyalty of a population to an entity
AU2004273813A1 (en) 2003-09-12 2005-03-31 Access Business Group International Llc Program for regulating health conditions
JP5235274B2 (en) * 2003-10-15 2013-07-10 株式会社サインポスト Method and apparatus for determining disease risk
EP2944957B1 (en) 2003-12-17 2018-08-22 Fred Hutchinson Cancer Research Center Methods and materials for canine breed identification
US7306562B1 (en) 2004-04-23 2007-12-11 Medical Software, Llc Medical risk assessment method and program product
CN101061384A (en) * 2004-10-21 2007-10-24 拜尔保健有限公司 Sensor-dispensing device and mechanism for extracting sensor
US8076065B2 (en) * 2005-05-19 2011-12-13 Synergenz Bioscience Limited Methods and compositions for assessment of pulmonary function and disorders
WO2006123943A1 (en) * 2005-05-20 2006-11-23 Synergenz Bioscience Limited Methods of analysis of polymorphisms and uses thereof
JP2007034845A (en) * 2005-07-28 2007-02-08 Genesis Healthcare Kk Health support information provision method, health support information provision device and health support information provision program
US20070038487A1 (en) * 2005-08-15 2007-02-15 Mccarthy William K Senior assisted living eligibility system
GB2430072B (en) * 2005-08-18 2008-02-13 Mark Jones Teaching apparatus
KR101068605B1 (en) * 2006-11-30 2011-09-30 아크레이 가부시키가이샤 Primer set for amplification of nat2 gene, reagent for amplification of nat2 gene comprising the same, and use of the same
US7540841B2 (en) * 2006-12-15 2009-06-02 General Electric Company System and method for in-situ mental health monitoring and therapy administration
US20080228698A1 (en) 2007-03-16 2008-09-18 Expanse Networks, Inc. Creation of Attribute Combination Databases
JP4410269B2 (en) * 2007-03-28 2010-02-03 株式会社東芝 Nucleic acid primer set, kit for detecting genotype of N-acetyltransferase 2 (NAT2), and detection method using the primer set
US20090043752A1 (en) * 2007-08-08 2009-02-12 Expanse Networks, Inc. Predicting Side Effect Attributes
US20090092693A1 (en) * 2007-10-05 2009-04-09 Mady Attila Platelet manipulation to prevent and treat endovascular disease and its sequelae, to prevent and treat arrhythmias and to prevent malignancy
EP2210226A4 (en) * 2007-10-12 2013-11-06 Patientslikeme Inc Self-improving method of using online communities to predict health-related outcomes
WO2010019056A1 (en) * 2008-08-11 2010-02-18 Macfarlane Engel & Associates Improved treatment method and device
US8200509B2 (en) 2008-09-10 2012-06-12 Expanse Networks, Inc. Masked data record access
US20100063835A1 (en) * 2008-09-10 2010-03-11 Expanse Networks, Inc. Method for Secure Mobile Healthcare Selection
US20100063865A1 (en) * 2008-09-10 2010-03-11 Expanse Networks, Inc. Masked Data Provider Profiling
US7917438B2 (en) * 2008-09-10 2011-03-29 Expanse Networks, Inc. System for secure mobile healthcare selection
US20100076988A1 (en) * 2008-09-10 2010-03-25 Expanse Networks, Inc. Masked Data Service Profiling
US20100070292A1 (en) * 2008-09-10 2010-03-18 Expanse Networks, Inc. Masked Data Transaction Database
CN102300578A (en) * 2008-12-01 2011-12-28 延寿有限责任公司 Methods And Compositions For Altering Health, Wellbeing, And Lifespan
US20100169313A1 (en) * 2008-12-30 2010-07-01 Expanse Networks, Inc. Pangenetic Web Item Feedback System
US20100169338A1 (en) * 2008-12-30 2010-07-01 Expanse Networks, Inc. Pangenetic Web Search System
US8255403B2 (en) 2008-12-30 2012-08-28 Expanse Networks, Inc. Pangenetic web satisfaction prediction system
US8386519B2 (en) 2008-12-30 2013-02-26 Expanse Networks, Inc. Pangenetic web item recommendation system
US8108406B2 (en) 2008-12-30 2012-01-31 Expanse Networks, Inc. Pangenetic web user behavior prediction system
US8463554B2 (en) 2008-12-31 2013-06-11 23Andme, Inc. Finding relatives in a database
CA2758481C (en) 2009-04-30 2018-03-20 Patientslikeme, Inc. Systems and methods for encouragement of data submission in online communities
EP2577538A1 (en) 2010-05-25 2013-04-10 The Regents of the University of California Bambam: parallel comparative analysis of high-throughput sequencing data
US9646134B2 (en) 2010-05-25 2017-05-09 The Regents Of The University Of California Bambam: parallel comparative analysis of high-throughput sequencing data
US20130138447A1 (en) * 2010-07-19 2013-05-30 Pathway Genomics Genetic based health management apparatus and methods
US20130151270A1 (en) * 2011-12-12 2013-06-13 Pathway Genomics Genetic Based Health Management Systems for Weight and Nutrition Control
US8762167B2 (en) * 2010-07-27 2014-06-24 Segterra Inc. Methods and systems for generation of personalized health plans
US8688477B1 (en) 2010-09-17 2014-04-01 National Assoc. Of Boards Of Pharmacy Method, system, and computer program product for determining a narcotics use indicator
WO2012138002A1 (en) * 2011-04-08 2012-10-11 Chang Man-Ik Mentor service terminal, mentor service method, and recording medium on which the method is recorded
USD731510S1 (en) 2012-06-06 2015-06-09 Omicia, Inc. Display screen or portion thereof with a graphical user interface
US9996889B2 (en) 2012-10-01 2018-06-12 International Business Machines Corporation Identifying group and individual-level risk factors via risk-driven patient stratification
US9974512B2 (en) 2014-03-13 2018-05-22 Convergence Medical, Llc Method, system, and computer program product for determining a patient radiation and diagnostic study score
EP3507722A4 (en) 2016-09-02 2020-03-18 FutureVault Inc. Automated document filing and processing methods and systems
WO2018039774A1 (en) 2016-09-02 2018-03-08 FutureVault Inc. Systems and methods for sharing documents
EP3507721B1 (en) 2016-09-02 2022-11-23 FutureVault Inc. Real-time document filtering systems and methods
US20190104758A1 (en) * 2017-10-09 2019-04-11 Anuthep Benja-Athon Preemptions of Chronic Diseases
US11894139B1 (en) 2018-12-03 2024-02-06 Patientslikeme Llc Disease spectrum classification
USD923032S1 (en) * 2019-06-03 2021-06-22 Abb Power Grids Switzerland Ag Display screen with an animated graphical user interface for power control system
WO2022016271A1 (en) * 2020-07-23 2022-01-27 The DNA Company Inc. Systems and methods for determining a physiological profile using genetic information
US11735310B2 (en) 2020-12-29 2023-08-22 Kpn Innovations, Llc. Systems and methods for generating a parasitic infection nutrition program

Citations (65)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4218738A (en) * 1978-05-05 1980-08-19 International Business Machines Corporation Method for authenticating the identity of a user of an information system
US4358535A (en) * 1980-12-08 1982-11-09 Board Of Regents Of The University Of Washington Specific DNA probes in diagnostic microbiology
US4513023A (en) * 1983-02-23 1985-04-23 Union Carbide Corporation Method of constructing thin electroluminescent lamp assemblies
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4816653A (en) * 1986-05-16 1989-03-28 American Telephone And Telegraph Company Security file system for a portable data carrier
US4959860A (en) * 1989-02-07 1990-09-25 Compaq Computer Corporation Power-on password functions for computer system
US5012514A (en) * 1990-06-26 1991-04-30 Paul Renton Hard drive security system
US5307263A (en) * 1992-11-17 1994-04-26 Raya Systems, Inc. Modular microprocessor-based health monitoring system
US5508199A (en) * 1989-01-03 1996-04-16 The United States Of America As Represented By The Department Of Health And Human Services P450db1 clones for identifying humans with genetic defect in drug metabolism
US5545527A (en) * 1994-07-08 1996-08-13 Visible Genetics Inc. Method for testing for mutations in DNA from a patient sample
US5550021A (en) * 1990-02-07 1996-08-27 Board Of Regents, The University Of Texas System Allelic diagnosis of susceptibility to compulsive disorder
US5569212A (en) * 1994-07-22 1996-10-29 Raya Systems, Inc. Apparatus for electrically determining injection doses in syringes
US5585232A (en) * 1992-07-06 1996-12-17 President And Fellows Of Harvard College Methods and diagnostic kits for determining toxicity utilizing E. coli stress promoters fused to reporter genes
US5601435A (en) * 1994-11-04 1997-02-11 Intercare Method and apparatus for interactively monitoring a physiological condition and for interactively providing health related information
US5604803A (en) * 1994-06-03 1997-02-18 Sun Microsystems, Inc. Method and apparatus for secure remote authentication in a public network
US5648482A (en) * 1990-06-22 1997-07-15 Hoffmann-La Roche Inc. Primers targeted to CYP2D6 gene for detecting poor metabolizers of drugs
US5678571A (en) * 1994-05-23 1997-10-21 Raya Systems, Inc. Method for treating medical conditions using a microprocessor-based video game
US5686246A (en) * 1995-08-03 1997-11-11 Kornman; Kenneth S. Detecting genetic predisposition to periodontal disease
US5708825A (en) * 1995-05-26 1998-01-13 Iconovex Corporation Automatic summary page creation and hyperlink generation
US5782814A (en) * 1994-07-22 1998-07-21 Raya Systems, Inc. Apparatus for determining and recording injection doses in syringes using electrical inductance
US5794219A (en) * 1996-02-20 1998-08-11 Health Hero Network, Inc. Method of conducting an on-line auction with bid pooling
US5822737A (en) * 1996-02-05 1998-10-13 Ogram; Mark E. Financial transaction system
US5828943A (en) * 1994-04-26 1998-10-27 Health Hero Network, Inc. Modular microprocessor-based diagnostic measurement apparatus and method for psychological conditions
US5832448A (en) * 1996-10-16 1998-11-03 Health Hero Network Multiple patient monitoring system for proactive health management
US5834044A (en) * 1995-06-07 1998-11-10 Mars, Incorporated Method of making a health food product containing anti-oxidants
US5837832A (en) * 1993-06-25 1998-11-17 Affymetrix, Inc. Arrays of nucleic acid probes on biological chips
US5844108A (en) * 1990-06-22 1998-12-01 Roche Molecular Systems, Inc. Primers targeted to NAT2 gene for detection of poor metabolizers of drugs
US5858659A (en) * 1995-11-29 1999-01-12 Affymetrix, Inc. Polymorphism detection
US5879163A (en) * 1996-06-24 1999-03-09 Health Hero Network, Inc. On-line health education and feedback system using motivational driver profile coding and automated content fulfillment
US5891622A (en) * 1996-09-30 1999-04-06 Oxford Biomedical Research, Inc. Assessment of oxidative stress in vivo
US5897493A (en) * 1997-03-28 1999-04-27 Health Hero Network, Inc. Monitoring system for remotely querying individuals
US5899855A (en) * 1992-11-17 1999-05-04 Health Hero Network, Inc. Modular microprocessor-based health monitoring system
US5908467A (en) * 1996-05-31 1999-06-01 International Business Machines Corporation System and method for displaying file parameters
US5913310A (en) * 1994-05-23 1999-06-22 Health Hero Network, Inc. Method for diagnosis and treatment of psychological and emotional disorders using a microprocessor-based video game
US5920859A (en) * 1997-02-05 1999-07-06 Idd Enterprises, L.P. Hypertext document retrieval system and method
US5920871A (en) * 1989-06-02 1999-07-06 Macri; Vincent J. Method of operating a general purpose digital computer for use in controlling the procedures and managing the data and information used in the operation of clinical (medical) testing and screening laboratories
US5918603A (en) * 1994-05-23 1999-07-06 Health Hero Network, Inc. Method for treating medical conditions using a microprocessor-based video game
US5933136A (en) * 1996-12-23 1999-08-03 Health Hero Network, Inc. Network media access control system for encouraging patient compliance with a treatment plan
US5940801A (en) * 1994-04-26 1999-08-17 Health Hero Network, Inc. Modular microprocessor-based diagnostic measurement apparatus and method for psychological conditions
US5950634A (en) * 1996-11-29 1999-09-14 Nikken Foods Co., Ltd. Method for assessing oxidative stress and its control in humans
US5951300A (en) * 1997-03-10 1999-09-14 Health Hero Network Online system and method for providing composite entertainment and health information
US5955111A (en) * 1996-10-08 1999-09-21 Hartford Hospital Methods and compositions for inducing production of stress proteins
US5956501A (en) * 1997-01-10 1999-09-21 Health Hero Network, Inc. Disease simulation system and method
US5985559A (en) * 1997-04-30 1999-11-16 Health Hero Network System and method for preventing, diagnosing, and treating genetic and pathogen-caused disease
US5997476A (en) * 1997-03-28 1999-12-07 Health Hero Network, Inc. Networked system for interactive communication and remote monitoring of individuals
US6004744A (en) * 1991-03-05 1999-12-21 Molecular Tool, Inc. Method for determining nucleotide identity through extension of immobilized primer
US6013431A (en) * 1990-02-16 2000-01-11 Molecular Tool, Inc. Method for determining specific nucleotide variations by primer extension in the presence of mixture of labeled nucleotides and terminators
US6023686A (en) * 1996-02-20 2000-02-08 Health Hero Network Method for conducting an on-line bidding session with bid pooling
US6045996A (en) * 1993-10-26 2000-04-04 Affymetrix, Inc. Hybridization assays on oligonucleotide arrays
US6046317A (en) * 1997-12-19 2000-04-04 Hormos Medical Oy, Ltd. DNA molecule encoding a mutant prepro-neuropeptide Y, a mutant signal peptide, and uses thereof
US6048689A (en) * 1997-03-28 2000-04-11 Gene Logic, Inc. Method for identifying variations in polynucleotide sequences
US6068615A (en) * 1994-07-22 2000-05-30 Health Hero Network, Inc. Inductance-based dose measurement in syringes
US6074821A (en) * 1994-05-26 2000-06-13 Mcgill University CDNA for human methylenetetrahydrofolate reductase
US6134552A (en) * 1997-10-07 2000-10-17 Sap Aktiengesellschaft Knowledge provider with logical hyperlinks
US6133039A (en) * 1998-02-09 2000-10-17 Washinton University In vivo method for determination of oxidative stress
US6167386A (en) * 1998-06-05 2000-12-26 Health Hero Network, Inc. Method for conducting an on-line bidding session with bid pooling
US6168563B1 (en) * 1992-11-17 2001-01-02 Health Hero Network, Inc. Remote health monitoring and maintenance system
US6186145B1 (en) * 1994-05-23 2001-02-13 Health Hero Network, Inc. Method for diagnosis and treatment of psychological and emotional conditions using a microprocessor-based virtual reality simulator
US6210272B1 (en) * 1997-12-22 2001-04-03 Health Hero Network, Inc. Multi-player interactive electronic game for health education
US6240393B1 (en) * 1998-06-05 2001-05-29 Health Pro Network, Inc. Aggregating and pooling weight loss information in a communication system with feedback
US6330426B2 (en) * 1994-05-23 2001-12-11 Stephen J. Brown System and method for remote education using a memory card
US6334778B1 (en) * 1994-04-26 2002-01-01 Health Hero Network, Inc. Remote psychological diagnosis and monitoring system
US20020146698A1 (en) * 2001-04-05 2002-10-10 Genelink, Inc. Kits and methods for assessing oxidative stress
US20030073612A1 (en) * 2001-04-05 2003-04-17 Genelink, Incorporated Kits and methods for assessing skin health

Family Cites Families (81)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3616923A1 (en) * 1986-05-20 1987-11-26 Nattermann A & Cie NEW PHARMACEUTICAL USE OF 2-PHENYL-1,2-BENZISOSELENAZOLE-3 (2H) -ON
US5032501A (en) * 1989-02-10 1991-07-16 Virginia Mason Research Center DNA probes to vntr loci
US5310653A (en) 1989-10-24 1994-05-10 Board Of Regents, The University Of Texas System Tumor marker protein and antibodies thereto for cancer risk assessment or diagnosis
US5773215A (en) 1989-10-24 1998-06-30 Board Of Regents, The University Of Texas System Tumor marker protein for cancer risk assessment
US5376526A (en) 1992-05-06 1994-12-27 The Board Of Trustees Of The Leland Stanford Junior University Genomic mismatch scanning
US6048837A (en) 1994-08-17 2000-04-11 The Rockefeller University OB polypeptides as modulators of body weight
US6350730B1 (en) 1994-08-17 2002-02-26 The Rockefeller University OB polypeptides and modified forms as modulators of body weight
US6001968A (en) 1994-08-17 1999-12-14 The Rockefeller University OB polypeptides, modified forms and compositions
US6429290B1 (en) 1994-08-17 2002-08-06 The Rockefeller University OB polypeptides, modified forms and derivatives
US6471956B1 (en) 1994-08-17 2002-10-29 The Rockefeller University Ob polypeptides, modified forms and compositions thereto
US6124448A (en) 1994-08-17 2000-09-26 The Rockfeller University Nucleic acid primers and probes for the mammalian OB gene
US6124439A (en) 1994-08-17 2000-09-26 The Rockefeller University OB polypeptide antibodies and method of making
US20020107211A1 (en) 1995-06-07 2002-08-08 The Rockefeller University Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof
US6309853B1 (en) 1994-08-17 2001-10-30 The Rockfeller University Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof
US5849989A (en) 1994-10-07 1998-12-15 Ontogeny, Inc. Insulin promoter factor, and uses related thereto
CA2251785A1 (en) * 1995-11-15 1997-05-22 Genelink, Inc. Non-invasive identification system
US5936810A (en) 1996-02-14 1999-08-10 Hitachi, Ltd. Magnetoresistive effect head
CA2290947A1 (en) 1996-05-28 1997-12-04 Fred I. Chasalow Phosphocholinate cardenolides
US6040155A (en) 1996-08-28 2000-03-21 Kay; John Multifunctional protein and DNA sequence encoding same
US6274310B1 (en) 1997-06-24 2001-08-14 The General Hospital Corporation Compositions and methods for detecting pancreatic disease
US6524814B1 (en) 1997-08-28 2003-02-25 Phairson Medical, Inc. Enzyme and DNA sequence encoding krill-derived multifunctional protein
US6284219B1 (en) 1998-06-30 2001-09-04 Phenome Sciences Inc. In vivo determination of metabolic function for use in therapy management
US6664062B1 (en) 1998-07-20 2003-12-16 Nuvelo, Inc. Thymidylate synthase gene sequence variances having utility in determining the treatment of disease
US20020039990A1 (en) 1998-07-20 2002-04-04 Stanton Vincent P. Gene sequence variances in genes related to folate metabolism having utility in determining the treatment of disease
US6410508B1 (en) 1998-10-07 2002-06-25 Med College Georgia Res Inst Glucose-dependent insulinotropic peptide for use as an osteotropic hormone
US20020102581A1 (en) 1999-02-19 2002-08-01 Hageman Gregory S. Diagnostics and therapeutics for ocular disorders
US20020123061A1 (en) 1999-03-01 2002-09-05 Florrye Cleveland Multiple use non-species specific genetic material collection and storage kit
IL131707A0 (en) * 1999-05-31 2001-03-19 Yissum Res Dev Co Acetylcholinesterase-derived peptides and uses thereof
KR20020016833A (en) 1999-06-15 2002-03-06 뉴트리-로직스, 인크. Nutrient Formulations for Disease Reduction, and Related Treatment and Component Screening Methods
US6585995B1 (en) 1999-09-21 2003-07-01 Hanson Stephen R Methods and compositions for treating platelet-related disorders
US20020048763A1 (en) 2000-02-04 2002-04-25 Penn Sharron Gaynor Human genome-derived single exon nucleic acid probes useful for gene expression analysis
CN100384875C (en) * 2000-02-25 2008-04-30 牛津格莱克科学(英国)有限公司 Protein
EP1266225A2 (en) * 2000-03-20 2002-12-18 Oxford GlycoSciences (UK) Limited Diagnosis and treatment of breast cancer
AU2001253597A1 (en) * 2000-04-12 2001-10-30 Minerva Biotechnologies Corporation Treatment of neurodegenerative disease
US20020052761A1 (en) 2000-05-11 2002-05-02 Fey Christopher T. Method and system for genetic screening data collection, analysis, report generation and access
US6475736B1 (en) 2000-05-23 2002-11-05 Variagenics, Inc. Methods for genetic analysis of DNA using biased amplification of polymorphic sites
JP4310599B2 (en) * 2000-07-05 2009-08-12 東洋紡績株式会社 Method for detecting nucleotide polymorphisms
AU2001273233A1 (en) 2000-07-06 2002-01-21 Deltagen, Inc. Transgenic mice containing cx2 gene disruptions
US20030077604A1 (en) * 2000-10-27 2003-04-24 Yongming Sun Compositions and methods relating to breast specific genes and proteins
AU2002239409A1 (en) * 2000-11-20 2003-11-17 Diadexus, Inc. Compositions and methods relating to colon specific genes and proteins
EP1349934A2 (en) 2000-11-20 2003-10-08 Diadexus, Inc. Compositions and methods relating to breast specific genes and proteins
EP1407016A2 (en) 2000-11-20 2004-04-14 Diadexus, Inc. Compositions and methods relating to breast specific genes and proteins
AU2002239538A1 (en) 2000-12-06 2002-06-18 Deltagen, Inc. Transgenic mice containing glucagon receptor gene disruptions
US20030113727A1 (en) * 2000-12-06 2003-06-19 Girn Kanwaljit Singh Family history based genetic screening method and apparatus
US20030069199A1 (en) * 2000-12-07 2003-04-10 Hanan Polansky Treatment methods based on microcompetition for a limiting GABP complex
US20030068616A1 (en) * 2000-12-07 2003-04-10 Hanan Polansky Drug discovery assays based on microcompetition for a limiting GABP complex
US20030104358A1 (en) * 2000-12-07 2003-06-05 Hanan Polansky Diagnosis methods based on microcompetition for a limiting GABP complex
US20020116731A1 (en) 2000-12-11 2002-08-22 Allen Keith D. Transgenic mice containing retinoid X receptor interacting protein gene disruptions
US20020137203A1 (en) 2000-12-13 2002-09-26 Wiles Michael V. Transgenic mice containing alpha-endosulfine gene disruptions
US7054758B2 (en) * 2001-01-30 2006-05-30 Sciona Limited Computer-assisted means for assessing lifestyle risk factors
WO2002064611A1 (en) * 2001-02-13 2002-08-22 Diadexus, Inc. Compositions and methods relating to breast specific genes and proteins
WO2002066605A2 (en) * 2001-02-15 2002-08-29 Diadexus, Inc. Compositions and methods relating to breast specific genes and proteins
WO2002070542A2 (en) * 2001-03-02 2002-09-12 Caritas St. Elizabeth's Medical Center Of Boston, Inc. Band 3 antigenic peptides, malaria polypeptides and uses thereof
US6756063B2 (en) * 2001-03-29 2004-06-29 Zoltan Laboratories, Llc Methods and compositions for the treatment of human and animal cancers
WO2002088753A1 (en) * 2001-04-26 2002-11-07 Mcgill University Individualization of therapy with erectile dysfunction agents
US20030077222A1 (en) * 2001-05-07 2003-04-24 Mcgill University Individualization of therapy with analgesics
WO2002092002A2 (en) * 2001-05-11 2002-11-21 The Burnham Institute Screening, diagnostic and therapeutic methods relating to riz
CA2448253A1 (en) * 2001-05-25 2002-11-28 Genset S.A. Human cdnas and proteins and uses thereof
US6867189B2 (en) * 2001-07-26 2005-03-15 Genset S.A. Use of adipsin/complement factor D in the treatment of metabolic related disorders
US7229774B2 (en) * 2001-08-02 2007-06-12 Regents Of The University Of Michigan Expression profile of prostate cancer
US20030040002A1 (en) * 2001-08-08 2003-02-27 Ledley Fred David Method for providing current assessments of genetic risk
US20030092013A1 (en) * 2001-08-16 2003-05-15 Vitivity, Inc. Diagnosis and treatment of vascular disease
US20030096248A1 (en) * 2001-09-04 2003-05-22 Vitivity, Inc. Diagnosis and treatment of vascular disease
US20030099958A1 (en) * 2001-09-05 2003-05-29 Vitivity, Inc. Diagnosis and treatment of vascular disease
US20030121067A1 (en) * 2001-09-24 2003-06-26 Brennan Thomas J. V1a arginine vasopressin receptor disruptions, compositions and methods relating thereto
US20030115617A1 (en) * 2001-09-24 2003-06-19 Allen Keith D. Thyrotropin releasing hormone receptor disruptions, compositions and methods relating thereto
US20030099957A1 (en) * 2001-09-28 2003-05-29 Vitivity, Inc. Diagnosis and treatment of vascular disease
US20030087244A1 (en) * 2001-10-09 2003-05-08 Vitivity, Inc Diagnosis and treatment of vascular disease
CA2470431A1 (en) * 2001-12-17 2003-06-26 Metabolex, Inc. Compositions and methods for diagnosing and treating diabetes, insulin resistance and dyslipidemia
JP2005514931A (en) * 2002-01-04 2005-05-26 メタボレックス インコーポレーティッド Nucleic acid encoding a G protein-coupled receptor involved in islet cell signaling
US20040014097A1 (en) * 2002-05-06 2004-01-22 Mcglennen Ronald C. Genetic test apparatus and method
JP2006507801A (en) * 2002-06-10 2006-03-09 メタボレックス インコーポレーティッド Methods and compositions for the treatment and diagnosis of diabetes
US20040106647A1 (en) * 2002-06-28 2004-06-03 Schneider Michael D. Modulators of Cdk9 as a therapeutic target in cardiac hypertrophy
WO2004002226A1 (en) * 2002-06-28 2004-01-08 Baylor College Of Medicine Modulators of cdk9 as a therapeutic target in cardiac hypertrophy
US20040063120A1 (en) * 2002-07-10 2004-04-01 The Regents Of The University Of Michigan Expression profile of lung cancer
US20040023225A1 (en) * 2002-08-05 2004-02-05 Vitivity, Inc. Methods and compositions for identifying risk factors for abnormal lipid levels and the diseases and disorders associated therewith
US20040115701A1 (en) * 2002-08-30 2004-06-17 Comings David E Method for risk assessment for polygenic disorders
US20040043389A1 (en) * 2002-09-04 2004-03-04 Vitivity, Inc. Methods and compositions for identifying risk factors for abnormal lipid levels and the diseases and disorders associated therewith
EP1547009A1 (en) * 2002-09-20 2005-06-29 Board Of Regents The University Of Texas System Computer program products, systems and methods for information discovery and relational analyses
US20040115708A1 (en) * 2002-10-28 2004-06-17 Ardies C. Murray Method of screening agents for the treatment and prevention of cancer and cachexia and the new use of specific agents for the treatment and prevention of cancer and cachexia
US20040110241A1 (en) * 2002-12-06 2004-06-10 Segal Mark S. Materials and methods for monitoring vascular endothelial function

Patent Citations (71)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4218738A (en) * 1978-05-05 1980-08-19 International Business Machines Corporation Method for authenticating the identity of a user of an information system
US4358535A (en) * 1980-12-08 1982-11-09 Board Of Regents Of The University Of Washington Specific DNA probes in diagnostic microbiology
US4358535B1 (en) * 1980-12-08 1986-05-13
US4513023A (en) * 1983-02-23 1985-04-23 Union Carbide Corporation Method of constructing thin electroluminescent lamp assemblies
US4683202B1 (en) * 1985-03-28 1990-11-27 Cetus Corp
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195B1 (en) * 1986-01-30 1990-11-27 Cetus Corp
US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4816653A (en) * 1986-05-16 1989-03-28 American Telephone And Telegraph Company Security file system for a portable data carrier
US5508199A (en) * 1989-01-03 1996-04-16 The United States Of America As Represented By The Department Of Health And Human Services P450db1 clones for identifying humans with genetic defect in drug metabolism
US4959860A (en) * 1989-02-07 1990-09-25 Compaq Computer Corporation Power-on password functions for computer system
US5920871A (en) * 1989-06-02 1999-07-06 Macri; Vincent J. Method of operating a general purpose digital computer for use in controlling the procedures and managing the data and information used in the operation of clinical (medical) testing and screening laboratories
US5550021A (en) * 1990-02-07 1996-08-27 Board Of Regents, The University Of Texas System Allelic diagnosis of susceptibility to compulsive disorder
US6013431A (en) * 1990-02-16 2000-01-11 Molecular Tool, Inc. Method for determining specific nucleotide variations by primer extension in the presence of mixture of labeled nucleotides and terminators
US5844108A (en) * 1990-06-22 1998-12-01 Roche Molecular Systems, Inc. Primers targeted to NAT2 gene for detection of poor metabolizers of drugs
US5648482A (en) * 1990-06-22 1997-07-15 Hoffmann-La Roche Inc. Primers targeted to CYP2D6 gene for detecting poor metabolizers of drugs
US5012514A (en) * 1990-06-26 1991-04-30 Paul Renton Hard drive security system
US6004744A (en) * 1991-03-05 1999-12-21 Molecular Tool, Inc. Method for determining nucleotide identity through extension of immobilized primer
US5585232A (en) * 1992-07-06 1996-12-17 President And Fellows Of Harvard College Methods and diagnostic kits for determining toxicity utilizing E. coli stress promoters fused to reporter genes
US5899855A (en) * 1992-11-17 1999-05-04 Health Hero Network, Inc. Modular microprocessor-based health monitoring system
US5307263A (en) * 1992-11-17 1994-04-26 Raya Systems, Inc. Modular microprocessor-based health monitoring system
US6168563B1 (en) * 1992-11-17 2001-01-02 Health Hero Network, Inc. Remote health monitoring and maintenance system
US5837832A (en) * 1993-06-25 1998-11-17 Affymetrix, Inc. Arrays of nucleic acid probes on biological chips
US6045996A (en) * 1993-10-26 2000-04-04 Affymetrix, Inc. Hybridization assays on oligonucleotide arrays
US5828943A (en) * 1994-04-26 1998-10-27 Health Hero Network, Inc. Modular microprocessor-based diagnostic measurement apparatus and method for psychological conditions
US6334778B1 (en) * 1994-04-26 2002-01-01 Health Hero Network, Inc. Remote psychological diagnosis and monitoring system
US5940801A (en) * 1994-04-26 1999-08-17 Health Hero Network, Inc. Modular microprocessor-based diagnostic measurement apparatus and method for psychological conditions
US5918603A (en) * 1994-05-23 1999-07-06 Health Hero Network, Inc. Method for treating medical conditions using a microprocessor-based video game
US5678571A (en) * 1994-05-23 1997-10-21 Raya Systems, Inc. Method for treating medical conditions using a microprocessor-based video game
US6186145B1 (en) * 1994-05-23 2001-02-13 Health Hero Network, Inc. Method for diagnosis and treatment of psychological and emotional conditions using a microprocessor-based virtual reality simulator
US6330426B2 (en) * 1994-05-23 2001-12-11 Stephen J. Brown System and method for remote education using a memory card
US5913310A (en) * 1994-05-23 1999-06-22 Health Hero Network, Inc. Method for diagnosis and treatment of psychological and emotional disorders using a microprocessor-based video game
US6074821A (en) * 1994-05-26 2000-06-13 Mcgill University CDNA for human methylenetetrahydrofolate reductase
US5604803A (en) * 1994-06-03 1997-02-18 Sun Microsystems, Inc. Method and apparatus for secure remote authentication in a public network
US5545527A (en) * 1994-07-08 1996-08-13 Visible Genetics Inc. Method for testing for mutations in DNA from a patient sample
US5782814A (en) * 1994-07-22 1998-07-21 Raya Systems, Inc. Apparatus for determining and recording injection doses in syringes using electrical inductance
US5569212A (en) * 1994-07-22 1996-10-29 Raya Systems, Inc. Apparatus for electrically determining injection doses in syringes
US6068615A (en) * 1994-07-22 2000-05-30 Health Hero Network, Inc. Inductance-based dose measurement in syringes
US5601435A (en) * 1994-11-04 1997-02-11 Intercare Method and apparatus for interactively monitoring a physiological condition and for interactively providing health related information
US6144837A (en) * 1994-11-04 2000-11-07 Health Hero Network, Inc. Method and apparatus for interactively monitoring a physiological condition and for interactively providing health-related information
US5708825A (en) * 1995-05-26 1998-01-13 Iconovex Corporation Automatic summary page creation and hyperlink generation
US5834044A (en) * 1995-06-07 1998-11-10 Mars, Incorporated Method of making a health food product containing anti-oxidants
US5686246A (en) * 1995-08-03 1997-11-11 Kornman; Kenneth S. Detecting genetic predisposition to periodontal disease
US5858659A (en) * 1995-11-29 1999-01-12 Affymetrix, Inc. Polymorphism detection
US5822737A (en) * 1996-02-05 1998-10-13 Ogram; Mark E. Financial transaction system
US5794219A (en) * 1996-02-20 1998-08-11 Health Hero Network, Inc. Method of conducting an on-line auction with bid pooling
US6023686A (en) * 1996-02-20 2000-02-08 Health Hero Network Method for conducting an on-line bidding session with bid pooling
US5908467A (en) * 1996-05-31 1999-06-01 International Business Machines Corporation System and method for displaying file parameters
US5879163A (en) * 1996-06-24 1999-03-09 Health Hero Network, Inc. On-line health education and feedback system using motivational driver profile coding and automated content fulfillment
US5891622A (en) * 1996-09-30 1999-04-06 Oxford Biomedical Research, Inc. Assessment of oxidative stress in vivo
US5955111A (en) * 1996-10-08 1999-09-21 Hartford Hospital Methods and compositions for inducing production of stress proteins
US5832448A (en) * 1996-10-16 1998-11-03 Health Hero Network Multiple patient monitoring system for proactive health management
US5950634A (en) * 1996-11-29 1999-09-14 Nikken Foods Co., Ltd. Method for assessing oxidative stress and its control in humans
US5933136A (en) * 1996-12-23 1999-08-03 Health Hero Network, Inc. Network media access control system for encouraging patient compliance with a treatment plan
US6233539B1 (en) * 1997-01-10 2001-05-15 Health Hero Network, Inc. Disease simulation system and method
US5956501A (en) * 1997-01-10 1999-09-21 Health Hero Network, Inc. Disease simulation system and method
US5920859A (en) * 1997-02-05 1999-07-06 Idd Enterprises, L.P. Hypertext document retrieval system and method
US5951300A (en) * 1997-03-10 1999-09-14 Health Hero Network Online system and method for providing composite entertainment and health information
US6048689A (en) * 1997-03-28 2000-04-11 Gene Logic, Inc. Method for identifying variations in polynucleotide sequences
US5897493A (en) * 1997-03-28 1999-04-27 Health Hero Network, Inc. Monitoring system for remotely querying individuals
US5997476A (en) * 1997-03-28 1999-12-07 Health Hero Network, Inc. Networked system for interactive communication and remote monitoring of individuals
US6368273B1 (en) * 1997-03-28 2002-04-09 Health Hero Network, Inc. Networked system for interactive communication and remote monitoring of individuals
US5985559A (en) * 1997-04-30 1999-11-16 Health Hero Network System and method for preventing, diagnosing, and treating genetic and pathogen-caused disease
US6134552A (en) * 1997-10-07 2000-10-17 Sap Aktiengesellschaft Knowledge provider with logical hyperlinks
US6046317A (en) * 1997-12-19 2000-04-04 Hormos Medical Oy, Ltd. DNA molecule encoding a mutant prepro-neuropeptide Y, a mutant signal peptide, and uses thereof
US6210272B1 (en) * 1997-12-22 2001-04-03 Health Hero Network, Inc. Multi-player interactive electronic game for health education
US6133039A (en) * 1998-02-09 2000-10-17 Washinton University In vivo method for determination of oxidative stress
US6167386A (en) * 1998-06-05 2000-12-26 Health Hero Network, Inc. Method for conducting an on-line bidding session with bid pooling
US6240393B1 (en) * 1998-06-05 2001-05-29 Health Pro Network, Inc. Aggregating and pooling weight loss information in a communication system with feedback
US20020146698A1 (en) * 2001-04-05 2002-10-10 Genelink, Inc. Kits and methods for assessing oxidative stress
US20030073612A1 (en) * 2001-04-05 2003-04-17 Genelink, Incorporated Kits and methods for assessing skin health

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8676608B2 (en) 2007-02-14 2014-03-18 Genelex Corporation Genetic data analysis and database tools
US20090055217A1 (en) * 2007-08-23 2009-02-26 Grichnik Anthony J Method and system for identifying and communicating a health risk
US8260636B2 (en) 2007-08-31 2012-09-04 Caterpillar Inc. Method and system for prioritizing communication of a health risk
US20090062621A1 (en) * 2007-08-31 2009-03-05 Grichnik Anthony J Method and system for prioritizing communication of a health risk
US20100169023A1 (en) * 2008-12-30 2010-07-01 Searete Llc, A Limited Liability Corporation Of State Of Delaware Computational methods and systems for suggesting modulators of CYP450 as treatment options
US8073633B2 (en) 2008-12-30 2011-12-06 The Invention Science Fund I, Llc Computational methods and systems for suggesting modulators of CYP450 as treatment options
US8073632B2 (en) 2008-12-30 2011-12-06 The Invention Science Fund I, Llc Computational methods and systems for treatment in relation to modulation of CYP450 enzyme activity
US20100169022A1 (en) * 2008-12-30 2010-07-01 Searete Llc, A Limited Liability Corporation Of The State Of Delaware Computational methods and systems for treatment in relation to modulation of CYP450 enzyme activity
US8315815B2 (en) 2008-12-30 2012-11-20 The Invention Science Fund I, Llc Computational methods and systems for suggesting modulators of CYP450 as treatment options
US8321151B2 (en) 2008-12-30 2012-11-27 The Invention Science Fund I, Llc Computational methods and systems for treatment in relation to modulation of CYP450 enzyme activity
US10210312B2 (en) 2013-02-03 2019-02-19 Youscript Inc. Systems and methods for quantification and presentation of medical risk arising from unknown factors
US11302431B2 (en) 2013-02-03 2022-04-12 Invitae Corporation Systems and methods for quantification and presentation of medical risk arising from unknown factors
CN109430731A (en) * 2018-10-31 2019-03-08 河北农业大学 A kind of formula and processing technology of high-quality meat mincing recombination mutton dried meat

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STCB Information on status: application discontinuation

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