U.S. patent application number 13/668842 was filed with the patent office on 2013-05-23 for kits and methods for assessing skin health.
This patent application is currently assigned to GeneLink, Inc.. The applicant listed for this patent is GeneLink, Inc.. Invention is credited to John R. DePhillipo, Robert P. Ricciardi.
Application Number | 20130130247 13/668842 |
Document ID | / |
Family ID | 32028981 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130130247 |
Kind Code |
A1 |
DePhillipo; John R. ; et
al. |
May 23, 2013 |
Kits and Methods for Assessing Skin Health
Abstract
The invention relates to kits and methods for assessing skin
health for a human and the human's susceptibility to skin
disorders. The methods involve assessing occurrence in the human's
genome of one or more polymorphisms (e.g., single nucleotide
polymorphisms) that occur in one or more genes associated disclosed
herein and that are associated with a disorder in humans. Preferred
assessment and scoring methods are disclosed, as are kits for
performing the methods.
Inventors: |
DePhillipo; John R.;
(Margate, NJ) ; Ricciardi; Robert P.; (Kennet
Square, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GeneLink, Inc.; |
Margate |
NJ |
US |
|
|
Assignee: |
GeneLink, Inc.
Margate
NJ
|
Family ID: |
32028981 |
Appl. No.: |
13/668842 |
Filed: |
November 5, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11731180 |
Mar 30, 2007 |
8313930 |
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13668842 |
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10247935 |
Sep 20, 2002 |
7211383 |
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11731180 |
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09826522 |
Apr 5, 2001 |
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10247935 |
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PCT/US02/10682 |
Apr 5, 2002 |
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09826522 |
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60289169 |
May 7, 2001 |
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60350517 |
Oct 22, 2001 |
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60335426 |
Oct 24, 2001 |
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60336815 |
Dec 5, 2001 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 1/6876 20130101;
C12Q 1/6883 20130101; C12Q 2600/156 20130101 |
Class at
Publication: |
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-23. (canceled)
24. A method of assessing skin health of a human, the method
comprising assessing occurrence in the human's genome of
disorder-associated polymorphisms in at least two genes selected
from the group consisting of a) the gene which encodes
mitochondrial manganese superoxide dismutase (MnSOD); b) the gene
which encodes cytoplasmic copper/zinc superoxide dismutase (CZSOD);
c) the gene which encodes catalase; d) the gene which encodes human
glutathione peroxidase (hGPX1); e) the gene which encodes
glutathione S transferase P1 (GSTP1); f) the gene which encodes
NAD(P)H:quinone oxidoreductase; g) the gene which encodes epoxide
hydrolase; h) the gene which encodes tumor necrosis factor alpha
(TNF-alpha); i) the gene which encodes NADH/NADPH oxidase p22
subunit (the phox gene); j) the gene which encodes nitric oxide
synthase; k) the gene which encodes cytochrome P450; l) the gene
which encodes matrix metalloproteinase 1 (MMP-1); and m) the gene
which encodes profilagrin, whereby occurrence of any of the
disorder-associated polymorphisms is an indication that the human
has poorer skin health than a human whose genome does not comprise
any of the disorder-associated polymorphisms, and whereby
occurrence of a plurality of the disorder-associated polymorphisms
is an indication that the human has even poorer skin health than a
human whose genome does not comprise the disorder-associated
polymorphisms, wherein the method further comprises calculating a
skin health score by summing, for each of the selected genes in
which a disorder-associated polymorphism occurs in the human's
genome, the product of a constant and a correlation factor, wherein
the correlation factor represents the fraction of humans
heterozygous or homozygous for the disorder-associated polymorphism
who exhibit the corresponding disorder, whereby the skin health
score represents the relative susceptibility of the human to a skin
disorder.
25. The method of claim 24, wherein the same constant is used for
each selected gene.
26. A method of assessing skin health of a human, the method
comprising assessing occurrence in the human's genome of
disorder-associated polymorphisms in at least two genes selected
from the group consisting of a) the gene which encodes
mitochondrial manganese superoxide dismutase (MnSOD); b) the gene
which encodes cytoplasmic copper/zinc superoxide dismutase (CZSOD);
c) the gene which encodes catalase; d) the gene which encodes human
glutathione peroxidase (hGPX1); e) the gene which encodes
glutathione S transferase P1 (GSTP1); f) the gene which encodes
NAD(P)H:quinone oxidoreductase; g) the gene which encodes epoxide
hydrolase; h) the gene which encodes tumor necrosis factor alpha
(TNF-alpha), i) the gene which encodes NADH/NADPH oxidase p22
subunit (the phox gene); j) the gene which encodes nitric oxide
synthase; k) the gene which encodes cytochrome P450; l) the gene
which encodes matrix metalloproteinase 1 (MMP-1); and m) the gene
which encodes profilagrin, whereby occurrence of any of the
disorder-associated polymorphisms is an indication that the human
has poorer skin health than a human whose genome does not comprise
any of the disorder-associated polymorphisms, and whereby
occurrence of a plurality of the disorder-associated polymorphisms
is an indication that the human has even poorer skin health than a
human whose genome does not comprise the disorder-associated
polymorphisms, wherein each of the disorder-associated
polymorphisms for which occurrence is assessed is a single
nucleotide polymorphism (SNP).
27. The method of claim 26, wherein occurrence of a SNP is assessed
by annealing a nucleic acid derived from the human's genome with a
primer that is complementary to the region adjacent the SNP on its
3' side, extending the primer using a polymerase in order to add a
nucleotide residue complementary to the SNP to the primer, and
detecting the identity of the nucleotide residue complementary to
the SNP.
28. The method of claim 27, wherein the nucleotide residue is a
non-extendable residue.
29. A method of assessing the likelihood that a human will develop
a skin disorder, the method comprising assessing occurrence in the
human's genome of disorder-associated polymorphisms in at least two
genes selected from the group consisting of a) the gene which
encodes mitochondrial MnSOD; b) the gene which encodes cytoplasmic
CZSOD; c) the gene which encodes catalase; d) the gene which
encodes hGPX1; e) the gene which encodes GSTP1; f) the gene which
encodes NAD(P)H:quinone oxidoreductase; g) the gene which encodes
epoxide hydrolase; h) the gene which encodes TNF-alpha; i) the phox
gene; j) the gene which encodes nitric oxide synthase; k) the gene
which encodes cytochrome P450; l) the gene which encodes MMP-1; and
m) the gene which encodes profilagrin, whereby occurrence of any of
the disorder-associated polymorphisms is an indication that the
human is more susceptible to the skin disorder than a human whose
genome does not comprise the disorder-associated polymorphism, and
whereby occurrence of a plurality of the disorder-associated
polymorphisms is an indication that the human is even more
susceptible to the skin disorder than a human whose genome does not
comprise the disorder-associated polymorphisms.
30. The method of claim 29, comprising assessing occurrence of at
least two disorder-associated polymorphisms selected from the group
consisting of A) a polymorphism in the open reading frame encoding
mitochondrial MnSOD; B) a polymorphism in the open reading frame
encoding cytoplasmic CZSOD; C) a polymorphism in the promoter
region of the gene encoding catalase; D) a polymorphism in the open
reading frame encoding hGPX1; E) a polymorphism in the open reading
frame encoding GSTP1; F) a polymorphism in the open reading frame
encoding NAD(P)H:quinone oxidoreductase; G) a polymorphism in the
open reading frame encoding epoxide hydrolase; H) a polymorphism in
the promoter region of the gene encoding TNF-alpha; I) a
polymorphism in the open reading frame of the phox gene; J) a
polymorphism in the open reading frame encoding nitric oxide
synthase; K) a polymorphism in the 5' flanking region of the gene
encoding cytochrome P450; and L) a polymorphism in the promoter
region of the gene encoding MMP-1.
31. The method of claim 29, comprising assessing occurrence of a
first polymorphism selected from the group consisting of i) a
polymorphism manifested as occurrence of a codon encoding alanine
at amino acid residue 9 of mitochondrial MnSOD; ii) a polymorphism
manifested as occurrence of a codon encoding valine at amino acid
residue 9 of mitochondrial MnSOD; iii) a polymorphism manifested as
occurrence of a codon encoding isoleucine at amino acid residue 58
of mitochondrial MnSOD; iv) a polymorphism manifested as occurrence
of a codon encoding thymine at amino acid residue 58 of
mitochondrial MnSOD; v) a polymorphism manifested as occurrence of
a codon encoding valine at amino acid residue 7 of cytoplasmic
CZSOD; vi) a polymorphism manifested as occurrence of a codon
encoding glutamic acid at amino acid residue 7 of cytoplasmic
CZSOD; vii) a polymorphism manifested as occurrence of a codon
encoding cysteine at amino acid residue 6 of cytoplasmic CZSOD;
viii) a polymorphism manifested as occurrence of a codon encoding
phenylalanine at amino acid residue 6 of cytoplasmic CZSOD; ix) a
polymorphism manifested as occurrence of a cytosine residue at
nucleotide residue 262 of the catalase gene; x) a polymorphism
manifested as occurrence of a thymine residue at nucleotide residue
262 of the catalase gene; xi) a polymorphism manifested as
occurrence of a codon encoding proline at amino acid residue 198 of
hGPX1; xii) a polymorphism manifested as occurrence of a codon
encoding leucine at amino acid residue 198 of hGPX1; xiii) a
polymorphism manifested as occurrence of a codon encoding valine at
amino acid residue 105 of GSTP1; xiv) a polymorphism manifested as
occurrence of a codon encoding isoleucine at amino acid residue 105
of GSTP1; xv) a polymorphism manifested as occurrence of a cytosine
residue at nucleotide residue 242 of the gene encoding
NAD(P)H:quinone oxidoreductase; xvi) a polymorphism manifested as
occurrence of a thymine residue at nucleotide residue 242 of the
gene encoding NAD(P)H:quinone oxidoreductase; xvii) a polymorphism
manifested as occurrence of a thymine residue at nucleotide residue
113 in exon 3 of the gene which encodes epoxide hydrolase; xviii) a
polymorphism manifested as occurrence of a cytosine residue at
nucleotide residue 113 in exon 3 of the gene which encodes epoxide
hydrolase; xix) a polymorphism manifested as occurrence of an
adenine residue at nucleotide residue -238 of the gene which
encodes TNF-alpha; xx) a polymorphism manifested as occurrence of
an adenine residue at nucleotide residue -308 of the gene which
encodes TNF-alpha; xxi) a polymorphism manifested as occurrence of
a cytosine residue at nucleotide residue 242 of the phox gene;
xxii) a polymorphism manifested as occurrence of a thymine residue
at nucleotide residue 242 of the phox gene; xxiii) a polymorphism
manifested as occurrence of a 27 base nucleotide residue between
nucleotide residues 5130 and 5511 of the gene encoding nitric oxide
synthase; xxiv) a polymorphism manifested as non-occurrence of a 27
nucleotide residue repeat between nucleotide residues 5130 and 5511
of the gene encoding nitric oxide synthase; xxv) a polymorphism
manifested as occurrence of an adenine residue at nucleotide
residue -290 of the gene encoding cytochrome P450; xxvi) a
polymorphism manifested as occurrence of a guanine residue at
nucleotide residue -290 of the gene encoding cytochrome P450;
xxvii) a polymorphism manifested as occurrence of a single guanine
residue at nucleotide residue -1607 of the human gene encoding
MMP-1; and xxviii) a polymorphism manifested as occurrence of a two
consecutive guanine residues at a site including nucleotide residue
-1607 of the human gene encoding MMP-1.
32. A method of selecting a dose of a skin protective composition
for administration to a human, the method comprising assessing
occurrence in the human's genome of disorder-associated
polymorphisms in at least two genes selected from the group
consisting of a) the gene which encodes mitochondrial MnSOD; b) the
gene which encodes cytoplasmic CZSOD; c) the gene which encodes
catalase; d) the gene which encodes hGPX1; e) the gene which
encodes GSTP1; f) the gene which encodes NAJNP)H:quinone
oxidoreductase; g) the gene which encodes epoxide hydrolase; h) the
gene which encodes tumor necrosis factor alpha (TNF-alpha); i) the
phox gene; j) the gene which encodes nitric oxide synthase; k) the
gene which encodes cytochrome P450; l) the gene which encodes
MMP-1; and m) the gene which encodes profilagrin, whereby
occurrence of any of the disorder-associated polymorphisms is an
indication that a greater dose of the composition should be
administered to the human; and selecting a dose of the composition
based on occurrence of the disorder-associated polymorphisms.
33. A kit for assessing relative susceptibility of a human to a
skin disorder, the kit comprising reagents for assessing occurrence
in the human's genome of disorder-associated polymorphisms in at
least two genes selected from the group consisting of a) the gene
which encodes mitochondria' MnSOD; b) the gene which encodes
cytoplasmic CZSOD; c) the gene which encodes catalase; d) the gene
which encodes hGPX1; e) the gene which encodes GSTP1; f) the gene
which encodes NAD(P)H:quinone oxidoreductase; g) the gene which
encodes epoxide hydrolase; h) the gene which encodes tumor necrosis
factor alpha (TNF-alpha); i) the phox gene; j) the gene which
encodes nitric oxide synthase; k) the gene which encodes cytochrome
P450; l) the gene which encodes MMP-1; and m) the gene which
encodes profilagrin.
34. The kit of claim 33, wherein the reagents comprise first
oligonucleotides that anneal with higher stringency with the
disorder-associated polymorphisms than with corresponding
non-disorder-associated polymorphisms.
35. The kit of claim 34, wherein each of the first oligonucleotides
is attached to a support.
36. The kit of claim 35, wherein each of the first oligonucleotides
is attached to the same support.
37. The kit of claim 35, wherein each of the first oligonucleotides
is attached to a different support.
38. The kit of claim 34, wherein the first oligonucleotides are
molecular beacon oligonucleotides.
39. The kit of claim 34, wherein the kit further comprises second
oligonucleotides that anneal with higher stringency with the
non-disorder-associated polymorphisms than with corresponding
disorder-associated polymorphisms.
40. The kit of claim 39, wherein the first and second
oligonucleotides are spectrally distinct molecular beacon
oligonucleotide pairs.
41. The kit of claim 33, wherein the reagents comprise primers that
are complementary to the region adjacent a characteristic residue
of the disorder-associated-polymorphism for amplifying at least the
characteristic residue.
42. The kit of claim 41, further comprising a polymerase capable of
extending the primers by adding a nucleotide residue complementary
to the characteristic residue.
43. The kit of claim 42, further comprising a non-extendable
nucleotide residue.
44. The kit of claim 33, further comprising an instructional
material which includes a numerical value representing the product
of a constant and a correlation factor, wherein the correlation
factor represents the fraction of humans heterozygous or homozygous
for the disorder-associated polymorphism who exhibit the
corresponding disorder.
45. The kit of claim 44, wherein the same constant is used for each
selected gene.
46. The kit of claim 33, wherein the kit comprises reagents for
assessing occurrence in the human's genome of at least two
polymorphisms selected from the group consisting of I) a
polymorphism manifested as a change from an alanine residue to a
valine residue at amino acid residue 9 of mitochondrial MnSOD; II)
a polymorphism manifested as a change from an isoleucine residue to
a thymine residue at amino acid residue 58 of mitochondrial MnSOD;
III) a polymorphism manifested as a change from a valine residue to
a glutamic acid residue at amino acid residue 7 of cytoplasmic
CZSOD; IV) a polymorphism manifested as a change from a cysteine
residue to a phenylalanine residue at amino acid residue 6 of
cytoplasmic CZSOD; V) a polymorphism manifested as a change from a
cytosine residue to a thymine residue at nucleotide residue -262 of
the catalase gene; VI) a polymorphism in the hGPX1 gene manifested
as a change from a proline residue to a leucine residue at amino
acid residue 198 of glutathione peroxidase; VII) a polymorphism in
the GSTP1 gene manifested as a change from a valine residue to an
isoleucine residue at amino acid residue 105 of glutathione
S-transferase P1; VIII) a polymorphism manifested as a change from
a cytosine residue to a thymine residue at nucleotide residue 242
of the gene encoding NAD(P)H:quinone oxidoreductase; IX) a
polymorphism manifested as a change from a thymine residue to a
cytosine residue at nucleotide residue 113 in exon 3 of the gene
which encodes epoxide hydrolase; X) a polymorphism manifested as a
change to an adenine residue at nucleotide residue 238 of the gene
which encodes TNF-alpha; XI) a polymorphism manifested as a change
to an adenine residue at nucleotide residue -308 of the gene which
encodes TNF-alpha; XII) a polymorphism manifested as a change from
a cytosine residue to a thymine residue at nucleotide residue 242
of the phox gene; XIII) a polymorphism manifested as a 27
nucleotide residue repeat between nucleotide residues 5130 and 5511
of the gene encoding nitric oxide synthase; XIV) a polymorphism
manifested as a change from an adenine residue to a guanine residue
at nucleotide residue -290 of the gene encoding cytochrome P450;
and XV) a polymorphism manifested as occurrence of two consecutive
guanine residues at a site including nucleotide residue -1607 of
the human gene encoding MMP-1.
47. (canceled)
48. A method of selecting a dose of a skin protective agent for
administration to a human in a nutritional product, the method
comprising assessing occurrence in the human's genome of
disorder-associated polymorphisms in at least two genes selected
from the group consisting of a) the gene which encodes
mitochondrial MnSOD; b) the gene which encodes cytoplasmic CZSOD;
c) the gene which encodes catalase; d) the gene which encodes hGPX
1; e) the gene which encodes GSTP1; f) the gene which encodes
NAD(P)H:quinone oxidoreductase; g) the gene which encodes epoxide
hydrolase; h) the gene which encodes TNF-alpha; i) the phox gene;
j) the gene which encodes nitric oxide synthase; k) the gene which
encodes cytochrome P450; l) the gene which encodes MMP-1; and m)
the gene which encodes profilagrin, whereby occurrence of any of
the disorder-associated polymorphisms is an indication that a
greater dose of the agent should be administered to the human in
the nutritional product; and selecting a dose of the agent for the
nutritional product based on occurrence of the disorder-associated
polymorphisms.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Application No. PCT/US02/10682 (filed 5 Apr. 2002) and of U.S.
patent application Ser. No. 09/826,522 (filed 5 Apr. 2001), and is
entitled to priority pursuant to 35 U.S.C. .sctn.119(e) to U.S.
provisional patent applications Nos. 60/289,169 (filed 7 May 2001),
60/350,517 (filed 22 Oct. 2001), 60/335,426 (filed 24 Oct. 2001),
and 60/336,815 (filed 5 Dec. 2001).
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
REFERENCE TO A MICROFICHE APPENDIX
[0003] Not applicable.
BACKGROUND OF THE INVENTION
[0004] 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.
[0005] 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.
[0006] 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. For example,
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.
[0007] Many human genes occur in a variety of forms which differ in
at least minor ways. Heterogeneity in human genes is believed to
have arisen, in part, from minor, non-fatal mutations that have
occurred in the genome over time. In some instances, 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. However, 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). Other known
polymorphic forms include those in which the sequence of larger
(e.g., 2-1000 residues) portions of a gene exhibits numerous
sequence differences and those which differ by the presence or
absence of portion of a gene.
[0008] Numerous disorders and physiological states have been
correlated with occurrence of one or more alternative forms of an
individual gene in the genome of a human who exhibits the disorder
or physiological state. For example, Kimura et al. (2000, Am. J.
Ophthalmol. 130:769-773) discloses an association between
occurrence of a SNP of the manganese superoxide dismutase gene and
a form of macular degeneration.
[0009] Associations between individual disorders and individual
genetic polymorphisms are known. However, 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.
[0010] 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.
BRIEF SUMMARY OF THE INVENTION
[0011] The invention relates to a method of assessing skin health
in a human. The method comprises assessing occurrence in the
human's genome of disorder-associated polymorphisms in at least two
(three, four, five, six, eight, ten, fifteen, or even all) genes
selected from the group consisting of [0012] genes which encode an
enzyme that catalyzes conversion of a toxic oxygen species to a
less toxic oxygen species; [0013] genes which encode a protein that
provides protection against oxidative stress; [0014] genes which
encode a protein that induces production of a toxic oxygen species;
[0015] genes which encode a protein that indirectly affects
oxidative stress; [0016] genes which encode a protein for which the
level of expression of the protein is associated with oxidative
stress; [0017] genes which encode a component of the human DNA
repair system; and [0018] genes which encode a protein associated
with production of a toxic oxygen species by a macrophage or
polymorphonuclear neutrophilic granulocyte.
[0019] It has been discovered that this method is particularly
useful for assessing skin health when the genes are selected from
the group consisting of [0020] a) the gene which encodes
mitochondrial manganese superoxide dismutase (MnSOD); [0021] b) the
gene which encodes cytoplasmic copper/zinc superoxide dismutase
(CZSOD); [0022] c) the gene which encodes catalase; [0023] d) the
gene which encodes human glutathione peroxidase (hGPX1); [0024] e)
the gene which encodes glutathione S transferase P1 (GSTP1) [0025]
f) the gene which encodes NAD(P)H:quinone oxidoreductase; [0026] g)
the gene which encodes epoxide hydrolase; [0027] h) the gene which
encodes tumor necrosis factor alpha (TNF-alpha); [0028] i) the gene
which encodes NADH/NADPH oxidase p22 subunit (the phox gene);
[0029] j) the gene which encodes nitric oxide synthase; [0030] k)
the gene which encodes cytochrome P450; [0031] l) the gene which
encodes matrix metalloproteinase 1 (MMP-1); and [0032] m) the gene
which encodes profilagrin. Occurrence of a disorder-associated
polymorphism in any of these genes is an indication that the human
has poorer skin health than a human whose genome does not comprise
the disorder-associated polymorphism, and occurrence of a plurality
of disorder-associated polymorphisms is an indication that the
human has even poorer skin health than a human whose genome
comprises only one of the disorder-associated polymorphisms (and
greater still than an individual whose genome does not comprise one
of these disorder-associated polymorphisms).
[0033] 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). When the method is used to assess the
advisability that a human should employ a skin care product,
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.
[0034] For example, occurrence of at least two (three, four, five,
six, eight, ten, or fifteen or more) disorder-associated
polymorphisms can be assessed, where the polymorphisms are selected
from the group consisting of [0035] A) a polymorphism in the open
reading frame encoding mitochondrial MnSOD; [0036] B) a
polymorphism in the open reading frame encoding cytoplasmic CZSOD;
[0037] C) a polymorphism in the promoter region of the gene
encoding catalase; [0038] D) a polymorphism in the open reading
frame of the hGPX1 gene; [0039] E) a polymorphism in the open
reading frame encoding glutathione S transferase P1 (GSTP1); [0040]
F) a polymorphism in the open reading frame encoding
NAD(P)H:quinone oxidoreductase; [0041] G) a polymorphism in the
open reading frame encoding epoxide hydrolase; [0042] H) a
polymorphism in the promoter region of the gene encoding TNF-alpha;
[0043] I) a polymorphism in the open reading frame of the phox
gene; [0044] J) a polymorphism in the open reading frame encoding
nitric oxide synthase; [0045] K) a polymorphism in the 5' flanking
region of the gene encoding cytochrome P450; and [0046] L) a
polymorphism in the promoter region of the gene encoding MMP-1.
[0047] Preferably, occurrence of all known polymorphisms at
individual sites (e.g., both of two known alternative forms or all
three forms of a polymorphism known to exist in three alternative
forms) is assessed within an individual's genome, so that the
individual's genotype for the polymorphism as that site can be
completely known. For example, appropriate polymorphisms that can
be assessed in the genes listed above include the following [0048]
i) a polymorphism manifested as occurrence of a codon encoding
alanine at amino acid residue 9 (i.e., in the signal sequence) of
MnSOD; [0049] ii) a polymorphism manifested as occurrence of a
codon encoding valine at amino acid residue 9 (i.e., in the signal
sequence) of MnSOD; [0050] iii) a polymorphism manifested as
occurrence of a codon encoding isoleucine at amino acid residue 58
of MnSOD; [0051] iv) a polymorphism manifested as occurrence of a
codon encoding thymine at amino acid residue 58 of MnSOD; [0052] v)
a polymorphism manifested as occurrence of a codon encoding valine
at amino acid residue 7 of CZSOD; [0053] vi) a polymorphism
manifested as occurrence of a codon encoding glutamic acid at amino
acid residue 7 of CZSOD; [0054] vii) a polymorphism manifested as
occurrence of a codon encoding cysteine at amino acid residue 6 of
CZSOD; [0055] viii) a polymorphism manifested as occurrence of a
codon encoding phenylalanine at amino acid residue 6 of CZSOD;
[0056] ix) a polymorphism manifested as occurrence of a cytosine
residue at nucleotide residue -262 (i.e., in the promoter region)
of the catalase gene; [0057] x) a polymorphism manifested as
occurrence of a thymine residue at nucleotide residue -262 (i.e.,
in the promoter region) of the catalase gene; [0058] xi) a
polymorphism manifested as occurrence of a codon encoding proline
at amino acid residue 198 of glutathione peroxidase (hGPX1); [0059]
xii) a polymorphism manifested as occurrence of a codon encoding
leucine at amino acid residue 198 of glutathione peroxidase
(hGPX1); [0060] xiii) a polymorphism manifested as occurrence of a
codon encoding valine at amino acid residue 105 of glutathione
S-transferase P1 (GSTP1); [0061] xiv) a polymorphism manifested as
occurrence of a codon encoding isoleucine at amino acid residue 105
of glutathione S-transferase P1 (GSTP1); [0062] xv) a polymorphism
manifested as occurrence of a cytosine residue at nucleotide
residue 242 (i.e., in the coding region) of the gene encoding
NAD(P)H:quinone oxidoreductase; [0063] xvi) a polymorphism
manifested as occurrence of a thymine residue at nucleotide residue
242 (i.e., in the coding region) of the gene encoding
NAD(P)H:quinone oxidoreductase; [0064] xvii) a polymorphism
manifested as occurrence of a thymine residue at nucleotide residue
113 in exon 3 of the gene which encodes epoxide hydrolase (i.e.,
resulting in a tyrosine residue in epoxide hydrolase); [0065]
xviii) a polymorphism manifested as occurrence of a cytosine
residue at nucleotide residue 113 in exon 3 of the gene which
encodes epoxide hydrolase (i.e., resulting in a histidine residue
in epoxide hydrolase); [0066] xix) a polymorphism manifested as
occurrence of an adenine residue at nucleotide residue -238 (i.e.,
in the promoter region) of the gene which encodes TNF-alpha (i.e.,
the TNF-alpha promoter variant designated TNF2); [0067] xx) a
polymorphism manifested as occurrence of an adenine residue at
nucleotide residue -308 (i.e., in the promoter region) of the gene
which encodes TNF-alpha (i.e., the TNF-alpha promoter variant
designated TNF3); [0068] xxi) a polymorphism manifested as
occurrence of a cytosine residue at nucleotide residue 242 (i.e.,
in the coding region) of the phox gene encoding the NADH/NADPH
oxidase p22 subunit; [0069] xxii) a polymorphism manifested as
occurrence of a thymine residue at nucleotide residue 242 (i.e., in
the coding region) of the phox gene encoding the NADH/NADPH oxidase
p22 subunit; [0070] xxiii) a polymorphism manifested as a 27
nucleotide residue repeat in intron 4 (i.e., between nucleotide
residues 5130 and 5511) of the gene encoding nitric oxide synthase;
[0071] xxiv) a polymorphism manifested as absence of a 27
nucleotide residue repeat in intron 4 (i.e., between nucleotide
residues 5130 and 5511) of the gene encoding nitric oxide synthase;
[0072] xxv) a polymorphism manifested as occurrence of an adenine
residue at nucleotide residue -290 (i.e., in the 5'-flanking
region) of the gene encoding cytochrome P450 (i.e., the
polymorphism designated the CYP3A4 cytochrome P450 variant); [0073]
xxvi) a polymorphism manifested as occurrence of a guanine residue
at nucleotide residue -290 (i.e., in the 5'-flanking region) of the
gene encoding cytochrome P450 (i.e., the polymorphism designated
the CYP3A4 cytochrome P450 variant); [0074] xxvii) a polymorphism
manifested as occurrence of a single guanine residue at nucleotide
residue -1607 of the human gene encoding MMP-1; and [0075] xxviii)
a polymorphism manifested as occurrence of a two consecutive
guanine residues at a site including nucleotide residue -1607 of
the human gene encoding MMP-1.
[0076] Occurrence of an individual disorder-associated polymorphism
can be assessed by first contacting a nucleic acid derived from the
human's genome with a first oligonucleotide that anneals with
higher stringency with the disorder-associated polymorphism than
with a corresponding non-disorder-associated polymorphism and then
assessing annealing of the first oligonucleotide and the nucleic
acid. Annealing of the first oligonucleotide and the nucleic acid
is an indication that the human's genome comprises the
disorder-associated polymorphism.
[0077] Occurrence of an individual disorder-associated polymorphism
can be further assessed by contacting the nucleic acid with a
second oligonucleotide that anneals with higher stringency with a
non-disorder-associated polymorphism than with the corresponding
disorder-associated polymorphism and assessing annealing of the
second oligonucleotide and the nucleic acid. Annealing of the
second oligonucleotide and the nucleic acid is an indication that
the human's genome comprises the non-disorder-associated
polymorphism. By assessing occurrence of both disorder-associated
and non-disorder associated polymorphisms in an individual's
genome, one can assess whether the individual is likely homologous
for the non-disorder-associated polymorphism, homologous for the
disorder-associated polymorphism, or heterozygous for the
disorder-associated polymorphism and non-disorder-associated
polymorphisms. This information can inform selection of an
appropriate agent or intervention and an appropriate dose,
duration, or intensity of the agent or intervention for improving
skin health or alleviating, inhibiting, or preventing a skin
disorder.
[0078] A skin health score can be calculated by summing, for each
of the selected genes in which a disorder-associated polymorphism
occurs in the human's genome, the product of a constant and a
correlation factor. The correlation factor can be one or it can,
for example, represent the fraction of humans heterozygous or
homozygous for the disorder-associated polymorphism who exhibit the
corresponding disorder. The skin health score represents the
relative susceptibility of the human to a skin disorder.
[0079] The same methods can be used to assess the likelihood that a
human will develop a skin disorder. Occurrence of any of the
disorder-associated polymorphisms is an indication that the human
is more susceptible to the skin disorder than a human whose genome
does not comprise the polymorphism, and occurrence of a plurality
of the disorder-associated polymorphisms is an indication that the
human is even more susceptible to the skin disorder than a human
whose genome does not comprise the polymorphisms.
[0080] These methods can also be used to select a dose of a skin
protective composition or other prophylactic or therapeutic
composition for administration to a human. Occurrence of any of the
disorder-associated polymorphisms is an indication that a greater
dose of the composition should be administered to the human. The
dose of the composition can be selected based on occurrence of the
polymorphisms.
[0081] The invention further relates to a kit for assessing
relative susceptibility of a human to a skin disorder. The kit
comprises reagents for assessing occurrence in the human's genome
of disorder-associated polymorphisms in at least two (three, four,
five, six, eight, ten, or fifteen or more) of the genes disclosed
herein.
[0082] In another aspect, the invention relates to a method of
assessing the advisability that a human should employ a nutritional
product comprising a skin protective agent or other prophylactic or
therapeutic composition. The method comprises assessing occurrence
in the human's genome of disorder-associated polymorphisms in at
least two (three, four, five, six, eight, ten, fifteen, or more) of
the genes disclosed herein. Occurrence of any of the
disorder-associated polymorphisms is an indication that it is more
advisable for the human to employ the nutritional product than a
human whose genome does not comprise the polymorphism, and
occurrence of a plurality of the disorder-associated polymorphisms
is an indication that it is even more advisable that the human
should employ the nutritional product than a human whose genome
does not comprise the polymorphisms.
[0083] In still another aspect, the invention relates to a method
of selecting a dose of a skin protective agent for administration
to a human in a nutritional product. The method comprises assessing
occurrence in the human's genome of disorder-associated
polymorphisms in at least two of the genes disclosed herein.
Occurrence of any of the polymorphisms is an indication that a
greater dose of the agent should be administered to the human in
the nutritional product. The dose of the agent for the nutritional
product can be selected based on occurrence of the
polymorphisms.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0084] The foregoing summary, as well as the following detailed
description of preferred embodiments of the invention, will be
better understood when read in conjunction with the appended
drawings. The invention is not limited to the precise arrangements
and instrumentalities shown.
[0085] FIGS. 1A and 1B are images which depict examples of results
that can be obtained by analyzing occurrence of polymorphisms in
several genes. The results shown in FIG. 1A are derived from a
hypothetical first human, and those shown in FIG. 1B are derived
from a hypothetical second human. Circles represent different
polymorphisms of the gene indicated to the left of the row of
circles. Filled circles indicate the presence of the polymorphism.
Non-filled circles indicate the absence of the polymorphism.
Numbers below each circle represent a correlation factor for the
polymorphism and a disease or disorder (i.e., not necessarily a
skin disease or disorder).
DETAILED DESCRIPTION OF THE INVENTION
[0086] The invention relates to kits and methods for assessing skin
health in a human by assessing occurrence in the human's genome of
genetic polymorphisms that are associated with disorders (i.e., any
type of disorder, whether a disorder of the skin or not). To better
characterize the human's genetic content, occurrence of polymorphic
forms (of the same genes) that are not associated with a disorder
can also be assessed, so that one can determine whether the human
is 1) homozygous for the disorder-associated polymorphism at a
genomic site, 2) heterozygous for disorder-associated and
non-disorder-associated polymorphisms at that site, or 3)
homozygous for non-disorder-associated polymorphisms at that site.
Assessments of genomic polymorphism content in two or more (and
preferably in three, four, five, six, eight, ten, fifteen, or more)
of the genes identified herein as being significant to skin health
can be combined to indicate the skin health of the human. This
assessment of skin health can be used to predict the likelihood
that the human will develop, is developing, is predisposed to
develop, or has already developed a skin disorder.
[0087] Crudely simplified, the methods involve determining whether
multiple polymorphisms that have been associated (by the inventors
or by others) with a human disorder (i.e., a disease or
pathological state, whether of the skin or not) occur in the genome
of the human being tested. In some embodiments, the number of
polymorphisms that occur in the human's genome are summed to yield
a value; the higher the value is, the greater the susceptibility of
the human to skin disorders is assessed to be (i.e., the poorer the
human's skin health is assessed to be). In other embodiments, a
weighting factor is assigned to each polymorphism tested, and the
weighting factors of polymorphisms that occur in the human's genome
are summed to yield a value that represents relative skin health
(e.g., as assessed by susceptibility to skin disorders). The
weighting factor can represent the product of a constant assigned
to the gene in which the corresponding polymorphism occurs and a
correlation factor that describes how informative occurrence of the
polymorphism is for occurrence of the disorder with which it is
associated. The weighting factor can also be influenced by whether
the human is homozygous or heterozygous for the disorder-associated
polymorphism. The invention includes a variety of alternative
methods and kits for performing the methods, as described in
greater detail herein.
Definitions
[0088] As used in this disclosure, the following terms have the
meanings associated with them in this section.
[0089] A "polymorphism" in a gene is one of the alternative forms
of a portion of the gene that are known to occur in the human
population. For example, many genes are known to exhibit single
nucleotide polymorphic forms, whereby the identity of a single
nucleotide residue of the gene differs among the forms. Each of the
polymorphic forms represents a single polymorphism, as the term is
used herein. Other known polymorphic 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 mRNA splicing patterns.
[0090] A "single nucleotide polymorphism" ("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.
[0091] A "disorder-associated" polymorphism is an alternative form
of a portion of a gene, wherein occurrence of the alternative form
in the genome of a human has been correlated with exhibition by the
human of a disease or a pathological state, whether the disease or
pathological state affects the skin, another tissue, or multiple
tissues.
[0092] A "non-disorder-associated" polymorphism is an alternative
form of a portion of a gene for which no significant positive
correlation has been made between occurrence of the alternative
form in the genome and occurrence of a disease or a pathological
state. Non-disorder-associated polymorphisms are sometimes
designated "neutral" polymorphisms in the art.
[0093] A disorder-associated polymorphism and a
non-disorder-associated polymorphism "correspond" with one another
if the two polymorphisms are two alternative forms of the same
portion of the gene. By way of example, if the identity of residue
100 of a gene is adenine in a disorder-associated polymorphism of
the gene and cytosine in a non-disorder-associated polymorphism of
the gene, then the two polymorphisms correspond with one another.
It is understood that there may be three or more corresponding
polymorphisms when there are more than two alternative forms of the
same portion of the gene.
[0094] A "characteristic residue" of a polymorphism is a nucleotide
residue, the identity of which is known to vary among the
alternative forms corresponding to the polymorphism.
[0095] A "skin disorder" is a pathological condition characterized
by dysfunction, (e.g., inflammation, necrosis, abnormal
proliferation, reduced elasticity, defective renewal, irritation,
or infection) of some portion of the skin.
[0096] "Skin health" is a measure of the absence of a skin disorder
in an individual human (i.e., characterized by normal skin function
and appearance) and the likelihood that the individual will
continue to exhibit absence of a skin disorder.
[0097] "Toxic oxygen species" include, in approximate order of
decreasing reactivity, hydroxyl radicals, superoxide radicals,
nitric oxide, peroxy nitrite (ONOO.sup.-; the product of a reaction
between nitric oxide and superoxide radical), and hydrogen
peroxide. Ordinary diatomic oxygen is not a toxic oxygen species,
as the term is used herein.
[0098] "Oxidative damage" refers to chemical reaction of a normal
cellular component (e.g., DNA, a protein, or a lipid) with a toxic
oxygen species, whereby at least one normal function of the
component is inhibited or eliminated. The terms "oxidative damage"
and "oxidative stress" are used interchangeably herein.
[0099] A "molecular beacon oligonucleotide" is a single-stranded
oligonucleotides having a fluorescent label (e.g., rhodamine, FAM,
TET, VIC, JOE, or HEX) attached to the 5'-end thereof and a
fluorescence quencher (e.g., TAMRA or DABCYL) attached to the
3'-end thereof (or vice versa), as described (Kostrikis et al.,
1998, Science 279:1228-1229).
[0100] Two molecular beacon oligonucleotides are "spectrally
distinct" if they can be differentially detected using
spectrophotometric or spectrofluorimetric methods. Examples of
characteristics that can be used to differentiate spectrally
distinct oligonucleotides include absorption or excitation
wavelength, emission wavelength, and fluorescent lifetime.
[0101] An "instructional material" is a publication, a recording, a
diagram, or any other medium of expression which can be used to
communicate how to use a kit described herein, numerical values for
weighting the significance of various polymorphisms that are
detectable using the kit, or both. The instructional material of
the kit of the invention can, for example, be affixed to a
container which contains a kit of the invention or be shipped
together with a container which contains the kit. Alternatively,
the instructional material can be shipped separately from the
container with the intention that the instructional material and
the kit be used cooperatively by the recipient.
[0102] The "stringency" with which two polynucleotides anneal means
the relative likelihood that the polynucleotides will anneal in a
solution as the conditions of the solution become less favorable
for annealing. Examples of stringent conditions are known in the
art and can be found in available references (e.g., Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989,
6.3.1-6.3.6). Aqueous and non-aqueous annealing methods are
described in that reference and either can be used. In general, a
first pair of polynucleotides anneal with higher stringency than a
second pair if the first pair is more likely to anneal (or remain
annealed) as one or more of the salt concentration, temperature,
and detergent concentration are increased.
[0103] With respect to a disorder, a "correlation factor" for a
disorder-associated polymorphism is the fractions of humans who are
heterozygous or homozygous for the polymorphism who exhibit the
disorder. The correlation factor can, alternatively, be based
solely on those who are heterozygous, solely on those who are
homozygous, or on those who are either heterozygous or
homozygous.
[0104] A "non-extendable" nucleotide residue is a nucleotide
residue that is capable of being added to a polynucleotide by a
polymerase (i.e., by extension of the polynucleotide in association
with a complement thereof, catalyzed by the polymerase) and that,
upon addition to the polynucleotide, renders the polynucleotide
incapable of being further extended by the polymerase.
Description
[0105] The invention relates to kits and methods for assessing the
skin health of a human by assessing occurrence in the human's
genome of genetic polymorphisms that are associated with disorders
(i.e., skin disorders or other disorders). Unlike other methods
that predict susceptibility to a disorder based on occurrence of a
particular polymorphism in a particular gene, a panel of
informative genes and polymorphisms is disclosed herein. Using two
or more of the genes in this panel, one can assess the
susceptibility of a human to a skin disorder, even if the skin
disorder has not been specifically associated with occurrence of a
polymorphism in the panel.
[0106] It has been discovered an individual's skin health can be
assessed by determining the polymorphic forms of certain genes that
are present in the individual's genome. The genes which are
assessed are disclosed herein. Assessment of disorder-associated
polymorphisms in two or more of these genes (preferably three,
four, five, six, eight, ten, fifteen, or more of these genes) in a
human's genome is predictive of the human's skin health. The
greater the number of these genes in which occurrence of
disorder-associated polymorphisms is assessed, the greater the
precision of the methods for predicting the human's skin health is
likely to be. Occurrence in the individual's genome of other
polymorphisms (e.g., ones known to be associated with occurrence of
the skin disorder of interest) can also be assessed concurrently or
sequentially.
[0107] Skin disorders for which the kits and methods described
herein are useful include inflammatory disorders (e.g., contact
dermatitis, uticaria, atopic dermatitis, psoriasis, lichen planus,
cutaneous lupus erythematosus, pemphigus, and scleroderma, sun
damage (e.g., reddening and sun burn), infectious diseases (e.g.,
bacterial and viral infections), and skin tumors (e.g., keratoses,
squamous cell carcinomas, basal cell carcinomas, melanomas, and
Kaposi's sarcoma).
[0108] Susceptibility of an individual to a skin disorder can be
affected by oxidative stress that skin cells experience. Several of
the genes having polymorphic forms that are informative for skin
health encode proteins that modulate the body's response to or
protection from oxidative stress. For example, genes which protect
against oxidative stress include genes which encode an enzyme that
catalyzes conversion of a toxic oxygen species to a less toxic
oxygen species, genes that encode a protein that directly provides
protection against oxidative damage, genes which encode a protein
that indirectly provides protection against oxidative damage, genes
which encode a component of the human DNA repair system, and genes
(not necessarily included within the preceding groups) which are
associated with inducible production of reactive oxygen species in
immune cells following microbial infection.
[0109] Polymorphisms have been identified in some, if not all, of
the numerous genes that encode components of the human DNA repair
system. Disorder-associated polymorphisms in these genes can be
informative for the skin health of an individual (e.g., for
susceptibility of the individual to a skin disorder). Examples of
these genes include those which encode apurinic and apyrimidinic
endonucleases, enzymes that catalyze excision of nucleotide
residues damaged by ultraviolet radiation, and enzymes that
catalyze site specific-recombination. Many such genes are known,
and include those listed in Wood et al., 2001, Science
291(5507):1284-1289.
[0110] Skin comprises immune cells and acts as a first line of
defense against microbial invasion. Genes that induce production of
reactive oxygen species in immune cells following microbial
infection include genes (e.g., genes which encode components of the
human phagocyte-specific NADPH-oxidase complex) associated with
respiratory burst (sometimes designated oxidative burst) phenomena
of macrophages and polymorphonuclear nucleophilic granulocytes,
whereby toxic oxygen species are produced in response to invasion
of a tissue by a microbe (e.g., a protozoan, or a bacterium such as
a Pseudomonas, Salmonella, or Serratia bacterium or a known
pathogen such as Bacillus anthracis, Escherichia coli, or
Staphylococcus aureus). Also included within this group are genes
which are known to be aberrant in patients afflicted with disorders
that inhibit or abolish antimicrobial activities of macrophages
(e.g., chronic granulomatous disease). Disorder-associated
polymorphisms in substantially any of these genes can be
informative of the susceptibility of the individual to a skin
disorder, particularly a skin infection or inflammatory skin
disorder. Identification of individuals in whom such polymorphisms
occur (e.g., using the methods described herein) can be used, for
example, to assess whether an individual has an elevated risk for
developing a skin disorder and whether some disorder inhibits
intervention should be undertaken.
[0111] It is not critical that the gene in which the occurrence of
a polymorphism occurs is recognized as being directly or indirectly
involved in a skin disorder. It is sufficient that an association
can be made between either the level of expression of the gene or
the sequence of the gene product and skin health of humans.
[0112] Skin disorders include allergic reactions, such as hives and
contact dermatitis. Genes that encode enzymes that catalyze
reactions responsible for decreasing electrophilic potential of
allergens (or their metabolites), a process designated
biotransformation of allergens, can affect the skin health of a
human. Members of the glutathione S-transferase (GST) family of
enzymes, such as GSTP1, participate in the biotransformation of
allergens. These enzymes also catalyze interconversions among
reactive forms of oxygen. Occurrence of one or more polymorphism in
one of these GST genes can be used to assess skin health of an
individual.
[0113] Another protein involved in production of toxic oxygen
species by components of the immune system in response to allergen
exposure is TNF-alpha. Allen et al. (2000, Immunogenetics
51:201-205) described a polymorphism that occurs at nucleotide
residue -308 (i.e., in the promoter region) of the gene that
encodes TNF-alpha. This polymorphism can be one of those assessed
as described herein.
[0114] Among enzymes that catalyze conversion of a toxic oxygen
species to a less toxic oxygen species, four are of particular
relevance, namely mitochondrial MnSOD, cytoplasmic CZSOD, catalase
(CAT), and glutathione peroxidase (GP). Polymorphisms that occur in
these genes are known to be associated with various disorders (see,
e.g., Kimura et al., 2000, Am. J. Ophthalmol. 130:769-773).
Occurrence of disorder-associated polymorphisms in at least one
(and preferably two, three, or all) of these four genes should be
assessed in the methods described herein, given the importance of
these genes. Similarly, the kits described herein preferably
include reagents for detecting disorder-associated polymorphisms in
at least one (and preferably two, three, or all) of these four
genes. In addition, the significance of occurrence of
disorder-associated polymorphisms in these genes can be applied by
assigning a greater weighting factor to disorder-associated
polymorphisms of these genes than to disorder-associated
polymorphisms in other genes disclosed herein.
[0115] Genes in which disorder-associated polymorphisms occur that
are useful for assessing the skin health of an individual include
[0116] genes which encode an enzyme that catalyzes conversion of a
toxic oxygen species to a less toxic oxygen species; [0117] genes
which encode a protein that provides protection against oxidative
stress; [0118] genes which encode a protein that induces production
of a toxic oxygen species; [0119] genes which encode a protein that
indirectly affects oxidative stress; [0120] genes which encode a
protein for which the level of expression of the protein is
associated with oxidative stress; [0121] genes which encode a
component of the human DNA repair system; and [0122] genes which
encode a protein associated with production of a toxic oxygen
species by a macrophage or polymorphonuclear neutrophilic
granulocyte.
[0123] It has been discovered that the following genes are of
particular relevance to skin health: [0124] a) the gene which
encodes mitochondrial MnSOD; [0125] b) the gene which encodes
cytoplasmic CZSOD; [0126] c) the gene which encodes catalase;
[0127] d) the gene which encodes hGPX1; [0128] e) the gene which
encodes GSTP1 [0129] f) the gene which encodes NAD(P)H:quinone
oxidoreductase; [0130] g) the gene which encodes epoxide hydrolase;
[0131] h) the gene which encodes TNF-alpha; [0132] i) the phox
gene; [0133] j) the gene which encodes nitric oxide synthase;
[0134] k) the gene which encodes cytochrome P450; [0135] l) the
gene which encodes MMP-1; and [0136] m) the gene which encodes
profilagrin.
[0137] Occurrence in a patient's genome of a disorder-associated
polymorphism in one of genes a)-l) is an indication that the
patient is at a greater risk of developing a skin disorder (or is
already afflicted with the disorder) than a human whose genome does
not include the disorder-associated polymorphism. Occurrence of
multiple disorder-associated polymorphisms in these genes in a
patient's genome is an indication that that patient is at greater
risk for developing a skin disorder (i.e., has poorer skin health)
than a human in whose genome fewer (or none) of the
disorder-associated polymorphisms occur. Thus, there is a
cumulative effect of disorder-associated polymorphisms in the genes
identified herein on the skin health of the human in which they
occur.
[0138] Occurrence of two copies of the same disorder-associated
polymorphism in the same human (i.e., homozygosity for the
disorder-associated polymorphism) is an indication that the human
is at a greater risk for developing a skin disorder (i.e., has
poorer skin health) than a human in whom only a single copy of the
polymorphism occurs (i.e., an individual heterozygous for the
disorder-associated polymorphism). Homozygosity for the
disorder-associated polymorphism can be accounted for by more
heavily weighting occurrence of two copies of the
disorder-associated polymorphism than occurrence of only a single
copy (e.g., by multiplying the significance associated with
occurrence of the disorder-associated polymorphism by a factor such
as two, five, ten or another value).
[0139] Although the invention is not limited to the particular
disorder-associated polymorphisms in the genes identified herein,
it is recognized that disorder-associated polymorphisms that occur
in particular portions of the genes can be more significant
indicators of skin health than disorder-associated polymorphisms
that occur in particular portions of the genes. Thus,
disorder-associated polymorphisms that occur in the following
regions of the indicated genes can be weighted more heavily than
disorder-associated polymorphisms that occur in other portions of
the genes. These polymorphisms include [0140] A)
disorder-associated polymorphisms in the open reading frame
encoding mitochondrial MnSOD; [0141] B) disorder-associated
polymorphisms in the open reading frame encoding cytoplasmic CZSOD;
[0142] C) disorder-associated polymorphisms in the promoter region
of the gene encoding catalase; [0143] D) disorder-associated
polymorphisms in the open reading frame encoding hGPX1; [0144] E)
disorder-associated polymorphisms in the open reading frame
encoding GSTP1; [0145] F) disorder-associated polymorphisms in the
open reading frame encoding NAD(P)H:quinone oxidoreductase; [0146]
G) disorder-associated polymorphisms in the open reading frame
encoding epoxide hydrolase; [0147] H) disorder-associated
polymorphisms in the promoter region of the gene encoding
TNF-alpha; [0148] I) disorder-associated polymorphisms in the open
reading frame of the phox gene; [0149] J) disorder-associated
polymorphisms in the open reading frame encoding nitric oxide
synthase; [0150] K) disorder-associated polymorphisms in the 5'
flanking region of the gene encoding cytochrome P450; and [0151] L)
disorder-associated polymorphisms in the promoter region of the
gene encoding MMP-1.
[0152] Occurrence of any of a number of particular polymorphisms
can be assayed in order to assess an individual's skin health. A
non-limiting list of such polymorphisms include the following:
[0153] I) a polymorphism manifested as a change from an alanine
residue to a valine residue at amino acid residue 9 (i.e., in the
signal sequence) of mitochondrial MnSOD; [0154] II) a polymorphism
manifested as a change from an isoleucine residue to a thymine
residue at amino acid residue 58 of mitochondrial MnSOD; [0155]
III) a polymorphism manifested as a change from a valine residue to
a glutamic acid residue at amino acid residue 7 of cytoplasmic
CZSOD; [0156] IV) a polymorphism manifested as a change from a
cysteine residue to a phenylalanine residue at amino acid residue 6
of cytoplasmic CZSOD; [0157] V) a polymorphism manifested as a
change from a cytosine residue to a thymine residue at nucleotide
residue -262 (i.e., in the promoter region) of the catalase gene;
[0158] VI) a polymorphism in the hGPX1 gene manifested as a change
from a proline residue to a leucine residue at amino acid residue
198 of glutathione peroxidase; [0159] VII) a polymorphism in the
GSTP1 gene manifested as a change from a valine residue to an
isoleucine residue at amino acid residue 105 of glutathione
S-transferase P1; [0160] VIII) a polymorphism manifested as a
change from a cytosine residue to a thymine residue at nucleotide
residue 242 (i.e., in the coding region) of the gene encoding
NAD(P)H:quinone oxidoreductase; [0161] IX) a polymorphism
manifested as a change from a thymine residue to a cytosine residue
at nucleotide residue 113 in exon 3 of the gene which encodes
epoxide hydrolase (i.e., effecting change of from a tyrosine
residue to a histidine residue in epoxide hydrolase); [0162] X) a
polymorphism manifested as a change to an adenine residue at
nucleotide residue -238 (i.e., in the promoter region) of the gene
which encodes TNF-alpha (i.e., the TNF-alpha promoter variant
designated TNF2); [0163] XI) a polymorphism manifested as a change
to an adenine residue at nucleotide residue -308 (i.e., in the
promoter region) of the gene which encodes TNF-alpha (i.e., the
TNF-alpha promoter variant designated TNF3); [0164] XII) a
polymorphism manifested as a change from a cytosine residue to a
thymine residue at nucleotide residue 242 (i.e., in the coding
region) of the phox gene encoding the NADH/NADPH oxidase p22
subunit; [0165] XIII) a polymorphism manifested as a 27 nucleotide
residue repeat in intron 4 (i.e., between nucleotide residues 5130
and 5511) of the gene encoding nitric oxide synthase; [0166] XIV) a
polymorphism manifested as a change from an adenine residue to a
guanine residue at nucleotide residue -290 (i.e., in the
5'-flanking region) of the gene encoding cytochrome P450 (i.e., the
polymorphism designated the CYP3A4 cytochrome P450 variant); and
[0167] XV) a polymorphism manifested as occurrence of a two
consecutive guanine residues at a site including nucleotide residue
-1607 of the human gene encoding MMP-1.
[0168] Occurrence of a disorder-associated polymorphism in an
individual's genome can be assessed in any of a variety of ways. In
one embodiment, a simple test (e.g., primer extension, PCR
amplification, or molecular beacon oligonucleotide-binding) is used
to determine whether or not the disorder-associated polymorphism
occurs in the individual's genome (i.e., without regard to copy
number). In another embodiment, a test is used to determine whether
the individual's genome includes a non-disorder-associated
polymorphism corresponding to a known disorder-associated
polymorphism in a gene disclosed herein (i.e., as an indication
that the individual is at least heterozygous for the
non-disorder-associated polymorphism). In yet another embodiment, a
test (i.e., using multiple probes or primers) is used that is able
to detect both disorder-associated polymorphisms and
non-disorder-associated polymorphisms in two, three, four, or more
genes disclosed herein. Using such a test, one can determine both
occurrence of a disorder-associated polymorphism in an individual's
genome and whether the individual is homozygous or heterozygous for
the disorder-associated polymorphism. This test also permits
`checking` of results, since it can both account for all known
polymorphic forms and indicate when a previously uncharacterized
polymorphism occurs at or near the site of a known
polymorphism.
[0169] In a kit or method for detecting both disorder-associated
polymorphisms and non-disorder-associated polymorphisms known for
the genes disclosed herein, one or more (preferably at least two,
three, four, five, six, eight, ten, or fifteen or more) of the
following polymorphisms can be assessed: [0170] i) a polymorphism
manifested as occurrence of a codon encoding alanine at amino acid
residue 9 (i.e., in the signal sequence) of mitochondrial MnSOD;
[0171] ii) a polymorphism manifested as occurrence of a codon
encoding valine at amino acid residue 9 (i.e., in the signal
sequence) of mitochondrial MnSOD; [0172] iii) a polymorphism
manifested as occurrence of a codon encoding isoleucine at amino
acid residue 58 of mitochondrial MnSOD; [0173] iv) a polymorphism
manifested as occurrence of a codon encoding thymine at amino acid
residue 58 of mitochondrial MnSOD; [0174] v) a polymorphism
manifested as occurrence of a codon encoding valine at amino acid
residue 7 of cytoplasmic CZSOD; [0175] vi) a polymorphism
manifested as occurrence of a codon encoding glutamic acid at amino
acid residue 7 of cytoplasmic CZSOD; [0176] vii) a polymorphism
manifested as occurrence of a codon encoding cysteine at amino acid
residue 6 of cytoplasmic CZSOD; [0177] viii) a polymorphism
manifested as occurrence of a codon encoding phenylalanine at amino
acid residue 6 of cytoplasmic CZSOD; [0178] ix) a polymorphism
manifested as occurrence of a cytosine residue at nucleotide
residue -262 (i.e., in the promoter region) of the catalase gene;
[0179] x) a polymorphism manifested as occurrence of a thymine
residue at nucleotide residue -262 (i.e., in the promoter region)
of the catalase gene; [0180] xi) a polymorphism in the hGPX1 gene
manifested as occurrence of a codon encoding proline at amino acid
residue 198 of glutathione peroxidase; [0181] xii) a polymorphism
in the hGPX1 gene manifested as occurrence of a codon encoding
leucine at amino acid residue 198 of glutathione peroxidase; [0182]
xiii) a polymorphism in the GSTP1 gene manifested as occurrence of
a codon encoding valine at amino acid residue 105 of glutathione
S-transferase P1; [0183] xiv) a polymorphism in the GSTP1 gene
manifested as occurrence of a codon encoding isoleucine at amino
acid residue 105 of glutathione S-transferase P1; [0184] xv) a
polymorphism manifested as occurrence of a cytosine residue at
nucleotide residue 242 (i.e., in the coding region) of the gene
encoding NAD(P)H:quinone oxidoreductase; [0185] xvi) a polymorphism
manifested as occurrence of a thymine residue at nucleotide residue
242 (i.e., in the coding region) of the gene encoding
NAD(P)H:quinone oxidoreductase; [0186] xvii) a polymorphism
manifested as occurrence of a thymine residue at nucleotide residue
113 in exon 3 of the gene which encodes epoxide hydrolase (i.e.,
resulting in a tyrosine residue in epoxide hydrolase); [0187]
xviii) a polymorphism manifested as occurrence of a cytosine
residue at nucleotide residue 113 in exon 3 of the gene which
encodes epoxide hydrolase (i.e., resulting in a histidine residue
in epoxide hydrolase); [0188] xix) a polymorphism manifested as
occurrence of an adenine residue at nucleotide residue -238 (i.e.,
in the promoter region) of the gene which encodes TNF-alpha (i.e.,
the TNF-alpha promoter variant designated TNF2); [0189] xx) a
polymorphism manifested as occurrence of an adenine residue at
nucleotide residue -308 (i.e., in the promoter region) of the gene
which encodes TNF-alpha (i.e., the TNF-alpha promoter variant
designated TNF3); [0190] xxi) a polymorphism manifested as
occurrence of a cytosine residue at nucleotide residue 242 (i.e.,
in the coding region) of the phox gene encoding the NADH/NADPH
oxidase p22 subunit; [0191] xxii) a polymorphism manifested as
occurrence of a thymine residue at nucleotide residue 242 (i.e., in
the coding region) of the phox gene encoding the NADH/NADPH oxidase
p22 subunit; [0192] xxiii) a polymorphism manifested as a 27
nucleotide residue repeat in intron 4 (i.e., between nucleotide
residues 5130 and 5511) of the gene encoding nitric oxide synthase;
[0193] xxiv) a polymorphism manifested as absence of a 27
nucleotide residue repeat in intron 4 (i.e., between nucleotide
residues 5130 and 5511) of the gene encoding nitric oxide synthase;
[0194] xxv) a polymorphism manifested as occurrence of an adenine
residue at nucleotide residue -290 (i.e., in the 5'-flanking
region) of the gene encoding cytochrome P450 (i.e., the
polymorphism designated the CYP3A4 cytochrome P450 variant); [0195]
xxvi) a polymorphism manifested as occurrence of a guanine residue
at nucleotide residue -290 (i.e., in the 5'-flanking region) of the
gene encoding cytochrome P450 (i.e., the polymorphism designated
the CYP3A4 cytochrome P450 variant); [0196] xxvii) a polymorphism
manifested as occurrence of a single guanine residue at nucleotide
residue -1607 of the human gene encoding MMP-1; and [0197] xxviii)
a polymorphism manifested as occurrence of a two consecutive
guanine residues at a site including nucleotide residue -1607 of
the human gene encoding MMP-1.
[0198] Another important set of polymorphisms that can be assessed
in order to determine an overall skin health score for a human are
disorder-associated polymorphisms that occur in the human
profilagrin gene. Numerous polymorphic forms of these gene are
known, and the associations of each of these forms with one or more
disorders is not yet fully characterized. Of course, whenever a
profilagrin polymorphic form is or becomes associated with a
disorder, occurrence of that disorder-associated polymorphic form
of the profilagrin gene can be used to assess skin health in a
human. Known profilagrin polymorphisms include SNPs and
filagrin-polymer-length polymorphisms. This latter term refers to
the number of filagrin polypeptides into which the profilagrin
polypeptide is post-translationally cleaved in a human cell.
Various individual humans are known to harbor profilagrin genes
that encode a profilagrin with at least 9 to 12 filagrin units.
[0199] An important aspect of this invention is that human skin
health (e.g., susceptibility to a skin disorder such as psoriasis,
eczema, a skin cancer, or a bacterial infection) can be associated
with occurrence in the human's genome of a disorder-associated
polymorphism in one of the genes described herein--even if there is
no known biochemical or physiological association between
occurrence of the polymorphism and skin health or incidence of a
skin disorder. Put another way, the present inventors have
discovered that genes and polymorphisms disclosed herein are
predictive indicators of the state of an individual human's skin
health. By assessing whether or not disorder-associated
polymorphisms occur in the genes identified herein in an individual
(and how many such polymorphisms occur in those genes), one can
assess the individual's skin health (e.g., as manifested as the
likelihood that the individual has, or will develop a skin
disorder).
[0200] If it is determined that an individual has poor skin health
(e.g., because multiple disorder-associated polymorphisms occur in
the individual's genome in the genes disclosed herein), then the
individual can be encouraged to make changes to improve their skin
health, skin appearance, or to reduce the likelihood of developing
skin disorders. Such changes can include use of skin protective
compositions (e.g., nutritional formulas including anti-oxidants,
sunscreens, and topical or system corticosteroids), use of cosmetic
compositions, improving nutrition, and avoiding sunlight.
Determination that an individual has relatively poor skin health
can also be used as an indication that the individual should be
monitored more closely than others for development of skin
disorders.
[0201] Early detection of a predisposition to develop a skin
disorder can enable an individual (or the individual's physician)
to take steps to delay, inhibit, alleviate (i.e., reduce the
severity of), or even prevent the disorder. The appropriate steps
for treating and preventing skin disorders are well known and
include modifying diet, exercise, and intake or topical application
of nutrients and pharmaceuticals. Palliative, therapeutic, and
prophylactic methods are known for many skin disorders, and these
can be undertaken once a patient's susceptibility to the disorder
is known. Thus, the kits and methods described herein permit a skin
disorder to be treated, inhibited, or prevented. The kits and
methods described herein allow these interventions to be made at an
early stage of the skin disorder (when treatment is often most
effective), or even before the disorder is symptomatically
manifested.
[0202] It was not previously appreciated that detection in a
human's genome of two or more disorder-associated polymorphisms in
the genes disclosed herein is indicative that the human exhibits
poorer skin health, manifested as greater susceptibility to skin
disorders than individuals having a genome containing fewer (or
none) of these disorder-associated polymorphisms. Previous studies
are believed to have recognized only association between a
polymorphism in only individual genes identified herein and a
particular disorder. The inventors believe that they are the first
to describe methods and kits for assessing a human's susceptibility
to skin disorders based on occurrence in the human of certain
polymorphisms that are not recognized as being associated with the
individual skin disorder.
[0203] It has been discovered that disorder-associated
polymorphisms that occur in the genes identified herein as a)-l)
can be used to assess both an individual's skin health and the
likelihood that the individual will develop (or is currently
afflicted with) a skin disorder. In one embodiment of the kits and
method described herein, occurrence of disorder-associated
polymorphisms (and/or non-disorder-associated polymorphisms) is
assessed in two or more of the genes identified herein as a)-l),
such as occurrence of a disorder-associated polymorphisms
identified herein as A)-L). By way of example, the kit or method
can involve assessing occurrence of multiple polymorphisms
identified herein as i)-xxviii).
Methods of Assessing Skin Health
[0204] The invention includes a method of assessing the skin health
(e.g., relative susceptibility to one or more skin disorders) of a
human. Skin health can be calculated relative to a hypothetical
human whose genome does not contain a single disorder-associated
polymorphism in a gene disclosed herein. Alternatively,
susceptibility can be calculated relative to another human who may
have one or more different disorder-associated polymorphism than
the human being assessed. In practice, the basis upon which raw
susceptibility scores are calculated is immaterial, so long as the
same basis is used for all humans whose scores are to be compared
(i.e., so that the scores are relatable to one another).
[0205] The relative skin health of a human can be used to assess
the risks and benefits of a variety of compositions, conditions,
and interventions. In one embodiment, the skin health of a human
can be used to determine whether the human would benefit by
supplementing nutritional intake with a composition that contains
one or more vitamins, minerals, or other skin protective agents.
Numerous skin protective agents are known and additional agents are
certain to be discovered over time. The usefulness of the kits and
methods disclosed herein does not depend on the identity of the
particular agent. Examples of skin protective agents include
vitamins (especially anti-oxidant vitamins), minerals,
naturally-occurring amino acids, derivatives of naturally-occurring
amino acids, plant extracts, and conventional skin care products
(e.g., skin softening and moisturizing lotions, Aloe extracts, and
the like). Anti-oxidant vitamins are preferably administered to
skin in a protein-complexed form (e.g., using preparations such as
the VITAZYME.RTM. vitamin preparations sold by Arch Personal Care
Products, L.P. of South Plainfield, N.J.). Similarly, skin
protective minerals such as manganese and selenium are also
preferably administered to skin in a protein-complexed form (e.g.,
using preparations such as the ACQUA-BIOMIN.TM. mineral
preparations sold by Arch Personal Care Products, L.P.). Useful
skin protective plant extracts include gape polyphenols and
naturally active botanicals (NABs) such as NAB Pikea robusta (red
algae) extract, NAB fennel seed (Foeniculum vulgare) extract, and
NAB red clover (Trifollum Pratense) leaf extract. Useful
naturally-occurring amino acids and derivatives thereof include
glycine, glutamine, N-acetylcysteine, and trimethylglycine.
Furthermore, the skin health, as assessed using a kit or method as
described herein, can indicate an appropriate dose of such an agent
for an individual patient.
[0206] The skin protective agent that is administered to an
individual subject can be determined by the overall skin health
score, by observing the genes in which disorder-associated
polymorphisms occur, or both.
[0207] For example, if a disorder-associated polymorphism occurs in
the subject's MnSOD gene, then a manganese-containing skin
protective agent, a zinc-containing skin protective agent, or a
manganese- and zinc-containing skin protective agent (e.g., one of
the ACQUA BIOMIN.TM. products) can be applied to the subject's skin
to inhibit or alleviate skin disorders.
[0208] If a disorder-associated polymorphism occurs in the
subject's glutathione peroxidase gene, then a skin protective agent
comprising one or more of selenium, grape polyphenols,
N-acetylcysteine, glutamine, glycine, or NAB fennel seed can be
applied to the subject's skin to inhibit or alleviate skin
disorders.
[0209] If a disorder-associated polymorphism occurs in the
subject's microsomal epoxide hydrolase gene, then a skin protective
agent comprising one or more of N-acetylcysteine, trimethylglycine,
an anti-oxidant vitamine (e.g., one of the VITAZYME.RTM. products),
NAB Pikea robusta, and NAB fennel seed can be applied to the
subject's skin to inhibit or alleviate skin disorders.
[0210] If a disorder-associated polymorphism occurs in the
subject's tumor necrosis factor-alpha gene, then a skin protective
agent comprising one or both NAB Pikea robusta and NAB red clover
leaf can be applied to the subject's skin to inhibit or alleviate
skin disorders.
[0211] Skin health of a human is determined by assessing occurrence
in the human's genome of disorder-associated polymorphisms in a
plurality of genes disclosed herein (e.g., 2, 3, 4, 6, 8, 10, 15,
or more genes). Occurrence of a disorder-associated polymorphism in
one of these genes is an indication that the human has a greater
susceptibility to skin disorders and poorer skin health than a
human in whose genome the polymorphism does not occur. Occurrence
of two or more such polymorphisms in the human's genome indicates
that the human exhibits even greater susceptibility to skin
disorders (and poorer skin health).
[0212] Occurrence of each disorder-associated polymorphism in a
gene disclosed herein is not necessarily equally indicative of
susceptibility to skin disorders and poorer skin health. In order
to account for differences in the significance of various
disorder-associated polymorphisms, a weighting factor can be
assigned to each polymorphism detected in the methods and kits
described herein. As indicated above, some genes have a more
significant role in skin health in humans than others. Generally,
disorder-associated polymorphisms that occur in one of these genes
are more significant than polymorphisms that occur in genes having
less significant roles in skin health. Thus, a greater weighting
factor can be assigned to these polymorphisms than to others. By
way of example, the weighting factor assigned to these
polymorphisms can be 1 to 10 times greater than the weighting
factor assigned to disorder-associated polymorphisms in other
genes.
[0213] Another factor which can influence the significance that is
assigned to occurrence of a disorder-associated polymorphism in a
human's genome is the degree to which the polymorphism is
correlated with the corresponding disorder. Some disorders are
highly correlated with occurrence of a genetic polymorphism, and
other disorders exhibit lower correlation with a polymorphism. When
a polymorphism is reported to be associated with a disorder (i.e.,
with a disease or pathological condition), a degree of correlation
between the polymorphism and the disorder can be determined or
obtained from reports in the literature. One useful way of
calculating a factor that describes correlation between a
polymorphism and a disorder is to calculate an odds ratio that
describes the likelihood that an individual in whose genome the
disorder-associate polymorphism 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 polymorphism, odds ratios calculated for
homozygous individuals can also be used, if they are available.
Odds ratios can be calculated as described in the art.
[0214] For a disorder-associated polymorphism, the odds ratio can
be calculated as follows. First, the odds of being afflicted with
the disorder are calculated for a first population in which the
polymorphism occurs by dividing the number of afflicted individuals
in the first population by the total number of individuals in the
first population. Second, the odds of being afflicted with the
disorder are calculated for a first population in whom the
polymorphism does not occur by dividing the number of afflicted
individuals in the second population by the total number of
individuals in the second population. Third, the odds ratio is
calculated by dividing the odds for the first population by the
odds for the second population. If the odds ratio is greater than
one, then this is an indication that occurrence of the polymorphism
is associated with occurrence of the disorder. Furthermore, the
magnitude of the odds ratio is an indication of the significance of
the association.
[0215] A skin health score for a human can be determined as
follows. A significance score can be assigned to each
disorder-associated polymorphism that is detected in the human's
genome using a method or kit described herein. The significance
score is a constant (e.g., 1.00), and is multiplied by any
significance factor (e.g., 1-10) and by any correlation factor that
is available. If information is available which describes the
correlation between homozygosity for the polymorphism and the
corresponding disorder, then that correlation factor should be used
in place of the correlation factor for mere occurrence of the
polymorphism, at least if the method or kit is used to rule out
occurrence in the subject's genome of corresponding
disorder-non-associated polymorphisms. If significance and
correlation factors are not available, then values of 1.00 should
be assigned to each. The skin health score is determined by summing
the significance score for each disorder-associated polymorphism
that is detected using the method or kit. This skin health score
can be compared with the values obtained from other subjects, or it
can be compared with the value (i.e., zero) which would be expected
to occur in a human whose genome does not include any
disorder-associated polymorphism in a gene disclosed herein. A high
skin health score corresponds to poor skin health. Thus, for two
individuals having different skin health scores, the individual
having the lower score has better skin health than the individual
having the higher score.
[0216] The method used to assess occurrence of any particular
disorder-associated polymorphism (or disorder-non-associated
polymorphism) is not critical. Numerous methods of detecting
occurrence of a polymorphism are known in the art, and
substantially any of those methods can be used in the kits and
methods described herein. Naturally, the reagents included in the
kit will vary depending on the method to be used to detect the
polymorphisms. Examples of some suitable polymorphism detection
methods are provided below.
[0217] In one embodiment, a pair of oligonucleotide primers are
used to amplify a portion of the gene that includes a polymorphic
region. Detection of one or more of the polymorphisms that occur at
the polymorphic region can be achieved by contacting the amplified
portion with an oligonucleotide having a sequence such that it will
anneal under stringent conditions with the amplified portion only
if one polymorphism occurs at the portion, but will not anneal with
the amplified portion if another polymorphism 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. An example
of stringent conditions is hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more washes in 0.2.times.SSC, 0.1% (w/v) SDS at 50.degree.
C.
[0218] In an alternative embodiment, one or more molecular beacon
oligonucleotides are used to detect polymorphisms
(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 polymorphisms are known to
occur).
[0219] 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 hairpin regions, whereby
the probe can self-anneal to form a hairpin structure. The 5'- and
3'-ends are brought into close association when the hairpin
structure forms. The probe also comprises a targeting portion which
is selected to be complementary to a target sequence (e.g. a single
polymorphism of a gene disclosed herein). The targeting portion and
at least one of the hairpin regions are located in close proximity
to one another, meaning that the targeting portion either overlaps
the hairpin region or flanks it, having no more than about 5
nucleotide residues therebetween.
[0220] If the hairpin regions of the molecular beacon probe anneal
with one another, then the probe does not fluoresce, because the
hairpin structure forms and the fluorescence quencher attached to
one end of the probe quenches fluorescence of the label attached to
the other end of the probe. If the targeting portion of the probe
anneals with a region of a nucleic acid having the target sequence,
then formation of the hairpin structure is inhibited, the
fluorescence quencher is not brought into association with the
fluorescent label, and the probe fluoresces. Multiple molecular
beacon probes can be used in a single reaction mixture, and
fluorescence associated with the probes can be differentiated if
the molecular beacon probes are spectrally distinct.
[0221] Thus, in this embodiment, 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 polymorphism of
a gene disclosed herein. If the polymorphism to be detected is a
single nucleotide polymorphism (SNP), then the target region
includes, and preferably is approximately centered around, the
nucleotide residue at which the polymorphism occurs. More
preferably, two such probes are used, one having a targeting region
completely complementary to the target region of one polymorphism
of the gene (e.g., one of two polymorphisms of a particular SNP),
and the other having a targeting region completely complementary to
the target region of a corresponding polymorphism of the gene
(e.g., the other polymorphism of the SNP if there are only two
polymorphic forms), so that occurrence of disorder-associated and
non-disorder-associated polymorphisms can be simultaneously
determined.
[0222] In yet another embodiment of how polymorphisms in gene
disclosed herein can be assessed, oligonucleotide primers which are
complementary to a region adjacent a characteristic residue of the
polymorphism are extended using a polymerase enzyme, and the
identity of the nucleotide residue that is added to the primer in
the position complementary to the characteristic residue is
determined. The primer can be extended in the presence of
non-extendable nucleotide residues in order to ensure that a
limited number of nucleotide residues (or only one) are
incorporated into the primer. Methods of this type are known in the
art (e.g., the SNP-IT.RTM. technology of Orchid Biocomputer, Inc.)
and are described, for example in U.S. Pat. Nos. 6,013,431 and
6,004,744.
Methods of Assessing Susceptibility to Individual Skin
Disorders
[0223] An patient's skin health score is predictive of the
patient's susceptibility to individual skin disorders (a higher
score indicating a greater susceptibility to such disorders). The
rate or likelihood of development and progression of skin disorders
can be estimated by assessing the skin health (i.e., determining a
skin health score) of a patient. The rate or likelihood of
development and progression of the particular skin disorders
disclosed herein can be estimated by assessing occurrence of the
disorder-associated polymorphisms disclosed herein.
[0224] The individual skin disorders for which susceptibility can
be assessed using these methods are not limited to those disclosed
herein. The methods can be used to assess susceptibility to
substantially any skin disorder. However, it is likely that
congenital skin defects which lead to development of aberrant skin
in utero or during the first few years of life are unlikely to be
associated with the disorder-associated polymorphisms described
herein.
Kits for Assessing Skin Health
[0225] The invention includes a kit for assessing the skin health
of a human and/or the susceptibility of the human to a skin
disorder. The kit contains reagents for performing one or more of
the methods described herein. The reagents used in certain
embodiments of the methods described herein are indicated above.
Reagents useful for performing those methods using a variety of
alternative sample preparation and polymorphism detection methods
or chemistries are apparent to the skilled artisan.
[0226] Kits for detecting polymorphisms 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 polymorphisms in at least two (or at least
three, four, six, eight, ten, or fifteen or more) genes disclosed
herein.
[0227] In one embodiment, the kit includes a plurality of
oligonucleotides which anneal under stringent conditions with a
disorder-associated polymorphism 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-polymorphism.
Each of the oligonucleotides can be attached to a surface in order
to facilitate handling of the oligonucleotide. The oligonucleotides
can be linked with a plurality of surfaces (e.g., oligonucleotides
for a particular polymorphism being attached to a particle discrete
from a particle to which oligonucleotides for another polymorphism
are attached), or they can be attached to discrete regions of a
single surface (e.g., a glass or silicon surface having
oligonucleotides attached at defined locations thereon, as in the
GENECHIP.TM. device of Affymetrix, Inc.). Annealing between
individual oligonucleotides and the polymorphism corresponding
thereto can be detected using standard methods. The kit can also
comprise oligonucleotides that are useful as molecular beacon
probes or as extendable primers.
[0228] In one embodiment, the kit further comprises a DNA
collection kit or apparatus, such as that described in co-pending
U.S. Pat. No. 6,291,171. Advantageously, DNA collected using the
kit or apparatus can be stored or archived, and subjected to
additional testing as previously unknown disorder-associated
polymorphisms are discovered in the genes disclosed herein, or as
the significance of previously unappreciated polymorphisms is
realized.
[0229] The invention also relates to a method of assessing the
advisability that a human should consume or apply a nutritional
product comprising a skin protective agent such as those described
above. The method is performed as described herein for assessing
the skin health of a human. If poorer skin health is detected in
the human (i.e., relative to a human not having a
disorder-associated polymorphism in a gene identified herein), then
it is more advisable the human should consume or apply a
nutritional product comprising the skin protective agent. A greater
skin health score (i.e., corresponding to poorer skin health) in a
human correlates with an increased advisability that the human
should use such a nutritional product, and also indicates that a
greater dose of the skin agent(s) should be included in the
nutritional product.
[0230] It will be appreciated by those skilled in the art that
changes can made to the embodiments described above without
departing from the broad inventive concept thereof.
[0231] The disclosure of every patent, patent application, and
publication cited herein is hereby incorporated herein by reference
in its entirety.
[0232] This invention is not limited to the particular embodiments
disclosed, and includes modifications within the spirit and scope
of the present invention as defined by the appended claims.
* * * * *