Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• Skin is the largest and most visible organ of the human body, and is also among the tissues most exposed to environmental stresses, hazards, and pathogens. Skin is a multi- layered tissue, primarily composed of the epidermis and dermis, and includes several accessory structures, such as sweat glands, sebaceous glands, and hair follicles. Skin serves many functions. For example, it is a protective barrier to external insults (e.g., heat, chemicals, bacteria), is involved in thermoregulation, inhibits dehydration, and performs sensory functions. Skin is also a bioreactor that produces various hormones and lipids that enter the body's circulation. A variety of immune cells function in skin as a first line of defense against bacterial or viral invasion and to maintain immune surveillance in skin and nearby body tissues. For these reasons, establishment and maintenance of good skin health is important to human health.
• external insults e.g., heat, chemicals, bacteria
• Skin health is also important for aesthetic reasons. Many people are deeply concerned about the appearance of their skin. A healthy skin appearance is maintained by a combination of cleaning, nutrition, and application of therapeutic and cosmetic products. However, overuse of skin care products can degrade skin health and appearance. Often, individuals employ trial-and-error techniques to identify skin care products (and doses thereof) that produce a desirable skin appearance. More precise methods are needed for identifying compositions (and suitable amounts of such compositions) that will enhance the health and appearance of an individual's skin. These methods would preferably be tailored to identify useful compositions and dosages for individuals. The present invention satisfies this need.
• Many skin disorders can be alleviated, inhibited, or even prevented by maintaining a high degree of skin health or by timely intervention with appropriate skin-affecting agents.
• such intervention can include consuming or topically applying skin care products, modulating sun exposure, adjusting diet, consuming nutritional or pharmaceutical products known to be effective against skin disorders, and undergoing heightened medical monitoring.
• These changes are often not made, owing to the expense or inconvenience of the changes and an individual's subjective belief that he or she is not at high risk for skin disorders.
• Improved assessment of skin health can help to identify individuals at risk for developing skin disorders and permit more informed decisions to be made regarding whether lifestyle changes or other interventions are justified.
• Heterogeneity in human genes is believed to have arisen, in part, from minor, non-fatal mutations that have occurred in the genome over time.
• differences between alternative forms of a gene are manifested as differences in the amino acid sequence of a protein encoded by the gene. Some amino acid sequence differences can alter the reactivity or substrate specificity of the protein. Differences between alternative forms of a gene can also affect the degree to which (if at all) the gene is expressed.
• many heterogeneities that occur in human genes appear not to be correlated with any particular phenotype. Known heterogeneities include, for example, single nucleotide polymorphisms (i.e., alternative forms of a gene having a difference at a single nucleotide residue).
• disorders can usually result from polymorphisms in any of a relatively large number of genes, and as a result, assessing the polymorphic form(s) of any single gene that occur in a human's genome is usually not predictive of the likelihood that the human will develop the disorder.
• a need remains for a method of assessing an individual's skin health or predisposition to develop skin disorders. Such assessment could be used to identify types and amounts of therapeutic, inhibitory, or preventive compositions or interventions that can be used to alleviate, inhibit, or prevent skin disorders. The invention satisfies these needs.
• Substantially the same method can be used to assess the advisability that a human should employ a skin care product, such as one comprising a skin protective ingredient or a vitamin (e.g., one of vitamins C and E).
• a skin care product such as one comprising a skin protective ingredient or a vitamin (e.g., one of vitamins C and E).
• occurrence of one or more disorder- associated polymorphisms in any of genes a)-l is an indication that it is more advisable for the human to use the product than when the individual's genome does not comprise disorder- associated polymorphisms in any of these genes.
• polymo ⁇ hic forms include alternative forms in which multiple consecutive or closely-spaced, non-consecutive nucleotide residues vary in sequence, forms which differ by the presence or absence of a single nucleotide residue or a small number of nucleotide residues, and forms which exhibit different niRNA splicing patterns.
• a "single nucleotide polymo ⁇ hism" (“SNP”) is one of the alternative forms of a portion of a gene that vary only in the identity of a single nucleotide residue in that portion.
• Genes in which disorder-associated polymo ⁇ hisms occur that are useful for assessing the skin health of an individual include: 1) genes which encode an enzyme that catalyzes conversion of a toxic oxygen species to a less toxic oxygen species; 2) genes winch encode a protein that provides protection against oxidative stress; 3) genes which encode a protein that induces production of a toxic oxygen species; 4) genes which encode a protein that indirectly affects oxidative stress; 5) genes which encode a protein for which the level of expression of the protein is associated with oxidative stress; 6) genes which encode a component of the human DNA repair system; and 7) genes which encode a protein associated with production of a toxic oxygen species by a macrophage or polymo ⁇ honuclear neutrophilic granulocyte.
• human skin health e.g., susceptibility to a skin disorder such as psoriasis, eczema, a skin cancer, or a bacterial infection
• a skin disorder such as psoriasis, eczema, a skin cancer, or a bacterial infection
• a disorder-associated polymo ⁇ hism in one of the genes described herein - even if there is no known biochemical or physiological association between occurrence of the polymo ⁇ hism and skin health or incidence of a skin disorder.
• genes and polymo ⁇ hisms disclosed herein are predictive indicators of the state of an individual human's skin health.
• One useful way of calculating a factor that describes correlation between a polymo ⁇ hism and a disorder is to calculate an odds ratio that describes the likelihood that an individual in whose genome the disorder-associate polymo ⁇ hism occurs will exhibit or develop the disorder. Because the kits and methods described herein can be used to detect whether the human is homozygous for the disease-associated polymo ⁇ hism, odds ratios calculated for homozygous individuals can also be used, if they are available. Odds ratios can be calculated as described in the art. [0080] For a disorder-associated polymo ⁇ hism, the odds ratio can be calculated as follows.
• any particular disorder-associated polymo ⁇ hism or disorder-non-associated polymo ⁇ hism
• Numerous methods of detecting occurrence of a polymo ⁇ hism are known in the art, and substantially any of those methods can be used in the kits and methods described herein.
• the reagents included in the kit will vary depending on the method to be used to detect the polymo ⁇ hisms. Examples of some suitable polymo ⁇ hism detection methods are provided below.
• a pair of ohgonucleotide primers are used to amplify a portion of the gene that includes a polymo ⁇ hic region.
• Detection of one or more of the polymo ⁇ hisms that occur at the polymo ⁇ hic region can be achieved by contacting the amplified portion with an ohgonucleotide having a sequence such that it will anneal under stringent conditions with the amplified portion only if one polymo ⁇ hism occurs at the portion, but will not anneal with the amplified portion if another polymo ⁇ hism occurs at that portion.
• Various acceptable stringent conditions are known in the art, and can be modified by the skilled artisan as appropriate to any particular amplified portion/oligonucleotide pair.
• one or more molecular beacon oligonucleotides are used to detect polymo ⁇ hisms (disorder-associated, non-disorder-associated, or both) in a sample that contains a copy of the subject's genome, a fraction of the subject's genome, or amplification products generated from the subject's genome (e.g., amplified portions of one or more of the genes disclosed herein in which polymo ⁇ hisms are known to occur).
• Molecular beacon probes are single-stranded oligonucleotides having a fluorescent label (e.g., rhodamine, FAM, TET, VIC, JOE, or HEX) attached at or near the 5'-end thereof and a fluorescence quencher (e.g., TAMRA or DABCYL) attached at or near the 3'-end thereof (or vice versa), as described (Kostrikis et al., 1998, Science 279:1228-1229).
• the sequence of each molecular beacon probe is selected to include two complementary hai ⁇ in regions, whereby the probe can self-anneal to form a hai ⁇ in structure.
• the 5'-and 3'-ends are brought into close association when the hai ⁇ in structure forms.
• the probe also comprises a targeting portion which is selected to be complementary to a target sequence (e.g., a single polymo ⁇ hism of a gene disclosed herein).
• the targeting portion and at least one of the hai ⁇ in regions are located in close proximity to one another, meaning that the targeting portion either overlaps the hai ⁇ in region or flanks it, having no more than about 5 nucleotide residues therebetween.
• one or more molecular beacon probes are used, each having targeting portion which is complementary to a target region (e.g., 20 to 40 nucleotide residues, more preferably 20 to 30 residues) of one polymo ⁇ hism of a gene disclosed herein. If the polymo ⁇ hism to be detected is a single nucleotide polymo ⁇ hism (SNP), then the target region includes, and preferably is approximately centered around, the nucleotide residue at which the polymo ⁇ hism occurs.
• SNP single nucleotide polymo ⁇ hism
• Kits for detecting polymo ⁇ hisms in individual genes are known in the art, and the kit of the invention can have similar components. However, a critical feature of the kit is that it includes reagents that permit its user to detect disorder-associated polymo ⁇ hisms in at least two (or at least three, four, six, eight, ten, or fifteen or more) genes disclosed herein. [0095] In one embodiment, the kit includes a plurality of oligonucleotides which anneal under stringent conditions with a disorder-associated polymo ⁇ hism of one of the genes (e.g., one of the genes identified herein as being of particular relevance for skin health), but not with a non-disorder associated-polymo ⁇ hism.

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• Chemical & Material Sciences (AREA)
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• 2002
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