U.S. patent application number 13/893224 was filed with the patent office on 2013-11-28 for cxcr4 as a susceptibility locus in juvenile idiopathic arthritis (jia) and methods of use thereof for the treatment and diagnosis of the same.
The applicant listed for this patent is The Children's Hospital of Philadelphia. Invention is credited to Terri H. Finkel, Hakon Hakonarson, Haitao Zhang.
Application Number | 20130315931 13/893224 |
Document ID | / |
Family ID | 46051593 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130315931 |
Kind Code |
A1 |
Finkel; Terri H. ; et
al. |
November 28, 2013 |
CXCR4 as a Susceptibility Locus in Juvenile Idiopathic Arthritis
(JIA) and Methods of Use Thereof for the Treatment and Diagnosis of
the Same
Abstract
Compositions and methods useful for the diagnosis and treatment
of juvenile idiopathic arthritis are disclosed.
Inventors: |
Finkel; Terri H.;
(Wynnewood, PA) ; Zhang; Haitao; (Philadelphia,
PA) ; Hakonarson; Hakon; (Malvern, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Children's Hospital of Philadelphia |
Philadelphia |
PA |
US |
|
|
Family ID: |
46051593 |
Appl. No.: |
13/893224 |
Filed: |
May 13, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US11/60430 |
Nov 11, 2011 |
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13893224 |
|
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61412775 |
Nov 11, 2010 |
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Current U.S.
Class: |
424/172.1 ;
506/10; 506/14; 506/16; 506/7 |
Current CPC
Class: |
G01N 33/5047 20130101;
C12Q 2600/136 20130101; C12Q 2600/156 20130101; G01N 33/5041
20130101; C12Q 1/6883 20130101; C12Q 2600/118 20130101; C12Q
2600/158 20130101; G01N 2800/102 20130101 |
Class at
Publication: |
424/172.1 ;
506/7; 506/10; 506/16; 506/14 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
[0002] Pursuant to 35 U.S.C. .sctn.202(c) it is acknowledged that
the U.S. Government has rights in the invention described, which
was made in part with funds from the National Institutes of Health,
Grant Number 5RC1AR058606-02.
Claims
1. A method for detecting an increased risk for developing juvenile
idiopathic arthritis (JIA) in a test subject, comprising, a)
obtaining a nucleic acid sample from said subject and determining
whether said sample contains at least one SNP or mutation
identified in the CXCR4 locus wherein if said SNP or mutation is
detected, said patient has an increased risk for developing JIA,
wherein said SNP or mutation containing nucleic acid is selected
from the group consisting of i) SNPs provided in Table 4; ii) a SNP
in LD with those listed in Table 4; iii) a non synonymous mutation;
and iv) a stop gain mutation.
2. The method as claimed in claim 1, wherein the target nucleic
acid is amplified prior to detection.
3. The method of claim 1, wherein the step of detecting the
presence of said SNP or mutation is performed using a process
selected from the group consisting of detection of specific
hybridization, measurement of allele size, restriction fragment
length polymorphism analysis, allele-specific hybridization
analysis, single base primer extension reaction, and sequencing of
an amplified polynucleotide.
4. The method as claimed in claim 1, wherein in the target nucleic
acid is DNA.
5. The method of claim 1, wherein nucleic acids comprising said SNP
or mutation are obtained from an isolated cell of a human test
subject.
6. A method for identifying therapeutic agents which alter immune
cell function or signaling, comprising a) providing cells
expressing at least one SNP or mutation containing nucleic acid as
claimed in claim 1; b) providing cells which express the cognate
wild type sequences corresponding to the SNP or mutation containing
nucleic acid of step a); c) contacting the cells of steps a) and b)
with a test agent and d) analyzing whether said agent alters immune
signaling or function of cells of step a) relative to those of step
b), thereby identifying agents which alter immune cell signaling or
function.
7. The method of claim 6 wherein said agent is selected from the
group consisting of agents listed in Table 12.
8. The method of claim 6 wherein said therapeutic has efficacy for
the treatment of JIA or other related aberrant immune dysfunction
disorders.
9. A method for the treatment of JIA in a patient in need thereof
comprising administration of an effective amount of the agent
identified by claim 6.
10. The method of claim 9, wherein said agent modulates the
inflammatory process.
11. The method of claim 9, wherein said agent modulates cytokine
release.
12. A multiplex SNP or mutation panel comprising isolated nucleic
acids informative of the presence of JIA, wherein said panel
contains the nucleic acids provided in Table 4 or a SNV selected
from the group consisting of NM.sub.--001008540:c.C1049A:p.S350Y,
NM.sub.--001008540:c.A169C:p.I57L, NM.sub.--001008540:c.C19G:p.L7V,
and NM.sub.--001008540:c.T14A:p.L5X stop codon.
13. A vector comprising at least one of the SNP-containing nucleic
acids of claim 12.
14. A host cell comprising the vector of claim 13.
15. A solid support comprising the JIA related SNP containing
nucleic acid of claim 12.
16. A kit for performing the method of claim 1, comprising a
multiplex SNP panel comprising nucleic acids informative of the
presence of JIA in an isolated nucleic acid sample, wherein said
panel contains the nucleic acids provided in Table 4.
17. The kit of claim 16, wherein said panel is affixed to a solid
support.
18. The kit of claim 16, wherein said panel is provided in silico.
Description
[0001] This application is a continuation in part of
PCT/US2011/60430 filed Nov. 11, 2011 which in turn claims priority
to U.S. Provisional Application 61/412,775 filed Nov. 11, 2010, the
entire contents being incorporated herein by reference as though
set forth in full.
FIELD OF THE INVENTION
[0003] This invention relates to the fields of genetics and the
diagnosis of juvenile idiopathic arthritis (JIA). More
specifically, the invention provides compositions and methods
useful for the diagnosis and treatment of JIA.
BACKGROUND OF THE INVENTION
[0004] Several publications and patent documents are cited through
the specification in order to describe the state of the art to
which this invention pertains. Each of these citations is
incorporated herein by reference as though set forth in full.
[0005] Pediatric arthritis is the leading cause of acquired
disability in children, afflicting about one in 1000 children
worldwide,.sup.1 all ethnicities and both genders, with onset as
early as the first year of life. Classification schemes for
pediatric arthritis are under evolution, akin to the recent
classification changes for adult rheumatoid arthritis; juvenile
rheumatoid arthritis (JRA) is the term used historically in North
America, while juvenile idiopathic arthritis (JIA) is the preferred
name elsewhere, and is now used increasingly worldwide. JIA is
defined as a group of chronic arthritides of unknown etiology,
occurring in children from 0 to 16 years of age..sup.3 Morbidity
associated with JIA can be life-long--with as many as 50% of
children with JIA entering adulthood with active disease.sup.1--and
represents a significant medical, financial, and emotional burden
for patients, for their families, and for society. In the United
States alone, arthritis and rheumatic diseases impact more than 46
million adults and 300,000 children, at a cost of $128 billion
annually in direct and indirect medical costs. Multiple studies
have shown that adults with JIA have lower rates of employment than
matched controls, and health related quality of life is diminished
in adults with JIA, particularly in those with active
disease..sup.4 Prompt recognition of the disease is important in
preventing permanent disability, however, lack of specific
confirmatory testing often delays diagnosis. The optimal management
of JIA remains complicated and poorly defined, despite recent
advances in therapy.sup.1; important side effects of many of the
newer therapeutic agents are increasingly being recognized,
although associated risk factors for the development of these
adverse events remain unknown.
[0006] The etiology of JIA is largely unknown. To our knowledge,
there are no data supporting a major role for environmental
exposures;.sup.5 this does not preclude a role of the environment
in the pathogenesis of JIA, but research to identify environmental
risk factors is lacking. On the other hand, a genetic component has
been implicated from twin and family studies:.sup.6 monozygotic
twins have a concordance rate between 25% and 40%; the calculated
sibling recurrence risk ratio (.lamda.s=15-30) is similar to that
calculated for type I diabetes; sibling pairs tend to show
concordance for age of onset, subtype and course; and a subset of
patients with JIA exhibits a heritable predisposition to develop
this disease with an autosomal dominant pattern of inheritance.
[0007] Yet, the genetic etiology of JIA remains elusive. JIA is an
example of a complex phenotype that is likely to be determined by
the net result of interactions between multiple genetic and
environmental factors. Previous attempts at identifying the genetic
basis of this disease through candidate gene studies have met with
limited success. The major histocompatibility complex (MHC), in
particular, the HLA-DRB1 locus, has been established as having the
strongest influence on susceptibility to JIA,.sup.7 contributing
.about.20% of the proportion of sibling recurrent risk..sup.8
Non-MHC loci are important as well, although candidate gene studies
have only convincingly demonstrated associations for a few loci,
and only four (PTPN22, STAT4, IL2-IL21, and IL2RA) have shown
association in the same direction in two or more
studies..sup.6,9-11 An inherent problem with candidate gene
association studies is their reliance on a suspected
disease-causing gene(s), whose identification derives from a
particular biological hypothesis regarding pathogenesis of the
disease or from previous work with related diseases. Since the
pathophysiological mechanisms underlying JIA are unknown, continued
use of the hypothesis-driven candidate gene association approach is
likely to miss many important genetic risk factors for the disease.
In view of all the foregoing it is clear a need exists to further
characterize and elucidate the genetic and molecular mechanisms
underlying this devastating disease.
SUMMARY OF THE INVENTION
[0008] In accordance with the present invention, methods are
provided for the diagnosis and treatment of JIA. An exemplary
method entails detecting the presence of at least one, two, three,
four, five, six or all of the JIA associated CXCR4 SNPs or
mutations in a target polynucleotide wherein if said (s) is/are
present, said patient has an increased risk for developing JIA.
[0009] In one aspect of the present invention, a method for
detecting a propensity for developing juvenile idiopathic arthritis
(JIA) in a patient in need thereof is provided. An exemplary method
entails detecting the presence of at least one SNP containing
nucleic acid in a target polynucleotide, said SNP being informative
of a the presence of an JIA associated alteration in the CXCR4 gene
wherein if said SNP is present, said patient has an increased risk
for developing JIA, wherein said SNP containing nucleic acid is
provided in Table 4.
[0010] In another embodiment of the invention a method for
identifying agents which alter immune cell function or signaling is
provided. Such a method comprises providing cells expressing at
least one nucleic acid comprising the JIA CXCR4 SNPs of the
invention, (step a); providing cells which express the cognate wild
type sequences which lack the SNP (step b); contacting the cells
from each sample with a test agent and analyzing whether said agent
alters immune signaling or function of cells of step a) relative to
those of step b), thereby identifying agents which alter immune
cell signaling or function. Methods of treating JIA patients via
administration of test agents identified using the methods
described herein are also encompassed by the present invention.
[0011] The invention also provides at least one isolated JIA
related SNP-containing nucleic acid selected from the group listed
in Table 4 and includes any SNP in linkage disequilibrium (LD) with
these SNPs. In one embodiment, a multiplex SNP panel containing all
of the informative SNPs from the tables provided herein and any of
their LD associated SNPs is disclosed. Such SNP containing nucleic
acids which indicate the presence of JIA associated nucleic acids
may optionally be contained in a suitable expression vector for
expression in immune cells. Alternatively, they may be immobilized
on a solid support. In yet another alternative, the panel may be
provided in silico.
[0012] According to yet another aspect of the present invention,
there is provided a method of treating JIA in a patient determined
to have at least one prescribed single nucleotide polymorphism
indicative of the presence of an JIA, as described hereinbelow, by
administering to the patient a therapeutically effective amount of
at least one member of the agents listed in Table 12. This method
provides a test and treat paradigm, whereby a patient's genetic
profile is used to personalize treatment with therapeutics targeted
towards specific immunological defects found in individuals
exhibiting JIA. Such a test and treat model may benefit up to 50%
of patients with JIA with greater efficacy and fewer side effects
than non-personalized treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. Genome-wide association results for the 2q21 region.
Manhattan plot showing the -log.sub.10(p values) of SNPs from the
GWAS of the discovery cohort. p values down to 10.sup.-20 only are
shown for optimal resolution; p values for the MHC on chromosome 6
reached 2.63.times.10.sup.-23.
[0014] FIG. 2. Association results for genotyped and imputed SNPs
in the 2q21 region. Genotyped (triangle) and imputed (circles) SNPs
are plotted with their combined p values in the three cohorts typed
genome-wide (discovery and replication cohorts 1 and 2). SNPs are
colored on the basis of their correlation with rs953387 (red:
r.sup.2.gtoreq.0.8; orange: 0.5.ltoreq.r.sup.2.ltoreq.0.8; yellow:
0.2.ltoreq.r.sup.2<0.5). Estimated recombination rates from
HapMap data are plotted to reflect the local linkage disequilibrium
(LD) structure.
[0015] FIG. 3. Representation of the chromosome 2q21 associated
interval. The figure shows pairwise r.sup.2 LD values of the top 10
SNPs from the HapMap CEU population (URLs). The CXCR4 gene is drawn
to scale in relation to the associated SNPs.
[0016] FIG. 4. Representation of the PTPN22 linkage-disequilibrium
(LD) blocks. The figure shows pairwise r.sup.2 LD values from the
HapMap CEU population. The PTPN22 gene is drawn to scale in
relation to the associated SNPs.
[0017] FIG. 5. The most significantly associated genotyped and
imputed SNPs (p<5.times.10.sup.-8 in the combined meta-analysis)
in the vicinity of CXCR4 on chromosome 2q21.
[0018] FIG. 6. CXCR4 expression levels stratified by SNP genotype.
The SNP genotypes of rs953387 and rs1016269 are associated with
CXCR4 transcript levels by quantitative PCR (eQTL) in immortalized
B-cell lines (from 30 CEU children) and T-cells (from umbilical
cords of 75 individuals of Western European origin),
respectively.
[0019] FIG. 7. CXCR4 tissue-specific gene expression levels (probe
identifiers: 217028_at, 211919_s_at, and 209201_x_at), based on the
GNF SymAtlas database on 79 human tissues. Expression of CXCR4 is
most prominent in the CD-annotated T-cells, B-cells, NK and
dendritic cells.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The genome-wide association approach serves the critical
need for a more comprehensive and unbiased strategy to identify
causal genes related to JIA. We have assembled a large cohort of
JIA patients--`large`, given the relatively low prevalence of
JIA--for a genome-wide association study (GWAS), which requires no
a priori assumptions regarding pathology. We have taken a novel
approach to discovery of JIA susceptibility loci by focusing on the
phenotypic commonality amongst our patients, that is, chronic
inflammation of the joints. We report a GWAS of JIA designed to
address the hypothesis that clinically different phenotypes share
common susceptibility loci, which confer a risk in childhood for
inflammatory arthritis. This approach is supported by the
intriguing discovery of common disease susceptibility loci across
multiple autoimmune diseases..sup.9,12,13
[0021] To identify risk factors underlying JIA, we performed a
genome-wide association study and replication in 1166 JIA cases and
9500 unrelated controls of European ancestry. Two epidemiological
JIA cohorts were combined and designated as the discovery cohort
and three as de novo independent replication cohorts. Variants at
the CXCR4 locus on 2q21 reached genome-wide significance in the
discovery cohort for association with JIA, and were confirmed in
our independent replication cohorts (combined
p=1.03.times.10.sup.-13 for rs953387, near CXCR4). Participants
with the minor allelic variant of rs953387 (OR 0.59, 95% CI
0.50-0.69) and with associated variants in close linkage
disequilibrium with the rs953387 minor allele were protected from
JIA, with about two-fold lower risk of JIA than non-carriers. CXCR4
expression was correlated with the genotype of rs953387 in
lymphoblastoid cell lines (p=0.014) and T-cells (p=0.0054). This is
the first GWAS of JIA to discover a genome-wide significant
susceptibility locus outside of the major histocompatibility
complex (MHC), and the first genetic evidence implicating CXCR4 in
the pathogenesis of autoimmune disease. This cell-surface chemokine
receptor has already been targeted in other diseases and may serve
as a tractable therapeutic target for this crippling pediatric
arthritis.
I. Definitions
[0022] For purposes of the present invention, "a" or "an" entity
refers to one or more of that entity; for example, "a cDNA" refers
to one or more cDNA or at least one cDNA. As such, the terms "a" or
"an," "one or more" and "at least one" can be used interchangeably
herein. It is also noted that the terms "comprising," "including,"
and "having" can be used interchangeably. Furthermore, a compound
"selected from the group consisting of" refers to one or more of
the compounds in the list that follows, including mixtures (i.e.
combinations) of two or more of the compounds. According to the
present invention, an isolated, or biologically pure molecule is a
compound that has been removed from its natural milieu. As such,
"isolated" and "biologically pure" do not necessarily reflect the
extent to which the compound has been purified. An isolated
compound of the present invention can be obtained from its natural
source, can be produced using laboratory synthetic techniques or
can be produced by any such chemical synthetic route.
[0023] The term "genetic alteration" as used herein refers to a
change from the wild-type or reference sequence of one or more
nucleic acid molecules. Genetic alterations include without
limitation, base pair substitutions, additions and deletions of at
least one nucleotide from a nucleic acid molecule of known
sequence.
[0024] A "single nucleotide polymorphism (SNP)" refers to a change
in which a single base in the DNA differs from the usual base at
that position. These single base changes are called SNPs or
"snips." Millions of SNP's have been cataloged in the human genome.
Some SNPs such as that which causes sickle cell are responsible for
disease. Other SNPs are normal variations in the genome.
[0025] "JIA-associated SNP" or "JIA-associated specific marker" is
a SNP or marker which is associated with an increased or decreased
risk of developing JIA not found normal patients who do not have
this disease. Such markers may include but are not limited to
nucleic acids, proteins encoded thereby, or other small molecules.
Thus, the phrase "JIA-associated SNP containing nucleic acid" is
encompassed by the above description.
[0026] "CXCR-4" is an alpha-chemokine receptor specific for
stromal-derived-factor-1 (SDF-1 also called CXCL12), a molecule
possessing potent chemotactic activity for lymphocytes. This
receptor is one of several chemokine receptors that HIV isolates
can use to infect CD4+ T cells. CXCR4 is upregulated during the
implantation window in natural and hormone replacement therapy
cycles in the endometrium, producing, in presence of a human
blastocyst, a surface polarization of the CXCR4 receptors
suggesting that this receptor is implicated in the adhesion phase
of human implantation. CXCR4's ligand SDF-1 is known to be
important in hematopoietic stem cell homing to the bone marrow and
in hematopoietic stem cell quiescence.
[0027] The term "solid matrix" as used herein refers to any format,
such as beads, microparticles, a microarray, the surface of a
microtitration well or a test tube, a dipstick or a filter. The
material of the matrix may be polystyrene, cellulose, latex,
nitrocellulose, nylon, polyacrylamide, dextran or agarose.
[0028] The phrase "consisting essentially of" when referring to a
particular nucleotide or amino acid means a sequence having the
properties of a given SEQ ID NO:. For example, when used in
reference to an amino acid sequence, the phrase includes the
sequence per se and molecular modifications that would not affect
the functional and novel characteristics of the sequence.
[0029] "Target nucleic acid" as used herein refers to a previously
defined region of a nucleic acid present in a complex nucleic acid
mixture wherein the defined wild-type region contains at least one
known nucleotide variation which may or may not be associated with
JIA. The nucleic acid molecule may be isolated from a natural
source by cDNA cloning or subtractive hybridization or synthesized
manually. The nucleic acid molecule may be synthesized manually by
the triester synthetic method or by using an automated DNA
synthesizer.
[0030] With regard to nucleic acids used in the invention, the term
"isolated nucleic acid" is sometimes employed. This term, when
applied to DNA, refers to a DNA molecule that is separated from
sequences with which it is immediately contiguous (in the 5' and 3'
directions) in the naturally occurring genome of the organism from
which it was derived. For example, the "isolated nucleic acid" may
comprise a DNA molecule inserted into a vector, such as a plasmid
or virus vector, or integrated into the genomic DNA of a prokaryote
or eukaryote. An "isolated nucleic acid molecule" may also comprise
a cDNA molecule. An isolated nucleic acid molecule inserted into a
vector is also sometimes referred to herein as a recombinant
nucleic acid molecule.
[0031] With respect to RNA molecules, the term "isolated nucleic
acid" primarily refers to an RNA molecule encoded by an isolated
DNA molecule as defined above. Alternatively, the term may refer to
an RNA molecule that has been sufficiently separated from RNA
molecules with which it would be associated in its natural state
(i.e., in cells or tissues), such that it exists in a
"substantially pure" form.
[0032] By the use of the term "enriched" in reference to nucleic
acid it is meant that the specific DNA or RNA sequence constitutes
a significantly higher fraction (2-5 fold) of the total DNA or RNA
present in the cells or solution of interest than in normal cells
or in the cells from which the sequence was taken. This could be
caused by a person by preferential reduction in the amount of other
DNA or RNA present, or by a preferential increase in the amount of
the specific DNA or RNA sequence, or by a combination of the two.
However, it should be noted that "enriched" does not imply that
there are no other DNA or RNA sequences present, just that the
relative amount of the sequence of interest has been significantly
increased.
[0033] The term "complementary" describes two nucleotides that can
form multiple favorable interactions with one another. For example,
adenine is complementary to thymine as they can form two hydrogen
bonds. Similarly, guanine and cytosine are complementary since they
can form three hydrogen bonds. Thus if a nucleic acid sequence
contains the following sequence of bases, thymine, adenine, guanine
and cytosine, a "complement" of this nucleic acid molecule would be
a molecule containing adenine in the place of thymine, thymine in
the place of adenine, cytosine in the place of guanine, and guanine
in the place of cytosine. Because the complement can contain a
nucleic acid sequence that forms optimal interactions with the
parent nucleic acid molecule, such a complement can bind with high
affinity to its parent molecule.
[0034] With respect to single stranded nucleic acids, particularly
oligonucleotides, the term "specifically hybridizing" refers to the
association between two single-stranded nucleotide molecules of
sufficiently complementary sequence to permit such hybridization
under pre-determined conditions generally used in the art
(sometimes termed "substantially complementary"). In particular,
the term refers to hybridization of an oligonucleotide with a
substantially complementary sequence contained within a
single-stranded DNA or RNA molecule of the invention, to the
substantial exclusion of hybridization of the oligonucleotide with
single-stranded nucleic acids of non-complementary sequence. For
example, specific hybridization can refer to a sequence which
hybridizes to any JIA specific marker gene or nucleic acid, but
does not hybridize to other nucleotides. Also polynucleotide which
"specifically hybridizes" may hybridize only to a single specific
marker, such as an JIA-specific marker shown in the Tables
contained herein. Appropriate conditions enabling specific
hybridization of single stranded nucleic acid molecules of varying
complementarity are well known in the art.
[0035] For instance, one common formula for calculating the
stringency conditions required to achieve hybridization between
nucleic acid molecules of a specified sequence homology is set
forth below (Sambrook et al., Molecular Cloning, Cold Spring Harbor
Laboratory (1989):
T.sub.m=81.5.degree. C.+16.6 Log [Na+]+0.41(% G+C)-0.63(%
formamide)-600/#bp in duplex
As an illustration of the above formula, using [Na+]=[0.368] and
50% formamide, with GC content of 42% and an average probe size of
200 bases, the T.sub.m is 57.degree. C. The T.sub.m of a DNA duplex
decreases by 1-1.5.degree. C. with every 1% decrease in homology.
Thus, targets with greater than about 75% sequence identity would
be observed using a hybridization temperature of 42.degree. C.
[0036] The stringency of the hybridization and wash depend
primarily on the salt concentration and temperature of the
solutions. In general, to maximize the rate of annealing of the
probe with its target, the hybridization is usually carried out at
salt and temperature conditions that are 20-25.degree. C. below the
calculated T.sub.m of the hybrid. Wash conditions should be as
stringent as possible for the degree of identity of the probe for
the target. In general, wash conditions are selected to be
approximately 12-20.degree. C. below the T.sub.m of the hybrid. In
regards to the nucleic acids of the current invention, a moderate
stringency hybridization is defined as hybridization in
6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/ml
denatured salmon sperm DNA at 42.degree. C., and washed in
2.times.SSC and 0.5% SDS at 55.degree. C. for 15 minutes. A high
stringency hybridization is defined as hybridization in
6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/ml
denatured salmon sperm DNA at 42.degree. C., and washed in
1.times.SSC and 0.5% SDS at 65.degree. C. for 15 minutes. A very
high stringency hybridization is defined as hybridization in
6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/ml
denatured salmon sperm DNA at 42.degree. C., and washed in
0.1.times.SSC and 0.5% SDS at 65.degree. C. for 15 minutes.
[0037] The term "oligonucleotide," as used herein is defined as a
nucleic acid molecule comprised of two or more ribo- or
deoxyribonucleotides, preferably more than three. The exact size of
the oligonucleotide will depend on various factors and on the
particular application and use of the oligonucleotide.
Oligonucleotides, which include probes and primers, can be any
length from 3 nucleotides to the full length of the nucleic acid
molecule, and explicitly include every possible number of
contiguous nucleic acids from 3 through the full length of the
polynucleotide. Preferably, oligonucleotides are at least about 10
nucleotides in length, more preferably at least 15 nucleotides in
length, more preferably at least about 20 nucleotides in
length.
[0038] The term "probe" as used herein refers to an
oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA,
whether occurring naturally as in a purified restriction enzyme
digest or produced synthetically, which is capable of annealing
with or specifically hybridizing to a nucleic acid with sequences
complementary to the probe. A probe may be either single-stranded
or double-stranded. The exact length of the probe will depend upon
many factors, including temperature, source of probe and use of the
method. For example, for diagnostic applications, depending on the
complexity of the target sequence, the oligonucleotide probe
typically contains 15-25 or more nucleotides, although it may
contain fewer nucleotides. The probes herein are selected to be
complementary to different strands of a particular target nucleic
acid sequence. This means that the probes must be sufficiently
complementary so as to be able to "specifically hybridize" or
anneal with their respective target strands under a set of
pre-determined conditions. Therefore, the probe sequence need not
reflect the exact complementary sequence of the target. For
example, a non-complementary nucleotide fragment may be attached to
the 5' or 3' end of the probe, with the remainder of the probe
sequence being complementary to the target strand. Alternatively,
non-complementary bases or longer sequences can be interspersed
into the probe, provided that the probe sequence has sufficient
complementarity with the sequence of the target nucleic acid to
anneal therewith specifically.
[0039] The term "primer" as used herein refers to an
oligonucleotide, either RNA or DNA, either single-stranded or
double-stranded, either derived from a biological system, generated
by restriction enzyme digestion, or produced synthetically which,
when placed in the proper environment, is able to functionally act
as an initiator of template-dependent nucleic acid synthesis. When
presented with an appropriate nucleic acid template, suitable
nucleoside triphosphate precursors of nucleic acids, a polymerase
enzyme, suitable cofactors and conditions such as a suitable
temperature and pH, the primer may be extended at its 3' terminus
by the addition of nucleotides by the action of a polymerase or
similar activity to yield a primer extension product. The primer
may vary in length depending on the particular conditions and
requirement of the application. For example, in diagnostic
applications, the oligonucleotide primer is typically 15-25 or more
nucleotides in length. The primer must be of sufficient
complementarity to the desired template to prime the synthesis of
the desired extension product, that is, to be able anneal with the
desired template strand in a manner sufficient to provide the 3'
hydroxyl moiety of the primer in appropriate juxtaposition for use
in the initiation of synthesis by a polymerase or similar enzyme.
It is not required that the primer sequence represent an exact
complement of the desired template. For example, a
non-complementary nucleotide sequence may be attached to the 5' end
of an otherwise complementary primer. Alternatively,
non-complementary bases may be interspersed within the
oligonucleotide primer sequence, provided that the primer sequence
has sufficient complementarity with the sequence of the desired
template strand to functionally provide a template-primer complex
for the synthesis of the extension product.
[0040] Polymerase chain reaction (PCR) has been described in U.S.
Pat. Nos. 4,683,195, 4,800,195, and 4,965,188, the entire
disclosures of which are incorporated by reference herein.
[0041] The term "vector" relates to a single or double stranded
circular nucleic acid molecule that can be infected, transfected or
transformed into cells and replicate independently or within the
host cell genome. A circular double stranded nucleic acid molecule
can be cut and thereby linearized upon treatment with restriction
enzymes. An assortment of vectors, restriction enzymes, and the
knowledge of the nucleotide sequences that are targeted by
restriction enzymes are readily available to those skilled in the
art, and include any replicon, such as a plasmid, cosmid, bacmid,
phage or virus, to which another genetic sequence or element
(either DNA or RNA) may be attached so as to bring about the
replication of the attached sequence or element. A nucleic acid
molecule of the invention can be inserted into a vector by cutting
the vector with restriction enzymes and ligating the two pieces
together.
[0042] Many techniques are available to those skilled in the art to
facilitate transformation, transfection, or transduction of the
expression construct into a prokaryotic or eukaryotic organism. The
terms "transformation", "transfection", and "transduction" refer to
methods of inserting a nucleic acid and/or expression construct
into a cell or host organism. These methods involve a variety of
techniques, such as treating the cells with high concentrations of
salt, an electric field, or detergent, to render the host cell
outer membrane or wall permeable to nucleic acid molecules of
interest, microinjection, PEG-fusion, and the like.
[0043] The term "promoter element" describes a nucleotide sequence
that is incorporated into a vector that, once inside an appropriate
cell, can facilitate transcription factor and/or polymerase binding
and subsequent transcription of portions of the vector DNA into
mRNA. In one embodiment, the promoter element of the present
invention precedes the 5' end of the JIA specific marker nucleic
acid molecule such that the latter is transcribed into mRNA. Host
cell machinery then translates mRNA into a polypeptide.
[0044] Those skilled in the art will recognize that a nucleic acid
vector can contain nucleic acid elements other than the promoter
element and the JIA specific marker nucleic acid molecule. These
other nucleic acid elements include, but are not limited to,
origins of replication, ribosomal binding sites, nucleic acid
sequences encoding drug resistance enzymes or amino acid metabolic
enzymes, and nucleic acid sequences encoding secretion signals,
localization signals, or signals useful for polypeptide
purification.
[0045] A "replicon" is any genetic element, for example, a plasmid,
cosmid, bacmid, plastid, phage or virus, that is capable of
replication largely under its own control. A replicon may be either
RNA or DNA and may be single or double stranded.
[0046] An "expression operon" refers to a nucleic acid segment that
may possess transcriptional and translational control sequences,
such as promoters, enhancers, translational start signals (e.g.,
ATG or AUG codons), polyadenylation signals, terminators, and the
like, and which facilitate the expression of a polypeptide coding
sequence in a host cell or organism.
[0047] As used herein, the terms "reporter," "reporter system",
"reporter gene," or "reporter gene product" shall mean an operative
genetic system in which a nucleic acid comprises a gene that
encodes a product that when expressed produces a reporter signal
that is a readily measurable, e.g., by biological assay,
immunoassay, radio immunoassay, or by colorimetric, fluorogenic,
chemiluminescent or other methods. The nucleic acid may be either
RNA or DNA, linear or circular, single or double stranded,
antisense or sense polarity, and is operatively linked to the
necessary control elements for the expression of the reporter gene
product. The required control elements will vary according to the
nature of the reporter system and whether the reporter gene is in
the form of DNA or RNA, but may include, but not be limited to,
such elements as promoters, enhancers, translational control
sequences, poly A addition signals, transcriptional termination
signals and the like.
[0048] The introduced nucleic acid may or may not be integrated
(covalently linked) into nucleic acid of the recipient cell or
organism. In bacterial, yeast, plant and mammalian cells, for
example, the introduced nucleic acid may be maintained as an
episomal element or independent replicon such as a plasmid.
Alternatively, the introduced nucleic acid may become integrated
into the nucleic acid of the recipient cell or organism and be
stably maintained in that cell or organism and further passed on or
inherited to progeny cells or organisms of the recipient cell or
organism. Finally, the introduced nucleic acid may exist in the
recipient cell or host organism only transiently.
[0049] The term "selectable marker gene" refers to a gene that when
expressed confers a selectable phenotype, such as antibiotic
resistance, on a transformed cell.
[0050] The term "operably linked" means that the regulatory
sequences necessary for expression of the coding sequence are
placed in the DNA molecule in the appropriate positions relative to
the coding sequence so as to effect expression of the coding
sequence. This same definition is sometimes applied to the
arrangement of transcription units and other transcription control
elements (e.g. enhancers) in an expression vector.
[0051] The terms "recombinant organism", or "transgenic organism"
refer to organisms which have a new combination of genes or nucleic
acid molecules. A new combination of genes or nucleic acid
molecules can be introduced into an organism using a wide array of
nucleic acid manipulation techniques available to those skilled in
the art. The term "organism" relates to any living being comprised
of a least one cell. An organism can be as simple as one eukaryotic
cell or as complex as a mammal. Therefore, the phrase "a
recombinant organism" encompasses a recombinant cell, as well as
eukaryotic and prokaryotic organism.
[0052] The term "isolated protein" or "isolated and purified
protein" is sometimes used herein. This term refers primarily to a
protein produced by expression of an isolated nucleic acid molecule
of the invention. Alternatively, this term may refer to a protein
that has been sufficiently separated from other proteins with which
it would naturally be associated, so as to exist in "substantially
pure" form. "Isolated" is not meant to exclude artificial or
synthetic mixtures with other compounds or materials, or the
presence of impurities that do not interfere with the fundamental
activity, and that may be present, for example, due to incomplete
purification, addition of stabilizers, or compounding into, for
example, immunogenic preparations or pharmaceutically acceptable
preparations.
[0053] A "specific binding pair" comprises a specific binding
member (sbm) and a binding partner (bp) which have a particular
specificity for each other and which in normal conditions bind to
each other in preference to other molecules. Examples of specific
binding pairs are antigens and antibodies, ligands and receptors
and complementary nucleotide sequences. The skilled person is aware
of many other examples. Further, the term "specific binding pair"
is also applicable where either or both of the specific binding
member and the binding partner comprise a part of a large molecule.
In embodiments in which the specific binding pair comprises nucleic
acid sequences, they will be of a length to hybridize to each other
under conditions of the assay, preferably greater than 10
nucleotides long, more preferably greater than 15 or 20 nucleotides
long.
[0054] "Sample" or "patient sample" or "biological sample"
generally refers to a sample which may be tested for a particular
molecule, preferably a JIA specific marker molecule, such as a
marker described hereinbelow. Samples may include but are not
limited to cells, body fluids, including blood, serum, plasma,
cerebral spinal fluid, urine, synovial fluid, saliva, tears,
pleural fluid and the like.
[0055] The terms "agent" and "compound" are used interchangeably
herein and denote a chemical compound, a mixture of chemical
compounds, a biological macromolecule, or an extract made from
biological materials such as bacteria, plants, fungi, or animal
(particularly mammalian) cells or tissues. Biological
macromolecules include siRNA, shRNA, antisense oligonucleotides,
peptides, peptide/DNA complexes, and any nucleic acid based
molecule which exhibits the capacity to modulate the activity of
the CNV or SNP-containing nucleic acids described herein or their
encoded proteins. Agents and compounds may also be referred to as
"test agents" or "test compounds" which are evaluated for potential
biological activity by inclusion in screening assays described
hereinbelow.
[0056] The term "modulate" as used herein refers to
increasing/promoting or decreasing/inhibiting a particular
cellular, biological or signaling function associated with the
normal activities of the genetic alteration containing molecules
described herein or the proteins encoded thereby. For example, the
term modulate refers to the ability of a test compound or test
agent to interfere with signaling or activity of a gene or protein
of the present invention. Alternatively, the term may refer to
augmentation of the activity of such a protein.
II. Methods of Using JIA-Associated CXCR4 SNPs for Diagnosing a
Propensity for the Development of JIA
[0057] JIA-related SNP-containing CXCR4 nucleic acids, including
but not limited to those listed below may be used for a variety of
purposes in accordance with the present invention. JIA-associated
SNP-containing DNA, RNA, or fragments thereof may be used as probes
to detect the presence of and/or expression of JIA specific
markers. Methods in which JIA specific marker nucleic acids may be
utilized as probes for such assays include, but are not limited to:
(1) in situ hybridization; (2) Southern hybridization (3) northern
hybridization; and (4) assorted amplification reactions such as
polymerase chain reactions (PCR).
[0058] Further, assays for detecting JIA-associated SNPs may be
conducted on any type of biological sample, including but not
limited to body fluids (including synovial fluid, blood, urine,
serum, gastric lavage, cerebral spinal fluid), any type of cell
(such as brain cells, white blood cells, mononuclear cells, fetal
cells in maternal circulation) or body tissue.
[0059] Clearly, JIA-associated SNP-containing nucleic acids,
vectors expressing the same, JIA SNP-containing marker proteins and
anti-JIA specific marker antibodies of the invention can be used to
detect JIA associated SNPs in body tissue, cells, or fluid, and
alter JIA SNP-containing CXCR4 marker protein expression for
purposes of assessing the genetic and protein interactions involved
in the development of JIA.
[0060] In most embodiments for screening for JIA-associated SNPs,
the JIA-associated SNP-containing nucleic acid in the sample will
initially be amplified, e.g. using PCR, to increase the amount of
the templates as compared to other sequences present in the sample.
This allows the target sequences to be detected with a high degree
of sensitivity if they are present in the sample. This initial step
may be avoided by using highly sensitive array techniques that are
important in the art.
[0061] Alternatively, new detection technologies can overcome this
limitation and enable analysis of small samples containing as
little as 1 .mu.g of total RNA. Using Resonance Light Scattering
(RLS) technology, as opposed to traditional fluorescence
techniques, multiple reads can detect low quantities of mRNAs using
biotin labeled hybridized targets and anti-biotin antibodies.
Another alternative to PCR amplification involves planar wave guide
technology (PWG) to increase signal-to-noise ratios and reduce
background interference. Both techniques are commercially available
from Qiagen Inc. (USA).
[0062] In another embodiment, the sequence information for the
CXCR4 SNPs associated with JIA of the invention are stored in a
computer readable medium and the patients genetic information has
already been obtained and is also stored in a computer readable
medium. In this embodiment, the diagnostic method entails a
comparison of this control and patient sequence information in
silico in order to diagnose an increased risk for developing
JIA.
[0063] Any of the aforementioned techniques may be used to detect
or quantify JIA-associated SNP marker expression and accordingly,
diagnose an increased risk for developing the same.
III. Kits and Articles of Manufacture
[0064] Any of the aforementioned products can be incorporated into
a kit which may contain a JIA-associated SNP specific CXCR4 marker
polynucleotide or one or more such markers immobilized on a Gene
Chip, an oligonucleotide, a polypeptide, a peptide, an antibody, a
label, marker, reporter, a pharmaceutically acceptable carrier, a
physiologically acceptable carrier, instructions for use, a
container, a vessel for administration, an assay substrate, or any
combination thereof.
IV. Methods of Using JIA-Associated CXCR4 SNPs for the Development
of Therapeutic Agents
[0065] Since the SNPs identified herein have been associated with
the etiology of JIA, methods for identifying agents that modulate
the activity of the CXCR4 gene and its encoded products containing
such SNPs should result in the generation of efficacious
therapeutic agents for the treatment of this disorder.
[0066] The CXCR4 locus provides a suitable target for the rational
design of therapeutic agents. Small nucleic acid molecules or
peptide molecules corresponding to these regions may be used to
advantage in the design of therapeutic agents that effectively
modulate the activity of the encoded proteins.
[0067] Molecular modeling should facilitate the identification of
specific organic molecules with capacity to bind to the active site
of the proteins encoded by the SNP-containing nucleic acids based
on conformation or key amino acid residues required for function. A
combinatorial chemistry approach will be used to identify molecules
with greatest activity and then iterations of these molecules will
be developed for further cycles of screening. Molecules available
for testing in this screening assay, include without limitation,
those provided in Table 12. Table 12 provides suitable drug
candidates, their commercial sources and reported mechanisms of
action.
[0068] The polypeptides or fragments employed in drug screening
assays may either be free in solution, affixed to a solid support
or within a cell. One method of drug screening utilizes eukaryotic
or prokaryotic host cells which are stably transformed with
recombinant polynucleotides expressing the polypeptide or fragment,
preferably in competitive binding assays. Such cells, either in
viable or fixed form, can be used for standard binding assays. One
may determine, for example, formation of complexes between the
polypeptide or fragment and the agent being tested, or examine the
degree to which the formation of a complex between the polypeptide
or fragment and a known substrate is interfered with by the agent
being tested.
[0069] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
for the encoded polypeptides and is described in detail in Geysen,
PCT published application WO 84/03564, published on Sep. 13, 1984.
Briefly stated, large numbers of different, small peptide test
compounds, such as those described above, are synthesized on a
solid substrate, such as plastic pins or some other surface. The
peptide test compounds are reacted with the target polypeptide and
washed. Bound polypeptide is then detected by methods well known in
the art.
[0070] A further technique for drug screening involves the use of
host eukaryotic cell lines or cells (such as described above) which
have a nonfunctional or altered JIA CXCR4 gene. These host cell
lines or cells are defective at the polypeptide level. The host
cell lines or cells are grown in the presence of drug compound.
Altered immune signaling or function of the host cells is measured
to determine if the compound is capable of regulating this function
in the defective cells. Host cells contemplated for use in the
present invention include but are not limited to bacterial cells,
fungal cells, insect cells, mammalian cells, and plant cells.
However, mammalian cells, particularly immune cells are preferred.
The JIA-associated SNP encoding CXCR4 DNA molecules may be
introduced singly into such host cells or in combination to assess
the phenotype of cells conferred by such expression. Methods for
introducing DNA molecules are also well known to those of ordinary
skill in the art. Such methods are set forth in Ausubel et al.
eds., Current Protocols in Molecular Biology, John Wiley &
Sons, NY, N.Y. 1995, the disclosure of which is incorporated by
reference herein.
[0071] A wide variety of expression vectors are available that can
be modified to express the novel DNA sequences of this invention.
The specific vectors exemplified herein are merely illustrative,
and are not intended to limit the scope of the invention.
Expression methods are described by Sambrook et al. Molecular
Cloning: A Laboratory Manual or Current Protocols in Molecular
Biology 16.3-17.44 (1989). Expression methods in Saccharomyces are
also described in Current Protocols in Molecular Biology
(1989).
[0072] Suitable vectors for use in practicing the invention include
prokaryotic vectors such as the pNH vectors (Stratagene Inc., 11099
N. Torrey Pines Rd., La Jolla, Calif. 92037), pET vectors (Novogen
Inc., 565 Science Dr., Madison, Wis. 53711) and the pGEX vectors
(Pharmacia LKB Biotechnology Inc., Piscataway, N.J. 08854).
Examples of eukaryotic vectors useful in practicing the present
invention include the vectors pRc/CMV, pRc/RSV, and pREP
(Invitrogen, 11588 Sorrento Valley Rd., San Diego, Calif. 92121);
pcDNA3.1/V5&His (Invitrogen); baculovirus vectors such as
pVL1392, pVL1393, or pAC360 (Invitrogen); and yeast vectors such as
YRP17, YIP5, and YEP24 (New England Biolabs, Beverly, Mass.), as
well as pRS403 and pRS413 Stratagene Inc.); Picchia vectors such as
pHIL-D1 (Phillips Petroleum Co., Bartlesville, Okla. 74004);
retroviral vectors such as PLNCX and pLPCX (Clontech); and
adenoviral and adeno-associated viral vectors.
[0073] Promoters for use in expression vectors of this invention
include promoters that are operable in prokaryotic or eukaryotic
cells. Promoters that are operable in prokaryotic cells include
lactose (lac) control elements, bacteriophage lambda (pL) control
elements, arabinose control elements, tryptophan (trp) control
elements, bacteriophage T7 control elements, and hybrids thereof.
Promoters that are operable in eukaryotic cells include Epstein
Barr virus promoters, adenovirus promoters, SV40 promoters, Rous
Sarcoma Virus promoters, cytomegalovirus (CMV) promoters,
baculovirus promoters such as AcMNPV polyhedrin promoter, Picchia
promoters such as the alcohol oxidase promoter, and Saccharomyces
promoters such as the gal4 inducible promoter and the PGK
constitutive promoter may also be employed.
[0074] In addition, a vector of this invention may contain any one
of a number of various markers facilitating the selection of a
transformed host cell. Such markers include genes associated with
temperature sensitivity, drug resistance, or enzymes associated
with phenotypic characteristics of the host organisms.
[0075] Host cells expressing the HA-associated CXCR4 SNPs of the
present invention or functional fragments thereof provide a system
in which to screen potential compounds or agents for the ability to
modulate the development of JIA. Thus, in one embodiment, the
nucleic acid molecules of the invention may be used to create
recombinant cell lines for use in assays to identify agents which
modulate aspects of cellular metabolism associated with JIA and
aberrant immune cell function. Also provided herein are methods to
screen for compounds capable of modulating the function of proteins
encoded by CXCR4 SNP-containing nucleic acids.
[0076] Another approach entails the use of phage display libraries
engineered to express fragment of the polypeptides encoded by the
SNP-containing nucleic acids on the phage surface. Such libraries
are then contacted with a combinatorial chemical library under
conditions wherein binding affinity between the expressed peptide
and the components of the chemical library may be detected. U.S.
Pat. Nos. 6,057,098 and 5,965,456 provide methods and apparatus for
performing such assays.
[0077] The goal of rational drug design is to produce structural
analogs of biologically active polypeptides of interest or of small
molecules with which they interact (e.g., agonists, antagonists,
inhibitors) in order to fashion drugs which are, for example, more
active or stable forms of the polypeptide, or which, e.g., enhance
or interfere with the function of a polypeptide in vivo. See, e.g.,
Hodgson, (1991) Bio/Technology 9:19-21 It is also possible to
isolate a target-specific antibody, selected by a functional assay,
and then to solve its crystal structure. In principle, this
approach yields a pharmacore upon which subsequent drug design can
be based.
[0078] One can bypass protein crystallography altogether by
generating anti-idiotypic antibodies (anti-ids) to a functional,
pharmacologically active antibody. As a mirror image of a mirror
image, the binding site of the anti-ids would be expected to be an
analog of the original molecule. The anti-id could then be used to
identify and isolate peptides from banks of chemically or
biologically produced banks of peptides. Selected peptides would
then act as the pharmacore.
[0079] Thus, one may design drugs which have, e.g., improved
polypeptide activity or stability or which act as inhibitors,
agonists, antagonists, etc. of polypeptide activity. By virtue of
the availability of SNP-containing CXCR4 nucleic acid sequences
described herein, sufficient amounts of the encoded polypeptide may
be made available to perform such analytical studies as x-ray
crystallography. In addition, the knowledge of the protein sequence
provided herein will guide those employing computer modeling
techniques in place of, or in addition to x-ray
crystallography.
[0080] In another embodiment, the availability of JIA-associated
CXCR4 SNP-containing nucleic acids enables the production of
strains of laboratory mice carrying the JIA-associated SNPs
containing nucleic acid of the invention. Transgenic mice
expressing the JIA-associated SNP(s) of the invention provide a
model system in which to examine the role of the SNP containing
CXCR4 nucleic acids play in the development and progression towards
RA. Methods of introducing transgenes in laboratory mice are known
to those of skill in the art. Three common methods include: 1.
integration of retroviral vectors encoding the foreign gene of
interest into an early embryo; 2. injection of DNA into the
pronucleus of a newly fertilized egg; and 3. the incorporation of
genetically manipulated embryonic stem cells into an early embryo.
Production of the transgenic mice described above will facilitate
the molecular elucidation of the role that CXCR4 protein plays in
various cellular metabolic processes, including: aberrant immune
signaling molecule production and function. Such mice provide an in
vivo screening tool to study putative therapeutic drugs in a whole
animal model and are encompassed by the present invention.
[0081] The term "animal" is used herein to include all vertebrate
animals, except humans. It also includes an individual animal in
all stages of development, including embryonic and fetal stages. A
"transgenic animal" is any animal containing one or more cells
bearing genetic information altered or received, directly or
indirectly, by deliberate genetic manipulation at the subcellular
level, such as by targeted recombination or microinjection or
infection with recombinant virus. The term "transgenic animal" is
not meant to encompass classical cross-breeding or in vitro
fertilization, but rather is meant to encompass animals in which
one or more cells are altered by or receive a recombinant DNA
molecule. This molecule may be specifically targeted to a defined
genetic locus, be randomly integrated within a chromosome, or it
may be extrachromosomally replicating DNA. The term "germ cell line
transgenic animal" refers to a transgenic animal in which the
genetic alteration or genetic information was introduced into a
germ line cell, thereby conferring the ability to transfer the
genetic information to offspring. If such offspring, in fact,
possess some or all of that alteration or genetic information, then
they, too, are transgenic animals.
[0082] The alteration of genetic information may be foreign to the
species of animal to which the recipient belongs, or foreign only
to the particular individual recipient, or may be genetic
information already possessed by the recipient. In the last case,
the altered or introduced gene may be expressed differently than
the native gene. Such altered or foreign genetic information would
encompass the introduction of JIA-associated SNP-containing
nucleotide sequences.
[0083] The DNA used for altering a target gene may be obtained by a
wide variety of techniques that include, but are not limited to,
isolation from genomic sources, preparation of cDNAs from isolated
mRNA templates, direct synthesis, or a combination thereof.
[0084] A preferred type of target cell for transgene introduction
is the embryonal stem cell (ES). ES cells may be obtained from
pre-implantation embryos cultured in vitro (Evans et al., (1981)
Nature 292:154-156; Bradley et al., (1984) Nature 309:255-258;
Gossler et al., (1986) Proc. Natl. Acad. Sci. 83:9065-9069).
Transgenes can be efficiently introduced into the ES cells by
standard techniques such as DNA transfection or by
retrovirus-mediated transduction. The resultant transformed ES
cells can thereafter be combined with blastocysts from a non-human
animal. The introduced ES cells thereafter colonize the embryo and
contribute to the germ line of the resulting chimeric animal.
[0085] One approach to the problem of determining the contributions
of individual genes and their expression products is to use
isolated JIA-associated SNP CXCR4 genes as insertional cassettes to
selectively inactivate a wild-type gene in totipotent ES cells
(such as those described above) and then generate transgenic mice.
The use of gene-targeted ES cells in the generation of
gene-targeted transgenic mice was described, and is reviewed
elsewhere (Frohman et al., (1989) Cell 56:145-147; Bradley et al.,
(1992) Bio/Technology 10:534-539).
[0086] Techniques are available to inactivate or alter any genetic
region to a mutation desired by using targeted homologous
recombination to insert specific changes into chromosomal alleles.
However, in comparison with homologous extrachromosomal
recombination, which occurs at a frequency approaching 100%,
homologous plasmid-chromosome recombination was originally reported
to only be detected at frequencies between 10.sup.-6 and 10.sup.-3.
Nonhomologous plasmid-chromosome interactions are more frequent
occurring at levels 10.sup.5-fold to 10.sup.2 fold greater than
comparable homologous insertion.
[0087] To overcome this low proportion of targeted recombination in
murine ES cells, various strategies have been developed to detect
or select rare homologous recombinants. One approach for detecting
homologous alteration events uses the polymerase chain reaction
(PCR) to screen pools of transformant cells for homologous
insertion, followed by screening of individual clones.
Alternatively, a positive genetic selection approach has been
developed in which a marker gene is constructed which will only be
active if homologous insertion occurs, allowing these recombinants
to be selected directly. One of the most powerful approaches
developed for selecting homologous recombinants is the
positive-negative selection (PNS) method developed for genes for
which no direct selection of the alteration exists. The PNS method
is more efficient for targeting genes which are not expressed at
high levels because the marker gene has its own promoter.
Non-homologous recombinants are selected against by using the
Herpes Simplex virus thymidine kinase (HSV-TK) gene and selecting
against its nonhomologous insertion with effective herpes drugs
such as gancyclovir (GANC) or (1-(2-deoxy-2-fluoro-B-D
arabinofluranosyl)-5-iodou-racil, (FIAU). By this counter
selection, the number of homologous recombinants in the surviving
transformants can be increased. Utilizing JIA-associated
SNP-containing nucleic acid as a targeted insertional cassette
provides means to detect a successful insertion as visualized, for
example, by acquisition of immunoreactivity to an antibody
immunologically specific for the CXCR4 polypeptide encoded by
JIA-associated SNP nucleic acid and, therefore, facilitates
screening/selection of ES cells with the desired genotype.
[0088] As used herein, a knock-in animal is one in which the
endogenous murine gene, for example, has been replaced with human
JIA-associated CXCR4 SNP-containing gene of the invention. Such
knock-in animals provide an ideal model system for studying the
development of JIA.
[0089] As used herein, the expression of a JIA-associated
SNP-containing nucleic acid, a JIA-associated CXCR4 fusion protein
in which the SNP is encoded can be targeted in a "tissue specific
manner" or "cell type specific manner" using a vector in which
nucleic acid sequences encoding all or a portion of an
JIA-associated SNP are operably linked to regulatory sequences
(e.g., promoters and/or enhancers) that direct expression of the
encoded protein in a particular tissue or cell type. Such
regulatory elements may be used to advantage for both in vitro and
in vivo applications. Promoters for directing tissue specific
proteins are well known in the art and described herein.
[0090] Methods of use for the transgenic mice of the invention are
also provided herein. Transgenic mice into which a nucleic acid
containing the JIA-associated SNP or its encoded CXCR4 protein have
been introduced are useful, for example, to develop screening
methods to screen therapeutic agents to identify those capable of
modulating the development of JIA.
V. Pharmaceutical and Peptide Therapies
[0091] The elucidation of the role played by the JIA associated
CXCR4 SNPs described herein in immune cell signaling and function
facilitates the development of pharmaceutical compositions useful
for treatment and diagnosis of JIA. These compositions may
comprise, in addition to one of the above substances, a
pharmaceutically acceptable excipient, carrier, buffer, stabilizer
or other materials well known to those skilled in the art. Such
materials should be non-toxic and should not interfere with the
efficacy of the active ingredient. The precise nature of the
carrier or other material may depend on the route of
administration, e.g. oral, intravenous, cutaneous or subcutaneous,
nasal, intramuscular, intraperitoneal routes.
[0092] Whether it is a polypeptide, antibody, peptide, nucleic acid
molecule, small molecule or other pharmaceutically useful compound
according to the present invention that is to be given to an
individual, administration is preferably in a "prophylactically
effective amount" or a "therapeutically effective amount" (as the
case may be, although prophylaxis may be considered therapy), this
being sufficient to show benefit to the individual.
[0093] The following materials and methods are provided to
facilitate the practice of the present invention.
Subjects and Methods
Participants
[0094] We undertook a multicenter, genome-wide association study of
five epidemiological cohorts in the USA, Australia, and Norway
(Table 1). Our case cohorts were comprised of patients with JIA,
with onset of arthritis at <16 years of age. JIA diagnosis and
JIA subtype were determined according to the International League
of Associations for Rheumatology (ILAR) revised criteria.sup.3 and
confirmed using the JIA Calculator.TM. software (URLs),.sup.14 an
algorithm-based tool adapted from the ILAR criteria. A summary of
design and clinical characteristics for our study samples is shown
in Tables 1-3.
TABLE-US-00001 TABLE 1 Discovery and replication cohorts. The JIA
data set following standard quality control procedures and
exclusion of non-European ancestry is shown. JIA Data Set.sup.a
Purpose Cases Controls Total TSRHC + CMHC Discovery 388 2500 2888
CHOP Replication 1 182 2000 2182 MCRI Replication 2 154 2000 2154
OUH Replication 3 442 3000.sup.b 3442 Combined Meta-analysis 1166
9500 10,666 .sup.aJIA Data Set: TSRHC; Texas Scottish Rite Hospital
for Children, Dallas, Texas, USA; CMHC; Children's Mercy Hospitals
and Clinics, Kansas City, Missouri, USA; CHOP; The Children's
Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; MCRI;
Murdoch Childrens Research Institute, Melbourne, Australia; OUH;
Department of Rheumatology, Oslo University Hospital,
Rikshospitalet, Oslo, Norway. .sup.bOut-of-study controls provided
by the Wellcome Trust Case-Control Consortium.
TABLE-US-00002 TABLE 2 JIA subtypes in the study cohorts.
Discovery.sup.b (TSRHC + Replication 1 Replication 2 Replication 3
JIA Subtypes.sup.a CMHC) (CHOP) (MCRI) (OUH) CCHMC.sup.c Total
Oligoarthritis, 135 45 76 181 437 persistent + extended
Polyarthritis, RF 86 35 40 88 249 negative Oligoarthritis + Poly
221 80 116 269 814 1500 arthritis, RF negative Polyarthritis, RF 19
7 10 18 -- 54 positive Polyarthritis, RF 27 0 0 0 -- 27 unknown
Systemic arthritis 44 23 12 24 -- 103 Enthesitis-related 38 32 6 62
-- 138 arthritis Psoriatic arthritis 32 19 5 31 -- 87
Undifferentiated 7 21 5 38 -- 71 arthritis Total 388 182 154 442
814 1980 .sup.aRevised ILAR criteria..sup.1 For this study, a
patient was considered to be rheumatoid factor (RF)-negative based
on a single test; patients for whom the RF was indeterminant (27
patients) were excluded from subtype analysis. .sup.bJIA Data Set:
TSRHC; Texas Scottish Rite Hospital for Children, Dallas, Texas,
USA; CMHC; Children's Mercy Hospitals and Clinics, Kansas City,
Missouri, USA; CHOP; The Children's Hospital of Philadelphia,
Philadelphia, Pennsylvania, USA; MCRI; Murdoch Childrens Research
Institute, Melbourne, Australia; OUH; Department of Rheumatology,
Oslo University Hospital, Rikshospitalet, Oslo, Norway.
.sup.cCCHMC, Cincinnati Children's Hospital Medical Center;
controls for this dataset were comprised of 649 local and 2400
out-of-study controls (see Methods); proxy SNPs of the two JIA
subtypes from CCHMC were not included in the meta-analysis (Table
2).
TABLE-US-00003 TABLE 3 Demographic characterization of the
discovery and replication cohorts. The age at onset, gender, and
subtype distributions of all case cohorts are similar to those
reported previously for JIA..sup.1 % JIA Subtypes.sup.a female Age
(years).sup.b Oligoarthritis, persistent 73% 5.9 (2.9, 9.7)
Oligoarthritis, extended 82% 3.95 (2.4, 7.8) Polyarthritis, RF
negative 79% 7.48 (3.2, 11.3) Polyarthritis, RF positive 95% 13.5
(10.3, 15.1) Systemic arthritis 67% 6.6 (3.2, 11.1)
Enthesitis-related arthritis 40% 11.9 (9.0, 14.1) Psoriatic
arthritis 71% 9.9 (7.0, 13.2) Undifferentiated arthritis 55% 9.8
(4.5, 13.8) Total 68% 8.3 (3.8, 12.2) .sup.aRevised ILAR criteria.
.sup.1For this study, a patient was considered to be rheumatoid
factor (RF)-negative based on a single test; patients for whom the
RF was indeterminant (27 patients) were excluded from subtype
analysis. .sup.bAge (years): median (25% percentile, 75%
percentile)
[0095] The targeted discovery cohort prior to standard quality
control (QC) procedures and exclusion of non-European ancestry was
comprised of 464 subjects with JIA from Texas Scottish Rite
Hospital for Children (TSRHC; Dallas, Tex.) and Children's Mercy
Hospitals and Clinics (CMHC; Kansas City, Mo.) of self-reported
European ancestry. To attempt to replicate associations in the
discovery cohort, three independent case sample collections of JIA
patients, also of self-reported European ancestry, were studied.
One of the replication cohorts was comprised of 196 subjects from
the Children's Hospital of Philadelphia (CHOP; Philadelphia, Pa.).
A second independent replication cohort was comprised of 221
subjects from the Murdoch Childrens Research Institute (MCRI; Royal
Children's Hospital, Melbourne, Australia). A third independent
replication cohort was comprised of 504 subjects from Oslo
University Hospital (OUH; Oslo, Norway). A subset of subjects from
these sites has been described previously..sup.12,15,16,17 The
clinical data relating to case samples were collected from the JIA
Registry maintained within the CHOP Division of Rheumatology for
the CHOP cohort; clinical data relating to case samples from TSRHC,
CMHC, MCRI, and OUH were drawn from records provided by the
respective sites and stored in a de-identified database at the
Center for Applied Genomics of the CHOP Research Institute.
[0096] The control subjects used included 6500 unrelated and
disease-free children recruited from within the CHOP Healthcare
Network. Control subjects (average age 7.9.+-.6.0 SD years; 53.74%
males, 46.26% females) had no history of JIA or other chronic
illnesses and were screened as negative for a diagnosis of
autoimmune diseases, based on data from CHOP's electronic health
record and by intake questionnaires obtained by the recruiting
staff from the Center for Applied Genomics. All pediatric controls
passed stringent quality control (QC), as detailed below; post-QC,
controls were selected by matching algorithms on the basis of
multidimensional scaling (MDS) analysis,.sup.18,19. In brief,
although all subjects were of self-reported European American
ancestry, we combined >10,000 control subjects with cases and
performed MDS to infer a homogeneous group of subjects of European
ancestry. Cases from the third replication cohort (OUH) were
subsequently analyzed, using the 3000 well-characterized subjects
from the Wellcome Trust Case-Control Consortium (WTCCC.sup.19) as
controls.
[0097] Six cases from the CHOP cohort and eleven cases from the OUH
cohort were excluded due to a low call rate. In addition, 202 cases
were excluded due to MDS correction: 76 cases were removed from the
discovery cohort; 8 cases were removed from the first replication
cohort (CHOP); 67 cases were removed from the second replication
cohort (MCRI); and 51 cases were removed from the third replication
cohort (OUH), yielding 1166 ethnically-matched JIA cases and 9500
controls (Table 4).
TABLE-US-00004 TABLE 4 The most significantly associated SNPs in
the vicinity of CXCR4 on chromosome 2q21. Discovery (TSRHC + CMHC)
Replication 1 (CHOP) Minor/ Minor allele Minor allele
Position.sup.a Major frequency frequency CHR SNP (NCBI 36)
Alleles.sup.a Case Control p value OR.sup.b 95% CI Case Control p
value 2 rs953387 136623640 G/T 0.29 0.41 2.07E-10 0.59 0.50-0.69
0.35 0.41 0.01 2 rs1123848 136661499 T/C 0.28 0.39 3.89E-10 0.59
0.50-0.70 0.35 0.40 0.04 2 rs4954564 136611978 G/A 0.30 0.41
3.08E-09 0.61 0.52-0.72 0.35 0.42 0.01 2 rs10221893 136730076 C/T
0.39 0.51 4.58E-09 0.63 0.54-0.74 0.50 0.53 0.31 2 rs6430612
136722668 C/T 0.39 0.51 7.98E-09 0.64 0.55-0.74 0.49 0.53 0.17 2
rs1016269 136657132 A/G 0.15 0.24 4.01E-08 0.56 0.46-0.69 0.19 0.25
0.02 Sample size Number of 388 182 cases Number of controls 2500
2000 Total sample number 2888 2182 Replication 2 (MCRI) Replication
3(OUH) p value Minor allele Minor allele (Combined frequency
frequency Meta- CHR SNP Case Control p value Case Control p value
analysis) 2 rs953387 0.35 0.42 0.02 0.21 0.26 5.52E-04 1.03E-13 2
rs1123848 0.33 0.40 0.01 NA NA NA 6.11E-11 2 rs4954564 0.35 0.42
0.02 0.21 0.26 7.16E-04 3.80E-13 2 rs10221893 0.46 0.52 0.05 0.31
0.33 3.23E-01 7.32E-07 2 rs6430612 0.46 0.52 0.05 0.31 0.33
3.23E-01 3.84E-07 2 rs1016269 0.21 0.25 0.11 0.12 0.16 4.91E-03
4.85E-10 Sample size Number of 154 442 1166 cases Number of
controls 2000 3000 9500 Total sample number 2154 3442 10,666 p
value, basic allelic test p value; OR, odds ratio; CI, confidence
interval; p value (combined), meta-analysis p value in all 4
cohorts. .sup.aThe chromosome coordinates and allele designations
are on the basis of the forward strand of the NCBI 36 genome
assembly. .sup.bThe odds ratio is calculated with respect to the
minor allele.
TABLE-US-00005 TABLE 5 Association of rs953387 with the JIA
subtypes. Sample Minor/ Minor Allele Position.sup.b Number Major
Frequency Combined Meta-analysis JIA Subtypes.sup.a CHR SNP (NCBI
36) Case Control Alleles Case Control p value OR.sup.c 95% CI
Oligoarthritis, persistent + extended 2 rs953387 136623640 437 5000
G/T 0.28 0.32 3.42E-04 0.84 0.72-0.98 Polyarthritis, RF
negative.sup.# 2 rs953387 136623640 249 4000 G/T 0.27 0.30 4.09E-03
0.88 0.72-1.08 Oligoarthritis + Polyarthritis, RF negative.sup.d 2
rs953387 136623640 1500 9049 G/T 0.31 0.33 6.31E-05 0.90 0.83-0.98
Polyarthritis, RF positive 2 rs953387 136623640 54 3500 G/T 0.25
0.28 1.25E-02 0.85 0.55-1.31 Systemic arthritis 2 rs953387
136623640 103 4000 G/T 0.26 0.30 4.04E-03 0.82 0.6-1.12
Enthesitis-related arthritis 2 rs953387 136623640 138 4000 G/T 0.24
0.30 3.82E-04 0.73 0.55-0.96 Psoriatic arthritis 2 rs953387
136623640 87 3500 G/T 0.32 0.28 0.580 1.18 0.85-1.63
Undifferentiated arthritis 2 rs953387 136623640 71 3500 G/T 0.29
0.28 0.175 1.02 0.71-1.47 p value, basic allelic test p value; OR,
odds ratio; CI, confidence interval; p value (combined),
meta-analysis p value in all cohorts. .sup.aSubtype sample number
for each cohort is shown in Supplemental Table 1. For
Oligoarthritis + Polyarthritis, RF negative, we received a fifth
cohort of 814 cases and 3049 controls from Cincinnati Children's
Hospital Medical Center (CCHMC) genotyped on Affymetrix, and are
presenting the imputed data. .sup.bThe chromosome coordinates and
allele designations are on the basis of the forward strand of the
NCBI 36 genome assembly. .sup.cThe odds ratio is calculated with
respect to the minor allele. .sup.dRF status unknown for 27 samples
from CMHC. These were included in the analysis of the JIA discovery
cohort, but not in the subtype analysis.
[0098] A secondary association analysis of the various JIA subtypes
also was performed in 1139 of our 1166 JIA cases in comparison with
controls (Table 5). Excluded from our subtype analysis were 27 of
the CMHC cases; these participants fulfilled clinical criteria for
the polyarthritis subtype of JIA, but rheumatoid factor status was
unknown (Table 2). In this analysis, we additionally included
imputed data from a cohort of 814 subjects with either
oligoarthritis or polyarthritis, RF negative, in comparison with
3039 controls (639 of whom were local and 2400 out-of-study
controls.sup.20) from the Cincinnati Children's Hospital Medical
Center (CCHMC; Cincinnati, Ohio). Subjects from this site were
genotyped on the Affymetrix 6.0 platform and have been described
previously..sup.9
[0099] The study was approved by the institutional review boards of
TSRHC, CMHC, CHOP, MCRI, OUH, and CCHMC, and was compliant with
HIPAA regulations. Parental written informed consent was obtained
from all participants in this study for the purpose of DNA
collection and genotyping.
Procedures and Study Design
[0100] We performed GWAS in our discovery cohort (TSRHC+CMHC),
including only those individuals of inferred European ancestry,
followed by replication analysis of positive signals in three
independent cohorts, including patients from CHOP (cohort 1), MCRI
(cohort 2) and OUH (cohort 3) in keeping with GWAS
standards..sup.21,22 Population structure (genetic differences
within an apparently homogeneous population) was investigated using
MDS. A total of 1166 cases and 9500 controls were inferred as
having European ancestry using these procedures. Our genetic
association data are reported according to Strengthening the
Reporting of Genetic Association studies (STREGA)
guidelines..sup.23
Genotyping
[0101] All JIA cases were recruited in the U.S., Australia, or
Norway. JIA cases and U.S. controls in our discovery cohort were
genotyped using the Human610-Quad arrays (with 610,000 SNP
markers). The overall genotype call rate was 99.95% after QC in the
discovery cohort. JIA cases in the replication cohorts and their
matching controls were genotyped using either the Illumina
HumanHap550 BeadChip (with 550,000 single nucleotide polymorphism
[SNP] markers) or the Human610-Quad arrays (with 610,000 SNP
markers). While about 98% of SNPs are identical on these two
platforms, their performance differs; we, therefore, examined only
SNPs that demonstrated significance in the 610,000 discovery
dataset in this study. JIA cases and local controls from CCHMC were
genotyped using the Affymetrix Genome-Wide Human SNP Array 6.0, as
described previously..sup.9 The publicly available control data set
from the Wellcome Trust Case Control Consortium [WTCCC-1].sup.19
and Molecular Genetics of Schizophrenia, non-Gain.sup.20 were
genotyped using the Affymetrix GeneChip 500K Mapping Array Set or
the Affymetrix 6.0 GWAS Array, respectively.
[0102] The criteria for SNP selection for the discovery and
replication stages have been previously reported by our
laboratory.sup.24 and are described below. We used Markov Chain
Haplotyping (MACH; URLs) for genotype imputation on markers that
were not present in the genotyping platforms for our JIA
cohort..sup.25 The default two-step procedure was adopted for
imputation. Whole-genome genotype imputation was performed on the
autosomal markers on the basis of phased haplotypes (release 22)
for the HapMap CEU population (URLs). We removed all markers with
MACH r.sup.2 measure of <0.3, and zeroed out imputed genotypes
with a posterior probability of <0.9.
Quality Control
[0103] We applied QC filters to exclude unreliable samples prior to
association analysis. A sample was excluded if the genotype call
rate was <95% or if the sample showed excess or deficient
heterozygosity (inbreeding coefficient |F|>0.1). Cryptic
relatedness or erroneous duplicates were evaluated using pair-wise
identity-by-descent estimation, and the sample with lower genotype
call rate was removed from each identified relative pair. For this
analysis, we eliminated SNPs with genotype call rate <98%, with
minor allele frequency (MAF)<1% in either cases or controls, or
if there was significant departure from Hardy-Weinberg equilibrium
(p<0.0001). In the discovery cohort, there were 56,873 SNPs with
missing rate >2%. A total of 518,907 SNPs passed QC and were
included in analysis. In addition, we used these genotyped SNPs and
120 phased chromosomes from the HapMap CEU samples (HapMap release
22, NCBI build 36) to impute genotypes for untyped SNPs using MACH
1.0 software..sup.25 Imputed SNPs with MAF <0.01 in either cases
or controls and SNPs with poor imputation quality (r.sup.2<0.3)
were excluded.
Population Stratification
[0104] Patients with JIA in the discovery and replication cohorts
were genetically matched with unrelated controls of European
ancestry using the MDS algorithm employed in PLINK.sup.18 for
inferring population structure (URLs). To help with our
interpretation of the population genetics, we included 924
individuals from thirteen HapMap populations as positive controls
in the MDS analysis. Comparing self-identified ancestry with the
MDS-inferred ancestry confirmed the reliability of MDS to identify
genetically inferred individuals of European ancestry. Thus, the
probability of false-positive associations caused by potential
problems such as population stratification between cases and
control subjects was minimized. To confirm this, we created a
Quantile-Quantile plot (QQ-plot) of p values obtained by the
allelic test and calculated the inflation factor of
test-statistics, .lamda.GC, using WGAViewer (URLs). The genomic
inflation lambda in the three cohorts typed genome-wide (discovery
and replication cohorts 1 and 2) were 1.11, 1.00, and 1.03,
respectively. When we correct the chi-square statistics with the
lambda in the discovery cohort using the stringent genomic control
method, five SNPs still remain GW significant
(p<5.times.10.sup.-8) in the discovery cohort and the
replication cohorts are unaffected. These data suggest that the
identified association of rs953387 in the GWAS is not a false
positive but has a robust, true association with JIA.
Power Calculations
[0105] The statistical rationale for our sample size was as
follows: We evaluated the power to detect association of tagSNPs
with JIA using the software, Quanto (URLs) for a combined cohort of
1166 subjects and 9500 controls (at a significance level of
5.times.10.sup.-8). To correct for multiple testing and achieve a
family-wise error rate of 0.05, we fixed the significance level of
the test at 5.times.10.sup.-8 based on published criteria..sup.22
The size of the genetic relative risk, defined as the risk of
having JIA in the presence of a genetic risk variant was varied
from 1.1 to 2.5. We used a range of MAF at the SNPs of 0.05 to
0.45, and a log additive genetic model for the relative risk. As
expected, the power increased with increasing levels of genotype
relative risk (GRR) and marker allele frequencies. For example,
even for a rare SNP with MAF of 0.05, we would have >80% power
to detect association if GRR is >1.9. However, for a more common
SNP with MAF of 0.25 or more, we would have >80% to detect
association if GRR is >1.4. These calculations reflect our power
to detect a single variant; if there are multiple variants
influencing the trait (as we expect), the power to detect at least
one of them is much better..sup.26 We were thus confident that
given the available sample size we would have sufficient power to
detect genetic variants with a wide range of effects on risk of
developing JIA.
Gene Expression
[0106] WGAViewer (URLs) was used to test association between SNP
genotypes and gene expression variation (eQTL), quantified in
immortalized B-lymphocytes and T-cells, using the databases from
the Sanger Institute Genevar project.sup.27 (URLs) and the HapMap
and GenCord projects..sup.28,29 As described by Stranger et
al.,.sup.27,30,31 transcript levels were measured using Illumina's
human whole-genome expression (WG-6 version 1) arrays,.sup.32 which
contained 47,294 probes, with two or more unique oligonucleotide
probes per gene, in four technical replicates. Raw intensity values
were normalized on a log scale using a quantile normalization
method.sup.33 across the four replicates for each individual, to
obtain a single expression level per individual..sup.27,30,31 There
was a high degree of correlation in the transcript level
measurements generated on this genome-wide array within and between
arrays (r.sup.2=0.96-0.99) and with expression measurements
generated on Illumina's low-density (.about.700 genes) custom
arrays..sup.31
Statistical Analyses
[0107] For genotyped SNPs, association was tested by basic allelic
test (chi-square test) and the odds ratio was calculated with
respect to the minor allele using PLINK.sup.1. The estimated
genomic control inflation factor lambda (.lamda.).sup.34 an
indicator of potential population stratification, was calculated
using WGAViewer (URLs). For imputed SNPs, association was examined
using MACH 1.0 software.sup.25 for untyped SNPs that were in strong
linkage disequilibrium (LD) with markers present in the genotyping
platforms (r.sup.2>0.9) for our JIA cohort. The default two-step
procedure was adopted for imputation. For comparison of our
association results for JIA with the WTCCC rheumatoid arthritis
(RA) cohorts,.sup.19 we also used MACH to infer the genotype on
untyped markers.
[0108] The criteria for SNP selection at each stage were based on
the approach of Kugathasan and colleagues..sup.24 In stage 1 of
this study (discovery analysis), 518,907 genotyped SNPs were
studied. A significant finding was defined a priori by published
criteria.sup.22 as p<5.times.10.sup.-8. Criteria for selection
of SNPs for stage 2 (replication analysis) were a GW-significant p
value as well as multiple hits at the same locus, to avoid spurious
association. Replication was claimed when the direction of effect
was the same and the p value was lower than the locus-specific
threshold (0.05), after correction for multiple tests.
[0109] Near identical results were obtained in permutation
tests--often referred to as the gold standard for
randomization.sup.35--performed for the six genome-wide significant
SNPs in the discovery cohort. The corrected p value based on 10,000
permutations for the most significant SNP rs4954564 (nominal
p=0.009) in replication 1 is 0.0274 (corrected by the P_ACT program
as 0.027.sup.35), suggesting the effective number of independent
tests is about three instead of six. This SNP has a p value of
0.017 in replication 2 (no further correction is needed as we
tested only this one in replication cohort 2). The corrected p
value for the whole SNP set based on 10,000 permutations, testing
the null hypothesis that no SNP from this set is associated with
JIA, is 0.0274 and 0.0232 for replications 1 and 2, respectively
(0.027 and 0.021 by the P_ACT program). For such a SNP set
analysis, PLINK provides a similar permutation test but considering
mean instead of minimum. The p values reported by PLINK based on
10,000 permutations are 0.04 and 0.03 for replications 1 and 2,
respectively. Thus, no matter whether based on individual SNPs or
SNP sets, permutation-based tests, P_ACT, or PLINK, all tests
successfully confirm the association at the 0.05 level.
[0110] Meta-analysis is a powerful method for testing the
significance of a replication study, combining the results of
several studies that address a set of related research hypotheses.
For our meta-analysis, we used a weighted Z-score method with
METAL.sup.36 (URLs), which accounts for the direction of
association relative to a consistent reference allele. In this
method, p values are combined across studies; the weight for each
cohort is calculated by taking into account sample size and
direction of effect. All meta-analyses comply with MOOSE guidelines
(URLs).
[0111] To test association between SNP genotypes and gene
expression, we used publicly available data from genome-wide
expression analysis of quantitative trait loci (eQTL) of the 270
individuals genotyped in the HapMap Project (including 30 Caucasian
trios of Northern and Western European origin
[CEU].sup.27,28,30,31) and the 85 individuals of the GenCord
project (a collection of cell lines from umbilical cords of
individuals of Western European origin.sup.29). To associate SNP
genotypes with gene expression, a p-value was calculated by a
linear regression model..sup.30 Specifically, the additive effect
of a SNP allele was tested by coding the genotypes of the SNP as 0,
1, and 2 (corresponding to the counts of the minor allele in each
genotype) and performing a linear regression of this variable with
the gene expression values.
[0112] The following examples are provided to illustrate certain
embodiments of the invention. They are not intended to limit the
invention in any way.
Example I
[0113] The discovery analysis included 388 children with JIA and
2500 genetically matched unrelated controls of European ancestry
with high-quality SNP array data (Table 4 and Tables 1-3). We
observed genome-wide significant associations
(p<5.times.10.sup.-8) with JIA at two loci in the discovery
cohort (FIG. 1). In addition to a strong association with the MHC
on 6p21 (Table 6), six intergenic SNPs located on 2q21 reached
genome-wide significance (Table 4 and Table 7), with the most
significant marker being rs953387 (p=2.07.times.10.sup.-10; OR
0.59, 95% CI 0.50-0.69). Five of these SNPs still remained GW
significant (p<5.times.10.sup.-8) after correction for the
estimated genomic inflation lambda (.lamda.; an indicator of
potential population stratification) using the stringent genomic
control method (see Subjects and Methods, Population
stratification). The protective OR of 0.59 for the minor allele of
rs953387 corresponds to an OR of 1.70 for the major allele (which,
in this case, confers risk). Four additional SNPs at the same locus
had p values below 1.times.10.sup.-4 (Table 8). These 10 SNPs map
to three linkage disequilibrium blocks on 2q21 spanning the CXCR4
gene region (FIGS. 2 and 3).
TABLE-US-00006 TABLE 6 Genome-wide association results (p < 5
.times. 10.sup.-8) in the discover cohort) for the MHC p value
Minor/ (Combined Major p value Meta- CHR SNP BP.sup.b Gene
Alleles.sup.b Case_MAF Control_MAF (Discovery) OR.sup.c analysis) 6
rs2395148 32429532 C6orf10 T/G 0.095 0.025 2.63E-23 4.08 7.95E-38 6
rs2073048 32443411 C6orf10 T/C 0.242 0.119 1.31E-20 2.36 3.43E-26 6
rs7770048 32442732 C6orf10 T/C 0.242 0.119 1.51E-20 2.36 1.86E-26 6
rs4248166 32474399 BTNL2 C/T 0.296 0.160 3.03E-20 2.21 3.19E-26 6
rs2294884 32475237 BTNL2 C/A 0.299 0.164 8.38E-20 2.18 4.19E-25 6
rs13192471 32779081 near C/T 0.244 0.136 6.32E-15 2.05 3.70E-23
HLA- DQB1 6 rs6907322 32432923 C6orf10 A/G 0.295 0.177 7.60E-15
1.95 1.78E-18 6 rs1794275 32779226 near T/C 0.275 0.163 4.99E-14
1.94 4.80E-16 HLA- DQB1 6 rs9268365 32441417 C6orf10 T/G 0.296
0.181 5.06E-14 1.91 4.41E-18 6 rs10947262 32481290 BTNL2 T/C 0.174
0.088 9.91E-14 2.18 1.84E-18 6 rs7765379 32788906 near G/T 0.188
0.098 9.91E-14 2.13 8.27E-16 HLA- DQA2 6 rs3763313 32484449 near
C/A 0.284 0.180 1.29E-11 1.80 1.60E-14 BTNL2 6 rs2071286 32287874
NOTCH4 A/G 0.331 0.222 2.30E-11 1.74 2.17E-14 6 rs1265048 31189388
near G/A 0.457 0.338 9.57E-11 1.65 1.62E-10 C6orf15 6 rs1035798
32259200 AGER T/C 0.370 0.261 2.31E-10 1.67 1.76E-16 6 rs2395185
32541145 near T/G 0.197 0.306 5.74E-10 0.56 3.27E-18 HLA- DRA 6
rs411326 32319295 near A/G 0.342 0.244 7.39E-09 1.61 2.97E-06
NOTCH4 6 rs2516049 32678378 near G/A 0.196 0.295 1.05E-08 0.58
1.20E-19 HLA- DRB1 6 rs477515 32677669 near T/C 0.196 0.295
1.20E-08 0.58 1.46E-19 HLA- DRB1 6 rs2301226 33142574 HLA- T/C
0.214 0.137 1.52E-08 1.72 1.40E-11 DPA1 6 rs17576984 32320963 near
T/C 0.148 0.085 1.84E-08 1.87 1.72E-13 NOTCH4 6 rs570963 32397572
C6orf10 C/T 0.175 0.107 3.09E-08 1.78 5.09E-11 6 rs3868075 31275794
HCG27 C/T 0.501 0.397 4.40E-08 1.52 2.23E-04 6 rs4713447 31270942
near G/A 0.501 0.397 4.45E-08 1.52 2.28E-04 HCG27 BP, base pair
chromosome coordinates; MAF, minor allele frequencies in discovery
cohort; p value, basic allelic test p value; OR, odds ratio in
discovery cohort; p value (combined), meta-analysis p value in the
three cohorts typed genome-wide (discovery and replication cohorts
1 and 2). .sup.bThe chromosome coordinates and allele designations
are on the basis of the forward strand of the NCBI 36 genome
assembly. .sup.cThe odds ratio is calculated with respect to the
minor allele.
TABLE-US-00007 TABLE 7 Genotype counts for the six SNPs in the
vicinity of CXCR4 on chromosome 2q21 Discovery Replication 1
Replication 2 Replication 3 Minor/ (TSRHC + CMHC) (CHOP) (MCRI)
(OUH) Major Genotypes Genotypes Genotypes Genotypes Genotypes
Genotypes Genotypes Genotypes SNP Alleles in cases.sup.a in
controls in cases in controls in cases in controls in cases in
controls rs953387 G/T 35/154/199 422/1198/ 22/82/77 373/910/716
21/65/68 377/909/ 17/148/277 232/1086/1619 880 713 rs1123848 T/C
32/150/206 396/1172/ 21/84/77 350/902/747 18/64/72 360/889/
19/146/277 NA 932 751 rs4954564 G/A 37/158/193 429/1196/ 22/83/77
375/916/695 21/66/67 381/911/ 18/147/277 231/1083/1623 875 699
rs10221893 C/T 66/174/148 625/1287/ 37/68/38 473/944/583 38/67/49
493/926/ 34/209/199 392/1167/1373 588 581 rs6430612 C/T 66/174/148
620/1288/ 45/88/49 471/949/577 37/68/49 498/925/ 34/209/199
392/1165/1376 592 577 rs1016269 A/G 13/91/284 149/900/ 7/56/119
122/741/1137 6/51/97 132/718/ 7/95/340 104/756/2077 1451 1149
.sup.aThe genotype counts are listed as homozygous genotypes of
minor allele/heterozygous genotypes/homozygous genotypes of major
allele.
TABLE-US-00008 TABLE 8 Genome-wide association results for CXCR4(p
< 1 .times. 10.sup.-3 in the discovery cohort) in our JIA cohort
and in the WTCCC RA cohort p value Trend p Minor/ (Combined value
in Major p value Meta- WTCCC SNP BP.sup.a Alleles.sup.a Case_MAF
Control_MAF (Discovery) analysis) cohort rs953387 136623640 G/T
0.289 0.408 2.07E-10 2.87E-11 0.240 rs1123848 136661499 T/C 0.276
0.393 3.89E-10 6.11E-11 NA rs4954564 136611978 G/A 0.299 0.411
3.08E-09 8.58E-11 0.239 rs10221893 136730076 C/T 0.394 0.507
4.58E-09 9.31E-08 0.191 rs6430612 136722668 C/T 0.394 0.506
7.98E-09 4.08E-08 0.192 rs1016269 136657132 A/G 0.151 0.240
4.01E-08 1.90E-08 0.174 rs749873 136533558 C/T 0.340 0.436 4.22E-07
8.94E-07 0.786 rs2011946 136534086 G/T 0.325 0.408 1.03E-05
1.59E-06 0.846 rs932206 136541742 G/A 0.427 0.509 1.83E-05 4.53E-06
0.801 rs882300 136692725 A/G 0.454 0.377 4.37E-05 8.51E-05 0.929
rs12466743 136937875 G/A 0.057 0.100 1.37E-04 1.37E-04 0.047
rs16834223 136910906 G/A 0.058 0.101 1.38E-04 3.94E-04 0.045
rs4954599 136753856 G/A 0.246 0.313 1.58E-04 1.41E-04 0.548
rs2090660 136535189 A/G 0.229 0.294 2.17E-04 9.63E-06 0.692
rs6756490 136723546 A/G 0.166 0.223 3.87E-04 4.44E-04 0.418
rs4477975 136725476 G/A 0.166 0.222 4.49E-04 5.02E-04 0.419
rs953388 136623599 A/G 0.104 0.152 5.14E-04 2.50E-03 0.144
rs2056296 136603782 T/C 0.084 0.126 6.93E-04 3.87E-06 0.454
rs4074120 136743057 C/T 0.228 0.286 8.16E-04 8.16E-04 0.361 BP,
base pair chromosome coordinates; MAF, minor allele frequencies in
discovery cohort; p value, basic allelic test p value; p value
(combined), meta-analysis p value in the three cohorts typed
genome-wide (discovery and replication cohorts 1 and 2). .sup.aThe
chromosome coordinates and allele designations are on the basis of
the forward strand of the NCBI 36 genome assembly.
[0114] We next sought to replicate our 2q21 association signals in
several independent cohorts of JIA patients. The first cohort
included 182 JIA patients and an additional set of 2000 healthy
controls recruited from the Children's Hospital of Philadelphia
(CHOP; Philadelphia, Pa.; Replication 1; Table 1). Four SNPs showed
evidence of association, and with the same direction of effect, as
in the discovery cohort, with p values ranging from 0.01 to 0.04
(Table 4). To seek further evidence of replication, we examined a
second independent cohort of 154 cases from the Murdoch Childrens
Research Institute (MCRI; Melbourne, Australia; Replication 2;
Table 1). A further additional set of 2000 genetically matched
control samples from CHOP was utilized as a comparator for this
analysis. Evidence of association with the same direction of effect
was observed for five SNPs (p.ltoreq.0.05) (Table 4). The SNPs at
the CXCR4 locus survived correction in the two independent
replication cohorts (p=0.0274 and 0.0232 for the set of SNPs in
replication cohorts 1 and 2; see Subjects and Methods, Statistical
analysis) and direction of effect is the same. A third independent
cohort of 442 cases and 3000 controls from WTCCC replicates these
data (OUH; Oslo, Norway; Replication 3; Table 1), with p values of
10.sup.-4 to 10.sup.-3 for three SNPs at this locus (Table 4).
[0115] Combined meta-analysis of all four cohorts indicated that
four SNPs at the 2q21 locus were associated with JIA at a
genome-wide significant level, with p values ranging from
1.03.times.10.sup.-13 to 4.85.times.10.sup.-10 (Table 4). In
addition, three loci which previously have been implicated in JIA
and several other autoimmune diseases.sup.6,9,13--the PTPN22 locus
on 1p13, the IL2RA locus on 10p15, and the ANTXR2 locus on
4q21.21--were nominally associated with JIA:
p=1.77.times.10.sup.-5, OR 1.65, 95% CI: 1.31-2.07 for rs2476601
near PTPN22 in our discovery cohort and in the subsequent
meta-analysis (Tables 9 and FIG. 4); p=0.022, OR 0.84, 95% CI:
0.72-0.97 for rs706779 near IL2RA and p=0.0043, OR=0.72, 95% CI:
0.58-0.90 for rs17509015 near ANTXR2 in the discovery cohort (data
not shown).
[0116] To determine if other variants that did not meet genome-wide
significance criteria in the GWAS discovery cohort associate with
JIA, we subsequently evaluated all SNPs with association p values
<1.times.10.sup.-5 for replication, following whole-genome
imputation (see Subjects and Methods). Eight other non-MHC SNPs
were genome-wide significant, although all but one had nearby SNPs
in LD that did not support the association--and thus were discarded
as likely genotyping errors--and none of these SNPs replicated when
randomized for the number of tests performed (Table 10). Thus, the
most significant non-MHC association signals--and the only ones
that replicated--were those in the 2q21 region near CXCR4; rs953387
remained the most significantly associated SNP and another four
imputed SNPs were identified with p values <5.times.10.sup.-8,
in support of the 2q21 locus (Table 11 and FIG. 2). Examination of
the 2q21 region indicated that all genotyped and imputed SNPs with
p values below 5.times.10.sup.-8 reside within the same .about.110
kilobase (kb) LD block (FIG. 5), suggesting that these SNPs are
tagging the same variant(s). Taken together, several sources of
converging evidence firmly establish that common genetic variants
on 2q21 confer susceptibility to JIA.
TABLE-US-00009 TABLE 9 Genome-wide association results for PTPN22
(20 kb genomic regions on either side of the gene) in our JIA
cohort and in the WTCCC RA cohort p value Trend p Minor/ (Combined
value in Major p value Meta- WTCCC CHR SNP BP.sup.a Gene
Alleles.sup.a Case_MAF Control_MAF (Discovery) OR.sup.b analysis)
cohort 1 rs3827733 114140112 RSBN1 G/A 0.196 0.181 0.306 1.11 0.527
0.051 1 rs3811021 114158186 PTPN22 C/T 0.192 0.179 0.396 1.09 0.607
0.059 1 rs2476599 114164982 PTPN22 A/G 0.273 0.270 0.839 1.02 0.537
0.016 1 rs3789607 114167957 PTPN22 C/T 0.264 0.305 0.022 0.82 0.061
0.149 1 rs2476601 114179091 PTPN22 A/G 0.133 0.085 1.77E-5 1.65
6.18E-04 1.11E-16 1 rs1217407 114195271 PTPN22 A/G 0.271 0.245
0.127 1.14 0.385 2.30E-08 1 rs1217418 114202754 PTPN22 A/G 0.464
0.427 0.055 1.16 0.272 5.53E-04 1 rs6665194 114219366 between A/G
0.447 0.414 0.080 1.15 0.582 1.34E-04 PTPN22 and BCL2L15 1
rs12566340 114221851 BCL2L15 T/C 0.256 0.237 0.233 1.11 0.974
7.26E-09 1 rs7529353 114221985 BCL2L15 A/G 0.258 0.238 0.227 1.11
0.910 6.88E-09 1 rs2358994 114230984 BCL2L15 A/G 0.197 0.168 0.045
1.22 0.288 6.40E-12 1 rs1217394 114235182 near G/A 0.305 0.333
0.123 0.88 0.304 0.217 BCL2L15 1 rs1217392 114235493 between T/G
0.378 0.340 0.041 1.18 0.446 4.64E-05 BCL2L15 and AP4B1 BP, base
pair chromosome coordinates; MAF, minor allele frequencies in
discovery cohort; p value, basic allelic test p value; OR, odds
ratio in discovery cohort; p value (combined), meta-analysis p
value in the three cohorts typed genome-wide (discovery and
replication cohorts 1 and 2). .sup.aThe chromosome coordinates and
allele designations are on the basis of the forward strand of the
NCBI 36 genome assembly.
TABLE-US-00010 TABLE 10 Results of the Discovery and combined
analyses (p < 1 .times. 10.sup.-5), excluding the MHC and CXCR4
p value Minor/ (Combined Major p value Meta- CHR SNP BP.sup.a Gene
Alleles.sup.a Case_MAF Control_MAF (Discovery) OR.sup.b analysis) 7
rs7455060 118965081 near G/A 0.342 0.050 4.40E-152 9.85 2.55E-122
KCND2 4 rs4862110 183988023 near C/T 0.497 0.176 1.83E-89 4.65
3.57E-84 ODZ3 20 rs4814335 15001067 MACROD2 A/G 0.493 0.175
6.77E-87 4.59 1.41E-55 5 rs4957798 108472453 FER T/C 0.392 0.186
1.01E-38 2.83 2.49E-27 5 rs7726659 74513834 near G/A 0.081 0.013
2.19E-33 6.90 2.24E-19 GCNT4 12 rs7970177 13630255 GRIN2B T/C 0.228
0.088 1.21E-30 3.05 6.57E-18 8 rs2445610 128266270 near MYC G/A
0.432 0.331 3.97E-08 1.54 0.049 8 rs2456449 128262163 near MYC G/A
0.416 0.317 4.85E-08 1.54 0.029 18 rs6565965 73256562 near C/T
0.512 0.414 3.54E-07 1.48 1.67E-04 GALR1 14 rs2296322 64548701
FNTB, MAX C/A 0.187 0.123 8.13E-07 1.65 3.70E-05 3 rs11915523
108411619 near G/A 0.056 0.024 1.19E-06 2.36 1.01E-03 CCDC54 16
rs1129568 3423476 near C/T 0.362 0.453 2.09E-06 0.69 4.67E-06
ZNF597 8 rs2466031 128278791 near MYC G/A 0.467 0.378 2.68E-06 1.44
0.140 5 rs4958132 133019176 near C/T 0.316 0.239 4.58E-06 1.47
0.066 FSTL4 9 rs10867781 83423712 TLE1 T/C 0.381 0.300 5.11E-06
1.44 6.71E-04 10 rs2180563 33430188 near A/G 0.090 0.152 5.50E-06
0.55 0.254 NRP1 5 rs10491294 133018222 near C/T 0.316 0.240
5.51E-06 1.46 0.060 FSTL4 14 rs12891137 78693470 NRXN3 C/T 0.142
0.090 5.81E-06 1.67 2.82E-03 8 rs2456461 128251633 near MYC G/A
0.468 0.383 6.76E-06 1.42 0.238 7 rs11561808 16281469 LOC729920 C/T
0.197 0.136 6.86E-06 1.56 9.27E-05 6 rs12524299 155769924 NOX3 T/C
0.192 0.133 8.90E-06 1.56 8.33E-03 16 rs7196196 3351856 near C/A
0.407 0.493 9.46E-06 0.71 2.92E-04 OR2C1 BP, base pair chromosome
coordinates; MAF, minor allele frequencies in discovery cohort; p
value, basic allelic test p value; OR, odds ratio in discovery
cohort; p value (combined), meta-analysis p value in the three
cohorts typed genome-wide (discovery and replication cohorts 1 and
2). .sup.aThe chromosome coordinates and allele designations are on
the basis of the forward strand of the NCBI 36 genome assembly.
.sup.bThe odds ratio is calculated with respect to the minor
allele.
TABLE-US-00011 TABLE 11 Genome wide association results for imputed
SNPs (p < 1 .times. 10-4) in combined analysis in the vincinity
of CXCR4 in our JIA cohort p value Minor/ (Combined Major Case
Control p value Meta- CHR SNP BP.sup.a Type Alleles.sup.a MAF MAF
(Discovery) OR.sup.b analysis) 2 rs6716987 136679964 Imputed A/C
0.273 0.390 4.86E-10 0.59 1.38E-10 2 rs4954577 136665181 Imputed A
0.274 0.390 5.99E-10 0.59 1.78E-10 2 rs1519527 136684887 Imputed
A/G 0.150 0.239 3.90E-08 0.56 2.74E-08 2 rs4954579 136684945
Imputed A/G 0.150 0.239 4.04E-08 0.56 2.80E-08 2 rs13024450
136731081 Imputed C/T 0.394 0.504 1.17E-08 0.64 6.98E-08 2
rs4452212 136732461 Imputed G/A 0.394 0.504 1.17E-08 0.64 7.15E-08
2 rs13004902 136744150 Imputed A/G 0.394 0.504 1.17E-08 0.64
7.52E-08 2 rs12691881 136746138 Imputed A/G 0.394 0.504 1.24E-08
0.64 8.00E-08 2 rs13018756 136724705 Imputed C/T 0.394 0.502
2.41E-08 0.65 1.23E-07 2 rs4072435 136730371 Imputed C/T 0.394
0.502 2.13E-08 0.64 1.45E-07 2 rs11674937 136650999 Imputed C/G
0.174 0.264 1.38E-07 0.59 9.15E-07 2 rs9973445 136595086 Imputed
C/G 0.084 0.126 8.61E-04 0.64 5.23E-06 2 rs12615624 136438073
Imputed G/A 0.262 0.331 1.48E-04 0.72 9.90E-06 2 rs745500 136299662
Imputed G/A 0.269 0.340 9.28E-05 0.71 1.62E-05 2 rs7579771
136296730 Imputed A/T 0.269 0.340 8.49E-05 0.71 1.66E-05 2
rs3754686 136319746 Imputed T/C 0.268 0.341 6.36E-05 0.71 1.83E-05
2 rs3769005 136319836 Imputed C/G 0.268 0.341 6.36E-05 0.71
1.83E-05 2 rs4954490 136324701 Imputed G/A 0.268 0.341 6.65E-05
0.71 1.87E-05 2 rs892715 136293047 Imputed T/C 0.269 0.340 8.74E-05
0.71 2.22E-05 2 rs1435576 136358352 Imputed T/A 0.274 0.352
2.73E-05 0.69 2.42E-05 2 rs309125 136360025 Imputed T/C 0.274 0.352
2.86E-05 0.69 2.44E-05 2 rs7589832 136220571 Imputed C/A 0.194
0.256 1.90E-04 0.70 4.25E-05 2 rs632632 136354686 Imputed C/T 0.274
0.350 4.67E-05 0.70 4.85E-05 2 rs12619365 136114644 Imputed T/C
0.258 0.324 2.36E-04 0.73 6.02E-05 2 rs2839740 136365353 Imputed
T/G 0.158 0.219 9.84E-05 0.67 6.51E-05 2 rs7581814 136358063
Imputed G/C 0.158 0.219 9.64E-05 0.67 6.57E-05
[0117] JIA is a complex phenotype comprised of seven subtypes, as
defined by the revised ILAR criteria..sup.3 We analyzed each
subtype in our combined dataset of JIA subjects (Table 2) for
association signals at rs953387, the non-MHC SNP most significantly
associated with all JIA. Five of the seven subtypes showed the same
direction of allelic effect, with p values in all but the smallest
cohorts ranging from 3.42.times.10.sup.-4 to 4.09.times.10.sup.-3
(Table 5); combined analysis of two subtypes, oligoarthritis and RF
negative polyarthritis, with an independent cohort from Cincinnati
Children's Hospital Medical Center (CCHMC; Cincinnati, Ohio), also
showed evidence of association, with a p value of
6.31.times.10.sup.-5. Of interest, only marginal association of
CXCR4 was observed with adult rheumatoid arthritis (RA) in the
WTCCC cohort.sup.19 (Table 8). Comparable results were observed in
our subjects with rheumatoid factor positive polyarthritis, the JIA
subtype most similar to RA, although this remains to be evaluated
in a larger study.
[0118] To examine whether the SNP genotypes associate with
expression levels of CXCR4 mRNA, we explored the Sanger Institute
GENe Expression VARiation (Genevar) public database (URLs), which
profiles gene expression in immortalized B-lymphocyte samples from
HapMap populations..sup.28 The most significant SNP (rs953387)
among six GW-significant SNPs at the CXCR4 locus was assessed in 30
CEU children (see Subjects and Methods, Gene expression). The
genotypes of rs953387 associated with expression levels of CXCR4
(p=0.014; FIG. 6). Analysis of an independent dataset of T-cell
lines from umbilical cords of 75 individuals of Western European
origin.sup.29 also demonstrated association of genotypes of
rs1016269 (Table 1) with levels of CXCR4 expression (p=0.0054; FIG.
6). These data suggest that variants in or around CXCR4 regulate
mRNA expression of this chemokine receptor.
Discussion
[0119] We have identified and replicated common genetic variants on
2q21 that are associated with susceptibility to JIA in a combined
sample set of more than 1100 JIA subjects of European ancestry. We
propose that susceptibility to JIA is controlled by a number of
"master switches", which, like CXCR4, are identifiable by allelic
variants in a significantly increased proportion of JIA patients
compared to healthy controls. Our data support a role for altered
expression of CXCR4 in JIA pathogenesis, although an alternate,
testable hypothesis is that these common variants near CXCR4 serve
as markers for potentially more rare variants within the coding
region of CXCR4. Our results represent, to our knowledge, the first
unbiased genome-wide significant association of a common variant
with JIA outside of the MHC, and the first genetic demonstration of
a possible role for the chemokine receptor, CXCR4, in the
pathogenesis of autoimmune disease.
[0120] The only previously reported GWAS of JIA was limited by the
small size of the discovery cohort (279 samples), coupled with a
lack of matching controls and restricted capture of variants by the
genotyping platform; the study failed to identify association of
any genetic variants with JIA at genome-wide significant
levels..sup.37 A recent candidate gene study identified a number of
JIA susceptibility loci in a large case-control cohort;.sup.9 this
study--which was not designed as a GWAS--also suffered from
restricted variant capture of the genotyping platform (425
candidate non-MHC region SNPs), precluding discovery of novel JIA
predisposition factors like the CXCR4 chemokine receptor.
[0121] Chemokine receptors are critical regulators of cell
migration in immune surveillance, inflammation and development. The
G protein-coupled chemokine receptor, CXCR4, is expressed on the
surface of T-cells, B-cells, monocytes, neutrophils and dendritic
cells (FIG. 7), and is activated exclusively by CXCL12 (also known
as stromal-derived-factor-1, SDF-1), a small peptide mediator and
potent chemoattractant for leukocytes, including B- and T-cells.
CXCR4 and its ligand, CXCL12, have been shown to play a role in
B-cell production, myelopoiesis, integrin activation, angiogenesis,
and chemotaxis..sup.38 Intriguingly, the human immunodeficiency
virus (HIV-1) has usurped CXCR4's unique CXCL12 binding site,
exploiting CXCR4 as a co-receptor in later stages of HIV-1
infection, and CXCR4 antagonists have been explored as treatments
for HIV. Binding of CXCR4 to CXCL12 is also proposed to play a role
in cancer metastases, and CXCR4 antagonists are under study in
human clinical trials for solid and non-solid tumors..sup.38
Available therapeutic agents targeting the CXCR4-CXCL12 axis for
activation or inhibition include plerixafor (AMD3100), recombinant
CXCL12, and high-affinity CXCR4 and CXCL12 monoclonal antibodies,
some of which are already in use in the clinic but not approved for
use in children. The recent report of crystal structures of CXCR4
with small-molecule and cyclic peptide inhibitors.sup.39 provide
new opportunities for drug discovery efforts targeting this
receptor.
[0122] CXCR4 and CXCL12 have been implicated in the pathogenesis of
autoimmune diseases..sup.38,40 In mouse models of autoimmune
disease, modulation of CXCR4 alters trafficking of leukocytes to
peripheral organs and polarization of regulatory T cells, and
accelerates onset of disease..sup.41,42 This is consistent with our
data showing that a risk variant of CXCR4 correlates with decreased
expression of CXCR4. An alternate hypothesis is that the effect of
low CXCR4 expression is indirect and leads to a compensatory
increase of the CXCR4 ligand, CXCL12. Our preliminary data suggest
that the risk variant of CXCR4 correlates with increased expression
of CXCL12 (data not shown). This is consistent with models of
collagen-induced arthritis in which CXCL12 acts as a
pro-inflammatory factor in the pathogenesis of inflammatory
arthritis,.sup.43,44 and with human studies in which CXCL12
enhances cellular proliferation and cytokine expression by
peripheral blood T cells, upregulates expression of cytokines and
chemokines by fibroblast-like synoviocytes from patients with
RA,.sup.45 and mediates lymphocyte ingress into RA synovial tissue,
synovial neovascularisation, and osteoclastogenesis..sup.46
[0123] Our results indicate that gene variants at the CXCR4 locus
predispose to the development of JIA. We have uncovered common
tagging SNPs that confer strong effects on a genome-wide scale and
replicate in three independent cohorts of JIA patients. The
extensive literature surrounding the biology of CXCR4 and CXCL12 in
health and disease and the ready availability of targeted
therapeutic agents make CXCR4 a particularly attractive candidate
for further investigation of its pathogenic role and therapeutic
potential in JIA and other autoimmune diseases affecting
children.
[0124] Table 12 below provides a list of putative test agents that
can be screened in accordance with the present invention for
efficacy for the treatment and prevention of JIA.
TABLE-US-00012 Company Product Phase Description Indication MOA
Molecule Type Ablynx nv (ABLX (EBR)) ALX0651 PC ALX-0651 is a
nanobody acts by the inhibition of CXCR4. Cancer CXC Chemokine --
CXCR4 is a chemokine receptor that plays an important role in
Receptor 4 (CXCR4) cell movement, tumor growth and metastasis.
ALX-0651 is Antagonist being developed as an intravenous
formulation for the treatment of cancer. Genzyme AMD070 II AMD-070
is a new type of antiretroviral known as HIV entry AIDS/HIV CXC
Chemokine -- Corporation (GENZ) inhibitor that binds to CXCR4
chemokine receptors and Receptor 4 (CXCR4) prevents the relevant
HIV strains from entering and infecting Antagonist the target
cells. AMD070 is being developed as oral formulation for the
treatment of AIDS/HIV. Note: This product is added upon acquisition
of AnorMED Inc. Genzyme AMD3100 F AMD-3100 contains plerixafor, a
potential new agent for AIDS/HIV CXC Chemokine Small Corporation
(GENZ) peripheral blood stem cell transplant in cancer patients. It
Receptor 4 (CXCR4) blocks a specific cellular receptor, known as
the CXCR4 Antagonist chemokine receptor, which is present on white
blood cells and other immune cells. AMD-3100 was under development
for the treatment of AIDS/HIV. Note: This product is added upon
acquisition of AnorMED Inc. Affitech A (AFFI (OMX AT009 PC AT009 is
an antibody that targets CXCR4 and its ligand, SDF-1 Inflammation
CXC Chemokine -- Nordic Exchange (Stromal Derived Factor-1). AT009
is being developed for the Receptor 4 (CXCR4) Copenhagen))
treatment of inflammation. Antagonist Biokine Therapeutics BKT140
II BKT140 is a CXC Chemokine Receptor 4 (CXCR4) antagonist.
Multiple Myeloma CXC Chemokine -- Ltd. (Private) BKT140 is being
developed for the treatment of myeloma and (Myeloma) Receptor 4
(CXCR4) other hematological diseases. Antagonist Daiichi Sankyo
Company, CS3955 D CS-3955 is a CXCR4 (Chemokine Receptor 4)
antagonist. It AIDS/HIV CXC Chemokine -- Limited (Stock Code
Number: inhibits the binding between HIV and CXCR4. CS-3955 was
Receptor 4 (CXCR4) 4568 (TYO)) under development as an oral
formulation for the treatment of Antagonist HIV infection. Note:
This product is added upon the merger of Daiichi Pharmaceutical
Co., Ltd. with Sankyo Co., Ltd Kureha Corporation (formerly CS3955
D CS-3955 is a CXCR4 (Chemokine Receptor 4) antagonist. It AIDS/HIV
CXC Chemokine -- Kureha Chemical Industry inhibits the binding
between HIV and CXCR4. CS-3955 was Receptor 4 (CXCR4) Co., Ltd.)
(Stock Code under development as an oral formulation for the
treatment of Antagonist Number: 4023 (TYO)) HIV infection. British
Canadian CTCE0214 I CTCE-0214 is an analog of SDF-1 and agonist of
the SDF-1 Neutropenia CXC Chemokine -- BioSciences Corp. receptor,
CXCR4. It mobilizes blood and progenitor cells and Receptor 4
(CXCR4) (BCBC) (Private) enhances the survival and expansion of
cord blood cells. It Antagonist rapidly increases the number of
stem cells and white blood cells (WBC) for patients with
chemotherapy induced Neutropenia. CTCE-0214 is being developed as a
subcutaneous formulation for the treatment of neutropenia. British
Canadian CTCE0324 PC CTCE-0324 is a peptide agonist that is an
analog of SDF-1. It Peripheral CXC Chemokine -- BioSciences Corp.
binds to CXCR4 and induces a host of cellular responses, Vascular
Disease Receptor 4 (CXCR4) (BCBC) (Private) leading to the
development of tube-like structures and Agonist sprouting of new
blood vessels from existing ones. CTCE-0324 is being developed as
an injection for the treatment of vascular disease by inducing the
formation of new blood vessels to circumvent existing narrowed
blood vessels. British Canadian CTCE9908 PC CTCE-9908 is an analog
of stromal cell-derived factor 1 (SDF- Cancer CXC Chemokine --
BioSciences Corp. 1), acts as a competitive antagonist at CXCR4
receptors and Receptor 4 (CXCR4) (BCBC) (Private) inhibits the
metastasis of cancer cells. It destroys the primary Antagonist
tumors and delays the occurrence of secondary tumors. CTCE- 9908 is
being developed as an injection for the treatment of cancer
(ovarian cancer and prostrate cancer). British Canadian CTCE9908 II
CTCE-9908, a small protein is an analog of stromal cell- Liver
Cancer CXC Chemokine -- BioSciences Corp. derived factor 1 (SDF-1),
acts as a competitive antagonist at Receptor 4 (CXCR4) (BCBC)
(Private) CXCR4 receptors and inhibits the metastasis of cancer
cells. It Antagonist destroys the primary tumors and delays the
occurrence of secondary tumors. CTCE-9908 is being developed as an
injection for the treatment of liver cancer. ChemoCentryx, Inc.
(Private) CXCR4 PC CXCR4 antagonist is a high potency small
molecule antagonist Inflammation CXC Chemokine Small antagonist of
chemokine receptor target CXCR4, which is expressed by
(Inflammatory Receptor 4 (CXCR4) CHEMOCENTRYX both cancerous and
non-cancerous cell types. CXCR4 Disorders) Antagonist antagonist is
being developed for the treatment of inflammatory disorders.
Shanghai Targetdrug CXCR4 NA CXCR4 antagonist is a human chemokine
receptor blocker. It Rheumatoid CXC Chemokine -- Ltd. (Private)
antagonist blocks T cell migration in the inflamed rheumatoid
arthritis Arthritis Receptor 4 (CXCR4) TARGETDRUG synovium. It is
being developed for the treatment of Antagonist rheumatoid
arthritis. Shanghai Targetdrug CXCR4 NA CXCR4 antagonist is a human
chemokine receptor blocker. It Asthma CXC Chemokine -- Ltd.
(Private) antagonist decreases CD4+ and CD8+ T-cell recruitment and
airway Receptor 4 (CXCR4) TARGETDRUG hyperresponsiveness and
reduces the airway inflammation in It Antagonist is being developed
for the treatment of asthma. Northwest Biotherapeutics CXCR4
antibody PC CXCR4 antibody (CXC chemokine receptor 4 monoclonal
Cancer CXC Chemokine Large Inc (NWBO) NORTHWEST antibody) works by
inhibiting the tumor growth and induces cell Receptor 4 (CXCR4)
death (apoptosis). It also blocks the movement or chemotaxis
Antagonist and the invasion of cancer cells through other tissues.
CXCR4 antibody is being developed for the treatment of cancer.
Genzyme CXCR4 Inhibitor D CXCR4 is a chemokine receptor which is
present on white Cancer CXC Chemokine -- Corporation (GENZ) GENZYME
blood cells and plays a key regulatory role in the trafficking and
Receptor 4 (CXCR4) homing of cells involved in the immune system.
Chemokine Antagonist inhibitors block chemokine receptors, and
prevent the chemotaxis from occurring. CXCR4 Inhibitor was under
development for the treatment of cancer. Note: This product is
added upon acquisition of AnorMED Inc. GlaxoSmithKline plc (GSK
GSK812397 NA GSK812397 is a noncompetitive CXCR4 (CXC chemokine
AIDS/HIV (HIV-1 CXC Chemokine -- (LON)) receptor) antagonist that
acts by inhibiting the cellular entry of Infections) Receptor 4
(CXCR4) X4-tropic strains of HIV-1 and is being developed for the
Antagonist treatment of HIV-1 infections. Kureha Corporation
(formerly KRH3140 D KRH-3140 is an antiviral drug interferes with
HIV-1 entry into T AIDS/HIV CXC Chemokine -- Kureha Chemical
Industry cells by antagonizing CXC chemokine receptor 4. KRH-3140
Receptor 4 (CXCR4) Co., Ltd.) (Stock Code was under development as
oral formulation for the treatment of Antagonist Number: 4023
(TYO)) HIV/AIDS. Kureha Corporation (formerly KRH3955 D KRH-3955 is
an antiviral drug, which interferes with HIV-1 entry AIDS/HIV CXC
Chemokine -- Kureha Chemical Industry into T cells by antagonizing
CXC chemokine receptor 4. KRH- Receptor 4 (CXCR4) Co., Ltd.) (Stock
Code 3955 was under development as oral formulation for the
Antagonist Number: 4023 (TYO)) treatment of HIV/AIDS. Bristol-Myers
Squibb MDX1338 I MDX-1338 acts on chemokine receptor CXCR4,
expressed on Acute CXC Chemokine -- Company (BMY) tumor cells and
induce apoptosis and inhibit proliferation, Myelogenous Receptor 4
(CXCR4) angiogenesis and metastasis of tumor cells. MDX-1338 is
Leukemia Antagonist being developed for the treatment of
relapsed/refractory acute (Relapsed/Refractory myelogenous
leukemia. Note: This product is added upon the Acute acquisition of
Medarex Inc. Myelogenous Leukemia) Genzyme Mozobil PC Mozobil
contains plerixafor, a novel hematopoietic stem cell Autoimmune CXC
Chemokine Small Corporation (GENZ) (HSC) mobilizer. It blocks a
specific cellular receptor, known as Diseases Receptor 4 (CXCR4)
the CXCR4 chemokine receptor, triggering the movement of Antagonist
stem cells out of the bone marrow and into the circulating blood.
Mozobil is being developed as subcutaneous injection for the
treatment of solid organ transplant/autoimmune diseases. Note: This
product is added upon acquisition of AnorMED Inc. Genzyme Mozobil
PC Mozobil contains plerixafor, a novel hematopoietic stem cell
Kidney Disease CXC Chemokine Small Corporation (GENZ) (HSC)
mobilizer. It blocks a specific cellular receptor, known as
(Ischemic Renal Receptor 4 (CXCR4) the CXCR4 chemokine receptor,
triggering the movement of Disease) Antagonist stem cells out of
the bone marrow and into the circulating blood. Mozobil is being
developed for the treatment of ischemic renal disease. Genzyme
Mozobil M Mozobil is a novel hematopoietic stem cell (HSC)
mobilizer. It Transplantation CXC Chemokine Small Corporation
(GENZ) blocks a specific cellular receptor, known as the CXCR4
(Stem Cell Receptor 4 (CXCR4) chemokine receptor, triggering the
movement of stem cells out Transplantation in Antagonist of the
bone marrow and into the circulating blood. Mozobil Cancer)
injection is intended to be used in combination with granulocyte
colony stimulating factor (G-CSF) to mobilize hematopoietic stem
cells to the bloodstream for collection and subsequent autologous
transplantation in patients with non-Hodgkin's lymphoma (NHL) and
multiple myeloma (MM). It is available as subcutaneous injection
(20 mg/ml) Note: This product is added upon acquisition of AnorMED
Inc. Genzyme Mozobil D Mozobil is a novel stem cell mobilizer,
contains plerixafor as Transplantation CXC Chemokine Small
Corporation (GENZ) active ingredient. It acts by blocking a
specific cellular receptor, (Stem Cell Receptor 4 (CXCR4) known as
the CXCR4 chemokine receptor, triggering the Transplantation for
Antagonist movement of stem cells out of the bone marrow and into
the Heart Tissue circulating blood. Mozobil was under development
as Repair) subcutaneous injection for stem cell transplantation in
patients who have suffered heart attacks. Note: This product is
added upon acquisition of AnorMED Inc. Genzyme Mozobil with II
Mozobil is a novel stem cell mobilizer, contains plerixafor as
Acute CXC Chemokine -- Corporation (GENZ) Mitoxantrone, active
ingredient. It acts by blocking a specific cellular receptor,
Myelogenous Receptor 4 (CXCR4) Etoposide and known as the CXCR4
chemokine receptor, triggering the Leukemia Antagonist Cytarabine
movement of stem cells out of the bone marrow and into the
(Relapsed or circulating blood. Mozobil (intravenous) in
combination with Refractory Acute Mitoxantrone (intravenous),
Etoposide (intravenous) and Myelogenous Cytarabine (intravenous) is
being developed as subcutaneous Leukemia) injection for tumor
sensitization in relapsed or refractory acute myeloid leukemia.
Note: This product is added upon acquisition of AnorMED Inc.
Genzyme Mozobil with II Mozobil contains plerixafor that acts by
blocking a specific Chronic CXC Chemokine -- Corporation (GENZ)
Rituximab cellular receptor, known as the CXCR4 chemokine receptor,
Lymphocytic Receptor 4 (CXCR4) triggering the movement of stem
cells out of the bone marrow Leukemia Antagonist; Membrane- and
into the circulating blood. Rituximab is a genetically Spanning
4-Domains, engineered chimeric monoclonal antibody that targets a
Subfamily A, Member 1 receptor called CD20 found on some B-cells.
It induces lysis (MS4A1) Inhibitor through several proposed
mechanisms, work with elements of the human immune system to kill
CD20+ B cells through antibody-dependent toxicity (ADCC) and
complement- dependent cytotoxicity (CDC). Mozobil (subcutaneous),
Rituximab combination is being developed for the treatment of
chronic lymphocytic leukemia or small lymphocytic lymphoma. Genzyme
Mozobil with II Mozobil contains plerixafor that acts by blocking a
specific Multiple Myeloma 26S Proteasome -- Corporation (GENZ)
Velcade cellular receptor, known as the CXCR4 chemokine receptor,
(Relapsed or Inhibitor; CXC Chemokine triggering the movement of
stem cells out of the bone marrow Relapsed/Refractory Receptor 4
(CXCR4) and into the circulating blood. Velcade contains
bortezomib. Multiple Antagonist Bortezomib is a proteasome
inhibitor. Proteasomes are Myeloma) enzyme complexes which are
present in all cells which break down intracellular proteins in a
regulated manner in both
healthy and cancerous cells. Inhibition of the proteasome prevents
the regulated breakdown of these intracellular proteins, thereby
interfering with many of these varied functions. This disruption of
essential pathways in cancer cells can lead to cell death and
inhibit tumor growth. Mozobil (subcutaneous) in combination with
Velcade (bortezomib) (intravenous) is being developed for the
treatment of relapsed or relapsed/refractory multiple myeloma. NeED
pharma (Private) ND401 NA ND401 is a compound that blocks viral
infection by inhibition of AIDS/HIV CC Chemokine Receptor -- both
CC Chemokine receptors (CCR5) and CXC Chemokine 5 (CCR5)
Antagonist; CXC receptor (CXCR4) co-receptors. ND401 is being
developed for Chemokine Receptor 4 the treatment of HIV Infection.
(CXCR4) Antagonist Osprey Pharmaceuticals OPLCXCL12LPM NA
OPL-CXCL12-LPM, a leukocyte population modulator is a Ovarian
Cancer CXC Chemokine -- USA (OPUS) (Private) highly potent
inhibitor of CXCL12-CXCR4 signaling pathway. Receptor 4 (CXCR4)
Leukocyte population modulator is a recombinant fusion Antagonist
protein comprised of a receptor-binding chemokine moiety connected
to a cellular toxin via a peptide linker. OPL-CXCL12- LPM is being
developed based on LPM platform technology for the treatment of
ovarian cancer. Polyphor Ltd (Private) PO16326 PC POL-6326 is a
potent, selective and reversible CXCR4 inhibitor Wounds (Wound CXC
Chemokine -- which is based on Protein Epitope Mimetics (PEM)
technology. Healing) Receptor 4 (CXCR4) POL-6326 is being developed
for the regeneration of tissue in Antagonist wound healing process.
Polyphor Ltd (Private) PO16326 PC POL-6326 is a potent, selective
and reversible CXCR4 Inflammation CXC Chemokine -- inhibitor. It is
based on Protein Epitope Mimetics (PEM) Receptor 4 (CXCR4)
technology. POL-6326 is being developed for the treatment of
Antagonist inflammation. Polyphor Ltd (Private) POL6326 PC POL-6326
is a potent, selective and reversible CXCR4 Leukemia CXC Chemokine
-- inhibitor. It is based on Protein Epitope Mimetics (PEM)
Receptor 4 (CXCR4) technology. POL-6326 is being developed for the
treatment of Antagonist leukemia. Polyphor Ltd (Private) POL6326 II
POL-6326 is a potent, selective and reversible CXCR4 inhibitor
Transplantation CXC Chemokine -- which is based on Protein Epitope
Mimetics (PEM) technology. (Hematopoietic Receptor 4 (CXCR4)
Blockade of the CXCR4 receptor mobilizes hematopoietic stem Stem
Cell Antagonist cell from the bone marrow into the blood stream
where they Transplantation) can be harvested for transplant
supporting the treatment of blood or bone marrow diseases. POL-6326
is being developed as intravenous and subcutaneous formulations for
the treatment of transplantation. Angioblast Systems, SDF-1 PC
SDF-1 (Stromal derived factor 1) is a chemically synthesized
Cardiovascular CXC Chemokine -- Inc. (Private) ANGIOBLAST short
peptide based product known as chemokines, which is a Diseases
Receptor 4 (CXCR4) cytokine and antigen receptor inhibitor. It acts
by inducing the Agonist stem cell migration. Stromal derived factor
1 is being developed for the treatment of congestive heart failure
and myocardial infraction. Samaritan Pharmaceuticals SP01A III
SP-01A is an oral HIV entry inhibitor drug. It cripples HIV's
AIDS/HIV CC Chemokine Receptor -- Inc (SPHC) ability to enter cells
by blocking the proteins on human T Cells 5 (CCR5) Antagonist; CXC
that would, otherwise, facilitate HIV's entry into those cells. It
Chemokine Receptor 4 reduces intracellular cholesterol and
corticosteroid (CXCR4) Antagonist biosynthesis, which causes the
inability of lipid rafts in the cellular membrane to organize,
ultimately preventing fusion of an HIV receptor and both the CCR5
and CXCR4 cellular receptors. SP-01A is being developed for the
treatment of AIDS/HIV infection. TaiGen Biotechnology Co., TG0054
PC TG-0054 is a potent and selective chemokine receptor 4 Eye
Diseases CXC Chemokine -- Ltd. (Private) antagonist that
effectively mobilizes bone marrow (General) (Eye Receptor 4 (CXCR4)
stem/progenitor cells into peripheral circulation. These Diseases)
Antagonist stem/progenitor cells can perform tissue and vasculature
repair. TG-0054 is being developed for the treatment of eye
diseases like age related macular degeneration and diabetic
retinopathy. TaiGen Biotechnology Co., TG0054 I TG-0054 is a potent
and selective chemokine receptor 4 Cardiovascular CXC Chemokine --
Ltd. (Private) antagonist that effectively mobilizes bone marrow
Diseases Receptor 4 (CXCR4) stem/progenitor cells into peripheral
circulation. These Antagonist stem/progenitor cells can perform
tissue and vasculature repair. TG-0054 is being developed as an
intravenous formulation for the treatment of cardiovascular
diseases like intermittent claudication, myocardial infarction and
non- hemorrhagic stroke. TaiGen Biotechnology Co., TG0054 II TG0054
is a potent and selective chemokine receptor 4 Cancer (Stem Cell
CXC Chemokine -- Ltd. (Private) (CXCR4) antagonist that effectively
mobilizes bone marrow Transplantation In Receptor 4 (CXCR4)
stem/progenitor cells into peripheral circulation. These Cancer
Patients) Antagonist stem/progenitor cells can perform tissue and
vasculature repair. TG0054 is being developed as an intravenous
formulation for stem cell transplantation in cancer patients with
multiple myeloma, non-Hodgkin lymphoma and Hodgkin disease
patients.
Example 2
[0125] Through exome sequencing of the CXCR4 gene in 432 JIA cases
using next-generation sequencing technology, we have identified 3
nonsynonymous mutations (nsSNVs) and one stop/gain mutation (stop
codon) of which only one is found to exist in one individual in a
public sequencing database of 6,500 subjects. All 4 variants are
otherwise absent in our control cohort as well as the 1000 genome
project.
[0126] Moreover, in a nsSNV burden analysis of our case cohort in
comparison with controls and public databases combined of over
7,500 subjects, we observed that the rare variant burden of nsSNVs
that are predicted by SIFT and PolyPhren scores to be highly
damaging to the function of the CXCR4 protein are over 4.times.
more common in the JIA patients compared to controls (P=0.0175;
OR=4.661764 with 95% CI [1.121435 14.721356]).
[0127] These results suggest that these mutations, all of which are
predicted to be pathogenic to the function of the CXCR4 gene, are
highly relevant to the pathogenesis of JIA and strongly support
CXCR4 as an important disease gene and therapeutic target in
JIA.
TABLE-US-00013 Gene Exonic Function Amino Acid Change CXCR4
nonsynonymous SNV NM_001008540:c.C1049A:p.S350Y CXCR4 nonsynonymous
SNV NM_001008540:c.A169C:p.I57L* CXCR4 nonsynonymous SNV
NM_001008540:c.C19G:p.L7V CXCR4 stopgain SNV
(NM_001008540:c.T14A:p.L5X stop codon) *present in one control
subject of over 7,500 and this mutation is the least detrimental of
the four mutations identified.
[0128] The information herein above can be applied clinically to
patients for diagnosing an increased susceptibility for developing
JIA, and therapeutic intervention. A preferred embodiment of the
invention comprises clinical application of the information
described herein to a patient. Diagnostic compositions, including
microarrays, and methods can be designed to identify the genetic
alterations described herein in nucleic acids from a patient to
assess susceptibility for developing JIA. This can occur after a
patient arrives in the clinic; the patient has blood drawn, and
using the diagnostic methods described herein, a clinician can
detect one or more indicative SNPs or mutations described in the
present application. The information obtained from the patient
sample, which can optionally be amplified prior to assessment, will
be used to diagnose a patient with an increased or decreased
susceptibility for developing JIA. Kits for performing the
diagnostic method of the invention are also provided herein. Such
kits comprise a microarray comprising at least one, two, three,
four, five, six or all of the SNPs provided herein in and the
necessary reagents for assessing the patient samples as described
above.
[0129] The invention also provides a method of treating JIA in a
patient determined to have at least one prescribed single
nucleotide polymorphism indicative of the presence of JIA, by
administering to the patient a therapeutically effective amount of
at least one member of the agents listed in Table 12. This method
provides a test and treat paradigm, whereby a patient's genetic
profile is used to personalize treatment with therapeutics targeted
towards specific immunological defects found in individuals
exhibiting JIA. Such a test and treat model may benefit up to 50%
of patients with JIA with greater efficacy and fewer side effects
than non-personalized treatment.
[0130] The identity of SNPs and mutations present in the CXCR4 gene
and the patient results will indicate which variants are present,
and will identify those that possess an altered risk for developing
JIA. The information provided herein allows for therapeutic
intervention at earlier times in disease progression that
previously possible. Also as described herein above, SNP containing
CXCR4 genes involved in JIA pathogenesis have been identified which
provide novel targets for the development of new therapeutic agents
efficacious for the treatment of JIA.
Web Resources (URLs)
[0131] The URLs for data presented herein are as follows: JIA
Calculator, http://www.jra-research.org/JIAcalc/index.php PLINK,
http://pngu.mgh.harvard.edu/.about.purcell/plink/ WGAViewer,
http://people.genome.duke.edu/.about.dg48/WGAViewer/ MACH,
http://www.sph.umich.edu/csg/abecasis/MaCH/index.html Metal,
http://www.sph.umich.edu/csg/abecasis/Metal/index.html MOOSE,
http://jama.ama-assn.org/cgi/content/full/283/15/2008 Genevar,
http://www.sanger.ac.uk/esources/software/genevar/ HapMap,
http://hapmap.ncbi.nlm.nih.gov/ Quanto,
http://hydra.usc.edu/gxe/
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[0178] While certain of the preferred embodiments of the present
invention have been described and specifically exemplified above,
it is not intended that the invention be limited to such
embodiments. It will be apparent to one skilled in the art that
various changes and modifications can be made therein without
departing from the scope of the present invention, as set forth in
the following claims.
* * * * *
References