U.S. patent application number 14/479916 was filed with the patent office on 2015-04-23 for genetic polymorphisms associated with cardiovascular disorders and drug response, methods of detection and uses thereof.
The applicant listed for this patent is Celera Corporation. Invention is credited to James J. DEVLIN, Olga IAKOUBOVA.
Application Number | 20150111775 14/479916 |
Document ID | / |
Family ID | 37889578 |
Filed Date | 2015-04-23 |
United States Patent
Application |
20150111775 |
Kind Code |
A1 |
IAKOUBOVA; Olga ; et
al. |
April 23, 2015 |
GENETIC POLYMORPHISMS ASSOCIATED WITH CARDIOVASCULAR DISORDERS AND
DRUG RESPONSE, METHODS OF DETECTION AND USES THEREOF
Abstract
The present invention is based on the discovery of genetic
polymorphisms that are associated with cardiovascular disorders,
particularly acute coronary events such as myocardial infarction
and stroke, and genetic polymorphisms that are associated with
responsiveness of an individual having a cardiovascular disorder to
treatment of the disorder with statin. In particular, the present
invention relates to nucleic acid molecules containing the
polymorphisms, variant proteins encoded by such nucleic acid
molecules, reagents for detecting the polymorphic nucleic acid
molecules and proteins, and methods of using the nucleic acid and
proteins as well as methods of using reagents for their
detection.
Inventors: |
IAKOUBOVA; Olga;
(Pleasanton, CA) ; DEVLIN; James J.; (Lafayette,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Celera Corporation |
Alameda |
CA |
US |
|
|
Family ID: |
37889578 |
Appl. No.: |
14/479916 |
Filed: |
September 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12758663 |
Apr 12, 2010 |
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14479916 |
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11526137 |
Sep 22, 2006 |
7799530 |
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12758663 |
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60720274 |
Sep 23, 2005 |
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Current U.S.
Class: |
506/9 ;
435/6.11 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 2600/106 20130101; C12Q 2600/158 20130101; C12Q 2600/16
20130101; A61P 9/10 20180101; A61P 9/00 20180101; C12Q 2600/172
20130101; A61K 31/70 20130101; C12Q 1/6883 20130101; C12Q 2600/136
20130101; C12Q 2600/118 20130101 |
Class at
Publication: |
506/9 ;
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of identifying an individual having an altered risk for
developing an acute coronary event or a method of evaluating an
individual's likelihood of responding to statin treatment,
comprising detecting the presence or absence of one or more alleles
of a SNP in said individual's nucleic acids, wherein the SNP is
selected from any one of the nucleotide sequences of SEQ ID
NOS:121-200 and SEQ ID NOS:251-410, and the presence or absence of
the alleles are correlated with an individual's risk for developing
an acute coronary event or likelihood of responding to statin
treatment.
2. The method of claim 1, wherein the SNP is selected from the
group of nucleotide sequences consisting of SEQ ID NOs of 356, 282,
358, 270, 256, 252, 354, 357, 306, 363, 361, 295, 285, 290, 251,
293, 294, 305, 266, 409, 410, 284, 406, 276, 263, 271, 264, 353,
261, 399, and 265.
3. The method of claim 1, comprising contacting the nucleic acids
of the individual with a detection reagent, and determining which
nucleotide is present or absent at a specified SNP position.
4. The method of claim 3, in which the nucleic acids are prepared
from blood cells of the individual.
5. The method of claim 3, in which the detection reagent is a
polynucleotide probe.
6. The method of claim 5, in which the 3' end of the probe
hybridizes to the SNP in the nucleic acid.
7. The method of claim 5, in which the probe is labeled with a
reporter dye.
8. A method of detecting a single nucleotide polymorphism (SNP) in
a nucleic acid molecule, comprising contacting a test sample with a
reagent which specifically hybridizes to a SNP in any one of the
nucleotide sequences of SEQ ID NOS:1-60 and 121-410 under stringent
hybridization conditions, and detecting the formation of a
hybridized duplex.
9. The method of claim 8 in which detection is carried out by a
process selected from the group consisting of: allele-specific
probe hybridization, allele-specific primer extension,
allele-specific amplification, sequencing, 5' nuclease digestion,
molecular beacon assay, oligonucleotide ligation assay, size
analysis, and single-stranded conformation polymorphism.
10. A method of identifying an individual having an altered risk
for developing an acute coronary event or a method of evaluating an
individual's likelihood of responding to statin treatment,
comprising detecting the presence or absence of a polymorphism in
said individual's nucleic acids, wherein said polymorphism is in
linkage disequilibrium with a SNP in any one of the nucleotide
sequences of SEQ ID NOS:121-200 and SEQ ID NOS:251-410, and the
presence or absence of the polymorphism is correlated with an
individual's risk for developing an acute coronary event or
likelihood of responding to statin treatment.
11. The method of claim 10, wherein the SNP is selected from the
group of nucleotide sequences consisting of SEQ ID NOs of 356, 282,
358, 270, 256, 252, 354, 357, 306, 363, 361, 295, 285, 290, 251,
293, 294, 305, 266, 409, 410, 284, 406, 276, 263, 271, 264, 353,
261, 399, and 265.
12-27. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
non-provisional application Ser. No. 12/758,663, filed Apr. 12,
2010, which is a divisional application of U.S. non-provisional
application Ser. No. 11/526,137, filed Sep. 22, 2006 (issued as
U.S. Pat. No. 7,799,530 on Sep. 21, 2010), which claims priority to
U.S. provisional application Ser. No. 60/720,274, filed on Sep. 23,
2005, the contents of each of which are hereby incorporated by
reference in its entirety into this application.
FIELD OF THE INVENTION
[0002] The present invention is in the field of cardiovascular
disorders and drug response, particularly acute coronary events and
statin treatment of acute coronary events. In particular, the
present invention relates to specific single nucleotide
polymorphisms (SNPs) in the human genome, and their association
with acute coronary events and/or variability in responsiveness to
statin treatment (including preventive treatment) between different
individuals. The naturally occurring SNPs disclosed herein can be
used as targets for the design of diagnostic reagents and the
development of therapeutic agents, as well as for disease
association and linkage analysis. In particular, the SNPs of the
present invention are useful, for example, in identifying whether
an individual is likely to experience an acute coronary event
(either a first or recurrent acute coronary event), for predicting
the seriousness or consequences of an acute coronary event in an
individual, for determining the prognosis of an individual's
recovery from an acute coronary event, for evaluating the
likelihood of an individual's response of to statins for the
treatment/prevention of acute coronary events, for providing
clinically important information for the prevention and/or
treatment of acute coronary events, and for screening and selecting
therapeutic agents. The SNPs disclosed herein are also useful for
human identification applications. Methods, assays, kits, and
reagents for detecting the presence of these polymorphisms and
their encoded products are provided.
BACKGROUND OF THE INVENTION
[0003] Acute Coronary Events and Response to Statin Treatment
[0004] The present invention relates to SNPs that are associated
with the occurrence of cardiovascular disorders, particularly acute
coronary events such as myocardial infarction and stroke. The
present invention also relates to SNPs that are associated with
variability between different individuals in the responses to
treatment (including preventive treatments) of cardiovascular
disorders with statins (e.g., pravastatin).
[0005] Myocardial Infarction
[0006] Myocardial infarction (MI) is the most common cause of
mortality in developed countries. It is a multifactoral disease
that involves atherogenesis, thrombus formation and propagation.
Thrombosis can result in complete or partial occlusion of coronary
arteries. The luminal narrowing or blockage of coronary arteries
reduces oxygen and nutrient supply to the cardiac muscle (cardiac
ischemia), leading to myocardial necrosis and/or stunning. MI,
unstable angina, or sudden ischemic death are clinical
manifestations of cardiac muscle damage. All three endpoints are
part of the Acute Coronary Syndrome since the underlying mechanisms
of acute complications of atherosclerosis are considered to be the
same.
[0007] Atherogenesis, the first step of pathogenesis of MI, is a
complex interaction between blood elements, mechanical forces,
disturbed blood flow, and vessel wall abnormality. On the cellular
level, these include endothelial dysfunction, monocytes/macrophages
activation by modified lipoproteins, monocytes/macrophages
migration into the neointima and subsequent migration and
proliferation of vascular smooth muscle cells (VSMC) from the media
that results in plaque accumulation.
[0008] In recent years, an unstable (vulnerable) plaque was
recognized as an underlying cause of arterial thrombotic events and
MI. A vulnerable plaque is a plaque, often not stenotic, that has a
high likelihood of becoming disrupted or eroded, thus forming a
thrombogenic focus. Two vulnerable plaque morphologies have been
described. A first type of vulnerable plaque morphology is a
rupture of the protective fibrous cap. It can occur in plaques that
have distinct morphological features such as large and soft lipid
pool with distinct necrotic core and thinning of the fibrous cap in
the region of the plaque shoulders. Fibrous caps have considerable
metabolic activity. The imbalance between matrix synthesis and
matrix degradation thought to be regulated by inflammatory
mediators combined with VSMC apoptosis are the key underlying
mechanisms of plaque rupture. A second type of vulnerable plaque
morphology, known as "plaque erosion", can also lead to a fatal
coronary thrombotic event. Plaque erosion is morphologically
different from plaque rupture. Eroded plaques do not have fractures
in the plaque fibrous cap, only superficial erosion of the intima.
The loss of endothelial cells can expose the thrombogenic
subendothelial matrix that precipitates thrombus formation. This
process could be regulated by inflammatory mediators. The
propagation of the acute thrombi for both plaque rupture and plaque
erosion events depends on the balance between coagulation and
thrombolysis. MI due to a vulnerable plaque is a complex phenomenon
that includes: plaque vulnerability, blood vulnerability
(hypercoagulation, hypothrombolysis), and heart vulnerability
(sensitivity of the heart to ischemia or propensity for
arrhythmia).
[0009] Recurrent myocardial infarction (RMI) can generally be
viewed as a severe form of MI progression caused by multiple
vulnerable plaques that are able to undergo pre-rupture or a
pre-erosive state, coupled with extreme blood coagulability.
[0010] The incidence of MI is still high despite currently
available preventive measures and therapeutic intervention. More
than 1,500,000 people in the US suffer acute MI each year (many
without seeking help due to unrecognized MI), and one third of
these people die. The lifetime risk of coronary artery disease
events at age 40 years is 42.4% for men (one in two) and 24.9% for
women (one in four) (Lloyd-Jones D M; Lancet, 1999 353: 89-92).
[0011] The current diagnosis of MI is based on the levels of
troponin I or T that indicate the cardiac muscle progressive
necrosis, impaired electrocardiogram (ECG), and detection of
abnormal ventricular wall motion or angiographic data (the presence
of acute thrombi). However, due to the asymptomatic nature of 25%
of acute MIs (absence of atypical chest pain, low ECG sensitivity),
a significant portion of MIs are not diagnosed and therefore not
treated appropriately (e.g., prevention of recurrent MIs).
[0012] Despite a very high prevalence and lifetime risk of MI,
there are no good prognostic markers that can identify an
individual with a high risk of vulnerable plaques and justify
preventive treatments. MI risk assessment and prognosis is
currently done using classic risk factors or the recently
introduced Framingham Risk Index. Both of these assessments put a
significant weight on LDL levels to justify preventive treatment.
However, it is well established that half of all MIs occur in
individuals without overt hyperlipidemia. Hence, there is a need
for additional risk factors for predicting predisposition to
MI.
[0013] Other emerging risk factors are inflammatory biomarkers such
as C-reactive protein (CRP), ICAM-1, SAA, TNF .alpha.,
homocysteine, impaired fasting glucose, new lipid markers (ox LDL,
Lp-a, MAD-LDL, etc.) and pro-thrombotic factors (fibrinogen,
PAI-1). Despite showing some promise, these markers have
significant limitations such as low specificity and low positive
predictive value, and the need for multiple reference intervals to
be used for different groups of people (e.g., males-females,
smokers-non smokers, hormone replacement therapy users, different
age groups). These limitations diminish the utility of such markers
as independent prognostic markers for MI screening.
[0014] Genetics plays an important role in MI risk. Families with a
positive family history of MI account for 14% of the general
population, 72% of premature MIs, and 48% of all MIs (RR Williams,
Am J Cardiology, 2001; 87:129). In addition, replicated linkage
studies have revealed evidence of multiple regions of the genome
that are associated with MI and relevant to MI genetic traits,
including regions on chromosomes 14, 2, 3 and 7 (Broeckel U, Nature
Genetics, 2002; 30: 210; Harrap S, Arterioscler Thromb Vasc Biol,
2002; 22: 874-878, Shearman A, Human Molecular Genetics, 2000, 9;
9, 1315-1320), implying that genetic risk factors influence the
onset, manifestation, and progression of MI. Recent association
studies have identified allelic variants that are associated with
acute complications of coronary heart disease, including allelic
variants of the ApoE, ApoA5, Lpa, APOCIII, and Klotho genes.
[0015] Genetic markers such as single nucleotide polymorphisms are
preferable to other types of biomarkers. Genetic markers that are
prognostic for MI can be genotyped early in life and could predict
individual response to various risk factors. The combination of
serum protein levels and genetic predisposition revealed by genetic
analysis of susceptibility genes can provide an integrated
assessment of the interaction between genotypes and environmental
factors, resulting in synergistically increased prognostic value of
diagnostic tests.
[0016] Thus, there is an urgent need for novel genetic markers that
are predictive of predisposition to MI, particularly for
individuals who are unrecognized as having a predisposition to MI.
Such genetic markers may enable prognosis of MI in much larger
populations compared with the populations that can currently be
evaluated by using existing risk factors and biomarkers. The
availability of a genetic test may allow, for example, appropriate
preventive treatments for acute coronary events to be provided for
susceptible individuals (such preventive treatments may include,
for example, statin treatments and statin dose escalation, as well
as changes to modifiable risk factors), lowering of the thresholds
for ECG and angiography testing, and allow adequate monitoring of
informative biomarkers.
[0017] Moreover, the discovery of genetic markers associated with
MI will provide novel targets for therapeutic intervention or
preventive treatments of MI, and enable the development of new
therapeutic agents for treating MI and other cardiovascular
disorders.
[0018] Stroke
[0019] Stroke is a prevalent and serious disease. Stroke is the
most common cause of disability, the second leading cause of
dementia, and the third leading cause of mortality in the United
States. It affects 4.7 million individuals in the United States,
with 500,000 first attacks and 200,000 recurrent cases yearly.
Approximately one in four men and one in five women aged 45 years
will have a stroke if they live to their 85th year. About 25
percent of those who have a stroke die within a year. For that,
stroke is the third leading cause of mortality in the United States
and is responsible for 170,000 deaths a year. Among those who
survive the stroke attack, 30 to 50 percent do not regain
functional independence. Stroke therefore is the most common cause
of disability and the second leading cause of dementia.
[0020] Stroke occurs when an artery bringing oxygen or nutrients to
the brain either ruptures, causing the hemorrhagic type of strokes,
or gets occluded, causing the thrombotic/embolic strokes that are
collectively referred to as ischemic strokes. In each case, a
cascade of cellular changes due to ischemia or increased cranial
pressure leads to injuries or death of the brain cells. In the
United States, the majority (about 80-90%) of strokes are ischemic,
including 31% large-vessel thrombotic (also referred to as
large-vessel occlusive disease), 20% small-vessel thrombotic (also
referred to as small-vessel occlusive disease), and 32% embolic or
cardiogenic (caused by a clot originating from elsewhere in the
body, e.g., from blood pooling due to atrial fibrillation, or from
carotid artery stenosis). The ischemic form of stroke shares common
pathological etiology with atherosclerosis and thrombosis.
Approximately 10-20% of strokes are of the hemorrhagic type,
involving bleeding within or around the brain. Bleeding within the
brain is known as cerebral hemorrhage, which is often linked to
high blood pressure. Bleeding into the meninges surrounding the
brain is known as a subarachnoid hemorrhage, which could be caused
by a ruptured cerebral aneurysm, an arteriovenous malformation, or
a head injury. The hemorrhagic strokes, although less prevalent,
pose a greater danger. Whereas about 8 percent of ischemic strokes
result in death within 30 days, about 38 percent of hemorrhagic
strokes result in death within the same time period.
[0021] Known risk factors for stroke can be divided into modifiable
and non-modifiable risk factors. Older age, male sex, black or
Hispanic ethnicity, and family history of stroke are non-modifiable
risk factors. Modifiable risk factors include hypertension,
smoking, increased insulin levels, asymptomatic carotid disease,
cardiac vessel disease, and hyperlipidemia. Information derived
from the Dutch Twin Registry estimates the heritability of stroke
as 0.32 for stroke death and 0.17 for stroke hospitalization.
[0022] The acute nature of stroke leaves physicians with little
time to prevent or lessen the devastation of brain damage.
Strategies to diminish the impact of stroke include prevention and
treatment with thrombolytic and, possibly, neuroprotective agents.
The success of preventive measures will depend on the
identification of risk factors and means to modulate their
impact.
[0023] Although some risk factors for stroke are not modifiable,
such as age and family history, other underlying pathology or risk
factors of stroke such as atherosclerosis, hypertension, smoking,
diabetes, aneurysm, and atrial fibrillation, are chronic and
amenable to effective life-style, medical, and surgical treatments.
Early recognition of patients with these risk factors, and
especially those with a family history, with a non-invasive test of
genetic markers will enable physicians to target the highest risk
individuals for aggressive risk reduction.
[0024] Statin Treatment
[0025] Coronary heart disease (CHD) accounts for approximately
two-thirds of cardiovascular mortality in the United States, with
CHD accounting for 1 in every 5 deaths in 1998, which makes it the
largest single cause of morality (American Heart Association. 2001
Heart and Stroke Statistical Update. Dallas, Tex.: American Heart
Association. 2000). Stroke is the third leading cause of death,
accounting for 1 of every 15 deaths. Reduction of coronary and
cerebrovascular events and total mortality by treatment with
HMG-CoA reductase inhibitors (statins) has been demonstrated in a
number of randomized, double blinded, placebo controlled
prospective trials (Waters, D. D., What do the statin trials tell
us? Clin Cardiol, 2001. 24(8 Suppl): p. 1113-7, Singh, B. K. and J.
L. Mehta, Management of dyslipidemia in the primary prevention of
coronary heart disease. Curr Opin Cardiol, 2002. 17(5): p. 503-11).
These drugs have their primary effect through the inhibition of
hepatic cholesterol synthesis, thereby upregulating LDL receptor in
the liver. The resultant increase in LDL catabolism results in
decreased circulating LDL, a major risk factor for cardiovascular
disease. In addition, statins cause relatively small reductions in
triglyceride levels (5 to 10%) and elevations in HDL cholesterol (5
to 10%). In a 5 year primary intervention trial (WOSCOPS),
pravastatin decreased clinical events 29% compared to placebo in
hypercholesterolemic subjects, achieving a 26% reduction in
LDL-cholesterol (LDL-C) (Shepherd, J., et al., Prevention of
coronary heart disease with pravastatin in men with
hypercholesterolemia. West of Scotland Coronary Prevention Study
Group. N Engl J Med, 1995. 333(20): p. 1301-7). In a similar
primary prevention trial (AFCAPS/TexCAPS) (Downs, J. R., et al.,
Primary prevention of acute coronary events with lovastatin in men
and women with average cholesterol levels: results of
AFCAPS/TexCAPS. Air Force/Texas Coronary Atherosclerosis Prevention
Study. Jama, 1998. 279(20): p. 1615-22) in which subjects with
average cholesterol levels were treated with lovastatin, LDL-C was
reduced an average of 25% and events decreased by 37%. Secondary
prevention statin trials include the CARE (Sacks, F. M., et al.,
The effect of pravastatin on coronary events after myocardial
infarction in patients with average cholesterol levels. Cholesterol
and Recurrent Events Trial investigators. N Engl J Med, 1996.
335(14): p. 1001-9) and LIPID (treatment with pravastatin)
(Prevention of cardiovascular events and death with pravastatin in
patients with coronary heart disease and a broad range of initial
cholesterol levels. The Long-Term Intervention with pravastatin in
Ischaemic Disease (LIPID) Study Group. N Engl J Med, 1998. 339(19):
p. 1349-57), and 4S (treatment with simvastatin) (Randomised trial
of cholesterol lowering in 4444 patients with coronary heart
disease: the Scandinavian Simvastatin Survival Study (4S). Lancet,
1994. 344(8934): p. 1383-9) studies. In these trials, clinical
event risk was reduced from between 23% and 34% with achieved LDL-C
lowering ranging between 25% and 35%.
[0026] In addition to LDL-lowering, a variety of potential
non-lipid lowering effects have been suggested to play a role in
cardiovascular risk reduction by statins. These include
anti-inflammatory effects on various vascular cell types including
foam cell macrophages, improved endothelial responses, inhibition
of platelet reactivity thereby decreasing hypercoaguability, and
many others (Puddu, P., G. M. Puddu, and A. Muscari, Current
thinking in statin therapy. Acta Cardiol, 2001. 56(4): p. 225-31,
Albert, M. A., et al., Effect of statin therapy on C-reactive
protein levels: the pravastatin inflammation/CRP evaluation
(PRINCE): a randomized trial and cohort study. Jama, 2001. 286(1):
p. 64-70, Rosenson, R. S., Non-lipid-lowering effects of statins on
atherosclerosis. Curr Cardiol Rep, 1999. 1(3): p. 225-32, Dangas,
G., et al., pravastatin: an antithrombotic effect independent of
the cholesterol-lowering effect. Thromb Haemost, 2000. 83(5): p.
688-92, Crisby, M., Modulation of the inflammatory process by
statins. Drugs Today (Barc), 2003. 39(2): p. 137-43, Liao, J. K.,
Role of statin pleiotropism in acute coronary syndromes and stroke.
Int J Clin Pract Suppl, 2003 (134): p. 51-7). However, because
hypercholesterolemia is a factor in many of these additional
pathophysiologic mechanisms that are reversed by statins, many of
these statin benefits may be a consequence of LDL lowering.
[0027] Statins as a class of drug are generally well tolerated. The
most common side effects include a variety of muscle-related
complaints or myopathies. While the incidence of muscle side
effects are low, the most serious side effect, myositis with
rhabdomyolysis, is life threatening. This adverse effect has been
highlighted by the recent withdrawal of cerevastatin when the drug
was found to be associated with a relatively high level of
rhabdomyolysis-related deaths. In addition, the development of a
high dose sustained release formulation of simvastatin was
discontinued for rhabdomyolysis-related issues (Davidson, M. H., et
al., The efficacy and six-week tolerability of simvastatin 80 and
160 mg/day. Am J Cardiol, 1997. 79(1): p. 38-42).
[0028] Statins can be divided into two types according to their
physicochemical and pharmacokinetic properties. Statins such as
lovastatin, simvastatin, atorvastatin, and cerevastatin are
hydrophobic in nature and, as such, diffuse across membranes and
thus are highly cell permeable. Hydrophilic statins such as
pravastatin are more polar, such that they require specific cell
surface transporters for cellular uptake (Ziegler, K. and W.
Stunkel, Tissue-selective action of pravastatin due to
hepatocellular uptake via a sodium-independent bile acid
transporter. Biochim Biophys Acta, 1992. 1139(3): p. 203-9,
Yamazaki, M., et al., Na(+)-independent multispecific anion
transporter mediates active transport of pravastatin into rat
liver. Am J Physiol, 1993. 264(1 Pt 1): p. G36-44, Komai, T., et
al., Carrier-mediated uptake of pravastatin by rat hepatocytes in
primary culture. Biochem Pharmacol, 1992. 43(4): p. 667-70). The
latter statin utilizes a transporter, OATP2, whose tissue
distribution is confined to the liver and, therefore, they are
relatively hepato-specific inhibitors (Hsiang, B., et al., A novel
human hepatic organic anion transporting polypeptide (OATP2).
Identification of a liver-specific human organic anion transporting
polypeptide and identification of rat and human
hydroxymethylglutaryl-CoA reductase inhibitor transporters. J Biol
Chem, 1999. 274(52): p. 37161-8). The former statins, not requiring
specific transport mechanisms, are available to all cells and they
can directly impact a much broader spectrum of cells and tissues.
These differences in properties may influence the spectrum of
activities that each statin possesses. pravastatin, for instance,
has a low myopathic potential in animal models and myocyte cultures
compared to other hydrophobic statins (Masters, B. A., et al., In
vitro myotoxicity of the 3-hydroxy-3-methylglutaryl coenzyme A
reductase inhibitors, pravastatin, lovastatin, and simvastatin,
using neonatal rat skeletal myocytes. Toxicol Appl Pharmacol, 1995.
131(1): p. 163-74. Nakahara, K., et al., Myopathy induced by
HMG-CoA reductase inhibitors in rabbits: a pathological,
electrophysiological, and biochemical study. Toxicol Appl
Pharmacol, 1998. 152(1): p. 99-106, Reijneveld, J. C., et al.,
Differential effects of 3-hydroxy-3-methylglutaryl-coenzyme A
reductase inhibitors on the development of myopathy in young rats.
Pediatr Res, 1996. 39(6): p. 1028-35).
[0029] Cardiovascular mortality in developed countries has
decreased sharply in recent decades (Tunstall-Pedoe, H., et al.,
Estimation of contribution of changes in coronary care to improving
survival, event rates, and coronary heart disease mortality across
the WHO MONICA Project populations. Lancet, 2000. 355(9205): p.
688-700). This is likely due to the development and use of
efficacious hypertension, thrombolytic and lipid lowering therapies
(Kuulasmaa, K., et al., Estimation of contribution of changes in
classic risk factors to trends in coronary-event rates across the
WHO MONICA Project populations. Lancet, 2000. 355(9205): p.
675-87). Nevertheless, cardiovascular diseases remain the major
cause of death in industrialized countries, at least in part due to
the presence of highly prevalent risk factors and insufficient
treatment (Wong, M. D., et al., Contribution of major diseases to
disparities in mortality. N Engl J Med, 2002. 347(20): p. 1585-92).
Even with appropriate therapy, not all patients respond equally
well to statin treatment. Despite the overwhelming evidence that
statins decrease risk for cardiovascular disease, both in primary
and secondary intervention settings, statin therapy clearly only
achieves partial risk reduction. While a decrease in risk of 23 to
37% seen in the above trials is substantial and extremely important
clinically, the majority of events still are not prevented by
statin treatment. This is not surprising given the complexity of
cardiovascular disease etiology, which is influenced by genetics,
environment, and a variety of additional risk factors including
dyslipidemia, age, gender, hypertension, diabetes, obesity, and
smoking. It is reasonable to assume that all of these
multi-factorial risks modify statin responses and determine the
final benefit that each individual achieves from therapy.
Furthermore, with the increasing incidence of Type 2 diabetes and
obesity in Western countries (Flegal, K. M., et al., Prevalence and
trends in obesity among US adults, 1999-2000. Jama, 2002. 288(14):
p. 1723-7, Boyle, J. P., et al., Projection of diabetes burden
through 2050: impact of changing demography and disease prevalence
in the U.S. Diabetes Care, 2001. 24(11): p. 1936-40), which are two
major risk factors for coronary artery disease, and the emergence
of greater cardiovascular risk factors in the developing world
(Yusuf, S., et al., Global burden of cardiovascular diseases: Part
H: variations in cardiovascular disease by specific ethnic groups
and geographic regions and prevention strategies. Circulation,
2001. 104(23): p. 2855-64, Yusuf, S., et al., Global burden of
cardiovascular diseases: part I: general considerations, the
epidemiologic transition, risk factors, and impact of urbanization.
Circulation, 2001. 104(22): p. 2746-53), the need for ever more
effective treatment of CHD is predicted to steadily increase.
[0030] Thus, there is a growing need for ways to better identify
people who have the highest chance to benefit from statins, and
those who have the lowest risk of developing side-effects. As
indicated above, severe myopathies represent a significant risk for
a low percentage of the patient population. This would be
particularly true for patients that may be treated more
aggressively with statins in the future. There are currently at
least three studies in progress that are investigating whether
treatments aimed at lowering LDL-C to levels below current NCEP
goals by administering higher statin doses to patients further
reduces CHD risk or provides additional cardiovascular benefits
(reviewed in Clark, L. T., Treating dyslipidemia with statins: the
risk-benefit profile. Am Heart J, 2003. 145(3): p. 387-96). It is
possible that more aggressive statin therapy than is currently
standard practice will become the norm in the future if additional
benefit is observed in such trials. More aggressive statin therapy
will likely increase the incidence of the above adverse events as
well as elevate the cost of treatment. Thus, increased emphasis
will be placed on stratifying responder and non-responder patients
in order for maximum benefit-risk ratios to be achieved at the
lowest cost.
[0031] The Third Report of the Expert Panel on Detection,
Evaluation and Treatment of High Blood Cholesterol in Adults
(ATPIII) contains current recommendations for the management of
high serum cholesterol (Executive Summary of The Third Report of
The National Cholesterol Education Program (NCEP) Expert Panel on
Detection, Evaluation, And Treatment of High Blood Cholesterol In
Adults (Adult Treatment Panel III). Jama, 2001. 285(19): p.
2486-97). A meta-analysis of 38 primary and secondary prevention
trials found that for every 10% decrease in serum cholesterol, CHD
mortality was reduced by 15%. These guidelines took into account
additional risk factors beyond serum cholesterol when making
recommendations for lipid lowering strategies. After considering
additional risk factors and updated information on lipid lowering
clinical trials, more patients are classified in the highest risk
category of CHD or CHD risk equivalent than before and are
recommended to decrease their LDL to less than 100 mg/dl. As a
consequence, more aggressive therapy is recommended and drug
therapy is recommended for 36.5 million Americans. In implementing
these recommendations, cost-effectiveness of treatments is a
primary concern. In lower risk populations, the cost of reducing
one event may exceed $125,000 compared with around $25,000 per
event in a high-risk patient group (Singh, B. K. and J. L. Mehta,
Management of dyslipidemia in the primary prevention of coronary
heart disease. Curr Opin Cardiol, 2002. 17(5): p. 503-11). The cost
of preventing an event in a very low risk patient may exceed $1
million. In the context of cost-containment, further risk
stratification of patients will help to avoid unnecessary treatment
of patients. In addition to the various clinical endpoints that are
currently considered in determining overall risk, the determination
of who and who not to treat with statins based on "statin response"
genotypes could substantially increase the precision of these
determinations in the future.
[0032] Evidence from gene association studies is accumulating to
indicate that responses to drugs are, indeed, at least partly under
genetic control. As such, pharmacogenetics--the study of
variability in drug responses attributed to hereditary factors in
different populations--may significantly assist in providing
answers toward meeting this challenge (Roses, A. D.,
Pharmacogenetics and the practice of medicine. Nature, 2000.
405(6788): p. 857-65, Mooser, V., et al., Cardiovascular
pharmacogenetics in the SNP era. J Thromb Haemost, 2003. 1(7): p.
1398-1402, Humma, L. M. and S. G. Terra, Pharmacogenetics and
cardiovascular disease: impact on drug response and applications to
disease management. Am. J. Health Syst Pharm, 2002. 59(13): p.
1241-52). Numerous associations have been reported between selected
genotypes, as defined by SNPs and other sequence variations and
specific responses to cardiovascular drugs. Polymorphisms in
several genes have been suggested to influence responses to statins
including CETP (Kuivenhoven, J. A., et al., The role of a common
variant of the cholesteryl ester transfer protein gene in the
progression of coronary atherosclerosis. The Regression Growth
Evaluation Statin Study Group. N Engl J Med, 1998. 338(2): p.
86-93), beta-fibrinogen (de Maat, M. P., et al., -455G/A
polymorphism of the beta-fibrinogen gene is associated with the
progression of coronary atherosclerosis in symptomatic men:
proposed role for an acute-phase reaction pattern of fibrinogen.
REGRESS group. Arterioscler Thromb Vasc Biol, 1998. 18(2): p.
265-71), hepatic lipase (Zambon, A., et al., Common hepatic lipase
gene promoter variant determines clinical response to intensive
lipid-lowering treatment. Circulation, 2001. 103(6): p. 792-8,
lipoprotein lipase (Jukema, J. W., et al., The Asp9 Asn mutation in
the lipoprotein lipase gene is associated with increased
progression of coronary atherosclerosis. REGRESS Study Group,
Interuniversity Cardiology Institute, Utrecht, The Netherlands.
Regression Growth Evaluation Statin Study. Circulation, 1996.
94(8): p. 1913-8), glycoprotein IIIa (Bray, P. F., et al., The
platelet Pl(A2) and angiotensin-converting enzyme (ACE) D allele
polymorphisms and the risk of recurrent events after acute
myocardial infarction. Am J Cardiol, 2001. 88(4): p. 347-52),
stromelysin-1 (de Maat, M. P., et al., Effect of the stromelysin-1
promoter on efficacy of pravastatin in coronary atherosclerosis and
restenosis. Am J Cardiol, 1999. 83(6): p. 852-6), and
apolipoprotein E (Gerdes, L. U., et al., The apolipoprotein
epsilon4 allele determines prognosis and the effect on prognosis of
simvastatin in survivors of myocardial infarction: a substudy of
the Scandinavian simvastatin survival study. Circulation, 2000.
101(12): p. 1366-71, Pedro-Botet, J., et al., Apolipoprotein E
genotype affects plasma lipid response to atorvastatin in a gender
specific manner. Atherosclerosis, 2001. 158(1): p. 183-93). Some of
these variants were shown to effect clinical events while others
were associated with changes in surrogate endpoints. The CETP
variant alleles B1 and B2 were shown to be correlated with HDL
cholesterol levels. Patients with B1B1 and B1B2 genotypes have
lower HDL cholesterol and greater progression of
angiographically-determined atherosclerosis than B2B2 subjects when
on placebo during the pravastatin REGRESS clinical trial.
Furthermore, B1B1 and B1B2 had significantly less progression of
atherosclerosis when on pravastatin whereas B2B2 patients derived
no benefit. Similarly, beta-fibrinogen promoter sequence variants
were also associated with disease progression and response to
pravastatin in the same study as were Stomelysin-1 promoter
variants. In the Cholesterol and Recurrent Events (CARE) trial, a
pravastatin secondary intervention study, glycoprotein Ma variants
were also associated with clinical event response to pravastatin.
In all of the above cases, genetic subgroups of placebo-treated
patients with CHD were identified who had increased risk for major
coronary events. Treatment with pravastatin abolished the harmful
effects associated with the "riskier" genotype, while having little
effect on patients with genotypes that were associated with less
risk. Finally, the impact of the apolipoprotein .epsilon.4 genotype
on prognosis and the response to simvastatin or placebo was
investigated in the Scandanavian Simvastatin Survival Study
(Pedro-Botet, J., et al., Apolipoprotein E genotype affects plasma
lipid response to atorvastatin in a gender specific manner.
Atherosclerosis, 2001. 158(1): p. 183-93). Patients with at least
one apolipoprotein .epsilon.4 allele had a higher risk for all
cause death than those lacking the allele. As was the case with
pravastatin treatment, simvastatin reversed this detrimental effect
of the "riskier allele". These results suggest that, in general,
high-risk patients with ischemic heart disease derive the greatest
benefit from statin therapy. However, these initial observations
should be repeated in other cohorts to further support the
predictive value of these specific genotypes. Although it is likely
that additional genes beyond the five examples above impact the
final outcome of an individual's response to statins, these five
examples serve to illustrate that it is possible to identify genes
that associate with statin clinical responses that could be used to
predict which patients will benefit from statin treatment and which
will not.
[0033] SNPs
[0034] The genomes of all organisms undergo spontaneous mutation in
the course of their continuing evolution, generating variant forms
of progenitor genetic sequences (Gusella, Ann. Rev. Biochem. 55,
831-854 (1986)). A variant form may confer an evolutionary
advantage or disadvantage relative to a progenitor form or may be
neutral. In some instances, a variant form confers an evolutionary
advantage to the species and is eventually incorporated into the
DNA of many or most members of the species and effectively becomes
the progenitor form. Additionally, the effects of a variant form
may be both beneficial and detrimental, depending on the
circumstances. For example, a heterozygous sickle cell mutation
confers resistance to malaria, but a homozygous sickle cell
mutation is usually lethal. In many cases, both progenitor and
variant forms survive and co-exist in a species population. The
coexistence of multiple forms of a genetic sequence gives rise to
genetic polymorphisms, including SNPs.
[0035] Approximately 90% of all polymorphisms in the human genome
are SNPs. SNPs are single base positions in DNA at which different
alleles, or alternative nucleotides, exist in a population. The SNP
position (interchangeably referred to herein as SNP, SNP site, SNP
locus, SNP marker, or marker) is usually preceded by and followed
by highly conserved sequences of the allele (e.g., sequences that
vary in less than 1/100 or 1/1000 members of the populations). An
individual may be homozygous or heterozygous for an allele at each
SNP position. A SNP can, in some instances, be referred to as a
"cSNP" to denote that the nucleotide sequence containing the SNP is
an amino acid coding sequence.
[0036] A SNP may arise from a substitution of one nucleotide for
another at the polymorphic site. Substitutions can be transitions
or transversions. A transition is the replacement of one purine
nucleotide by another purine nucleotide, or one pyrimidine by
another pyrimidine. A transversion is the replacement of a purine
by a pyrimidine, or vice versa. A SNP may also be a single base
insertion or deletion variant referred to as an "indel" (Weber et
al., "Human diallelic insertion/deletion polymorphisms", Am J Hum
Genet 2002 October; 71(4):854-62).
[0037] A synonymous codon change, or silent mutation/SNP (terms
such as "SNP," "polymorphism," "mutation," "mutant," "variation"
and "variant" are used herein interchangeably), is one that does
not result in a change of amino acid due to the degeneracy of the
genetic code. A substitution that changes a codon coding for one
amino acid to a codon coding for a different amino acid (i.e., a
non-synonymous codon change) is referred to as a missense mutation.
A nonsense mutation results in a type of non-synonymous codon
change in which a stop codon is formed, thereby leading to
premature termination of a polypeptide chain and a truncated
protein. A read-through mutation is another type of non-synonymous
codon change that causes the destruction of a stop codon, thereby
resulting in an extended polypeptide product. While SNPs can be
bi-, tri-, or tetra-allelic, the vast majority of the SNPs are
bi-allelic, and are thus often referred to as "bi-allelic markers"
or "di-allelic markers."
[0038] As used herein, references to SNPs and SNP genotypes include
individual SNPs and/or haplotypes, which are groups of SNPs that
are generally inherited together. Haplotypes can have stronger
correlations with diseases or other phenotypic effects compared
with individual SNPs, and therefore may provide increased
diagnostic accuracy in some cases (Stephens et al. Science 293,
489-493, 20 Jul. 2001).
[0039] Causative SNPs are those SNPs that produce alterations in
gene expression or in the expression, structure, and/or function of
a gene product, and therefore are most predictive of a possible
clinical phenotype. One such class includes SNPs falling within
regions of genes encoding a polypeptide product, i.e. cSNPs. These
SNPs may result in an alteration of the amino acid sequence of the
polypeptide product (i.e., non-synonymous codon changes) and give
rise to the expression of a defective or other variant protein.
Furthermore, in the case of nonsense mutations, a SNP may lead to
premature termination of a polypeptide product. Such variant
products can result in a pathological condition, e.g., genetic
disease. Examples of genes in which a SNP within a coding sequence
causes a genetic disease include sickle cell anemia and cystic
fibrosis.
[0040] Causative SNPs do not necessarily have to occur in coding
regions; causative SNPs can occur in, for example, any genetic
region that can ultimately affect the expression, structure, and/or
activity of the protein encoded by a nucleic acid. Such genetic
regions include, for example, those involved in transcription, such
as SNPs in transcription factor binding domains, SNPs in promoter
regions, in areas involved in transcript processing, such as SNPs
at intron-exon boundaries that may cause defective splicing, or
SNPs in mRNA processing signal sequences such as polyadenylation
signal regions. Some SNPs that are not causative SNPs nevertheless
are in close association with, and therefore segregate with, a
disease-causing sequence. In this situation, the presence of a SNP
correlates with the presence of, or predisposition to, or an
increased risk in developing the disease. These SNPs, although not
causative, are nonetheless also useful for diagnostics, disease
predisposition screening, and other uses.
[0041] An association study of a SNP and a specific disorder
involves determining the presence or frequency of the SNP allele in
biological samples from individuals with the disorder of interest,
such as those individuals who respond to statin treatment
("responders") or those individuals who do not respond to statin
treatment ("non-responders"), and comparing the information to that
of controls (i.e., individuals who do not have the disorder;
controls may be also referred to as "healthy" or "normal"
individuals) who are preferably of similar age and race. The
appropriate selection of patients and controls is important to the
success of SNP association studies. Therefore, a pool of
individuals with well-characterized phenotypes is extremely
desirable.
[0042] A SNP may be screened in diseased tissue samples or any
biological sample obtained from a diseased individual, and compared
to control samples, and selected for its increased (or decreased)
occurrence in a specific phenotype, such as such as response or
non-response to statin treatment of cardiovascular disease. Once a
statistically significant association is established between one or
more SNP(s) and a pathological condition (or other phenotype) of
interest, then the region around the SNP can optionally be
thoroughly screened to identify the causative genetic
locus/sequence(s) (e.g., causative SNP/mutation, gene, regulatory
region, etc.) that influences the pathological condition or
phenotype. Association studies may be conducted within the general
population and are not limited to studies performed on related
individuals in affected families (linkage studies).
[0043] Clinical trials have shown that patient response to
treatment with pharmaceuticals is often heterogeneous. There is a
continuing need to improve pharmaceutical agent design and therapy.
In that regard, SNPs can be used to identify patients most suited
to therapy with particular pharmaceutical agents (this is often
termed "pharmacogenomics"). Similarly, SNPs can be used to exclude
patients from certain treatment due to the patient's increased
likelihood of developing toxic side effects or their likelihood of
not responding to the treatment. Pharmacogenomics can also be used
in pharmaceutical research to assist the drug development and
selection process. (Linder et al. (1997), Clinical Chemistry, 43,
254; Marshall (1997), Nature Biotechnology, 15, 1249; International
Patent Application WO 97/40462, Spectra Biomedical; and Schafer et
al. (1998), Nature Biotechnology, 16, 3).
SUMMARY OF THE INVENTION
[0044] The present invention relates to the identification of novel
SNPs, unique combinations of such SNPs, and haplotypes of SNPs that
are associated with cardiovascular disorders and/or drug response,
particularly acute coronary events (e.g., myocardial infarction)
and response to statins for the treatment (including preventive
treatment) of cardiovascular disorders such as acute coronary
events. The polymorphisms disclosed herein are directly useful as
targets for the design of diagnostic reagents and the development
of therapeutic agents for use in the diagnosis and treatment of
cardiovascular disorders and related pathologies, particularly
acute coronary events.
[0045] Based on the identification of SNPs associated with
cardiovascular disorders, particular acute coronary events, and/or
response to statin treatment, the present invention also provides
methods of detecting these variants as well as the design and
preparation of detection reagents needed to accomplish this task.
The invention specifically provides, for example, novel SNPs in
genetic sequences involved in cardiovascular disorders and/or
responsiveness to statin treatment, isolated nucleic acid molecules
(including, for example, DNA and RNA molecules) containing these
SNPs, variant proteins encoded by nucleic acid molecules containing
such SNPs, antibodies to the encoded variant proteins,
computer-based and data storage systems containing the novel SNP
information, methods of detecting these SNPs in a test sample,
methods of determining the risk of an individual of experiencing a
first or recurring acute coronary event, methods for prognosing the
severity or consequences of the acute coronary event, methods of
treating an individual who has an increased risk of experiencing an
acute coronary event, methods of identifying individuals who have
an altered (i.e., increased or decreased) likelihood of responding
to statin treatment of a cardiovascular disorder (e.g., an acute
coronary event) based on the presence or absence of one or more
particular nucleotides (alleles) at one or more SNP sites disclosed
herein or the detection of one or more encoded variant products
(e.g., variant mRNA transcripts or variant proteins), methods of
identifying individuals who are more or less likely to respond to a
treatment, particularly statin treatment of a cardiovascular
disorder such as an acute coronary event (or more or less likely to
experience undesirable side effects from a treatment, etc.),
methods of screening for compounds useful in the treatment of a
disorder associated with a variant gene/protein, compounds
identified by these methods, methods of treating disorders mediated
by a variant gene/protein, methods of using the novel SNPs of the
present invention for human identification, etc. The present
invention also provides methods for identifying individuals who
possess SNPs that are associated with an increased risk of
developing an acute coronary event such as MI, and yet can benefit
from being treated with statin because statin treatment can lower
their risk of developing an acute coronary event such as MI.
[0046] Since cardiovascular disorders/diseases share certain
similar features that may be due to common genetic factors that are
involved in their underlying mechanisms, the SNPs identified herein
as being particularly associated with acute coronary events and/or
statin response may be used as diagnostic/prognostic markers or
therapeutic targets for a broad spectrum of cardiovascular diseases
such as coronary heart disease (CHD), atherosclerosis,
cerebrovascular disease, congestive heart failure, congenital heart
disease, and pathologies and symptoms associated with various heart
diseases (e.g., angina, hypertension), as well as for predicting
responses to drugs other than statins that are used to treat
cardiovascular diseases.
[0047] The present invention further provides methods for selecting
or formulating a treatment regimen (e.g., methods for determining
whether or not to administer statin treatment to an individual
having cardiovascular disease, methods for selecting a particular
statin-based treatment regimen such as dosage and frequency of
administration of statin, or a particular form/type of statin such
as a particular pharmaceutical formulation or compound, methods for
administering an alternative, non-statin-based treatment to
individuals who are predicted to be unlikely to respond positively
to statin treatment, etc.), and methods for determining the
likelihood of experiencing toxicity or other undesirable side
effects from statin treatment, etc. The present invention also
provides methods for selecting individuals to whom a statin or
other therapeutic will be administered based on the individual's
genotype, and methods for selecting individuals for a clinical
trial of a statin or other therapeutic agent based on the genotypes
of the individuals (e.g., selecting individuals to participate in
the trial who are most likely to respond positively from the statin
treatment). The present invention further provides methods for
reducing the risk of an individual in developing cardiovascular
disorders such as MI or RMI, using statin treatment, when said
individual carries one or more SNPs identified herein as being
associated with such cardiovascular disorders.
[0048] In Tables 1-2, the present invention provides gene
information, transcript sequences (SEQ ID NOS:1-60), encoded amino
acid sequences (SEQ ID NOS:61-120), genomic sequences (SEQ ID
NOS:201-250), transcript-based context sequences (SEQ ID
NOS:121-200) and genomic-based context sequences (SEQ ID
NOS:251-410) that contain the SNPs of the present invention, and
extensive SNP information that includes observed alleles, allele
frequencies, populations/ethnic groups in which alleles have been
observed, information about the type of SNP and corresponding
functional effect, and, for cSNPs, information about the encoded
polypeptide product. The transcript sequences (SEQ ID NOS:1-60),
amino acid sequences (SEQ ID NOS:61-120), genomic sequences (SEQ ID
NOS:201-250), transcript-based SNP context sequences (SEQ ID
NOS:121-200), and genomic-based SNP context sequences (SEQ ID
NOS:251-410) are also provided in the Sequence Listing.
[0049] In a specific embodiment of the present invention, SNPs that
occur naturally in the human genome are provided as isolated
nucleic acid molecules. These SNPs are associated with
cardiovascular disorders, particular acute coronary events, and/or
response to statin treatment, such that they can have a variety of
uses in the diagnosis and/or treatment of cardiovascular disorders
and related pathologies and particularly in the treatment of
cardiovascular disorders with statins. One aspect of the present
invention relates to an isolated nucleic acid molecule comprising a
nucleotide sequence in which at least one nucleotide is a SNP
disclosed in Tables 3 and/or 4. In an alternative embodiment, a
nucleic acid of the invention is an amplified polynucleotide, which
is produced by amplification of a SNP-containing nucleic acid
template. In another embodiment, the invention provides for a
variant protein which is encoded by a nucleic acid molecule
containing a SNP disclosed herein.
[0050] In yet another embodiment of the invention, a reagent for
detecting a SNP in the context of its naturally-occurring flanking
nucleotide sequences (which can be, e.g., either DNA or mRNA) is
provided. In particular, such a reagent may be in the form of, for
example, a hybridization probe or an amplification primer that is
useful in the specific detection of a SNP of interest. In an
alternative embodiment, a protein detection reagent is used to
detect a variant protein that is encoded by a nucleic acid molecule
containing a SNP disclosed herein. A preferred embodiment of a
protein detection reagent is an antibody or an antigen-reactive
antibody fragment.
[0051] Various embodiments of the invention also provide kits
comprising SNP detection reagents, and methods for detecting the
SNPs disclosed herein by employing detection reagents. In a
specific embodiment, the present invention provides for a method of
identifying an individual having an increased or decreased risk of
developing a cardiovascular disorder (e.g. experiencing an acute
coronary event) by detecting the presence or absence of one or more
SNP alleles disclosed herein. The present invention also provides
methods for evaluating whether an individual is likely (or
unlikely) to respond to statin treatment of cardiovascular disease
by detecting the presence or absence of one or more SNP alleles
disclosed herein.
[0052] The nucleic acid molecules of the invention can be inserted
in an expression vector, such as to produce a variant protein in a
host cell. Thus, the present invention also provides for a vector
comprising a SNP-containing nucleic acid molecule,
genetically-engineered host cells containing the vector, and
methods for expressing a recombinant variant protein using such
host cells. In another specific embodiment, the host cells,
SNP-containing nucleic acid molecules, and/or variant proteins can
be used as targets in a method for screening and identifying
therapeutic agents or pharmaceutical compounds useful in the
treatment of cardiovascular diseases.
[0053] An aspect of this invention is a method for treating
cardiovascular disorders, particular acute coronary events, in a
human subject wherein said human subject harbors a SNP, gene,
transcript, and/or encoded protein identified in Tables 1-2, which
method comprises administering to said human subject a
therapeutically or prophylactically effective amount of one or more
agents (e.g. statins) counteracting the effects of the disorder,
such as by inhibiting (or stimulating) the activity of the gene,
transcript, and/or encoded protein identified in Tables 1-2.
[0054] Another aspect of this invention is a method for identifying
an agent useful in therapeutically or prophylactically treating
cardiovascular disorders, particular acute coronary events, in a
human subject wherein said human subject harbors a SNP, gene,
transcript, and/or encoded protein identified in Tables 1-2, which
method comprises contacting the gene, transcript, or encoded
protein with a candidate agent (e.g., statin) under conditions
suitable to allow formation of a binding complex between the gene,
transcript, or encoded protein and the candidate agent (such as a
statin) and detecting the formation of the binding complex, wherein
the presence of the complex identifies said agent.
[0055] Another aspect of this invention is a method for treating a
cardiovascular disorder in a human subject, which method
comprises:
[0056] (i) determining that said human subject harbors a SNP, gene,
transcript, and/or encoded protein identified in Tables 1-2,
and
[0057] (ii) administering to said subject a therapeutically or
prophylactically effective amount of one or more agents (such as a
statin) counteracting the effects of the disease.
[0058] Many other uses and advantages of the present invention will
be apparent to those skilled in the art upon review of the detailed
description of the preferred embodiments herein. Solely for clarity
of discussion, the invention is described in the sections below by
way of non-limiting examples.
DESCRIPTION OF THE FILES CONTAINED ON THE CD-R NAMED CD000002ORD
CDR
TABLE-US-LTS-CD-00001 LENGTHY TABLES The patent application
contains a lengthy table section. A copy of the table is available
in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150111775A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
[0059] The CD-R named CD000002ORD CDR contains the following five
text (ASCII) files:
[0060] 1) File SEQLIST_CD000002ORD.txt provides the Sequence
Listing. The Sequence Listing provides the transcript sequences
(SEQ ID NOS:1-60) and protein sequences (SEQ ID NOS:61-120) as
shown in Table 1, and genomic sequences (SEQ ID NOS:201-250) as
shown in Table 2, for each gene that contains one or more SNPs of
the present invention. Also provided in the Sequence Listing are
context sequences flanking each SNP, including both
transcript-based context sequences as shown in Table 1 (SEQ ID
NOS:121-200) and genomic-based context sequences as shown in Table
2 (SEQ ID NOS:251-410). The context sequences generally provide 100
bp upstream (5') and 100 bp downstream (3') of each SNP, with the
SNP in the middle of the context sequence, for a total of 200 bp of
context sequence surrounding each SNP. File SEQLIST_CD000002ORD.txt
is 1,451 KB in size, and was created on Sep. 21, 2006.
[0061] 2) File TABLE1_CD000002ORD.txt provides Table 1. File
TABLE1_CD000002ORD.txt is 96 KB in size, and was created on Sep.
21, 2006.
[0062] 3) File TABLE2_CD000002ORD.txt provides Table 2. File
TABLE2_CD000002ORD.txt is 122 KB in size, and was created on Sep.
21, 2006.
[0063] 4) File TABLE3_CD000002ORD.txt provides Table 3. File
TABLE3_CD000002ORD.txt is 1 KB in size, and was created on Sep. 21,
2006.
[0064] 5) File TABLE4_CD000002ORD.txt provides Table 4. File
TABLE4_CD000002ORD.txt is 1 KB in size, and was created on Sep. 21,
2006.
The material contained on the CD-R labeled CD000002ORDCDR is hereby
incorporated by reference pursuant to 37 CFR 1.77(b)(4).
DESCRIPTION OF TABLE 1 AND TABLE 2
[0065] Table 1 and Table 2 (both provided on the CD-R) disclose the
SNPs and associated gene/transcript/protein information of the
present invention. For each gene, Table 1 and Table 2 each provide
a header containing gene/transcript/protein information, followed
by a transcript and protein sequence (in Table 1) or genomic
sequence (in Table 2), and then SNP information regarding each SNP
found in that gene/transcript.
[0066] NOTE: SNPs may be included in both Table 1 and Table 2;
Table 1 presents the SNPs relative to their transcript sequences
and encoded protein sequences, whereas Table 2 presents the SNPs
relative to their genomic sequences (in some instances Table 2 may
also include, after the last gene sequence, genomic sequences of
one or more intergenic regions, as well as SNP context sequences
and other SNP information for any SNPs that lie within these
intergenic regions). SNPs can readily be cross-referenced between
Tables based on their hCV (or, in some instances, hDV)
identification numbers.
[0067] The gene/transcript/protein information includes: [0068] a
gene number (1 through n, where n=the total number of genes in the
Table) [0069] a Celera hCG and UID internal identification numbers
for the gene [0070] a Celera hCT and UID internal identification
numbers for the transcript (Table 1 only) [0071] a public Genbank
accession number (e.g., RefSeq NM number) for the transcript (Table
1 only) [0072] a Celera hCP and UID internal identification numbers
for the protein encoded by the hCT transcript (Table 1 only) [0073]
a public Genbank accession number (e.g., RefSeq NP number) for the
protein (Table 1 only) [0074] an art-known gene symbol [0075] an
art-known gene/protein name [0076] Celera genomic axis position
(indicating start nucleotide position-stop nucleotide position)
[0077] the number of the chromosome on which the gene is located
[0078] an OMIM (Online Mendelian Inheritance in Man; Johns Hopkins
University/NCBI) public reference number for obtaining further
information regarding the medical significance of each gene [0079]
alternative gene/protein name(s) and/or symbol(s) in the OMIM
entry.
[0080] NOTE: Due to the presence of alternative splice forms,
multiple transcript/protein entries can be provided for a single
gene entry in Table 1; i.e., for a single Gene Number, multiple
entries may be provided in series that differ in their
transcript/protein information and sequences.
[0081] Following the gene/transcript/protein information is a
transcript sequence and protein sequence (in Table 1), or a genomic
sequence (in Table 2), for each gene, as follows: [0082] transcript
sequence (Table 1 only) (corresponding to SEQ ID NOS:1-60 of the
Sequence Listing), with SNPs identified by their IUB codes
(transcript sequences can include 5' UTR, protein coding, and 3'
UTR regions)
[0083] NOTE: If there are differences between the nucleotide
sequence of the hCT transcript and the corresponding public
transcript sequence identified by the Genbank accession number, the
hCT transcript sequence (and encoded protein) is provided, unless
the public sequence is a RefSeq transcript sequence identified by
an NM number, in which case the RefSeq NM transcript sequence (and
encoded protein) is provided. However, whether the hCT transcript
or RefSeq NM transcript is used as the transcript sequence, the
disclosed SNPs are represented by their IUB codes within the
transcript. [0084] the encoded protein sequence (Table 1 only;
corresponding to SEQ ID NOS:61-120 of the Sequence Listing) [0085]
the genomic sequence of the gene (Table 2 only), including 6 kb on
each side of the gene boundaries (i.e., 6 kb on the 5' side of the
gene plus 6 kb on the 3' side of the gene; corresponding to SEQ ID
NOS:201-250 of the Sequence Listing).
[0086] After the last gene sequence, Table 2 may include additional
genomic sequences of intergenic regions (in such instances, these
sequences are identified as "Intergenic region:" followed by a
numerical identification number), as well as SNP context sequences
and other SNP information for any SNPs that lie within each
intergenic region (and such SNPs are identified as "INTERGENIC" for
SNP type).
[0087] NOTE: The transcript, protein, and transcript-based SNP
context sequences are provided in both Table 1 and in the Sequence
Listing. The genomic and genomic-based SNP context sequences are
provided in both Table 2 and in the Sequence Listing. SEQ ID NOS
are indicated in Table 1 for each transcript sequence (SEQ ID
NOS:1-60), protein sequence (SEQ ID NOS:61-120), and
transcript-based SNP context sequence (SEQ ID NOS:121-200), and SEQ
ID NOS are indicated in Table 2 for each genomic sequence (SEQ ID
NOS:201-250), and genomic-based SNP context sequence (SEQ ID
NOS:251-410).
[0088] The SNP information includes: [0089] context sequence (taken
from the transcript sequence in Table 1, and the genomic sequence
in Table 2) with the SNP represented by its IUB code, including 100
base pairs upstream (5') of the SNP position plus 100 base pairs
downstream (3') of the SNP position (the transcript-based SNP
context sequences in Table 1 are provided in the Sequence Listing
as SEQ ID NOS:121-200; the genomic-based SNP context sequences in
Table 2 are provided in the Sequence Listing as SEQ ID NOS:251-410)
[0090] Celera hCV internal identification number for the SNP (in
some instances, an "hDV" number is given instead of an "hCV"
number) [0091] SNP position (position of the SNP within the given
transcript sequence [Table 1] or within the given genomic sequence
[Table 2]) [0092] SNP source (may include any combination of one or
more of the following five codes, depending on which internal
sequencing projects and/or public databases the SNP has been
observed in: "Applera"=SNP observed during the re-sequencing of
genes and regulatory regions of 39 individuals, "Celera"=SNP
observed during shotgun sequencing and assembly of the Celera human
genome sequence, "Celera Diagnostics"=SNP observed during
re-sequencing of nucleic acid samples from individuals who have
cardiovascular disorders [e.g., experienced an acute coronary
event], and/or have undergone statin treatment, "dbSNP"=SNP
observed in the dbSNP public database, "HGBASE"=SNP observed in the
HGBASE public database, "HGMD"=SNP observed in the Human Gene
Mutation Database [HGMD] public database, "HapMap"=SNP observed in
the International HapMap Project public database, "CSNP"=SNP
observed in an internal Applied Biosystems [Foster City, Calif.]
database of coding SNPS [cSNPs])
[0093] NOTE: Multiple "Applera" source entries for a single SNP
indicate that the same SNP was covered by multiple overlapping
amplification products and the re-sequencing results (e.g.,
observed allele counts) from each of these amplification products
is being provided. [0094] population/allele/allele count
information in the format of
[population1(first_allele,count|second_allele,count)population2(first_all-
ele,count|second_allele,count) total (first_allele,total
count|second_allele,total count)]
[0095] NOTE: The information in this field includes
populations/ethnic groups in which particular SNP alleles have been
observed ("cau"=Caucasian, "his"=Hispanic, "chn"=Chinese, and
"afr"=African-American, "jpn"=Japanese, "ind"=Indian,
"mex"=Mexican, "ain"="American Indian, "cra"=Celera donor,
"no_pop"=no population information available), identified SNP
alleles, and observed allele counts (within each population group
and total allele counts), where available ("-" in the allele field
represents a deletion allele of an insertion/deletion ["indel"]
polymorphism, in which case the corresponding insertion allele,
which may be comprised of one or more nucleotides, is indicated in
the allele field on the opposite side of the "|"; "-" in the count
field indicates that allele count information is not
available).
[0096] NOTE: For SNPs of "Applera" SNP source, genes/regulatory
regions of 39 individuals (20 Caucasians and 19 African Americans)
were re-sequenced and, since each SNP position is represented by
two chromosomes in each individual (with the exception of SNPs on X
and Y chromosomes in males, for which each SNP position is
represented by a single chromosome), up to 78 chromosomes were
genotyped for each SNP position. Thus, the sum of the
African-American ("afr") allele counts is up to 38, the sum of the
Caucasian allele counts ("cau") is up to 40, and the total sum of
all allele counts is up to 78.
[0097] NOTE: Semicolons separate population/allele/count
information corresponding to each indicated SNP source; i.e., if
four SNP sources are indicated, such as "Celera", "dbSNP",
"HGBASE", and "HGMD", then population/allele/count information is
provided in four groups which are separated by semicolons and
listed in the same order as the listing of SNP sources, with each
population/allele/count information group corresponding to the
respective SNP source based on order; thus, in this example, the
first population/allele/count information group would correspond to
the first listed SNP source (Celera) and the third
population/allele/count information group separated by semicolons
would correspond to the third listed SNP source (HGBASE); if
population/allele/count information is not available for any
particular SNP source, then a pair of semicolons is still inserted
as a place-holder in order to maintain correspondence between the
list of SNP sources and the corresponding listing of
population/allele/count information. [0098] SNP type (e.g.,
location within gene/transcript and/or predicted functional effect)
["MISSENSE MUTATION"=SNP causes a change in the encoded amino acid
(i.e., a non-synonymous coding SNP); "SILENT MUTATION"=SNP does not
cause a change in the encoded amino acid (i.e., a synonymous coding
SNP); "STOP CODON MUTATION"=SNP is located in a stop codon;
"NONSENSE MUTATION"=SNP creates or destroys a stop codon; "UTR
5"=SNP is located in a 5' UTR of a transcript; "UTR 3"=SNP is
located in a 3' UTR of a transcript; "PUTATIVE UTR 5"=SNP is
located in a putative 5' UTR; "PUTATIVE UTR 3"=SNP is located in a
putative 3' UTR; "DONOR SPLICE SITE"=SNP is located in a donor
splice site (5' intron boundary); "ACCEPTOR SPLICE SITE"=SNP is
located in an acceptor splice site (3' intron boundary); "CODING
REGION"=SNP is located in a protein-coding region of the
transcript; "EXON"=SNP is located in an exon; "INTRON"=SNP is
located in an intron; "hmCS"=SNP is located in a human-mouse
conserved segment; "TFBS"=SNP is located in a transcription factor
binding site; "UNKNOWN"=SNP type is not defined; "INTERGENIC"=SNP
is intergenic, i.e., outside of any gene boundary] [0099] protein
coding information (Table 1 only) where relevant, in the format of
[protein SEQ ID NO:#, amino acid position, (amino acid-1, codon1)
(amino acid-2, codon2)]
[0100] NOTE: The information in this field includes the SEQ ID NO
of the encoded protein sequence, position of the amino acid residue
within the protein identified by the SEQ ID NO that is encoded by
the codon containing the SNP, amino acids (represented by
one-letter amino acid codes) that are encoded by the alternative
SNP alleles (in the case of stop codons, "X" is used for the
one-letter amino acid code), and alternative codons containing the
alternative SNP nucleotides which encode the amino acid residues
(thus, for example, for missense mutation-type SNPs, at least two
different amino acids and at least two different codons are
generally indicated; for silent mutation-type SNPs, one amino acid
and at least two different codons are generally indicated, etc.).
In instances where the SNP is located outside of a protein-coding
region (e.g., in a UTR region), "None" is indicated following the
protein SEQ ID NO.
DESCRIPTION OF TABLE 3 AND TABLE 4
[0101] Tables 3 and 4 (both provided on the CD-R) provide a list of
a subset of SNPs from Table 1 (in the case of Table 3) or Table 2
(in the case of Table 4) for which the SNP source falls into one of
the following three categories: 1) SNPs for which the SNP source is
only "Applera" and none other, 2) SNPs for which the SNP source is
only "Celera Diagnostics" and none other, and 3) SNPs for which the
SNP source is both "Applera" and "Celera Diagnostics" but none
other.
[0102] These SNPs have not been observed in any of the public
databases (dbSNP, HGBASE, and HGMD), and were also not observed
during shotgun sequencing and assembly of the Celera human genome
sequence (i.e., "Celera" SNP source). Tables 3 and 4 provide the
hCV identification number (or hDV identification number for SNPs
having "Celera Diagnostics" SNP source) and the SEQ ID NO of the
context sequence for each of these SNPs.
DESCRIPTION OF TABLES 5-21
[0103] Tables 5-21 provide the allelic frequencies and results of
statistical analyses for SNPs disclosed in Tables 1-4 (SNPs can be
cross-referenced between tables based on their hCV identification
numbers). The results shown in Tables 5-21 provide support for the
association of these SNPs with cardiovascular disorders,
particularly acute coronary events such as myocardial
infarction/recurrent MI, and/or the association of these SNPs with
drug response, such as statins administered as a preventive
treatment for acute coronary events. As one example, the
statistical results in Tables 5-21 show that the association of
these SNPs with risk of MI/RMI and/or response to statin treatment
in the prevention of MI is supported by P values <0.05 in
genotypic association tests.
[0104] Tables 5-10, 17 and 18 present statistics from the analysis
of the genetic polymorphism hCV3054799 in gene c6orf102. Table 5
lists the number of case and control samples genotyped in the CARE
and WOSCOPS studies, and the number of patients in each treatment
arm, placebo and pravastatin. Also shown in this table are the
frequencies of the major and minor alleles (i.e., higher and lower
frequency alleles, respectively). Table 6 shows the genotypic
frequencies for hCV3054799 in the CARE and WOSCOPS studies, while
Table 7 lists the number of cases and controls of combined
genotypes (major homozygous plus heterozygous, minor homozygous
plus heterozygous) that are used in calculating dominant and
recessive modes of association with MI risk.
[0105] The association of SNP hCV3054799 with MI risk and response
to statin treatment was evaluated using the allelic/genotypic
frequencies from the counts obtained in Tables 5-7. Table 8
presents statistical data for the association of hCV3054799 with MI
risk, in two replicated studies (in the case of the CARE study
patients, recurrent MI specifically). Because this SNP is also
associated with a positive response to statin treatment (see Table
10), statistics in Table 8 are presented for patients stratified by
treatment group (placebo vs. pravastatin), as well as for all
patients unstratified. The risk estimates in Table 8 are not
adjusted for conventional MI risk factors such as age, sex, and
smoking/non-smoking status.
[0106] Table 9 presents the MI risk estimates for placebo-treated
individuals that are heterozygous or homozygous for the rare
(minor) allele of SNP hCV3054799. Odds ratios for the minor
homozygous patients (Min hom) are derived from a comparison of the
risk estimate for minor with major homozygote carriers; odds ratios
for the heterozygous (Het) are derived from a comparison of the
risk estimate for heterozygote with major homozygote carriers. In
the CARE patients, risk estimates are justified by a stepwise
logistic regression analysis, which serves to adjust for
conventional risk factors such as age, sex, smoking/non-smoking
status, and baseline LDL and HDL. In the WOSCOPS patients, logistic
regression was done to adjust for baseline LDL and HDL. No
adjustment was done for the sex stratum in WOSCOPS because all
patients were male; likewise, no adjustment was done for smoking
status or age, because cases and controls in WOSCOPS were matched
for these parameters. This adjustment confirms the association of
hCV3054799 with MI risk, independent of conventional risk
factors.
[0107] Table 10 presents the statistical association of hCV3054799
with prevention of MI by statin treatment (i.e., response to
pravastatin as an MI-preventive treatment) in two replicated
studies. (In the CARE study, association of statins with prevention
of recurrent MI is presented.) Also shown are the risk estimates of
this SNP for MI/RMI in the placebo-treated patient group (data also
shown in Table 8).
[0108] Comparing Tables 8-10 shows that this c6orf102 SNP is
associated with MI risk AND response to statin treatment.
Individuals in the placebo groups in both the CARE and WOSCOPS
studies, who were heterozygous or homozygous for the minor allele,
had a significantly higher risk of experiencing an MI than
individuals who were homozygous for the major allele. Importantly,
these statistics indicate that CARE individuals who were
heterozygous or minor-allele homozygous were also significantly
protected by pravastatin treatment against an adverse coronary
event, relative to those individuals homozygous for the major
allele. Tables 17 and 18 provide further analysis showing the
association of hCV3054799 with MI risk and its association with
prevention of MI by statin treatment.
[0109] Tables 11-13 provide genotypic counts for other significant
polymorphisms identified in the CARE study, by treatment arm.
Tables 14 and 15 demonstrate the statistical associations of these
SNPs with RMI risk, in placebo-treated patients (Table 14) and in
all patients, unstratified by treatment arm (Table 15).
[0110] Table 16 shows significant markers from the CARE study and
the statistical associations of their genotypes with RMI prevention
by means of statin treatment (i.e., response to pravastatin as an
MI-preventive treatment). Results are justified as a significant
risk difference between placebo- and statin-treated groups, where
the Breslow Day P values are <0.05 for each SNP. Tables 19 and
20 provide additional analysis showing that individuals carrying
the markers listed in tables 19 and 20 can have their risk of
developing MI/RMI reduced by statin treatment, given that those
markers are MI/RMI-associated markers as shown in Table 14. There
are six markers in table 20.
[0111] Table 21 provides a list of the sample LD SNPs that are
related to and derived from an interrogated SNP. These LD SNPs are
provided as an example of the groups of SNPs which can also serve
as markers for disease association based on their being in LD with
the interrogated SNP. The criteria and process of selecting such LD
SNPs, including the calculation of the r.sup.2 value and the
r.sup.2 threshold value, are described in Example 3, below.
[0112] In Table 21, the column labeled "Interrogated SNP" presents
each marker as identified by its unique identifier, the hCV number.
The column labeled "Interrogated SNP rs" presents the publicly
known identifier rs number for the corresponding hCV number. The
column labeled "LD SNP" presents the hCV numbers of the LD SNPs
that are derived from their corresponding interrogated SNPs. The
column labeled "LD SNP rs" presents the publicly known rs number
for the corresponding hCV number. The column labeled "Power"
presents the level of power where the r.sup.2 threshold is set. For
example, when power is set at 51%, the threshold r.sup.2 value
calculated therefrom is the minimum r.sup.2 that an LD SNP must
have in reference to an interrogated SNP, in order for the LD SNP
to be classified as a marker capable of being associated with a
disease phenotype at greater than 51% probability. The column
labeled "Threshold r.sub.T.sup.2" presents the minimum value of
r.sup.2 that an LD SNP must meet in reference to an interrogated
SNP in order to qualify as an LD SNP. The column labeled "r.sup.2"
presents the actual r.sup.2 value of the LD SNP in reference to the
interrogated SNP to which it is related.
[0113] NOTE: SNPs can be cross-referenced between Tables 1-21 based
on the identification number (hCV number) provided for each
SNP.
[0114] Follows are descriptions of the column headings used in the
various Tables presented herein.
TABLE-US-00001 TABLES 5-9 Column Heading Definition Study CARE =
Cholesterol and Recurrent Events. WOSCOPS = West of Scotland
Coronary Prevention Study. Stratum Subpopulation used for analysis
(placebo- or pravastatin-treated, or all patients, unstratified by
treatment). All Count, Number of individuals analyzed in Case
Count, Cont each study, separated into Case and Control Count
groups according to MI status, or ALL samples combined. Allele
Particular SNP variant investigated. Case Freq, Total number of
chromosomes with Cont Freq each allele in each study, divided by
twice the total number of individuals tested (i.e., two alleles per
individual) for Cases or Controls. Major Allele Major = high
frequency allele. Minor Allele Minor = rare (low frequency) allele.
Genot Diploid genotypes present for this SNP. Major homozygous
Carriers of 0 rare alleles. Minor homozygous Carriers of 2 rare
alleles. Heterozygous Carriers of 1 rare allele. Maj hom + Het All
carriers of 0 or 1 rare allele. Het + Min hom All carriers of 1 or
2 rare alleles. Heterozygous Odds ratio of an MI event for a
carrier with 1 rare Carriers (Het vs. allele, using carriers with 0
rare alleles as a reference. Maj hom) OR ("95% CI" is the
confidence interval.) Dominant Odds ratio of an MI event for a
carrier with 1 or 2 rare ([Het + Min hom] alleles, using carriers
with 0 rare alleles as a reference. vs. Maj hom) OR ("95% CI" is
the confidence interval.) Recessive (Min OR of an MI event for a
carrier with 2 rare alleles, hom vs. [Maj using carriers with 0
rare alleles as a reference. hom + Het]) ("95% CI" is the
confidence interval.) OR Allelic Odds ratio of an MI event in those
OR patients with genotypes possessing the rare allele vs. patients
with no rare allele.
TABLE-US-00002 TABLE 10 Column Heading Description MI/RMI risk
estimation Odds ratio of an MI event with a (Het vs. Maj hom)
genotype containing one rare OR allele vs. odds of MI in a genotype
with no rare alleles. (Data from placebo-treated patients only.)
Risk Reduction by Statin Odds ratio of an MI event in
pravastatin-treated (Heterozygous carriers) vs. placebo-treated
patients. OR (Data from heterozygous carriers only.)
TABLE-US-00003 TABLES 11-21 Column Heading See Descriptions in
previous tables and the Examples
DESCRIPTION OF THE FIGURE
[0115] FIG. 1 provides a diagrammatic representation of a
computer-based discovery system containing the SNP information of
the present invention in computer readable form.
DETAILED DESCRIPTION OF THE INVENTION
[0116] The present invention provides SNPs associated with
cardiovascular disorders, particularly acute coronary events such
as myocardial infarction and stroke (including recurrent acute
coronary events such as recurrent myocardial infarction), and SNPs
that are associated with an individual's responsiveness to
therapeutic agents, particularly statins, that are used for the
treatment (including preventive treatment) of cardiovascular
disorders, particularly treatment of acute coronary events. The
present invention further provides nucleic acid molecules
containing these SNPs, methods and reagents for the detection of
the SNPs disclosed herein, uses of these SNPs for the development
of detection reagents, and assays or kits that utilize such
reagents. The statin response-associated SNPs disclosed herein are
useful for diagnosing, screening for, and evaluating response to
statin treatment of cardiovascular diseases in humans. Furthermore,
such SNPs and their encoded products are useful targets for the
development of therapeutic agents.
[0117] A large number of SNPs have been identified from
re-sequencing DNA from 39 individuals, and they are indicated as
"Applera" SNP source in Tables 1-2. Their allele frequencies
observed in each of the Caucasian and African-American ethnic
groups are provided. Additional SNPs included herein were
previously identified during shotgun sequencing and assembly of the
human genome, and they are indicated as "Celera" SNP source in
Tables 1-2. Furthermore, the information provided in Table 1-2,
particularly the allele frequency information obtained from 39
individuals and the identification of the precise position of each
SNP within each gene/transcript, allows haplotypes (i.e., groups of
SNPs that are co-inherited) to be readily inferred. The present
invention encompasses SNP haplotypes, as well as individual
SNPs.
[0118] Thus, the present invention provides individual SNPs
associated with cardiovascular disorders, particularly acute
coronary events, and SNPs associated with responsiveness to statin
for the treatment of cardiovascular diseases, as well as
combinations of SNPs and haplotypes in genetic regions associated
with cardiovascular disorders and/or statin response,
polymorphic/variant transcript sequences (SEQ ID NOS:1-60) and
genomic sequences (SEQ ID NOS:201-250) containing SNPs, encoded
amino acid sequences (SEQ ID NOS: 61-120), and both
transcript-based SNP context sequences (SEQ ID NOS: 121-200) and
genomic-based SNP context sequences (SEQ ID NOS:251-410)
(transcript sequences, protein sequences, and transcript-based SNP
context sequences are provided in Table 1 and the Sequence Listing;
genomic sequences and genomic-based SNP context sequences are
provided in Table 2 and the Sequence Listing), methods of detecting
these polymorphisms in a test sample, methods of determining the
risk of an individual of having or developing a cardiovascular
disorder such as an acute coronary event, methods of determining
response to statin treatment of cardiovascular disease, methods of
screening for compounds useful for treating cardiovascular disease,
compounds identified by these screening methods, methods of using
the disclosed SNPs to select a treatment strategy, methods of
treating a disorder associated with a variant gene/protein (i.e.,
therapeutic methods), and methods of using the SNPs of the present
invention for human identification.
[0119] Since cardiovascular disorders/diseases share certain
similar features that may be due to common genetic factors that are
involved in their underlying mechanisms, the SNPs identified herein
as being particularly associated with acute coronary events and/or
statin response may be used as diagnostic/prognostic markers or
therapeutic targets for a broad spectrum of cardiovascular diseases
such as coronary heart disease (CHD), atherosclerosis,
cerebrovascular disease, congestive heart failure, congenital heart
disease, and pathologies and symptoms associated with various heart
diseases (e.g., angina, hypertension), as well as for predicting
responses to drugs other than statins that are used to treat
cardiovascular diseases.
[0120] The present invention further provides methods for selecting
or formulating a treatment regimen (e.g., methods for determining
whether or not to administer statin treatment to an individual
having cardiovascular disease, methods for selecting a particular
statin-based treatment regimen such as dosage and frequency of
administration of statin, or a particular form/type of statin such
as a particular pharmaceutical formulation or compound, methods for
administering an alternative, non-statin-based treatment to
individuals who are predicted to be unlikely to respond positively
to statin treatment, etc.), and methods for determining the
likelihood of experiencing toxicity or other undesirable side
effects from statin treatment, etc. The present invention also
provides methods for selecting individuals to whom a statin or
other therapeutic will be administered based on the individual's
genotype, and methods for selecting individuals for a clinical
trial of a statin or other therapeutic agent based on the genotypes
of the individuals (e.g., selecting individuals to participate in
the trial who are most likely to respond positively from the statin
treatment).
[0121] The present invention provides novel SNPs associated with
cardiovascular disorders and/or response to statin treatment, as
well as SNPs that were previously known in the art, but were not
previously known to be associated with cardiovascular disorders
and/or statin response. Accordingly, the present invention provides
novel compositions and methods based on the novel SNPs disclosed
herein, and also provides novel methods of using the known, but
previously unassociated, SNPs in methods relating to evaluating an
individual's likelihood of having or developing a cardiovascular
disorder, predicting the likelihood of an individual experiencing a
reoccurrence of a cardiovascular disorder (e.g., experiencing
recurrent myocardial infarctions) prognosing the severity of a
cardiovascular disorder in an individual, or prognosing an
individual's recovery from a cardiovascular disorder, and methods
relating to evaluating an individual's likelihood of responding to
statin treatment for cardiovascular disease. In Tables 1-2, known
SNPs are identified based on the public database in which they have
been observed, which is indicated as one or more of the following
SNP types: "dbSNP"=SNP observed in dbSNP, "HGBASE"=SNP observed in
HGBASE, and "HGMD"=SNP observed in the Human Gene Mutation Database
(HGMD). Novel SNPs for which the SNP source is only "Applera" and
none other, i.e., those that have not been observed in any public
databases and which were also not observed during shotgun
sequencing and assembly of the Celera human genome sequence (i.e.,
"Celera" SNP source), are indicated in Tables 3-4.
[0122] Particular SNP alleles of the present invention can be
associated with either an increased risk of having a cardiovascular
disorder (e.g., experiencing an acute coronary event) or of
responding to statin treatment of cardiovascular disease, or a
decreased likelihood of having a cardiovascular disorder or of
responding to statin treatment of cardiovascular disease. Thus,
whereas certain SNPs (or their encoded products) can be assayed to
determine whether an individual possesses a SNP allele that is
indicative of an increased likelihood of experiencing a coronary
event or of responding to statin treatment, other SNPs (or their
encoded products) can be assayed to determine whether an individual
possesses a SNP allele that is indicative of a decreased likelihood
of experiencing a coronary event or of responding to statin
treatment. Similarly, particular SNP alleles of the present
invention can be associated with either an increased or decreased
likelihood of having a reoccurrence of a cardiovascular disorder,
of fully recovering from a cardiovascular disorder, of experiencing
toxic effects from a particular treatment or therapeutic compound,
etc. The term "altered" may be used herein to encompass either of
these two possibilities (e.g., an increased or a decreased
risk/likelihood). SNP alleles that are associated with a decreased
risk of having or developing a cardiovascular disorder such as
myocardial infarction may be referred to as "protective" alleles,
and SNP alleles that are associated with an increased risk of
having or developing a cardiovascular disorder may be referred to
as "susceptibility" alleles, "risk" alleles, or "risk factors".
[0123] Those skilled in the art will readily recognize that nucleic
acid molecules may be double-stranded molecules and that reference
to a particular site on one strand refers, as well, to the
corresponding site on a complementary strand. In defining a SNP
position, SNP allele, or nucleotide sequence, reference to an
adenine, a thymine (uridine), a cytosine, or a guanine at a
particular site on one strand of a nucleic acid molecule also
defines the thymine (uridine), adenine, guanine, or cytosine
(respectively) at the corresponding site on a complementary strand
of the nucleic acid molecule. Thus, reference may be made to either
strand in order to refer to a particular SNP position, SNP allele,
or nucleotide sequence. Probes and primers, may be designed to
hybridize to either strand and SNP genotyping methods disclosed
herein may generally target either strand. Throughout the
specification, in identifying a SNP position, reference is
generally made to the protein-encoding strand, only for the purpose
of convenience.
[0124] References to variant peptides, polypeptides, or proteins of
the present invention include peptides, polypeptides, proteins, or
fragments thereof, that contain at least one amino acid residue
that differs from the corresponding amino acid sequence of the
art-known peptide/polypeptide/protein (the art-known protein may be
interchangeably referred to as the "wild-type", "reference", or
"normal" protein). Such variant peptides/polypeptides/proteins can
result from a codon change caused by a nonsynonymous nucleotide
substitution at a protein-coding SNP position (i.e., a missense
mutation) disclosed by the present invention. Variant
peptides/polypeptides/proteins of the present invention can also
result from a nonsense mutation, i.e. a SNP that creates a
premature stop codon, a SNP that generates a read-through mutation
by abolishing a stop codon, or due to any SNP disclosed by the
present invention that otherwise alters the structure,
function/activity, or expression of a protein, such as a SNP in a
regulatory region (e.g. a promoter or enhancer) or a SNP that leads
to alternative or defective splicing, such as a SNP in an intron or
a SNP at an exon/intron boundary. As used herein, the terms
"polypeptide", "peptide", and "protein" are used
interchangeably.
Isolated Nucleic Acid Molecules and SNP Detection Reagents &
Kits
[0125] Tables 1 and 2 provide a variety of information about each
SNP of the present invention that is associated with cardiovascular
disorders (e.g., acute coronary events such as myocardial
infarction and stroke) and/or responsiveness to statin treatment,
including the transcript sequences (SEQ ID NOS:1-60), genomic
sequences (SEQ ID NOS:201-250), and protein sequences (SEQ ID
NOS:61-120) of the encoded gene products (with the SNPs indicated
by IUB codes in the nucleic acid sequences). In addition, Tables 1
and 2 include SNP context sequences, which generally include 100
nucleotide upstream (5') plus 100 nucleotides downstream (3') of
each SNP position (SEQ ID NOS:121-200 correspond to
transcript-based SNP context sequences disclosed in Table 1, and
SEQ ID NOS:251-410 correspond to genomic-based context sequences
disclosed in Table 2), the alternative nucleotides (alleles) at
each SNP position, and additional information about the variant
where relevant, such as SNP type (coding, missense, splice site,
UTR, etc.), human populations in which the SNP was observed,
observed allele frequencies, information about the encoded protein,
etc.
[0126] Isolated Nucleic Acid Molecules
[0127] The present invention provides isolated nucleic acid
molecules that contain one or more SNPs disclosed Table 1 and/or
Table 2. Preferred isolated nucleic acid molecules contain one or
more SNPs identified in Table 3 and/or Table 4. Isolated nucleic
acid molecules containing one or more SNPs disclosed in at least
one of Tables 1-4 may be interchangeably referred to throughout the
present text as "SNP-containing nucleic acid molecules". Isolated
nucleic acid molecules may optionally encode a full-length variant
protein or fragment thereof. The isolated nucleic acid molecules of
the present invention also include probes and primers (which are
described in greater detail below in the section entitled "SNP
Detection Reagents"), which may be used for assaying the disclosed
SNPs, and isolated full-length genes, transcripts, cDNA molecules,
and fragments thereof, which may be used for such purposes as
expressing an encoded protein.
[0128] As used herein, an "isolated nucleic acid molecule"
generally is one that contains a SNP of the present invention or
one that hybridizes to such molecule such as a nucleic acid with a
complementary sequence, and is separated from most other nucleic
acids present in the natural source of the nucleic acid molecule.
Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule containing a SNP of the present invention, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or chemical precursors or
other chemicals when chemically synthesized. A nucleic acid
molecule can be fused to other coding or regulatory sequences and
still be considered "isolated." Nucleic acid molecules present in
non-human transgenic animals, which do not naturally occur in the
animal, are also considered "isolated". For example, recombinant
DNA molecules contained in a vector are considered "isolated".
Further examples of "isolated" DNA molecules include recombinant
DNA molecules maintained in heterologous host cells, and purified
(partially or substantially) DNA molecules in solution. Isolated
RNA molecules include in vivo or in vitro RNA transcripts of the
isolated SNP-containing DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0129] Generally, an isolated SNP-containing nucleic acid molecule
comprises one or more SNP positions disclosed by the present
invention with flanking nucleotide sequences on either side of the
SNP positions. A flanking sequence can include nucleotide residues
that are naturally associated with the SNP site and/or heterologous
nucleotide sequences. Preferably the flanking sequence is up to
about 500, 300, 100, 60, 50, 30, 25, 20, 15, 10, 8, or 4
nucleotides (or any other length in-between) on either side of a
SNP position, or as long as the full-length gene or entire
protein-coding sequence (or any portion thereof such as an exon),
especially if the SNP-containing nucleic acid molecule is to be
used to produce a protein or protein fragment.
[0130] For full-length genes and entire protein-coding sequences, a
SNP flanking sequence can be, for example, up to about 5 KB, 4 KB,
3 KB, 2 KB, 1 KB on either side of the SNP. Furthermore, in such
instances, the isolated nucleic acid molecule comprises exonic
sequences (including protein-coding and/or non-coding exonic
sequences), but may also include intronic sequences. Thus, any
protein coding sequence may be either contiguous or separated by
introns. The important point is that the nucleic acid is isolated
from remote and unimportant flanking sequences and is of
appropriate length such that it can be subjected to the specific
manipulations or uses described herein such as recombinant protein
expression, preparation of probes and primers for assaying the SNP
position, and other uses specific to the SNP-containing nucleic
acid sequences.
[0131] An isolated SNP-containing nucleic acid molecule can
comprise, for example, a full-length gene or transcript, such as a
gene isolated from genomic DNA (e.g., by cloning or PCR
amplification), a cDNA molecule, or an mRNA transcript molecule.
Polymorphic transcript sequences are provided in Table 1 and in the
Sequence Listing (SEQ ID NOS: 1-60), and polymorphic genomic
sequences are provided in Table 2 and in the Sequence Listing (SEQ
ID NOS:201-250). Furthermore, fragments of such full-length genes
and transcripts that contain one or more SNPs disclosed herein are
also encompassed by the present invention, and such fragments may
be used, for example, to express any part of a protein, such as a
particular functional domain or an antigenic epitope.
[0132] Thus, the present invention also encompasses fragments of
the nucleic acid sequences provided in Tables 1-2 (transcript
sequences are provided in Table 1 as SEQ ID NOS:1-60, genomic
sequences are provided in Table 2 as SEQ ID NOS:201-250,
transcript-based SNP context sequences are provided in Table 1 as
SEQ ID NO:121-200, and genomic-based SNP context sequences are
provided in Table 2 as SEQ ID NO:251-410) and their complements. A
fragment typically comprises a contiguous nucleotide sequence at
least about 8 or more nucleotides, more preferably at least about
12 or more nucleotides, and even more preferably at least about 16
or more nucleotides. Further, a fragment could comprise at least
about 18, 20, 22, 25, 30, 40, 50, 60, 80, 100, 150, 200, 250 or 500
(or any other number in-between) nucleotides in length. The length
of the fragment will be based on its intended use. For example, the
fragment can encode epitope-bearing regions of a variant peptide or
regions of a variant peptide that differ from the normal/wild-type
protein, or can be useful as a polynucleotide probe or primer. Such
fragments can be isolated using the nucleotide sequences provided
in Table 1 and/or Table 2 for the synthesis of a polynucleotide
probe. A labeled probe can then be used, for example, to screen a
cDNA library, genomic DNA library, or mRNA to isolate nucleic acid
corresponding to the coding region. Further, primers can be used in
amplification reactions, such as for purposes of assaying one or
more SNPs sites or for cloning specific regions of a gene.
[0133] An isolated nucleic acid molecule of the present invention
further encompasses a SNP-containing polynucleotide that is the
product of any one of a variety of nucleic acid amplification
methods, which are used to increase the copy numbers of a
polynucleotide of interest in a nucleic acid sample. Such
amplification methods are well known in the art, and they include
but are not limited to, polymerase chain reaction (PCR) (U.S. Pat.
Nos. 4,683,195; and 4,683,202; PCR Technology: Principles and
Applications for DNA Amplification, ed. H. A. Erlich, Freeman
Press, NY, NY, 1992), ligase chain reaction (LCR) (Wu and Wallace,
Genomics 4:560, 1989; Landegren et al., Science 241:1077, 1988),
strand displacement amplification (SDA) (U.S. Pat. Nos. 5,270,184;
and 5,422,252), transcription-mediated amplification (TMA) (U.S.
Pat. No. 5,399,491), linked linear amplification (LLA) (U.S. Pat.
No. 6,027,923), and the like, and isothermal amplification methods
such as nucleic acid sequence based amplification (NASBA), and
self-sustained sequence replication (Guatelli et al., Proc. Natl.
Acad. Sci. USA 87: 1874, 1990). Based on such methodologies, a
person skilled in the art can readily design primers in any
suitable regions 5' and 3' to a SNP disclosed herein. Such primers
may be used to amplify DNA of any length so long that it contains
the SNP of interest in its sequence.
[0134] As used herein, an "amplified polynucleotide" of the
invention is a SNP-containing nucleic acid molecule whose amount
has been increased at least two fold by any nucleic acid
amplification method performed in vitro as compared to its starting
amount in a test sample. In other preferred embodiments, an
amplified polynucleotide is the result of at least ten fold, fifty
fold, one hundred fold, one thousand fold, or even ten thousand
fold increase as compared to its starting amount in a test sample.
In a typical PCR amplification, a polynucleotide of interest is
often amplified at least fifty thousand fold in amount over the
unamplified genomic DNA, but the precise amount of amplification
needed for an assay depends on the sensitivity of the subsequent
detection method used.
[0135] Generally, an amplified polynucleotide is at least about 16
nucleotides in length. More typically, an amplified polynucleotide
is at least about 20 nucleotides in length. In a preferred
embodiment of the invention, an amplified polynucleotide is at
least about 30 nucleotides in length. In a more preferred
embodiment of the invention, an amplified polynucleotide is at
least about 32, 40, 45, 50, or 60 nucleotides in length. In yet
another preferred embodiment of the invention, an amplified
polynucleotide is at least about 100, 200, 300, 400, or 500
nucleotides in length. While the total length of an amplified
polynucleotide of the invention can be as long as an exon, an
intron or the entire gene where the SNP of interest resides, an
amplified product is typically up to about 1,000 nucleotides in
length (although certain amplification methods may generate
amplified products greater than 1000 nucleotides in length). More
preferably, an amplified polynucleotide is not greater than about
600-700 nucleotides in length. It is understood that irrespective
of the length of an amplified polynucleotide, a SNP of interest may
be located anywhere along its sequence.
[0136] In a specific embodiment of the invention, the amplified
product is at least about 201 nucleotides in length, comprises one
of the transcript-based context sequences or the genomic-based
context sequences shown in Tables 1-2. Such a product may have
additional sequences on its 5' end or 3' end or both. In another
embodiment, the amplified product is about 101 nucleotides in
length, and it contains a SNP disclosed herein. Preferably, the SNP
is located at the middle of the amplified product (e.g., at
position 101 in an amplified product that is 201 nucleotides in
length, or at position 51 in an amplified product that is 101
nucleotides in length), or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 15, or 20 nucleotides from the middle of the amplified product
(however, as indicated above, the SNP of interest may be located
anywhere along the length of the amplified product).
[0137] The present invention provides isolated nucleic acid
molecules that comprise, consist of, or consist essentially of one
or more polynucleotide sequences that contain one or more SNPs
disclosed herein, complements thereof, and SNP-containing fragments
thereof.
[0138] Accordingly, the present invention provides nucleic acid
molecules that consist of any of the nucleotide sequences shown in
Table 1 and/or Table 2 (transcript sequences are provided in Table
1 as SEQ ID NOS:1-60, genomic sequences are provided in Table 2 as
SEQ ID NOS:201-250, transcript-based SNP context sequences are
provided in Table 1 as SEQ ID NO:121-200, and genomic-based SNP
context sequences are provided in Table 2 as SEQ ID NO:251-410), or
any nucleic acid molecule that encodes any of the variant proteins
provided in Table 1 (SEQ ID NOS:61-120). A nucleic acid molecule
consists of a nucleotide sequence when the nucleotide sequence is
the complete nucleotide sequence of the nucleic acid molecule.
[0139] The present invention further provides nucleic acid
molecules that consist essentially of any of the nucleotide
sequences shown in Table 1 and/or Table 2 (transcript sequences are
provided in Table 1 as SEQ ID NOS:1-60, genomic sequences are
provided in Table 2 as SEQ ID NOS:201-250, transcript-based SNP
context sequences are provided in Table 1 as SEQ ID NO:121-250, and
genomic-based SNP context sequences are provided in Table 2 as SEQ
ID NO:251-410), or any nucleic acid molecule that encodes any of
the variant proteins provided in Table 1 (SEQ ID NOS:61-120). A
nucleic acid molecule consists essentially of a nucleotide sequence
when such a nucleotide sequence is present with only a few
additional nucleotide residues in the final nucleic acid
molecule.
[0140] The present invention further provides nucleic acid
molecules that comprise any of the nucleotide sequences shown in
Table 1 and/or Table 2 or a SNP-containing fragment thereof
(transcript sequences are provided in Table 1 as SEQ ID NOS:1-60,
genomic sequences are provided in Table 2 as SEQ ID NOS:201-250,
transcript-based SNP context sequences are provided in Table 1 as
SEQ ID NO:121-250, and genomic-based SNP context sequences are
provided in Table 2 as SEQ ID NO:251-410), or any nucleic acid
molecule that encodes any of the variant proteins provided in Table
1 (SEQ ID NOS:61-120). A nucleic acid molecule comprises a
nucleotide sequence when the nucleotide sequence is at least part
of the final nucleotide sequence of the nucleic acid molecule. In
such a fashion, the nucleic acid molecule can be only the
nucleotide sequence or have additional nucleotide residues, such as
residues that are naturally associated with it or heterologous
nucleotide sequences. Such a nucleic acid molecule can have one to
a few additional nucleotides or can comprise many more additional
nucleotides. A brief description of how various types of these
nucleic acid molecules can be readily made and isolated is provided
below, and such techniques are well known to those of ordinary
skill in the art (Sambrook and Russell, 2000, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, NY).
[0141] The isolated nucleic acid molecules can encode mature
proteins plus additional amino or carboxyl-terminal amino acids or
both, or amino acids interior to the mature peptide (when the
mature form has more than one peptide chain, for instance). Such
sequences may play a role in processing of a protein from precursor
to a mature form, facilitate protein trafficking, prolong or
shorten protein half-life, or facilitate manipulation of a protein
for assay or production. As generally is the case in situ, the
additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0142] Thus, the isolated nucleic acid molecules include, but are
not limited to, nucleic acid molecules having a sequence encoding a
peptide alone, a sequence encoding a mature peptide and additional
coding sequences such as a leader or secretory sequence (e.g., a
pre-pro or pro-protein sequence), a sequence encoding a mature
peptide with or without additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but untranslated sequences
that play a role in, for example, transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding,
and/or stability of mRNA. In addition, the nucleic acid molecules
may be fused to heterologous marker sequences encoding, for
example, a peptide that facilitates purification.
[0143] Isolated nucleic acid molecules can be in the form of RNA,
such as mRNA, or in the form DNA, including cDNA and genomic DNA,
which may be obtained, for example, by molecular cloning or
produced by chemical synthetic techniques or by a combination
thereof (Sambrook and Russell, 2000, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, NY). Furthermore,
isolated nucleic acid molecules, particularly SNP detection
reagents such as probes and primers, can also be partially or
completely in the form of one or more types of nucleic acid
analogs, such as peptide nucleic acid (PNA) (U.S. Pat. Nos.
5,539,082; 5,527,675; 5,623,049; 5,714,331). The nucleic acid,
especially DNA, can be double-stranded or single-stranded.
Single-stranded nucleic acid can be the coding strand (sense
strand) or the complementary non-coding strand (antisense strand).
DNA, RNA, or PNA segments can be assembled, for example, from
fragments of the human genome (in the case of DNA or RNA) or single
nucleotides, short oligonucleotide linkers, or from a series of
oligonucleotides, to provide a synthetic nucleic acid molecule.
Nucleic acid molecules can be readily synthesized using the
sequences provided herein as a reference; oligonucleotide and PNA
oligomer synthesis techniques are well known in the art (see, e.g.,
Corey, "Peptide nucleic acids: expanding the scope of nucleic acid
recognition", Trends Biotechnol. 1997 June; 15(6):224-9, and Hyrup
et al., "Peptide nucleic acids (PNA): synthesis, properties and
potential applications", Bioorg Med Chem. 1996 January; 4(1):5-23).
Furthermore, large-scale automated oligonucleotide/PNA synthesis
(including synthesis on an array or bead surface or other solid
support) can readily be accomplished using commercially available
nucleic acid synthesizers, such as the Applied Biosystems (Foster
City, Calif.) 3900 High-Throughput DNA Synthesizer or Expedite 8909
Nucleic Acid Synthesis System, and the sequence information
provided herein.
[0144] The present invention encompasses nucleic acid analogs that
contain modified, synthetic, or non-naturally occurring nucleotides
or structural elements or other alternative/modified nucleic acid
chemistries known in the art. Such nucleic acid analogs are useful,
for example, as detection reagents (e.g., primers/probes) for
detecting one or more SNPs identified in Table 1 and/or Table 2.
Furthermore, kits/systems (such as beads, arrays, etc.) that
include these analogs are also encompassed by the present
invention. For example, PNA oligomers that are based on the
polymorphic sequences of the present invention are specifically
contemplated. PNA oligomers are analogs of DNA in which the
phosphate backbone is replaced with a peptide-like backbone
(Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters,
4: 1081-1082 (1994), Petersen et al., Bioorganic & Medicinal
Chemistry Letters, 6: 793-796 (1996), Kumar et al., Organic Letters
3(9): 1269-1272 (2001), WO96/04000). PNA hybridizes to
complementary RNA or DNA with higher affinity and specificity than
conventional oligonucleotides and oligonucleotide analogs. The
properties of PNA enable novel molecular biology and biochemistry
applications unachievable with traditional oligonucleotides and
peptides.
[0145] Additional examples of nucleic acid modifications that
improve the binding properties and/or stability of a nucleic acid
include the use of base analogs such as inosine, intercalators
(U.S. Pat. No. 4,835,263) and the minor groove binders (U.S. Pat.
No. 5,801,115). Thus, references herein to nucleic acid molecules,
SNP-containing nucleic acid molecules, SNP detection reagents
(e.g., probes and primers), oligonucleotides/polynucleotides
include PNA oligomers and other nucleic acid analogs. Other
examples of nucleic acid analogs and alternative/modified nucleic
acid chemistries known in the art are described in Current
Protocols in Nucleic Acid Chemistry, John Wiley & Sons, N.Y.
(2002).
[0146] The present invention further provides nucleic acid
molecules that encode fragments of the variant polypeptides
disclosed herein as well as nucleic acid molecules that encode
obvious variants of such variant polypeptides. Such nucleic acid
molecules may be naturally occurring, such as paralogs (different
locus) and orthologs (different organism), or may be constructed by
recombinant DNA methods or by chemical synthesis. Non-naturally
occurring variants may be made by mutagenesis techniques, including
those applied to nucleic acid molecules, cells, or organisms.
Accordingly, the variants can contain nucleotide substitutions,
deletions, inversions and insertions (in addition to the SNPs
disclosed in Tables 1-2). Variation can occur in either or both the
coding and non-coding regions. The variations can produce
conservative and/or non-conservative amino acid substitutions.
[0147] Further variants of the nucleic acid molecules disclosed in
Tables 1-2, such as naturally occurring allelic variants (as well
as orthologs and paralogs) and synthetic variants produced by
mutagenesis techniques, can be identified and/or produced using
methods well known in the art. Such further variants can comprise a
nucleotide sequence that shares at least 70-80%, 80-85%, 85-90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity
with a nucleic acid sequence disclosed in Table 1 and/or Table 2
(or a fragment thereof) and that includes a novel SNP allele
disclosed in Table 1 and/or Table 2. Further, variants can comprise
a nucleotide sequence that encodes a polypeptide that shares at
least 70-80%, 80-85%, 85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity with a polypeptide sequence disclosed
in Table 1 (or a fragment thereof) and that includes a novel SNP
allele disclosed in Table 1 and/or Table 2. Thus, an aspect of the
present invention that is specifically contemplated are isolated
nucleic acid molecules that have a certain degree of sequence
variation compared with the sequences shown in Tables 1-2, but that
contain a novel SNP allele disclosed herein. In other words, as
long as an isolated nucleic acid molecule contains a novel SNP
allele disclosed herein, other portions of the nucleic acid
molecule that flank the novel SNP allele can vary to some degree
from the specific transcript, genomic, and context sequences shown
in Tables 1-2, and can encode a polypeptide that varies to some
degree from the specific polypeptide sequences shown in Table
1.
[0148] To determine the percent identity of two amino acid
sequences or two nucleotide sequences of two molecules that share
sequence homology, the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second amino acid or nucleic acid sequence for optimal
alignment and non-homologous sequences can be disregarded for
comparison purposes). In a preferred embodiment, at least 30%, 40%,
50%, 60%, 70%, 80%, or 90% or more of the length of a reference
sequence is aligned for comparison purposes. The amino acid
residues or nucleotides at corresponding amino acid positions or
nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein,
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0149] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. (Computational Molecular Biology, Lesk, A.
M., ed., Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, D. W., ed., Academic Press,
New York, 1993; Computer Analysis of Sequence Data, Part 1,
Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,
1994; Sequence Analysis in Molecular Biology, von Heinje, G.,
Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M Stockton Press, New York, 1991). In a
preferred embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch algorithm
(J. Mol. Biol. (48):444-453 (1970)) which has been incorporated
into the GAP program in the GCG software package, using either a
Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
[0150] In yet another preferred embodiment, the percent identity
between two nucleotide sequences is determined using the GAP
program in the GCG software package (Devereux, J., et al., Nucleic
Acids Res. 12(1):387 (1984)), using a NWSgapdna.CMP matrix and a
gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3,
4, 5, or 6. In another embodiment, the percent identity between two
amino acid or nucleotide sequences is determined using the
algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which
has been incorporated into the ALIGN program (version 2.0), using a
PAM120 weight residue table, a gap length penalty of 12, and a gap
penalty of 4.
[0151] The nucleotide and amino acid sequences of the present
invention can further be used as a "query sequence" to perform a
search against sequence databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches
can be performed with the NBLAST program, score=100, wordlength=12
to obtain nucleotide sequences homologous to the nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to the proteins of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. In addition to BLAST, examples of
other search and sequence comparison programs used in the art
include, but are not limited to, FASTA (Pearson, Methods Mol. Biol.
25, 365-389 (1994)) and KERR (Dufresne et al., Nat Biotechnol 2002
December; 20(12):1269-71). For further information regarding
bioinformatics techniques, see Current Protocols in Bioinformatics,
John Wiley & Sons, Inc., N.Y.
[0152] The present invention further provides non-coding fragments
of the nucleic acid molecules disclosed in Table 1 and/or Table 2.
Preferred non-coding fragments include, but are not limited to,
promoter sequences, enhancer sequences, intronic sequences, 5'
untranslated regions (UTRs), 3' untranslated regions, gene
modulating sequences and gene termination sequences. Such fragments
are useful, for example, in controlling heterologous gene
expression and in developing screens to identify gene-modulating
agents.
[0153] SNP Detection Reagents
[0154] In a specific aspect of the present invention, the SNPs
disclosed in Table 1 and/or Table 2, and their associated
transcript sequences (provided in Table 1 as SEQ ID NOS:1-60),
genomic sequences (provided in Table 2 as SEQ ID NOS:201-250), and
context sequences (transcript-based context sequences are provided
in Table 1 as SEQ ID NOS:121-200; genomic-based context sequences
are provided in Table 2 as SEQ ID NOS:251-410), can be used for the
design of SNP detection reagents. As used herein, a "SNP detection
reagent" is a reagent that specifically detects a specific target
SNP position disclosed herein, and that is preferably specific for
a particular nucleotide (allele) of the target SNP position (i.e.,
the detection reagent preferably can differentiate between
different alternative nucleotides at a target SNP position, thereby
allowing the identity of the nucleotide present at the target SNP
position to be determined). Typically, such detection reagent
hybridizes to a target SNP-containing nucleic acid molecule by
complementary base-pairing in a sequence specific manner, and
discriminates the target variant sequence from other nucleic acid
sequences such as an art-known form in a test sample. An example of
a detection reagent is a probe that hybridizes to a target nucleic
acid containing one or more of the SNPs provided in Table 1 and/or
Table 2. In a preferred embodiment, such a probe can differentiate
between nucleic acids having a particular nucleotide (allele) at a
target SNP position from other nucleic acids that have a different
nucleotide at the same target SNP position. In addition, a
detection reagent may hybridize to a specific region 5' and/or 3'
to a SNP position, particularly a region corresponding to the
context sequences provided in Table 1 and/or Table 2
(transcript-based context sequences are provided in Table 1 as SEQ
ID NOS:121-200; genomic-based context sequences are provided in
Table 2 as SEQ ID NOS:251-410). Another example of a detection
reagent is a primer which acts as an initiation point of nucleotide
extension along a complementary strand of a target polynucleotide.
The SNP sequence information provided herein is also useful for
designing primers, e.g. allele-specific primers, to amplify (e.g.,
using PCR) any SNP of the present invention.
[0155] In one preferred embodiment of the invention, a SNP
detection reagent is an isolated or synthetic DNA or RNA
polynucleotide probe or primer or PNA oligomer, or a combination of
DNA, RNA and/or PNA, that hybridizes to a segment of a target
nucleic acid molecule containing a SNP identified in Table 1 and/or
Table 2. A detection reagent in the form of a polynucleotide may
optionally contain modified base analogs, intercalators or minor
groove binders. Multiple detection reagents such as probes may be,
for example, affixed to a solid support (e.g., arrays or beads) or
supplied in solution (e.g., probe/primer sets for enzymatic
reactions such as PCR, RT-PCR, TaqMan assays, or primer-extension
reactions) to form a SNP detection kit.
[0156] A probe or primer typically is a substantially purified
oligonucleotide or PNA oligomer. Such oligonucleotide typically
comprises a region of complementary nucleotide sequence that
hybridizes under stringent conditions to at least about 8, 10, 12,
16, 18, 20, 22, 25, 30, 40, 50, 55, 60, 65, 70, 80, 90, 100, 120
(or any other number in-between) or more consecutive nucleotides in
a target nucleic acid molecule. Depending on the particular assay,
the consecutive nucleotides can either include the target SNP
position, or be a specific region in close enough proximity 5'
and/or 3' to the SNP position to carry out the desired assay.
[0157] Other preferred primer and probe sequences can readily be
determined using the transcript sequences (SEQ ID NOS:1-60),
genomic sequences (SEQ ID NOS:201-250), and SNP context sequences
(transcript-based context sequences are provided in Table 1 as SEQ
ID NOS:121-200; genomic-based context sequences are provided in
Table 2 as SEQ ID NOS:251-410) disclosed in the Sequence Listing
and in Tables 1-2. It will be apparent to one of skill in the art
that such primers and probes are directly useful as reagents for
genotyping the SNPs of the present invention, and can be
incorporated into any kit/system format.
[0158] In order to produce a probe or primer specific for a target
SNP-containing sequence, the gene/transcript and/or context
sequence surrounding the SNP of interest is typically examined
using a computer algorithm which starts at the 5' or at the 3' end
of the nucleotide sequence. Typical algorithms will then identify
oligomers of defined length that are unique to the gene/SNP context
sequence, have a GC content within a range suitable for
hybridization, lack predicted secondary structure that may
interfere with hybridization, and/or possess other desired
characteristics or that lack other undesired characteristics.
[0159] A primer or probe of the present invention is typically at
least about 8 nucleotides in length. In one embodiment of the
invention, a primer or a probe is at least about 10 nucleotides in
length. In a preferred embodiment, a primer or a probe is at least
about 12 nucleotides in length. In a more preferred embodiment, a
primer or probe is at least about 16, 17, 18, 19, 20, 21, 22, 23,
24 or 25 nucleotides in length. While the maximal length of a probe
can be as long as the target sequence to be detected, depending on
the type of assay in which it is employed, it is typically less
than about 50, 60, 65, or 70 nucleotides in length. In the case of
a primer, it is typically less than about 30 nucleotides in length.
In a specific preferred embodiment of the invention, a primer or a
probe is within the length of about 18 and about 28 nucleotides.
However, in other embodiments, such as nucleic acid arrays and
other embodiments in which probes are affixed to a substrate, the
probes can be longer, such as on the order of 30-70, 75, 80, 90,
100, or more nucleotides in length (see the section below entitled
"SNP Detection Kits and Systems").
[0160] For analyzing SNPs, it may be appropriate to use
oligonucleotides specific for alternative SNP alleles. Such
oligonucleotides which detect single nucleotide variations in
target sequences may be referred to by such terms as
"allele-specific oligonucleotides," "allele-specific probes," or
"allele-specific primers." The design and use of allele-specific
probes for analyzing polymorphisms is described in, e.g., Mutation
Detection A Practical Approach, ed. Cotton et al. Oxford University
Press, 1998; Saiki et al., Nature 324, 163-166 (1986); Dattagupta,
EP235,726; and Saiki, WO 89/11548.
[0161] While the design of each allele-specific primer or probe
depends on variables such as the precise composition of the
nucleotide sequences flanking a SNP position in a target nucleic
acid molecule, and the length of the primer or probe, another
factor in the use of primers and probes is the stringency of the
condition under which the hybridization between the probe or primer
and the target sequence is performed. Higher stringency conditions
utilize buffers with lower ionic strength and/or a higher reaction
temperature, and tend to require a more perfect match between
probe/primer and a target sequence in order to form a stable
duplex. If the stringency is too high, however, hybridization may
not occur at all. In contrast, lower stringency conditions utilize
buffers with higher ionic strength and/or a lower reaction
temperature, and permit the formation of stable duplexes with more
mismatched bases between a probe/primer and a target sequence. By
way of example and not limitation, exemplary conditions for high
stringency hybridization conditions using an allele-specific probe
are as follows: Prehybridization with a solution containing
5.times. standard saline phosphate EDTA (SSPE), 0.5% NaDodSO.sub.4
(SDS) at 55.degree. C., and incubating probe with target nucleic
acid molecules in the same solution at the same temperature,
followed by washing with a solution containing 2.times.SSPE, and
0.1% SDS at 55.degree. C. or room temperature.
[0162] Moderate stringency hybridization conditions may be used for
allele-specific primer extension reactions with a solution
containing, e.g., about 50 mM KCl at about 46.degree. C.
Alternatively, the reaction may be carried out at an elevated
temperature such as 60.degree. C. In another embodiment, a
moderately stringent hybridization condition suitable for
oligonucleotide ligation assay (OLA) reactions wherein two probes
are ligated if they are completely complementary to the target
sequence may utilize a solution of about 100 mM KCl at a
temperature of 46.degree. C.
[0163] In a hybridization-based assay, allele-specific probes can
be designed that hybridize to a segment of target DNA from one
individual but do not hybridize to the corresponding segment from
another individual due to the presence of different polymorphic
forms (e.g., alternative SNP alleles/nucleotides) in the respective
DNA segments from the two individuals. Hybridization conditions
should be sufficiently stringent that there is a significant
detectable difference in hybridization intensity between alleles,
and preferably an essentially binary response, whereby a probe
hybridizes to only one of the alleles or significantly more
strongly to one allele. While a probe may be designed to hybridize
to a target sequence that contains a SNP site such that the SNP
site aligns anywhere along the sequence of the probe, the probe is
preferably designed to hybridize to a segment of the target
sequence such that the SNP site aligns with a central position of
the probe (e.g., a position within the probe that is at least three
nucleotides from either end of the probe). This design of probe
generally achieves good discrimination in hybridization between
different allelic forms.
[0164] In another embodiment, a probe or primer may be designed to
hybridize to a segment of target DNA such that the SNP aligns with
either the 5' most end or the 3' most end of the probe or primer.
In a specific preferred embodiment that is particularly suitable
for use in a oligonucleotide ligation assay (U.S. Pat. No.
4,988,617), the 3' most nucleotide of the probe aligns with the SNP
position in the target sequence.
[0165] Oligonucleotide probes and primers may be prepared by
methods well known in the art. Chemical synthetic methods include,
but are limited to, the phosphotriester method described by Narang
et al., 1979, Methods in Enzymology 68:90; the phosphodiester
method described by Brown et al., 1979, Methods in Enzymology
68:109, the diethylphosphoamidate method described by Beaucage et
al., 1981, Tetrahedron Letters 22:1859; and the solid support
method described in U.S. Pat. No. 4,458,066.
[0166] Allele-specific probes are often used in pairs (or, less
commonly, in sets of 3 or 4, such as if a SNP position is known to
have 3 or 4 alleles, respectively, or to assay both strands of a
nucleic acid molecule for a target SNP allele), and such pairs may
be identical except for a one nucleotide mismatch that represents
the allelic variants at the SNP position. Commonly, one member of a
pair perfectly matches a reference form of a target sequence that
has a more common SNP allele (i.e., the allele that is more
frequent in the target population) and the other member of the pair
perfectly matches a form of the target sequence that has a less
common SNP allele (i.e., the allele that is rarer in the target
population). In the case of an array, multiple pairs of probes can
be immobilized on the same support for simultaneous analysis of
multiple different polymorphisms.
[0167] In one type of PCR-based assay, an allele-specific primer
hybridizes to a region on a target nucleic acid molecule that
overlaps a SNP position and only primes amplification of an allelic
form to which the primer exhibits perfect complementarity (Gibbs,
1989, Nucleic Acid Res. 17 2427-2448). Typically, the primer's
3'-most nucleotide is aligned with and complementary to the SNP
position of the target nucleic acid molecule. This primer is used
in conjunction with a second primer that hybridizes at a distal
site. Amplification proceeds from the two primers, producing a
detectable product that indicates which allelic form is present in
the test sample. A control is usually performed with a second pair
of primers, one of which shows a single base mismatch at the
polymorphic site and the other of which exhibits perfect
complementarity to a distal site. The single-base mismatch prevents
amplification or substantially reduces amplification efficiency, so
that either no detectable product is formed or it is formed in
lower amounts or at a slower pace. The method generally works most
effectively when the mismatch is at the 3'-most position of the
oligonucleotide (i.e., the 3'-most position of the oligonucleotide
aligns with the target SNP position) because this position is most
destabilizing to elongation from the primer (see, e.g., WO
93/22456). This PCR-based assay can be utilized as part of the
TaqMan assay, described below.
[0168] In a specific embodiment of the invention, a primer of the
invention contains a sequence substantially complementary to a
segment of a target SNP-containing nucleic acid molecule except
that the primer has a mismatched nucleotide in one of the three
nucleotide positions at the 3'-most end of the primer, such that
the mismatched nucleotide does not base pair with a particular
allele at the SNP site. In a preferred embodiment, the mismatched
nucleotide in the primer is the second from the last nucleotide at
the 3'-most position of the primer. In a more preferred embodiment,
the mismatched nucleotide in the primer is the last nucleotide at
the 3'-most position of the primer.
[0169] In another embodiment of the invention, a SNP detection
reagent of the invention is labeled with a fluorogenic reporter dye
that emits a detectable signal. While the preferred reporter dye is
a fluorescent dye, any reporter dye that can be attached to a
detection reagent such as an oligonucleotide probe or primer is
suitable for use in the invention. Such dyes include, but are not
limited to, Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5,
Cy7, Dabcyl, Edans, Eosin, Erythrosin, Fluorescein, 6-Fam, Tet,
Joe, Hex, Oregon Green, Rhodamine, Rhodol Green, Tamra, Rox, and
Texas Red.
[0170] In yet another embodiment of the invention, the detection
reagent may be further labeled with a quencher dye such as Tamra,
especially when the reagent is used as a self-quenching probe such
as a TaqMan (U.S. Pat. Nos. 5,210,015 and 5,538,848) or Molecular
Beacon probe (U.S. Pat. Nos. 5,118,801 and 5,312,728), or other
stemless or linear beacon probe (Livak et al., 1995, PCR Method
Appl. 4:357-362; Tyagi et al., 1996, Nature Biotechnology 14:
303-308; Nazarenko et al., 1997, Nucl. Acids Res. 25:2516-2521;
U.S. Pat. Nos. 5,866,336 and 6,117,635).
[0171] The detection reagents of the invention may also contain
other labels, including but not limited to, biotin for streptavidin
binding, hapten for antibody binding, and oligonucleotide for
binding to another complementary oligonucleotide such as pairs of
zipcodes.
[0172] The present invention also contemplates reagents that do not
contain (or that are complementary to) a SNP nucleotide identified
herein but that are used to assay one or more SNPs disclosed
herein. For example, primers that flank, but do not hybridize
directly to a target SNP position provided herein are useful in
primer extension reactions in which the primers hybridize to a
region adjacent to the target SNP position (i.e., within one or
more nucleotides from the target SNP site). During the primer
extension reaction, a primer is typically not able to extend past a
target SNP site if a particular nucleotide (allele) is present at
that target SNP site, and the primer extension product can be
detected in order to determine which SNP allele is present at the
target SNP site. For example, particular ddNTPs are typically used
in the primer extension reaction to terminate primer extension once
a ddNTP is incorporated into the extension product (a primer
extension product which includes a ddNTP at the 3'-most end of the
primer extension product, and in which the ddNTP is a nucleotide of
a SNP disclosed herein, is a composition that is specifically
contemplated by the present invention). Thus, reagents that bind to
a nucleic acid molecule in a region adjacent to a SNP site and that
are used for assaying the SNP site, even though the bound sequences
do not necessarily include the SNP site itself, are also
contemplated by the present invention.
[0173] SNP Detection Kits and Systems
[0174] A person skilled in the art will recognize that, based on
the SNP and associated sequence information disclosed herein,
detection reagents can be developed and used to assay any SNP of
the present invention individually or in combination, and such
detection reagents can be readily incorporated into one of the
established kit or system formats which are well known in the art.
The terms "kits" and "systems", as used herein in the context of
SNP detection reagents, are intended to refer to such things as
combinations of multiple SNP detection reagents, or one or more SNP
detection reagents in combination with one or more other types of
elements or components (e.g., other types of biochemical reagents,
containers, packages such as packaging intended for commercial
sale, substrates to which SNP detection reagents are attached,
electronic hardware components, etc.). Accordingly, the present
invention further provides SNP detection kits and systems,
including but not limited to, packaged probe and primer sets (e.g.,
TaqMan probe/primer sets), arrays/microarrays of nucleic acid
molecules, and beads that contain one or more probes, primers, or
other detection reagents for detecting one or more SNPs of the
present invention. The kits/systems can optionally include various
electronic hardware components; for example, arrays ("DNA chips")
and microfluidic systems ("lab-on-a-chip" systems) provided by
various manufacturers typically comprise hardware components. Other
kits/systems (e.g., probe/primer sets) may not include electronic
hardware components, but may be comprised of, for example, one or
more SNP detection reagents (along with, optionally, other
biochemical reagents) packaged in one or more containers.
[0175] In some embodiments, a SNP detection kit typically contains
one or more detection reagents and other components (e.g., a
buffer, enzymes such as DNA polymerases or ligases, chain extension
nucleotides such as deoxynucleotide triphosphates, and in the case
of Sanger-type DNA sequencing reactions, chain terminating
nucleotides, positive control sequences, negative control
sequences, and the like) necessary to carry out an assay or
reaction, such as amplification and/or detection of a
SNP-containing nucleic acid molecule. A kit may further contain
means for determining the amount of a target nucleic acid, and
means for comparing the amount with a standard, and can comprise
instructions for using the kit to detect the SNP-containing nucleic
acid molecule of interest. In one embodiment of the present
invention, kits are provided which contain the necessary reagents
to carry out one or more assays to detect one or more SNPs
disclosed herein. In a preferred embodiment of the present
invention, SNP detection kits/systems are in the form of nucleic
acid arrays, or compartmentalized kits, including
microfluidic/lab-on-a-chip systems.
[0176] SNP detection kits/systems may contain, for example, one or
more probes, or pairs of probes, that hybridize to a nucleic acid
molecule at or near each target SNP position. Multiple pairs of
allele-specific probes may be included in the kit/system to
simultaneously assay large numbers of SNPs, at least one of which
is a SNP of the present invention. In some kits/systems, the
allele-specific probes are immobilized to a substrate such as an
array or bead. For example, the same substrate can comprise
allele-specific probes for detecting at least 1; 10; 100; 1000;
10,000; 100,000 (or any other number in-between) or substantially
all of the SNPs shown in Table 1 and/or Table 2.
[0177] The terms "arrays", "microarrays", and "DNA chips" are used
herein interchangeably to refer to an array of distinct
polynucleotides affixed to a substrate, such as glass, plastic,
paper, nylon or other type of membrane, filter, chip, or any other
suitable solid support. The polynucleotides can be synthesized
directly on the substrate, or synthesized separate from the
substrate and then affixed to the substrate. In one embodiment, the
microarray is prepared and used according to the methods described
in U.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995
(Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14:
1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93:
10614-10619), all of which are incorporated herein in their
entirety by reference. In other embodiments, such arrays are
produced by the methods described by Brown et al., U.S. Pat. No.
5,807,522.
[0178] Nucleic acid arrays are reviewed in the following
references: Zammatteo et al., "New chips for molecular biology and
diagnostics", Biotechnol Annu Rev. 2002; 8:85-101; Sosnowski et
al., "Active microelectronic array system for DNA hybridization,
genotyping and pharmacogenomic applications", Psychiatr Genet. 2002
December; 12(4):181-92; Heller, "DNA microarray technology:
devices, systems, and applications", Annu Rev Biomed Eng. 2002;
4:129-53. Epub 2002 Mar. 22; Kolchinsky et al., "Analysis of SNPs
and other genomic variations using gel-based chips", Hum Mutat.
2002 April; 19(4):343-60; and McGall et al., "High-density genechip
oligonucleotide probe arrays", Adv Biochem Eng Biotechnol. 2002;
77:21-42.
[0179] Any number of probes, such as allele-specific probes, may be
implemented in an array, and each probe or pair of probes can
hybridize to a different SNP position. In the case of
polynucleotide probes, they can be synthesized at designated areas
(or synthesized separately and then affixed to designated areas) on
a substrate using a light-directed chemical process. Each DNA chip
can contain, for example, thousands to millions of individual
synthetic polynucleotide probes arranged in a grid-like pattern and
miniaturized (e.g., to the size of a dime). Preferably, probes are
attached to a solid support in an ordered, addressable array.
[0180] A microarray can be composed of a large number of unique,
single-stranded polynucleotides, usually either synthetic antisense
polynucleotides or fragments of cDNAs, fixed to a solid support.
Typical polynucleotides are preferably about 6-60 nucleotides in
length, more preferably about 15-30 nucleotides in length, and most
preferably about 18-25 nucleotides in length. For certain types of
microarrays or other detection kits/systems, it may be preferable
to use oligonucleotides that are only about 7-20 nucleotides in
length. In other types of arrays, such as arrays used in
conjunction with chemiluminescent detection technology, preferred
probe lengths can be, for example, about 15-80 nucleotides in
length, preferably about 50-70 nucleotides in length, more
preferably about 55-65 nucleotides in length, and most preferably
about 60 nucleotides in length. The microarray or detection kit can
contain polynucleotides that cover the known 5' or 3' sequence of a
gene/transcript or target SNP site, sequential polynucleotides that
cover the full-length sequence of a gene/transcript; or unique
polynucleotides selected from particular areas along the length of
a target gene/transcript sequence, particularly areas corresponding
to one or more SNPs disclosed in Table 1 and/or Table 2.
Polynucleotides used in the microarray or detection kit can be
specific to a SNP or SNPs of interest (e.g., specific to a
particular SNP allele at a target SNP site, or specific to
particular SNP alleles at multiple different SNP sites), or
specific to a polymorphic gene/transcript or genes/transcripts of
interest.
[0181] Hybridization assays based on polynucleotide arrays rely on
the differences in hybridization stability of the probes to
perfectly matched and mismatched target sequence variants. For SNP
genotyping, it is generally preferable that stringency conditions
used in hybridization assays are high enough such that nucleic acid
molecules that differ from one another at as little as a single SNP
position can be differentiated (e.g., typical SNP hybridization
assays are designed so that hybridization will occur only if one
particular nucleotide is present at a SNP position, but will not
occur if an alternative nucleotide is present at that SNP
position). Such high stringency conditions may be preferable when
using, for example, nucleic acid arrays of allele-specific probes
for SNP detection. Such high stringency conditions are described in
the preceding section, and are well known to those skilled in the
art and can be found in, for example, Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989),
6.3.1-6.3.6.
[0182] In other embodiments, the arrays are used in conjunction
with chemiluminescent detection technology. The following patents
and patent applications, which are all hereby incorporated by
reference, provide additional information pertaining to
chemiluminescent detection: U.S. patent application Ser. Nos.
10/620,332 and 10/620,333 describe chemiluminescent approaches for
microarray detection; U.S. Pat. Nos. 6,124,478, 6,107,024,
5,994,073, 5,981,768, 5,871,938, 5,843,681, 5,800,999, and
5,773,628 describe methods and compositions of dioxetane for
performing chemiluminescent detection; and U.S. published
application US2002/0110828 discloses methods and compositions for
microarray controls.
[0183] In one embodiment of the invention, a nucleic acid array can
comprise an array of probes of about 15-25 nucleotides in length.
In further embodiments, a nucleic acid array can comprise any
number of probes, in which at least one probe is capable of
detecting one or more SNPs disclosed in Table 1 and/or Table 2,
and/or at least one probe comprises a fragment of one of the
sequences selected from the group consisting of those disclosed in
Table 1, Table 2, the Sequence Listing, and sequences complementary
thereto, said fragment comprising at least about 8 consecutive
nucleotides, preferably 10, 12, 15, 16, 18, 20, more preferably 22,
25, 30, 40, 47, 50, 55, 60, 65, 70, 80, 90, 100, or more
consecutive nucleotides (or any other number in-between) and
containing (or being complementary to) a novel SNP allele disclosed
in Table 1 and/or Table 2. In some embodiments, the nucleotide
complementary to the SNP site is within 5, 4, 3, 2, or 1 nucleotide
from the center of the probe, more preferably at the center of said
probe.
[0184] A polynucleotide probe can be synthesized on the surface of
the substrate by using a chemical coupling procedure and an ink jet
application apparatus, as described in PCT application WO95/251116
(Baldeschweiler et al.) which is incorporated herein in its
entirety by reference. In another aspect, a "gridded" array
analogous to a dot (or slot) blot may be used to arrange and link
cDNA fragments or oligonucleotides to the surface of a substrate
using a vacuum system, thermal, UV, mechanical or chemical bonding
procedures. An array, such as those described above, may be
produced by hand or by using available devices (slot blot or dot
blot apparatus), materials (any suitable solid support), and
machines (including robotic instruments), and may contain 8, 24,
96, 384, 1536, 6144 or more polynucleotides, or any other number
which lends itself to the efficient use of commercially available
instrumentation.
[0185] Using such arrays or other kits/systems, the present
invention provides methods of identifying the SNPs disclosed herein
in a test sample. Such methods typically involve incubating a test
sample of nucleic acids with an array comprising one or more probes
corresponding to at least one SNP position of the present
invention, and assaying for binding of a nucleic acid from the test
sample with one or more of the probes. Conditions for incubating a
SNP detection reagent (or a kit/system that employs one or more
such SNP detection reagents) with a test sample vary. Incubation
conditions depend on such factors as the format employed in the
assay, the detection methods employed, and the type and nature of
the detection reagents used in the assay. One skilled in the art
will recognize that any one of the commonly available
hybridization, amplification and array assay formats can readily be
adapted to detect the SNPs disclosed herein.
[0186] A SNP detection kit/system of the present invention may
include components that are used to prepare nucleic acids from a
test sample for the subsequent amplification and/or detection of a
SNP-containing nucleic acid molecule. Such sample preparation
components can be used to produce nucleic acid extracts (including
DNA and/or RNA), proteins or membrane extracts from any bodily
fluids (such as blood, serum, plasma, urine, saliva, phlegm,
gastric juices, semen, tears, sweat, etc.), skin, hair, cells
(especially nucleated cells), biopsies, buccal swabs or tissue
specimens. The test samples used in the above-described methods
will vary based on such factors as the assay format, nature of the
detection method, and the specific tissues, cells or extracts used
as the test sample to be assayed. Methods of preparing nucleic
acids, proteins, and cell extracts are well known in the art and
can be readily adapted to obtain a sample that is compatible with
the system utilized. Automated sample preparation systems for
extracting nucleic acids from a test sample are commercially
available, and examples are Qiagen's BioRobot 9600, Applied
Biosystems' PRISM 6700, and Roche Molecular Systems' COBAS
AmpliPrep System.
[0187] Another form of kit contemplated by the present invention is
a compartmentalized kit. A compartmentalized kit includes any kit
in which reagents are contained in separate containers. Such
containers include, for example, small glass containers, plastic
containers, strips of plastic, glass or paper, or arraying material
such as silica. Such containers allow one to efficiently transfer
reagents from one compartment to another compartment such that the
test samples and reagents are not cross-contaminated, or from one
container to another vessel not included in the kit, and the agents
or solutions of each container can be added in a quantitative
fashion from one compartment to another or to another vessel. Such
containers may include, for example, one or more containers which
will accept the test sample, one or more containers which contain
at least one probe or other SNP detection reagent for detecting one
or more SNPs of the present invention, one or more containers which
contain wash reagents (such as phosphate buffered saline,
Tris-buffers, etc.), and one or more containers which contain the
reagents used to reveal the presence of the bound probe or other
SNP detection reagents. The kit can optionally further comprise
compartments and/or reagents for, for example, nucleic acid
amplification or other enzymatic reactions such as primer extension
reactions, hybridization, ligation, electrophoresis (preferably
capillary electrophoresis), mass spectrometry, and/or laser-induced
fluorescent detection. The kit may also include instructions for
using the kit. Exemplary compartmentalized kits include
microfluidic devices known in the art (see, e.g., Weigl et al.,
"Lab-on-a-chip for drug development", Adv Drug Deliv Rev. 2003 Feb.
24; 55(3):349-77). In such microfluidic devices, the containers may
be referred to as, for example, microfluidic "compartments",
"chambers", or "channels".
[0188] Microfluidic devices, which may also be referred to as
"lab-on-a-chip" systems, biomedical micro-electro-mechanical
systems (bioMEMs), or multicomponent integrated systems, are
exemplary kits/systems of the present invention for analyzing SNPs.
Such systems miniaturize and compartmentalize processes such as
probe/target hybridization, nucleic acid amplification, and
capillary electrophoresis reactions in a single functional device.
Such microfluidic devices typically utilize detection reagents in
at least one aspect of the system, and such detection reagents may
be used to detect one or more SNPs of the present invention. One
example of a microfluidic system is disclosed in U.S. Pat. No.
5,589,136, which describes the integration of PCR amplification and
capillary electrophoresis in chips. Exemplary microfluidic systems
comprise a pattern of microchannels designed onto a glass, silicon,
quartz, or plastic wafer included on a microchip. The movements of
the samples may be controlled by electric, electroosmotic or
hydrostatic forces applied across different areas of the microchip
to create functional microscopic valves and pumps with no moving
parts. Varying the voltage can be used as a means to control the
liquid flow at intersections between the micro-machined channels
and to change the liquid flow rate for pumping across different
sections of the microchip. See, for example, U.S. Pat. No.
6,153,073, Dubrow et al., and U.S. Pat. No. 6,156,181, Parce et
al.
[0189] For genotyping SNPs, an exemplary microfluidic system may
integrate, for example, nucleic acid amplification, primer
extension, capillary electrophoresis, and a detection method such
as laser induced fluorescence detection. In a first step of an
exemplary process for using such an exemplary system, nucleic acid
samples are amplified, preferably by PCR. Then, the amplification
products are subjected to automated primer extension reactions
using ddNTPs (specific fluorescence for each ddNTP) and the
appropriate oligonucleotide primers to carry out primer extension
reactions which hybridize just upstream of the targeted SNP. Once
the extension at the 3' end is completed, the primers are separated
from the unincorporated fluorescent ddNTPs by capillary
electrophoresis. The separation medium used in capillary
electrophoresis can be, for example, polyacrylamide,
polyethyleneglycol or dextran. The incorporated ddNTPs in the
single nucleotide primer extension products are identified by
laser-induced fluorescence detection. Such an exemplary microchip
can be used to process, for example, at least 96 to 384 samples, or
more, in parallel.
[0190] Uses of Nucleic Acid Molecules
[0191] The nucleic acid molecules of the present invention have a
variety of uses, especially in predicting an individual's risk for
developing a cardiovascular disorder (particularly the risk for
experiencing a first or recurrent acute coronary event such as a
myocardial infarction or stroke), for prognosing the progression of
a cardiovascular disorder in an individual (e.g., the severity or
consequences of an acute coronary event), in evaluating the
likelihood of an individual who has a cardiovascular disorder of
responding to treatment of the cardiovascular disorder with statin,
and/or predicting the likelihood that the individual will
experience toxicity or other undesirable side effects from the
statin treatment, etc. For example, the nucleic acid molecules are
useful as hybridization probes, such as for genotyping SNPs in
messenger RNA, transcript, cDNA, genomic DNA, amplified DNA or
other nucleic acid molecules, and for isolating full-length cDNA
and genomic clones encoding the variant peptides disclosed in Table
1 as well as their orthologs.
[0192] A probe can hybridize to any nucleotide sequence along the
entire length of a nucleic acid molecule provided in Table 1 and/or
Table 2. Preferably, a probe of the present invention hybridizes to
a region of a target sequence that encompasses a SNP position
indicated in Table 1 and/or Table 2. More preferably, a probe
hybridizes to a SNP-containing target sequence in a
sequence-specific manner such that it distinguishes the target
sequence from other nucleotide sequences which vary from the target
sequence only by which nucleotide is present at the SNP site. Such
a probe is particularly useful for detecting the presence of a
SNP-containing nucleic acid in a test sample, or for determining
which nucleotide (allele) is present at a particular SNP site
(i.e., genotyping the SNP site).
[0193] A nucleic acid hybridization probe may be used for
determining the presence, level, form, and/or distribution of
nucleic acid expression. The nucleic acid whose level is determined
can be DNA or RNA. Accordingly, probes specific for the SNPs
described herein can be used to assess the presence, expression
and/or gene copy number in a given cell, tissue, or organism. These
uses are relevant for diagnosis of disorders involving an increase
or decrease in gene expression relative to normal levels. In vitro
techniques for detection of mRNA include, for example, Northern
blot hybridizations and in situ hybridizations. In vitro techniques
for detecting DNA include Southern blot hybridizations and in situ
hybridizations (Sambrook and Russell, 2000, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y.).
[0194] Probes can be used as part of a diagnostic test kit for
identifying cells or tissues in which a variant protein is
expressed, such as by measuring the level of a variant
protein-encoding nucleic acid (e.g., mRNA) in a sample of cells
from a subject or determining if a polynucleotide contains a SNP of
interest.
[0195] Thus, the nucleic acid molecules of the invention can be
used as hybridization probes to detect the SNPs disclosed herein,
thereby determining whether an individual with the polymorphisms is
likely or unlikely to develop a cardiovascular disorder such as an
acute coronary event, or the likelihood that an individual will
respond positively to statin treatment of a cardiovascular
disorder. Detection of a SNP associated with a disease phenotype
provides a diagnostic tool for an active disease and/or genetic
predisposition to the disease.
[0196] Furthermore, the nucleic acid molecules of the invention are
therefore useful for detecting a gene (gene information is
disclosed in Table 2, for example) which contains a SNP disclosed
herein and/or products of such genes, such as expressed mRNA
transcript molecules (transcript information is disclosed in Table
1, for example), and are thus useful for detecting gene expression.
The nucleic acid molecules can optionally be implemented in, for
example, an array or kit format for use in detecting gene
expression.
[0197] The nucleic acid molecules of the invention are also useful
as primers to amplify any given region of a nucleic acid molecule,
particularly a region containing a SNP identified in Table 1 and/or
Table 2.
[0198] The nucleic acid molecules of the invention are also useful
for constructing recombinant vectors (described in greater detail
below). Such vectors include expression vectors that express a
portion of, or all of, any of the variant peptide sequences
provided in Table 1. Vectors also include insertion vectors, used
to integrate into another nucleic acid molecule sequence, such as
into the cellular genome, to alter in situ expression of a gene
and/or gene product. For example, an endogenous coding sequence can
be replaced via homologous recombination with all or part of the
coding region containing one or more specifically introduced
SNPs.
[0199] The nucleic acid molecules of the invention are also useful
for expressing antigenic portions of the variant proteins,
particularly antigenic portions that contain a variant amino acid
sequence (e.g., an amino acid substitution) caused by a SNP
disclosed in Table 1 and/or Table 2.
[0200] The nucleic acid molecules of the invention are also useful
for constructing vectors containing a gene regulatory region of the
nucleic acid molecules of the present invention.
[0201] The nucleic acid molecules of the invention are also useful
for designing ribozymes corresponding to all, or a part, of an mRNA
molecule expressed from a SNP-containing nucleic acid molecule
described herein.
[0202] The nucleic acid molecules of the invention are also useful
for constructing host cells expressing a part, or all, of the
nucleic acid molecules and variant peptides.
[0203] The nucleic acid molecules of the invention are also useful
for constructing transgenic animals expressing all, or a part, of
the nucleic acid molecules and variant peptides. The production of
recombinant cells and transgenic animals having nucleic acid
molecules which contain the SNPs disclosed in Table 1 and/or Table
2 allow, for example, effective clinical design of treatment
compounds and dosage regimens.
[0204] The nucleic acid molecules of the invention are also useful
in assays for drug screening to identify compounds that, for
example, modulate nucleic acid expression.
[0205] The nucleic acid molecules of the invention are also useful
in gene therapy in patients whose cells have aberrant gene
expression. Thus, recombinant cells, which include a patient's
cells that have been engineered ex vivo and returned to the
patient, can be introduced into an individual where the recombinant
cells produce the desired protein to treat the individual.
[0206] SNP Genotyping Methods
[0207] The process of determining which specific nucleotide (i.e.,
allele) is present at each of one or more SNP positions, such as a
SNP position in a nucleic acid molecule disclosed in Table 1 and/or
Table 2, is referred to as SNP genotyping. The present invention
provides methods of SNP genotyping, such as for use in evaluating
an individual's risk for developing a cardiovascular
disease--particularly an acute coronary event (such as myocardial
infarction or stroke) and for evaluating an individual's prognosis
for disease severity and recovery, for predicting the likelihood
that an individual who has previously experienced an acute coronary
event will experience one or more recurrent acute coronary events,
for implementing a preventive or treatment regimen for an
individual based on that individual having an increased
susceptibility for developing a cardiovascular disorder (e.g.,
increased risk for experiencing one or more myocardial infarctions
or strokes), in evaluating an individual's likelihood of responding
to statin treatment for cardiovascular disease, in selecting a
treatment regimen (e.g., in deciding whether or not to administer
statin treatment to an individual having a cardiovascular disease,
or in formulating or selecting a particular statin-based treatment
regimen such as dosage and/or frequency of administration of statin
treatment or choosing which form/type of statin to be administered
such as a particular pharmaceutical composition or compound, etc.),
determining the likelihood of experiencing toxicity or other
undesirable side effects from the statin treatment, or selecting
individuals for a clinical trial of a statin (e.g., selecting
individuals to participate in the trial who are most likely to
respond positively from the statin treatment), etc.
[0208] Nucleic acid samples can be genotyped to determine which
allele(s) is/are present at any given genetic region (e.g., SNP
position) of interest by methods well known in the art. The
neighboring sequence can be used to design SNP detection reagents
such as oligonucleotide probes, which may optionally be implemented
in a kit format. Exemplary SNP genotyping methods are described in
Chen et al., "Single nucleotide polymorphism genotyping:
biochemistry, protocol, cost and throughput", Pharmacogenomics J.
2003; 3(2):77-96; Kwok et al., "Detection of single nucleotide
polymorphisms", Curr Issues Mol Biol. 2003 April; 5(2):43-60; Shi,
"Technologies for individual genotyping: detection of genetic
polymorphisms in drug targets and disease genes", Am J
Pharmacogenomics. 2002; 2(3):197-205; and Kwok, "Methods for
genotyping single nucleotide polymorphisms", Annu Rev Genomics Hum
Genet 2001; 2:235-58. Exemplary techniques for high-throughput SNP
genotyping are described in Marnellos, "High-throughput SNP
analysis for genetic association studies", Curr Opin Drug Discov
Devel. 2003 May; 6(3):317-21. Common SNP genotyping methods
include, but are not limited to, TaqMan assays, molecular beacon
assays, nucleic acid arrays, allele-specific primer extension,
allele-specific PCR, arrayed primer extension, homogeneous primer
extension assays, primer extension with detection by mass
spectrometry, pyrosequencing, multiplex primer extension sorted on
genetic arrays, ligation with rolling circle amplification,
homogeneous ligation, OLA (U.S. Pat. No. 4,988,167), multiplex
ligation reaction sorted on genetic arrays, restriction-fragment
length polymorphism, single base extension-tag assays, and the
Invader assay. Such methods may be used in combination with
detection mechanisms such as, for example, luminescence or
chemiluminescence detection, fluorescence detection, time-resolved
fluorescence detection, fluorescence resonance energy transfer,
fluorescence polarization, mass spectrometry, and electrical
detection.
[0209] Various methods for detecting polymorphisms include, but are
not limited to, methods in which protection from cleavage agents is
used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes
(Myers et al., Science 230:1242 (1985); Cotton et al., PNAS 85:4397
(1988); and Saleeba et al., Meth. Enzymol. 217:286-295 (1992)),
comparison of the electrophoretic mobility of variant and wild type
nucleic acid molecules (Orita et al., PNAS 86:2766 (1989); Cotton
et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet.
Anal. Tech. Appl. 9:73-79 (1992)), and assaying the movement of
polymorphic or wild-type fragments in polyacrylamide gels
containing a gradient of denaturant using denaturing gradient gel
electrophoresis (DGGE) (Myers et al., Nature 313:495 (1985)).
Sequence variations at specific locations can also be assessed by
nuclease protection assays such as RNase and S1 protection or
chemical cleavage methods.
[0210] In a preferred embodiment, SNP genotyping is performed using
the TaqMan assay, which is also known as the 5' nuclease assay
(U.S. Pat. Nos. 5,210,015 and 5,538,848). The TaqMan assay detects
the accumulation of a specific amplified product during PCR. The
TaqMan assay utilizes an oligonucleotide probe labeled with a
fluorescent reporter dye and a quencher dye. The reporter dye is
excited by irradiation at an appropriate wavelength, it transfers
energy to the quencher dye in the same probe via a process called
fluorescence resonance energy transfer (FRET). When attached to the
probe, the excited reporter dye does not emit a signal. The
proximity of the quencher dye to the reporter dye in the intact
probe maintains a reduced fluorescence for the reporter. The
reporter dye and quencher dye may be at the 5' most and the 3' most
ends, respectively, or vice versa. Alternatively, the reporter dye
may be at the 5' or 3' most end while the quencher dye is attached
to an internal nucleotide, or vice versa. In yet another
embodiment, both the reporter and the quencher may be attached to
internal nucleotides at a distance from each other such that
fluorescence of the reporter is reduced.
[0211] During PCR, the 5' nuclease activity of DNA polymerase
cleaves the probe, thereby separating the reporter dye and the
quencher dye and resulting in increased fluorescence of the
reporter. Accumulation of PCR product is detected directly by
monitoring the increase in fluorescence of the reporter dye. The
DNA polymerase cleaves the probe between the reporter dye and the
quencher dye only if the probe hybridizes to the target
SNP-containing template which is amplified during PCR, and the
probe is designed to hybridize to the target SNP site only if a
particular SNP allele is present.
[0212] Preferred TaqMan primer and probe sequences can readily be
determined using the SNP and associated nucleic acid sequence
information provided herein. A number of computer programs, such as
Primer Express (Applied Biosystems, Foster City, Calif.), can be
used to rapidly obtain optimal primer/probe sets. It will be
apparent to one of skill in the art that such primers and probes
for detecting the SNPs of the present invention are useful in
screening for individuals who are susceptible to developing a
cardiovascular disorder (e.g., an acute coronary event) or in
screening individuals who have a cardiovascular disorder for their
likelihood of responding to statin treatment. These probes and
primers can be readily incorporated into a kit format. The present
invention also includes modifications of the Taqman assay well
known in the art such as the use of Molecular Beacon probes (U.S.
Pat. Nos. 5,118,801 and 5,312,728) and other variant formats (U.S.
Pat. Nos. 5,866,336 and 6,117,635).
[0213] Another preferred method for genotyping the SNPs of the
present invention is the use of two oligonucleotide probes in an
OLA (see, e.g., U.S. Pat. No. 4,988,617). In this method, one probe
hybridizes to a segment of a target nucleic acid with its 3' most
end aligned with the SNP site. A second probe hybridizes to an
adjacent segment of the target nucleic acid molecule directly 3' to
the first probe. The two juxtaposed probes hybridize to the target
nucleic acid molecule, and are ligated in the presence of a linking
agent such as a ligase if there is perfect complementarity between
the 3' most nucleotide of the first probe with the SNP site. If
there is a mismatch, ligation would not occur. After the reaction,
the ligated probes are separated from the target nucleic acid
molecule, and detected as indicators of the presence of a SNP.
[0214] The following patents, patent applications, and published
international patent applications, which are all hereby
incorporated by reference, provide additional information
pertaining to techniques for carrying out various types of OLA:
U.S. Pat. Nos. 6,027,889, 6,268,148, 5,494,810, 5,830,711, and
6,054,564 describe OLA strategies for performing SNP detection; WO
97/31256 and WO 00/56927 describe OLA strategies for performing SNP
detection using universal arrays, wherein a zipcode sequence can be
introduced into one of the hybridization probes, and the resulting
product, or amplified product, hybridized to a universal zip code
array; U.S. application US01/17329 (and Ser. No. 09/584,905)
describes OLA (or LDR) followed by PCR, wherein zipcodes are
incorporated into OLA probes, and amplified PCR products are
determined by electrophoretic or universal zipcode array readout;
U.S. applications 60/427818, 60/445636, and 60/445494 describe
SNPlex methods and software for multiplexed SNP detection using OLA
followed by PCR, wherein zipcodes are incorporated into OLA probes,
and amplified PCR products are hybridized with a zipchute reagent,
and the identity of the SNP determined from electrophoretic readout
of the zipchute. In some embodiments, OLA is carried out prior to
PCR (or another method of nucleic acid amplification). In other
embodiments, PCR (or another method of nucleic acid amplification)
is carried out prior to OLA.
[0215] Another method for SNP genotyping is based on mass
spectrometry. Mass spectrometry takes advantage of the unique mass
of each of the four nucleotides of DNA. SNPs can be unambiguously
genotyped by mass spectrometry by measuring the differences in the
mass of nucleic acids having alternative SNP alleles. MALDI-TOF
(Matrix Assisted Laser Desorption Ionization--Time of Flight) mass
spectrometry technology is preferred for extremely precise
determinations of molecular mass, such as SNPs. Numerous approaches
to SNP analysis have been developed based on mass spectrometry.
Preferred mass spectrometry-based methods of SNP genotyping include
primer extension assays, which can also be utilized in combination
with other approaches, such as traditional gel-based formats and
microarrays.
[0216] Typically, the primer extension assay involves designing and
annealing a primer to a template PCR amplicon upstream (5') from a
target SNP position. A mix of dideoxynucleotide triphosphates
(ddNTPs) and/or deoxynucleotide triphosphates (dNTPs) are added to
a reaction mixture containing template (e.g., a SNP-containing
nucleic acid molecule which has typically been amplified, such as
by PCR), primer, and DNA polymerase. Extension of the primer
terminates at the first position in the template where a nucleotide
complementary to one of the ddNTPs in the mix occurs. The primer
can be either immediately adjacent (i.e., the nucleotide at the 3'
end of the primer hybridizes to the nucleotide next to the target
SNP site) or two or more nucleotides removed from the SNP position.
If the primer is several nucleotides removed from the target SNP
position, the only limitation is that the template sequence between
the 3' end of the primer and the SNP position cannot contain a
nucleotide of the same type as the one to be detected, or this will
cause premature termination of the extension primer. Alternatively,
if all four ddNTPs alone, with no dNTPs, are added to the reaction
mixture, the primer will always be extended by only one nucleotide,
corresponding to the target SNP position. In this instance, primers
are designed to bind one nucleotide upstream from the SNP position
(i.e., the nucleotide at the 3' end of the primer hybridizes to the
nucleotide that is immediately adjacent to the target SNP site on
the 5' side of the target SNP site). Extension by only one
nucleotide is preferable, as it minimizes the overall mass of the
extended primer, thereby increasing the resolution of mass
differences between alternative SNP nucleotides. Furthermore,
mass-tagged ddNTPs can be employed in the primer extension
reactions in place of unmodified ddNTPs. This increases the mass
difference between primers extended with these ddNTPs, thereby
providing increased sensitivity and accuracy, and is particularly
useful for typing heterozygous base positions. Mass-tagging also
alleviates the need for intensive sample-preparation procedures and
decreases the necessary resolving power of the mass
spectrometer.
[0217] The extended primers can then be purified and analyzed by
MALDI-TOF mass spectrometry to determine the identity of the
nucleotide present at the target SNP position. In one method of
analysis, the products from the primer extension reaction are
combined with light absorbing crystals that form a matrix. The
matrix is then hit with an energy source such as a laser to ionize
and desorb the nucleic acid molecules into the gas-phase. The
ionized molecules are then ejected into a flight tube and
accelerated down the tube towards a detector. The time between the
ionization event, such as a laser pulse, and collision of the
molecule with the detector is the time of flight of that molecule.
The time of flight is precisely correlated with the mass-to-charge
ratio (m/z) of the ionized molecule. Ions with smaller m/z travel
down the tube faster than ions with larger m/z and therefore the
lighter ions reach the detector before the heavier ions. The
time-of-flight is then converted into a corresponding, and highly
precise, m/z. In this manner, SNPs can be identified based on the
slight differences in mass, and the corresponding time of flight
differences, inherent in nucleic acid molecules having different
nucleotides at a single base position. For further information
regarding the use of primer extension assays in conjunction with
MALDI-TOF mass spectrometry for SNP genotyping, see, e.g., Wise et
al., "A standard protocol for single nucleotide primer extension in
the human genome using matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry", Rapid Commun Mass Spectrom.
2003; 17(11):1195-202.
[0218] The following references provide further information
describing mass spectrometry-based methods for SNP genotyping:
Bocker, "SNP and mutation discovery using base-specific cleavage
and MALDI-TOF mass spectrometry", Bioinformatics. 2003 July; 19
Suppl 1:144-153; Storm et al., "MALDI-TOF mass spectrometry-based
SNP genotyping", Methods Mol Biol. 2003; 212:241-62; Jurinke et
al., "The use of MassARRAY technology for high throughput
genotyping", Adv Biochem Eng Biotechnol. 2002; 77:57-74; and
Jurinke et al., "Automated genotyping using the DNA MassArray
technology", Methods Mol Biol. 2002; 187:179-92.
[0219] SNPs can also be scored by direct DNA sequencing. A variety
of automated sequencing procedures can be utilized ((1995)
Biotechniques 19:448), including sequencing by mass spectrometry
(see, e.g., PCT International Publication No. WO94/16101; Cohen et
al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl.
Biochem. Biotechnol. 38:147-159 (1993)). The nucleic acid sequences
of the present invention enable one of ordinary skill in the art to
readily design sequencing primers for such automated sequencing
procedures. Commercial instrumentation, such as the Applied
Biosystems 377, 3100, 3700, 3730, and 3730x1 DNA Analyzers (Foster
City, Calif.), is commonly used in the art for automated
sequencing.
[0220] Other methods that can be used to genotype the SNPs of the
present invention include single-strand conformational polymorphism
(SSCP), and denaturing gradient gel electrophoresis (DGGE) (Myers
et al., Nature 313:495 (1985)). SSCP identifies base differences by
alteration in electrophoretic migration of single stranded PCR
products, as described in Orita et al., Proc. Nat. Acad.
Single-stranded PCR products can be generated by heating or
otherwise denaturing double stranded PCR products. Single-stranded
nucleic acids may refold or form secondary structures that are
partially dependent on the base sequence. The different
electrophoretic mobilities of single-stranded amplification
products are related to base-sequence differences at SNP positions.
DGGE differentiates SNP alleles based on the different
sequence-dependent stabilities and melting properties inherent in
polymorphic DNA and the corresponding differences in
electrophoretic migration patterns in a denaturing gradient gel
(Erlich, ed., PCR Technology, Principles and Applications for DNA
Amplification, W.H. Freeman and Co, New York, 1992, Chapter 7).
[0221] Sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can
also be used to score SNPs based on the development or loss of a
ribozyme cleavage site. Perfectly matched sequences can be
distinguished from mismatched sequences by nuclease cleavage
digestion assays or by differences in melting temperature. If the
SNP affects a restriction enzyme cleavage site, the SNP can be
identified by alterations in restriction enzyme digestion patterns,
and the corresponding changes in nucleic acid fragment lengths
determined by gel electrophoresis
[0222] SNP genotyping can include the steps of, for example,
collecting a biological sample from a human subject (e.g., sample
of tissues, cells, fluids, secretions, etc.), isolating nucleic
acids (e.g., genomic DNA, mRNA or both) from the cells of the
sample, contacting the nucleic acids with one or more primers which
specifically hybridize to a region of the isolated nucleic acid
containing a target SNP under conditions such that hybridization
and amplification of the target nucleic acid region occurs, and
determining the nucleotide present at the SNP position of interest,
or, in some assays, detecting the presence or absence of an
amplification product (assays can be designed so that hybridization
and/or amplification will only occur if a particular SNP allele is
present or absent). In some assays, the size of the amplification
product is detected and compared to the length of a control sample;
for example, deletions and insertions can be detected by a change
in size of the amplified product compared to a normal genotype.
[0223] SNP genotyping is useful for numerous practical
applications, as described below. Examples of such applications
include, but are not limited to, SNP-disease association analysis,
disease predisposition screening, disease diagnosis, disease
prognosis, disease progression monitoring, determining therapeutic
strategies based on an individual's genotype ("pharmacogenomics"),
developing therapeutic agents based on SNP genotypes associated
with a disease or likelihood of responding to a drug, stratifying a
patient population for clinical trial for a treatment regimen,
predicting the likelihood that an individual will experience toxic
side effects from a therapeutic agent, and human identification
applications such as forensics.
Analysis of Genetic Association Between SNPS and Phenotypic
Traits
[0224] SNP genotyping for disease diagnosis, disease predisposition
screening, disease prognosis, determining drug responsiveness
(pharmacogenomics), drug toxicity screening, and other uses
described herein, typically relies on initially establishing a
genetic association between one or more specific SNPs and the
particular phenotypic traits of interest.
[0225] Different study designs may be used for genetic association
studies (Modern Epidemiology, Lippincott Williams & Wilkins
(1998), 609-622). Observational studies are most frequently carried
out in which the response of the patients is not interfered with.
The first type of observational study identifies a sample of
persons in whom the suspected cause of the disease is present and
another sample of persons in whom the suspected cause is absent,
and then the frequency of development of disease in the two samples
is compared. These sampled populations are called cohorts, and the
study is a prospective study. The other type of observational study
is case-control or a retrospective study. In typical case-control
studies, samples are collected from individuals with the phenotype
of interest (cases) such as certain manifestations of a disease,
and from individuals without the phenotype (controls) in a
population (target population) that conclusions are to be drawn
from. Then the possible causes of the disease are investigated
retrospectively. As the time and costs of collecting samples in
case-control studies are considerably less than those for
prospective studies, case-control studies are the more commonly
used study design in genetic association studies, at least during
the exploration and discovery stage.
[0226] In both types of observational studies, there may be
potential confounding factors that should be taken into
consideration. Confounding factors are those that are associated
with both the real cause(s) of the disease and the disease itself,
and they include demographic information such as age, gender,
ethnicity as well as environmental factors. When confounding
factors are not matched in cases and controls in a study, and are
not controlled properly, spurious association results can arise. If
potential confounding factors are identified, they should be
controlled for by analysis methods explained below.
[0227] In a genetic association study, the cause of interest to be
tested is a certain allele or a SNP or a combination of alleles or
a haplotype from several SNPs. Thus, tissue specimens (e.g., whole
blood) from the sampled individuals may be collected and genomic
DNA genotyped for the SNP(s) of interest. In addition to the
phenotypic trait of interest, other information such as demographic
(e.g., age, gender, ethnicity, etc.), clinical, and environmental
information that may influence the outcome of the trait can be
collected to further characterize and define the sample set. In
many cases, these factors are known to be associated with diseases
and/or SNP allele frequencies. There are likely gene-environment
and/or gene-gene interactions as well. Analysis methods to address
gene-environment and gene-gene interactions (for example, the
effects of the presence of both susceptibility alleles at two
different genes can be greater than the effects of the individual
alleles at two genes combined) are discussed below.
[0228] After all the relevant phenotypic and genotypic information
has been obtained, statistical analyses are carried out to
determine if there is any significant correlation between the
presence of an allele or a genotype with the phenotypic
characteristics of an individual. Preferably, data inspection and
cleaning are first performed before carrying out statistical tests
for genetic association. Epidemiological and clinical data of the
samples can be summarized by descriptive statistics with tables and
graphs. Data validation is preferably performed to check for data
completion, inconsistent entries, and outliers. Chi-square tests
and t-tests (Wilcoxon rank-sum tests if distributions are not
normal) may then be used to check for significant differences
between cases and controls for discrete and continuous variables,
respectively. To ensure genotyping quality, Hardy-Weinberg
disequilibrium tests can be performed on cases and controls
separately. Significant deviation from Hardy-Weinberg equilibrium
(HWE) in both cases and controls for individual markers can be
indicative of genotyping errors. If HWE is violated in a majority
of markers, it is indicative of population substructure that should
be further investigated. Moreover, Hardy-Weinberg disequilibrium in
cases only can indicate genetic association of the markers with the
disease (Genetic Data Analysis, Weir B., Sinauer (1990)).
[0229] To test whether an allele of a single SNP is associated with
the case or control status of a phenotypic trait, one skilled in
the art can compare allele frequencies in cases and controls.
Standard chi-square tests and Fisher exact tests can be carried out
on a 2.times.2 table (2 SNP alleles.times.2 outcomes in the
categorical trait of interest). To test whether genotypes of a SNP
are associated, chi-square tests can be carried out on a 3.times.2
table (3 genotypes.times.2 outcomes). Score tests are also carried
out for genotypic association to contrast the three genotypic
frequencies (major homozygotes, heterozygotes and minor
homozygotes) in cases and controls, and to look for trends using 3
different modes of inheritance, namely dominant (with contrast
coefficients 2, -1, -1), additive (with contrast coefficients 1, 0,
-1) and recessive (with contrast coefficients 1, 1, -2). Odds
ratios for minor versus major alleles, and odds ratios for
heterozygote and homozygote variants versus the wild type genotypes
are calculated with the desired confidence limits, usually 95%.
[0230] In order to control for confounders and to test for
interaction and effect modifiers, stratified analyses may be
performed using stratified factors that are likely to be
confounding, including demographic information such as age,
ethnicity, and gender, or an interacting element or effect
modifier, such as a known major gene (e.g., APOE for Alzheimer's
disease or HLA genes for autoimmune diseases), or environmental
factors such as smoking in lung cancer. Stratified association
tests may be carried out using Cochran-Mantel-Haenszel tests that
take into account the ordinal nature of genotypes with 0, 1, and 2
variant alleles. Exact tests by StatXact may also be performed when
computationally possible. Another way to adjust for confounding
effects and test for interactions is to perform stepwise multiple
logistic regression analysis using statistical packages such as SAS
or R. Logistic regression is a model-building technique in which
the best fitting and most parsimonious model is built to describe
the relation between the dichotomous outcome (for instance, getting
a certain disease or not) and a set of independent variables (for
instance, genotypes of different associated genes, and the
associated demographic and environmental factors). The most common
model is one in which the logit transformation of the odds ratios
is expressed as a linear combination of the variables (main
effects) and their cross-product terms (interactions) (Applied
Logistic Regression, Hosmer and Lemeshow, Wiley (2000)). To test
whether a certain variable or interaction is significantly
associated with the outcome, coefficients in the model are first
estimated and then tested for statistical significance of their
departure from zero.
[0231] In addition to performing association tests one marker at a
time, haplotype association analysis may also be performed to study
a number of markers that are closely linked together. Haplotype
association tests can have better power than genotypic or allelic
association tests when the tested markers are not the
disease-causing mutations themselves but are in linkage
disequilibrium with such mutations. The test will even be more
powerful if the disease is indeed caused by a combination of
alleles on a haplotype (e.g., APOE is a haplotype formed by 2 SNPs
that are very close to each other). In order to perform haplotype
association effectively, marker-marker linkage disequilibrium
measures, both D' and R.sup.2, are typically calculated for the
markers within a gene to elucidate the haplotype structure. Recent
studies (Daly et al, Nature Genetics, 29, 232-235, 2001) in linkage
disequilibrium indicate that SNPs within a gene are organized in
block pattern, and a high degree of linkage disequilibrium exists
within blocks and very little linkage disequilibrium exists between
blocks. Haplotype association with the disease status can be
performed using such blocks once they have been elucidated.
[0232] Haplotype association tests can be carried out in a similar
fashion as the allelic and genotypic association tests. Each
haplotype in a gene is analogous to an allele in a multi-allelic
marker. One skilled in the art can either compare the haplotype
frequencies in cases and controls or test genetic association with
different pairs of haplotypes. It has been proposed (Schaid et al,
Am. J. Hum. Genet., 70, 425-434, 2002) that score tests can be done
on haplotypes using the program "haplo.score". In that method,
haplotypes are first inferred by EM algorithm and score tests are
carried out with a generalized linear model (GLM) framework that
allows the adjustment of other factors.
[0233] An important decision in the performance of genetic
association tests is the determination of the significance level at
which significant association can be declared when the p-value of
the tests reaches that level. In an exploratory analysis where
positive hits will be followed up in subsequent confirmatory
testing, an unadjusted p-value <0.1 (a significance level on the
lenient side) may be used for generating hypotheses for significant
association of a SNP with certain phenotypic characteristics of a
disease. It is preferred that a p-value <0.05 (a significance
level traditionally used in the art) is achieved in order for a SNP
to be considered to have an association with a disease. It is more
preferred that a p-value <0.01 (a significance level on the
stringent side) is achieved for an association to be declared. When
hits are followed up in confirmatory analyses in more samples of
the same source or in different samples from different sources,
adjustment for multiple testing will be performed as to avoid
excess number of hits while maintaining the experiment-wise error
rates at 0.05. While there are different methods to adjust for
multiple testing to control for different kinds of error rates, a
commonly used but rather conservative method is Bonferroni
correction to control the experiment-wise or family-wise error rate
(Multiple comparisons and multiple tests, Westfall et al, SAS
Institute (1999)). Permutation tests to control for the false
discovery rates, FDR, can be more powerful (Benjamini and Hochberg,
Journal of the Royal Statistical Society, Series B 57, 1289-1300,
1995, Resampling-based Multiple Testing, Westfall and Young, Wiley
(1993)). Such methods to control for multiplicity would be
preferred when the tests are dependent and controlling for false
discovery rates is sufficient as opposed to controlling for the
experiment-wise error rates.
[0234] In replication studies using samples from different
populations after statistically significant markers have been
identified in the exploratory stage, meta-analyses can then be
performed by combining evidence of different studies (Modern
Epidemiology, Lippincott Williams & Wilkins, 1998, 643-673). If
available, association results known in the art for the same SNPs
can be included in the meta-analyses.
[0235] Since both genotyping and disease status classification can
involve errors, sensitivity analyses may be performed to see how
odds ratios and p-values would change upon various estimates on
genotyping and disease classification error rates.
[0236] It has been well known that subpopulation-based sampling
bias between cases and controls can lead to spurious results in
case-control association studies (Ewens and Spielman, Am. J. Hum.
Genet. 62, 450-458, 1995) when prevalence of the disease is
associated with different subpopulation groups. Such bias can also
lead to a loss of statistical power in genetic association studies.
To detect population stratification, Pritchard and Rosenberg
(Pritchard et al. Am. J. Hum. Gen. 1999, 65:220-228) suggested
typing markers that are unlinked to the disease and using results
of association tests on those markers to determine whether there is
any population stratification. When stratification is detected, the
genomic control (GC) method as proposed by Devlin and Roeder
(Devlin et al. Biometrics 1999, 55:997-1004) can be used to adjust
for the inflation of test statistics due to population
stratification. GC method is robust to changes in population
structure levels as well as being applicable to DNA pooling designs
(Devlin et al. Genet. Epidem. 20001, 21:273-284).
[0237] While Pritchard's method recommended using 15-20 unlinked
microsatellite markers, it suggested using more than 30 biallelic
markers to get enough power to detect population stratification.
For the GC method, it has been shown (Bacanu et al. Am. J. Hum.
Genet. 2000, 66:1933-1944) that about 60-70 biallelic markers are
sufficient to estimate the inflation factor for the test statistics
due to population stratification. Hence, 70 intergenic SNPs can be
chosen in unlinked regions as indicated in a genome scan (Kehoe et
al. Hum. Mol. Genet. 1999, 8:237-245).
[0238] Once individual risk factors, genetic or non-genetic, have
been found for the predisposition to disease, the next step is to
set up a classification/prediction scheme to predict the category
(for instance, disease or no-disease) that an individual will be in
depending on his genotypes of associated SNPs and other non-genetic
risk factors. Logistic regression for discrete trait and linear
regression for continuous trait are standard techniques for such
tasks (Applied Regression Analysis, Draper and Smith, Wiley
(1998)). Moreover, other techniques can also be used for setting up
classification. Such techniques include, but are not limited to,
MART, CART, neural network, and discriminant analyses that are
suitable for use in comparing the performance of different methods
(The Elements of Statistical Learning, Hastie, Tibshirani &
Friedman, Springer (2002)).
[0239] Disease Diagnosis and Predisposition Screening
[0240] Information on association/correlation between genotypes and
disease-related phenotypes can be exploited in several ways. For
example, in the case of a highly statistically significant
association between one or more SNPs with predisposition to a
disease for which treatment is available, detection of such a
genotype pattern in an individual may justify immediate
administration of treatment, or at least the institution of regular
monitoring of the individual. Detection of the susceptibility
alleles associated with serious disease in a couple contemplating
having children may also be valuable to the couple in their
reproductive decisions. In the case of a weaker but still
statistically significant association between a SNP and a human
disease, immediate therapeutic intervention or monitoring may not
be justified after detecting the susceptibility allele or SNP.
Nevertheless, the subject can be motivated to begin simple
life-style changes (e.g., diet, exercise) that can be accomplished
at little or no cost to the individual but would confer potential
benefits in reducing the risk of developing conditions for which
that individual may have an increased risk by virtue of having the
susceptibility allele(s).
[0241] The SNPs of the invention may contribute to cardiovascular
disorders such as acute coronary events, or to responsiveness of an
individual to statin treatment, in different ways. Some
polymorphisms occur within a protein coding sequence and contribute
to disease phenotype by affecting protein structure. Other
polymorphisms occur in noncoding regions but may exert phenotypic
effects indirectly via influence on, for example, replication,
transcription, and/or translation. A single SNP may affect more
than one phenotypic trait. Likewise, a single phenotypic trait may
be affected by multiple SNPs in different genes.
[0242] As used herein, the terms "diagnose", "diagnosis", and
"diagnostics" include, but are not limited to any of the following:
detection of a cardiovascular disorders that an individual may
presently have, predisposition/susceptibility screening (e.g.,
determining whether an individual has an increased risk of
experiencing an acute coronary event in the future, or determining
whether an individual has a decreased risk of experiencing an acute
coronary event in the future), determining a particular type or
subclass of cardiovascular disorder in an individual known to
currently have or to have previously experienced a cardiovascular
disorder, confirming or reinforcing a previously made diagnosis of
a cardiovascular disorder, evaluating an individual's likelihood of
responding to statin treatment for cardiovascular disorders,
predisposition screening (e.g., evaluating an individual's
likelihood of responding to statin treatment if the individual were
to develop a cardiovascular disorder in the future), determining a
particular type or subclass of responder/non-responder in an
individual known to respond or not respond to statin treatment,
confirming or reinforcing a previously made classification of an
individual as a responder/non-responder to statin treatment,
pharmacogenomic evaluation of an individual to determine which
therapeutic strategy that individual is most likely to positively
respond to or to predict whether a patient is likely to respond to
a particular treatment such as statin treatment, predicting whether
a patient is likely to experience toxic effects from a particular
treatment or therapeutic compound, and evaluating the future
prognosis of an individual having a cardiovascular disorder. Such
diagnostic uses are based on the SNPs individually or in a unique
combination or SNP haplotypes of the present invention.
[0243] Haplotypes are particularly useful in that, for example,
fewer SNPs can be genotyped to determine if a particular genomic
region harbors a locus that influences a particular phenotype, such
as in linkage disequilibrium-based SNP association analysis.
[0244] Linkage disequilibrium (LD) refers to the co-inheritance of
alleles (e.g., alternative nucleotides) at two or more different
SNP sites at frequencies greater than would be expected from the
separate frequencies of occurrence of each allele in a given
population. The expected frequency of co-occurrence of two alleles
that are inherited independently is the frequency of the first
allele multiplied by the frequency of the second allele. Alleles
that co-occur at expected frequencies are said to be in "linkage
equilibrium". In contrast, LD refers to any non-random genetic
association between allele(s) at two or more different SNP sites,
which is generally due to the physical proximity of the two loci
along a chromosome. LD can occur when two or more SNPs sites are in
close physical proximity to each other on a given chromosome and
therefore alleles at these SNP sites will tend to remain
unseparated for multiple generations with the consequence that a
particular nucleotide (allele) at one SNP site will show a
non-random association with a particular nucleotide (allele) at a
different SNP site located nearby. Hence, genotyping one of the SNP
sites will give almost the same information as genotyping the other
SNP site that is in LD.
[0245] Various degrees of LD can be encountered between two or more
SNPs with the result being that some SNPs are more closely
associated (i.e., in stronger LD) than others. Furthermore, the
physical distance over which LD extends along a chromosome differs
between different regions of the genome, and therefore the degree
of physical separation between two or more SNP sites necessary for
LD to occur can differ between different regions of the genome.
[0246] For diagnostic purposes and similar uses, if a particular
SNP site is found to be useful for, for example, predicting an
individual's susceptibility to an acute coronary event or an
individual's response to statin treatment, then the skilled artisan
would recognize that other SNP sites which are in LD with this SNP
site would also be useful for predicting an individual's response
to statin treatment. Various degrees of LD can be encountered
between two or more SNPs with the result being that some SNPs are
more closely associated (i.e., in stronger LD) than others.
Furthermore, the physical distance over which LD extends along a
chromosome differs between different regions of the genome, and
therefore the degree of physical separation between two or more SNP
sites necessary for LD to occur can differ between different
regions of the genome. Thus, polymorphisms (e.g., SNPs and/or
haplotypes) that are not the actual disease-causing (causative)
polymorphisms, but are in LD with such causative polymorphisms, are
also useful. In such instances, the genotype of the polymorphism(s)
that is/are in LD with the causative polymorphism is predictive of
the genotype of the causative polymorphism and, consequently,
predictive of the phenotype (e.g., responder/non-responder to
statin treatment) that is influenced by the causative SNP(s).
Therefore, polymorphic markers that are in LD with causative
polymorphisms are useful as diagnostic markers, and are
particularly useful when the actual causative polymorphism(s)
is/are unknown.
[0247] Examples of polymorphisms that can be in LD with one or more
causative polymorphisms (and/or in LD with one or more
polymorphisms that have a significant statistical association with
a condition) and therefore useful for diagnosing the same condition
that the causative/associated SNP(s) is used to diagnose, include,
for example, other SNPs in the same gene, protein-coding, or mRNA
transcript-coding region as the causative/associated SNP, other
SNPs in the same exon or same intron as the causative/associated
SNP, other SNPs in the same haplotype block as the
causative/associated SNP, other SNPs in the same intergenic region
as the causative/associated SNP, SNPs that are outside but near a
gene (e.g., within 6 kb on either side, 5' or 3', of a gene
boundary) that harbors a causative/associated SNP, etc. Such useful
LD SNPs can be selected from among the SNPs disclosed in Tables
1-2, for example.
[0248] Linkage disequilibrium in the human genome is reviewed in:
Wall et al., "Haplotype blocks and linkage disequilibrium in the
human genome", Nat Rev Genet. 2003 August; 4(8):587-97; Garner et
al., "On selecting markers for association studies: patterns of
linkage disequilibrium between two and three diallelic loci", Genet
Epidemiol. 2003 January; 24(1):57-67; Ardlie et al., "Patterns of
linkage disequilibrium in the human genome", Nat Rev Genet. 2002
April; 3(4):299-309 (erratum in Nat Rev Genet 2002 July; 3(7):566);
and Remm et al., "High-density genotyping and linkage
disequilibrium in the human genome using chromosome 22 as a model";
Curr Opin Chem Biol. 2002 February; 6(1):24-30; Haldane J B S
(1919) The combination of linkage values, and the calculation of
distances between the loci of linked factors. J Genet 8:299-309;
Mendel, G. (1866) Versuche uber Pflanzen-Hybriden. Verhandlungen
des naturforschenden Vereines in Brunn [Proceedings of the Natural
History Society of Brunn]; Lewin B (1990) Genes IV. Oxford
University Press, New York, USA; Hartl D L and Clark A G (1989)
Principles of Population Genetics 2.sup.nd ed. Sinauer Associates,
Inc. Sunderland, Mass., USA; Gillespie J H (2004) Population
Genetics: A Concise Guide. 2.sup.nd ed. Johns Hopkins University
Press. USA; Lewontin R C (1964) The interaction of selection and
linkage. I. General considerations; heterotic models. Genetics
49:49-67; Hoel P G (1954) Introduction to Mathematical Statistics
2.sup.nd ed. John Wiley & Sons, Inc. New York, USA; Hudson R R
(2001) Two-locus sampling distributions and their application.
Genetics 159:1805-1817; Dempster A P, Laird N M, Rubin D B (1977)
Maximum likelihood from incomplete data via the EM algorithm. J R
Stat Soc 39:1-38; Excoffier L, Slatkin M (1995) Maximum-likelihood
estimation of molecular haplotype frequencies in a diploid
population. Mol Biol Evol 12(5):921-927; Tregouet D A, Escolano S,
Tiret L, Mallet A, Golmard J L (2004) A new algorithm for
haplotype-based association analysis: the Stochastic-EM algorithm.
Ann Hum Genet 68(Pt 2):165-177; Long A D and Langley C H (1999) The
power of association studies to detect the contribution of
candidate genetic loci to variation in complex traits. Genome
Research 9:720-731; Agresti A (1990) Categorical Data Analysis.
John Wiley & Sons, Inc. New York, USA; Lange K (1997)
Mathematical and Statistical Methods for Genetic Analysis.
Springer-Verlag New York, Inc. New York, USA; The International
HapMap Consortium (2003) The International HapMap Project. Nature
426:789-796; The International HapMap Consortium (2005) A haplotype
map of the human genome. Nature 437:1299-1320; Thorisson G A, Smith
A V, Krishnan L, Stein L D (2005), The International HapMap Project
Web Site. Genome Research 15:1591-1593; McVean G, Spencer C C A,
Chaix R (2005) Perspectives on human genetic variation from the
HapMap project. PLoS Genetics 1(4):413-418; Hirschhorn J N, Daly M
J (2005) Genome-wide association studies for common diseases and
complex traits. Nat Genet 6:95-108; Schrodi S J (2005) A
probabilistic approach to large-scale association scans: a
semi-Bayesian method to detect disease-predisposing alleles. SAGMB
4(1):31; Wang W Y S, Barratt B J, Clayton D G, Todd J A (2005)
Genome-wide association studies: theoretical and practical
concerns. Nat Rev Genet 6:109-118. Pritchard J K, Przeworski M
(2001) Linkage disequilibrium in humans: models and data. Am J Hum
Genet 69:1-14.
[0249] As discussed above, one aspect of the present invention is
the discovery that SNPs which are in certain LD distance with the
interrogated SNP can also be used as valid markers for identifying
an increased or decreased risks of having or developing CHD. As
used herein, the term "interrogated SNP" refers to SNPs that have
been found to be associated with an increased or decreased risk of
disease using genotyping results and analysis, or other appropriate
experimental method as exemplified in the working examples
described in this application. As used herein, the term "LD SNP"
refers to a SNP that has been characterized as a SNP associating
with an increased or decreased risk of diseases due to their being
in LD with the "interrogated SNP" under the methods of calculation
described in the application. Below, applicants describe the
methods of calculation with which one of ordinary skilled in the
art may determine if a particular SNP is in LD with an interrogated
SNP. The parameter r.sup.2 is commonly used in the genetics art to
characterize the extent of linkage disequilibrium between markers
(Hudson, 2001). As used herein, the term "in LD with" refers to a
particular SNP that is measured at above the threshold of a
parameter such as r.sup.2 with an interrogated SNP.
[0250] It is now common place to directly observe genetic variants
in a sample of chromosomes obtained from a population. Suppose one
has genotype data at two genetic markers located on the same
chromosome, for the markers A and B. Further suppose that two
alleles segregate at each of these two markers such that alleles
A.sub.1 and A.sub.2 can be found at marker A and alleles B.sub.1
and B.sub.2 at marker B. Also assume that these two markers are on
a human autosome. If one is to examine a specific individual and
find that they are heterozygous at both markers, such that their
two-marker genotype is A.sub.1A.sub.2B.sub.1B.sub.2, then there are
two possible configurations: the individual in question could have
the alleles A.sub.1B.sub.1 on one chromosome and A.sub.2B.sub.2 on
the remaining chromosome; alternatively, the individual could have
alleles A.sub.1B.sub.2 on one chromosome and A.sub.2B.sub.1 on the
other. The arrangement of alleles on a chromosome is called a
haplotype. In this illustration, the individual could have
haplotypes A.sub.1B.sub.1/A.sub.2B.sub.2 or
A.sub.1B.sub.2/A.sub.2B.sub.1 (see Hartl and Clark (1989) for a
more complete description). The concept of linkage equilibrium
relates the frequency of haplotypes to the allele frequencies.
[0251] Assume that a sample of individuals is selected from a
larger population. Considering the two markers described above,
each having two alleles, there are four possible haplotypes:
A.sub.1B.sub.1, A.sub.1B.sub.2, A.sub.2B.sub.1 and A.sub.2B.sub.2.
Denote the frequencies of these four haplotypes with the following
notation.
P.sub.11=freq(A.sub.1B.sub.1) (1)
P.sub.12=freq(A.sub.1B.sub.2) (2)
P.sub.21=freq(A.sub.2B.sub.1) (3)
P.sub.22=freq(A.sub.2B.sub.2) (4)
The allele frequencies at the two markers are then the sum of
different haplotype frequencies, it is straightforward to write
down a similar set of equations relating single-marker allele
frequencies to two-marker haplotype frequencies:
p.sub.1=freq(A.sub.1)=P.sub.11+P.sub.12 (5)
p.sub.2=freq(A.sub.2)=P.sub.21+P.sub.22 (6)
q.sub.1=freq(B.sub.1)=P.sub.11+P.sub.21 (7)
q.sub.2=freq(B.sub.2)=P.sub.12+P.sub.22 (8)
[0252] Note that the four haplotype frequencies and the allele
frequencies at each marker must sum to a frequency of 1.
P.sub.11+P.sub.12+P.sub.21+P.sub.22=1 (9)
p.sub.1+p.sub.2=1 (10)
q.sub.1+q.sub.2=1 (11)
If there is no correlation between the alleles at the two markers,
one would expect that the frequency of the haplotypes would be
approximately the product of the composite alleles. Therefore,
P.sub.11.apprxeq.p.sub.1q.sub.1 (12)
P.sub.12.apprxeq.p.sub.1q.sub.2 (13)
P.sub.21.apprxeq.p.sub.2q.sub.1 (14)
P.sub.22.apprxeq.p.sub.2q.sub.2 (15)
These approximating equations (12)-(15) represent the concept of
linkage equilibrium where there is independent assortment between
the two markers--the alleles at the two markers occur together at
random. These are represented as approximations because linkage
equilibrium and linkage disequilibrium are concepts typically
thought of as properties of a sample of chromosomes; and as such
they are susceptible to stochastic fluctuations due to the sampling
process. Empirically, many pairs of genetic markers will be in
linkage equilibrium, but certainly not all pairs.
[0253] Having established the concept of linkage equilibrium above,
applicants can now describe the concept of linkage disequilibrium
(LD), which is the deviation from linkage equilibrium. Since the
frequency of the A.sub.1B.sub.1 haplotype is approximately the
product of the allele frequencies for A.sub.1 and B.sub.1 under the
assumption of linkage equilibrium as stated mathematically in (12),
a simple measure for the amount of departure from linkage
equilibrium is the difference in these two quantities, D,
D=P.sub.11-p.sub.1q.sub.1 (16)
D=0 indicates perfect linkage equilibrium. Substantial departures
from D=0 indicates LD in the sample of chromosomes examined. Many
properties of D are discussed in Lewontin (1964) including the
maximum and minimum values that D can take. Mathematically, using
basic algebra, it can be shown that D can also be written solely in
terms of haplotypes:
D=P.sub.11P.sub.22-P.sub.12P.sub.21 (17)
If one transforms D by squaring it and subsequently dividing by the
product of the allele frequencies of A.sub.1, A.sub.2B.sub.1 and
B.sub.2, the resulting quantity, called r.sup.2, is equivalent to
the square of the Pearson's correlation coefficient commonly used
in statistics (e.g. Hoel, 1954).
r 2 = D 2 p 1 p 2 q 1 q 2 ( 18 ) ##EQU00001##
[0254] As with D, values of r.sup.2 close to 0 indicate linkage
equilibrium between the two markers examined in the sample set. As
values of r.sup.2 increase, the two markers are said to be in
linkage disequilibrium. The range of values that r.sup.2 can take
are from 0 to 1. r.sup.2=1 when there is a perfect correlation
between the alleles at the two markers.
[0255] In addition, the quantities discussed above are
sample-specific. And as such, it is necessary to formulate notation
specific to the samples studied. In the approach discussed here,
three types of samples are of primary interest: (i) a sample of
chromosomes from individuals affected by a disease-related
phenotype (cases), (ii) a sample of chromosomes obtained from
individuals not affected by the disease-related phenotype
(controls), and (iii) a standard sample set used for the
construction of haplotypes and calculation pairwise linkage
disequilibrium. For the allele frequencies used in the development
of the method described below, an additional subscript will be
added to denote either the case or control sample sets.
p.sub.1,cs=freq(A.sub.1 in cases) (19)
p.sub.2,cs=freq(A.sub.2 in cases) (20)
q.sub.1,cs=freq(B.sub.1 in cases) (21)
q.sub.2,cs=freq(B.sub.2 in cases) (22)
Similarly,
p.sub.1,ct=freq(A.sub.1 in controls) (23)
p.sub.2,ct=freq(A.sub.2 in controls) (24)
q.sub.1,ct=freq(B.sub.1 in controls) (25)
q.sub.2,ct=freq(B.sub.2 in controls) (26)
[0256] As a well-accepted sample set is necessary for robust
linkage disequilibrium calculations, data obtained from the
International HapMap project (The International HapMap Consortium
2003, 2005; Thorisson et al, 2005; McVean et al, 2005) can be used
for the calculation of pairwise r.sup.2 values. Indeed, the samples
genotyped for the International HapMap Project were selected to be
representative examples from various human sub-populations with
sufficient numbers of chromosomes examined to draw meaningful and
robust conclusions from the patterns of genetic variation observed.
The International HapMap project website (hapmap.org) contains a
description of the project, methods utilized and samples examined.
It is useful to examine empirical data to get a sense of the
patterns present in such data.
[0257] Haplotype frequencies were explicit arguments in equation
(18) above. However, knowing the 2-marker haplotype frequencies
requires that phase to be determined for doubly heterozygous
samples. When phase is unknown in the data examined, various
algorithms can be used to infer phase from the genotype data. This
issue was discussed earlier where the doubly heterozygous
individual with a 2-SNP genotype of A.sub.1A.sub.2B.sub.1B.sub.2
could have one of two different sets of chromosomes:
A.sub.1B.sub.1/A.sub.2B.sub.2 or A.sub.1B.sub.2/A.sub.2B.sub.1. One
such algorithm to estimate haplotype frequencies is the
expectation-maximization (EM) algorithm first formalized by
Dempster et al (1977). This algorithm is often used in genetics to
infer haplotype frequencies from genotype data (e.g. Excoffier and
Slatkin, 1995; Tregouet et al, 2004). It should be noted that for
the two-SNP case explored here, EM algorithms have very little
error provided that the allele frequencies and sample sizes are not
too small. The impact on r.sup.2 values is typically
negligible.
[0258] As correlated genetic markers share information,
interrogation of SNP markers in LD with a disease-associated SNP
marker can also have sufficient power to detect disease association
(Long and Langley, 1999). The relationship between the power to
directly find disease-associated alleles and the power to
indirectly detect disease-association was investigated by Pritchard
and Przeworski (2001). In a straight-forward derivation, it can be
shown that the power to detect disease association indirectly at a
marker locus in linkage disequilibrium with a disease-association
locus is approximately the same as the power to detect
disease-association directly at the disease-association locus if
the sample size is increased by a factor of
1 r 2 ##EQU00002##
(the reciprocal of equation 18) at the marker in comparison with
the disease-association locus.
[0259] Therefore, if one calculated the power to detect
disease-association indirectly with an experiment having N samples,
then equivalent power to directly detect disease-association (at
the actual disease-susceptibility locus) would necessitate an
experiment using approximately r.sup.2 N samples. This elementary
relationship between power, sample size and linkage disequilibrium
can be used to derive an r.sup.2 threshold value useful in
determining whether or not genotyping markers in linkage
disequilibrium with a SNP marker directly associated with disease
status has enough power to indirectly detect
disease-association.
[0260] To commence a derivation of the power to detect
disease-associated markers through an indirect process, define the
effective chromosomal sample size as
n = 4 N cs N ct N cs + N ct ; ( 27 ) ##EQU00003##
where N.sub.cs and N.sub.ct are the numbers of diploid cases and
controls, respectively. This is necessary to handle situations
where the numbers of cases and controls are not equivalent. For
equal case and control sample sizes, N.sub.cs=N.sub.ct=N, the value
of the effective number of chromosomes is simply n=2N--as expected.
Let power be calculated for a significance level .alpha. (such that
traditional P-values below .alpha. will be deemed statistically
significant). Define the standard Gaussian distribution function as
.PSI.(.cndot.). Mathematically,
.PHI. ( x ) = 1 2 .pi. .intg. - .infin. x - .theta. 2 2 .theta. (
28 ) ##EQU00004##
Alternatively, the following error function notation (Erf) may also
be used,
.PHI. ( x ) = 1 2 [ 1 + Erf ( x 2 ) ] ( 29 ) ##EQU00005##
[0261] For example, .PSI.(1.644854)=0.95. The value of r.sup.2 may
be derived to yield a pre-specified minimum amount of power to
detect disease association though indirect interrogation. Noting
that the LD SNP marker could be the one that is carrying the
disease-association allele, therefore that this approach
constitutes a lower-bound model where all indirect power results
are expected to be at least as large as those interrogated.
[0262] Denote by .beta. the error rate for not detecting truly
disease-associated markers. Therefore, 1-.beta. is the classical
definition of statistical power. Substituting the
Pritchard-Pzreworski result into the sample size, the power to
detect disease association at a significance level of .alpha. is
given by the approximation
1 - .beta. .apprxeq. .PHI. [ q 1 , cs - q 1 , ct q 1 , cs ( 1 - q 1
, cs ) + q 1 , ct ( 1 - q 1 , ct ) r 2 n - Z 1 - .alpha. / 2 ] ; (
30 ) ##EQU00006##
where Z.sub.u is the inverse of the standard normal cumulative
distribution evaluated at u (u .epsilon.(0,1)).
Z.sub.u=.PSI..sup.-1(u), where
.PSI.(.PSI..sup.-1(u))=.PSI..sup.-1(.PSI.(u))=u. For example,
setting .alpha.=0.05, and therefore 1-.alpha./2=0.975, we obtain
Z.sub.0.975=1.95996. Next, setting power equal to a threshold of a
minimum power of T,
T = .PHI. [ q 1 , cs - q 1 , ct q 1 , cs ( 1 - q 1 , cs ) + q 1 ,
ct ( 1 - q 1 , ct ) r 2 n - Z 1 - .alpha. / 2 ] ( 31 )
##EQU00007##
and solving for r.sup.2, the following threshold r.sup.2 is
obtained:
r T 2 = [ q 1 , cs ( 1 - q 1 , cs ) + q 1 , ct ( 1 - q 1 , ct ) ] n
( q 1 , cs - q 1 , ct ) 2 [ .PHI. - 1 ( T ) + Z 1 - .alpha. / 2 ]
Or , ( 32 ) r T 2 = ( Z T + Z 1 - .alpha. / 2 n ) [ q 1 , cs - ( q
1 , cs ) 2 + q 1 , ct - ( q 1 , ct ) 2 ( q 1 , cs - q 1 , ct ) 2 ]
( 33 ) ##EQU00008##
[0263] Suppose that r.sup.2 is calculated between an interrogated
SNP and a number of other SNPs with varying levels of LD with the
interrogated SNP. The threshold value r.sub.T.sup.2 is the minimum
value of linkage disequilibrium between the interrogated SNP and
the potential LD SNPs such that the LD SNP still retains a power
greater or equal to T for detecting disease-association. For
example, suppose that SNP rs200 is genotyped in a case-control
disease-association study and it is found to be associated with a
disease phenotype. Further suppose that the minor allele frequency
in 1,000 case chromosomes was found to be 16% in contrast with a
minor allele frequency of 10% in 1,000 control chromosomes. Given
those measurements one could have predicted, prior to the
experiment, that the power to detect disease association at a
significance level of 0.05 was quite high--approximately 98% using
a test of allelic association. Applying equation (32) one can
calculate a minimum value of r.sup.2 to indirectly assess disease
association assuming that the minor allele at SNP rs200 is truly
disease-predisposing for a threshold level of power. If one sets
the threshold level of power to be 80%, then r.sub.T.sup.2=0.489
given the same significance level and chromosome numbers as above.
Hence, any SNP with a pairwise r.sup.2 value with rs200 greater
than 0.489 is expected to have greater than 80% power to detect the
disease association. Further, this is assuming the conservative
model where the LD SNP is disease-associated only through linkage
disequilibrium with the interrogated SNP rs200.
[0264] The contribution or association of particular SNPs and/or
SNP haplotypes with disease phenotypes, such as susceptibility to
acute coronary events or responsiveness to statin treatment,
enables the SNPs of the present invention to be used to develop
superior diagnostic tests capable of identifying individuals who
express a detectable trait, such as predisposition to acute
coronary events or responder/non-responder to statin treatment, as
the result of a specific genotype, or individuals whose genotype
places them at an increased or decreased risk of developing a
detectable trait at a subsequent time as compared to individuals
who do not have that genotype. As described herein, diagnostics may
be based on a single SNP or a group of SNPs. Combined detection of
a plurality of SNPs (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 25, 30, 32, 48, 50, 64, 96,
100, or any other number in-between, or more, of the SNPs provided
in Table 1 and/or Table 2) typically increases the probability of
an accurate diagnosis. For example, the presence of a single SNP
known to correlate with response to statin treatment might indicate
a probability of 20% that an individual will respond to statin
treatment, whereas detection of five SNPs, each of which correlates
with response to statin treatment, might indicate a probability of
80% that an individual will respond to statin treatment. To further
increase the accuracy of diagnosis or predisposition screening,
analysis of the SNPs of the present invention can be combined with
that of other polymorphisms or other risk factors that correlate
with response to statin treatment, such as family history.
[0265] It will, of course, be understood by practitioners skilled
in the treatment or diagnosis of cardiovascular disorders that the
present invention generally does not intend to provide an absolute
identification of individuals who will or will not experience an
acute coronary event or develop another cardiovascular disorder, or
those individuals who will or will not respond to statin treatment
of cardiovascular disorders, but rather to indicate a certain
increased (or decreased) degree or likelihood of responding to
statin treatment based on statistically significant association
results. However, this information is extremely valuable as it can,
for example, indicate that an individual having a cardiovascular
disorder should follow a particular statin-based treatment regimen,
or should follow an alternative treatment regimen that does not
involve statin. This information can also be used to initiate
preventive treatments or to allow an individual carrying one or
more significant SNPs or SNP haplotypes to foresee warning signs
such as minor clinical symptoms of cardiovascular disease, or to
have regularly scheduled physical exams to monitor for
cardiovascular disorders in order to identify and begin treatment
of the disorder at an early stage. Particularly with diseases that
are extremely debilitating or fatal if not treated on time, the
knowledge of a potential predisposition to the disease or
likelihood of responding to available treatments, even if this
predisposition or likelihood is not absolute, would likely
contribute in a very significant manner to treatment efficacy.
[0266] The diagnostic techniques of the present invention may
employ a variety of methodologies to determine whether a test
subject has a SNP or a SNP pattern associated with an increased or
decreased risk of developing a detectable trait or whether the
individual suffers from a detectable trait as a result of a
particular polymorphism/mutation, including, for example, methods
which enable the analysis of individual chromosomes for
haplotyping, family studies, single sperm DNA analysis, or somatic
hybrids. The trait analyzed using the diagnostics of the invention
may be any detectable trait that is commonly observed in
cardiovascular disorders or during the course of statin
treatment.
[0267] Another aspect of the present invention relates to a method
of determining whether an individual is at risk (or less at risk)
of developing one or more traits or whether an individual expresses
one or more traits as a consequence of possessing a particular
trait-causing or trait-influencing allele. These methods generally
involve obtaining a nucleic acid sample from an individual and
assaying the nucleic acid sample to determine which nucleotide(s)
is/are present at one or more SNP positions, wherein the assayed
nucleotide(s) is/are indicative of an increased or decreased risk
of developing the trait or indicative that the individual expresses
the trait as a result of possessing a particular trait-causing or
trait-influencing allele.
[0268] In another embodiment, the SNP detection reagents of the
present invention are used to determine whether an individual has
one or more SNP allele(s) affecting the level (e.g., the
concentration of mRNA or protein in a sample, etc.) or pattern
(e.g., the kinetics of expression, rate of decomposition, stability
profile, Km, Vmax, etc.) of gene expression (collectively, the
"gene response" of a cell or bodily fluid). Such a determination
can be accomplished by screening for mRNA or protein expression
(e.g., by using nucleic acid arrays, RT-PCR, TaqMan assays, or mass
spectrometry), identifying genes having altered expression in an
individual, genotyping SNPs disclosed in Table 1 and/or Table 2
that could affect the expression of the genes having altered
expression (e.g., SNPs that are in and/or around the gene(s) having
altered expression, SNPs in regulatory/control regions, SNPs in
and/or around other genes that are involved in pathways that could
affect the expression of the gene(s) having altered expression, or
all SNPs could be genotyped), and correlating SNP genotypes with
altered gene expression. In this manner, specific SNP alleles at
particular SNP sites can be identified that affect gene
expression.
[0269] Pharmacogenomics and Therapeutics/Drug Development
[0270] The present invention provides methods for assessing the
pharmacogenomics of a subject harboring particular SNP alleles or
haplotypes to a particular therapeutic agent or pharmaceutical
compound, or to a class of such compounds. Pharmacogenomics deals
with the roles which clinically significant hereditary variations
(e.g., SNPs) play in the response to drugs due to altered drug
disposition and/or abnormal action in affected persons. See, e.g.,
Roses, Nature 405, 857-865 (2000); Gould Rothberg, Nature
Biotechnology 19, 209-211 (2001); Eichelbaum, Clin. Exp. Pharmcol.
Physiol. 23(10-11):983-985 (1996); and Linder, Clin. Chem.
43(2):254-266 (1997). The clinical outcomes of these variations can
result in severe toxicity of therapeutic drugs in certain
individuals or therapeutic failure of drugs in certain individuals
as a result of individual variation in metabolism. Thus, the SNP
genotype of an individual can determine the way a therapeutic
compound acts on the body or the way the body metabolizes the
compound. For example, SNPs in drug metabolizing enzymes can affect
the activity of these enzymes, which in turn can affect both the
intensity and duration of drug action, as well as drug metabolism
and clearance.
[0271] The discovery of SNPs in drug metabolizing enzymes, drug
transporters, proteins for pharmaceutical agents, and other drug
targets has explained why some patients do not obtain the expected
drug effects, show an exaggerated drug effect, or experience
serious toxicity from standard drug dosages. SNPs can be expressed
in the phenotype of the extensive metabolizer and in the phenotype
of the poor metabolizer. Accordingly, SNPs may lead to allelic
variants of a protein in which one or more of the protein functions
in one population are different from those in another population.
SNPs and the encoded variant peptides thus provide targets to
ascertain a genetic predisposition that can affect treatment
modality. For example, in a ligand-based treatment, SNPs may give
rise to amino terminal extracellular domains and/or other
ligand-binding regions of a receptor that are more or less active
in ligand binding, thereby affecting subsequent protein activation.
Accordingly, ligand dosage would necessarily be modified to
maximize the therapeutic effect within a given population
containing particular SNP alleles or haplotypes.
[0272] As an alternative to genotyping, specific variant proteins
containing variant amino acid sequences encoded by alternative SNP
alleles could be identified. Thus, pharmacogenomic characterization
of an individual permits the selection of effective compounds and
effective dosages of such compounds for prophylactic or therapeutic
uses based on the individual's SNP genotype, thereby enhancing and
optimizing the effectiveness of the therapy. Furthermore, the
production of recombinant cells and transgenic animals containing
particular SNPs/haplotypes allow effective clinical design and
testing of treatment compounds and dosage regimens. For example,
transgenic animals can be produced that differ only in specific SNP
alleles in a gene that is orthologous to a human disease
susceptibility gene.
[0273] Pharmacogenomic uses of the SNPs of the present invention
provide several significant advantages for patient care,
particularly in predicting an individual's predisposition to acute
coronary events and other cardiovascular disorders and in
predicting an individual's responsiveness to the use of statin for
treating cardiovascular disease. Pharmacogenomic characterization
of an individual, based on an individual's SNP genotype, can
identify those individuals unlikely to respond to treatment with a
particular medication and thereby allows physicians to avoid
prescribing the ineffective medication to those individuals. On the
other hand, SNP genotyping of an individual may enable physicians
to select the appropriate medication and dosage regimen that will
be most effective based on an individual's SNP genotype. This
information increases a physician's confidence in prescribing
medications and motivates patients to comply with their drug
regimens. Furthermore, pharmacogenomics may identify patients
predisposed to toxicity and adverse reactions to particular drugs
or drug dosages. Adverse drug reactions lead to more than 100,000
avoidable deaths per year in the United States alone and therefore
represent a significant cause of hospitalization and death, as well
as a significant economic burden on the healthcare system (Pfost
et. al., Trends in Biotechnology, August 2000.). Thus,
pharmacogenomics based on the SNPs disclosed herein has the
potential to both save lives and reduce healthcare costs
substantially.
[0274] Pharmacogenomics in general is discussed further in Rose et
al., "Pharmacogenetic analysis of clinically relevant genetic
polymorphisms", Methods Mol Med. 2003; 85:225-37. Pharmacogenomics
as it relates to Alzheimer's disease and other neurodegenerative
disorders is discussed in Cacabelos, "Pharmacogenomics for the
treatment of dementia", Ann Med. 2002; 34(5):357-79, Maimone et
al., "Pharmacogenomics of neurodegenerative diseases", Eur J
Pharmacol. 2001 Feb. 9; 413(1):11-29, and Poirier, "Apolipoprotein
E: a pharmacogenetic target for the treatment of Alzheimer's
disease", Mol Diagn. 1999 December; 4(4):335-41. Pharmacogenomics
as it relates to cardiovascular disorders is discussed in Siest et
al., "Pharmacogenomics of drugs affecting the cardiovascular
system", Clin Chem Lab Med. 2003 April; 41(4):590-9, Mukherjee et
al., "Pharmacogenomics in cardiovascular diseases", Prog Cardiovasc
Dis. 2002 May-June; 44(6):479-98, and Mooser et al.,
"Cardiovascular pharmacogenetics in the SNP era", J Thromb Haemost.
2003 July; 1(7):1398-402. Pharmacogenomics as it relates to cancer
is discussed in McLeod et al., "Cancer pharmacogenomics: SNPs,
chips, and the individual patient", Cancer Invest. 2003;
21(4):630-40 and Watters et al., "Cancer pharmacogenomics: current
and future applications", Biochim Biophys Acta. 2003 Mar. 17;
1603(2):99-111.
[0275] The SNPs of the present invention also can be used to
identify novel therapeutic targets for cardiovascular disorders.
For example, genes containing the disease-associated variants
("variant genes") or their products, as well as genes or their
products that are directly or indirectly regulated by or
interacting with these variant genes or their products, can be
targeted for the development of therapeutics that, for example,
treat the disease or prevent or delay disease onset. The
therapeutics may be composed of, for example, small molecules,
proteins, protein fragments or peptides, antibodies, nucleic acids,
or their derivatives or mimetics which modulate the functions or
levels of the target genes or gene products.
[0276] The SNP-containing nucleic acid molecules disclosed herein,
and their complementary nucleic acid molecules, may be used as
antisense constructs to control gene expression in cells, tissues,
and organisms. Antisense technology is well established in the art
and extensively reviewed in Antisense Drug Technology: Principles,
Strategies, and Applications, Crooke (ed.), Marcel Dekker, Inc.:
New York (2001). An antisense nucleic acid molecule is generally
designed to be complementary to a region of mRNA expressed by a
gene so that the antisense molecule hybridizes to the mRNA and
thereby blocks translation of mRNA into protein. Various classes of
antisense oligonucleotides are used in the art, two of which are
cleavers and blockers. Cleavers, by binding to target RNAs,
activate intracellular nucleases (e.g., RNaseH or RNase L) that
cleave the target RNA. Blockers, which also bind to target RNAs,
inhibit protein translation through steric hindrance of ribosomes.
Exemplary blockers include peptide nucleic acids, morpholinos,
locked nucleic acids, and methylphosphonates (see, e.g., Thompson,
Drug Discovery Today, 7 (17): 912-917 (2002)). Antisense
oligonucleotides are directly useful as therapeutic agents, and are
also useful for determining and validating gene function (e.g., in
gene knock-out or knock-down experiments).
[0277] Antisense technology is further reviewed in: Lavery et al.,
"Antisense and RNAi: powerful tools in drug target discovery and
validation", Curr Opin Drug Discov Devel. 2003 July; 6(4):561-9;
Stephens et al., "Antisense oligonucleotide therapy in cancer",
Curr Opin Mol Ther. 2003 April; 5(2):118-22; Kurreck, "Antisense
technologies. Improvement through novel chemical modifications",
Eur J Biochem. 2003 April; 270(8):1628-44; Dias et al., "Antisense
oligonucleotides: basic concepts and mechanisms", Mol Cancer Ther.
2002 March; 1(5):347-55; Chen, "Clinical development of antisense
oligonucleotides as anti-cancer therapeutics", Methods Mol Med.
2003; 75:621-36; Wang et al., "Antisense anticancer oligonucleotide
therapeutics", Curr Cancer Drug Targets. 2001 November;
1(3):177-96; and Bennett, "Efficiency of antisense oligonucleotide
drug discovery", Antisense Nucleic Acid Drug Dev. 2002 June;
12(3):215-24.
[0278] The SNPs of the present invention are particularly useful
for designing antisense reagents that are specific for particular
nucleic acid variants. Based on the SNP information disclosed
herein, antisense oligonucleotides can be produced that
specifically target mRNA molecules that contain one or more
particular SNP nucleotides. In this manner, expression of mRNA
molecules that contain one or more undesired polymorphisms (e.g.,
SNP nucleotides that lead to a defective protein such as an amino
acid substitution in a catalytic domain) can be inhibited or
completely blocked. Thus, antisense oligonucleotides can be used to
specifically bind a particular polymorphic form (e.g., a SNP allele
that encodes a defective protein), thereby inhibiting translation
of this form, but which do not bind an alternative polymorphic form
(e.g., an alternative SNP nucleotide that encodes a protein having
normal function).
[0279] Antisense molecules can be used to inactivate mRNA in order
to inhibit gene expression and production of defective proteins.
Accordingly, these molecules can be used to treat a disorder, such
as a cardiovascular disorder, characterized by abnormal or
undesired gene expression or expression of certain defective
proteins. This technique can involve cleavage by means of ribozymes
containing nucleotide sequences complementary to one or more
regions in the mRNA that attenuate the ability of the mRNA to be
translated. Possible mRNA regions include, for example,
protein-coding regions and particularly protein-coding regions
corresponding to catalytic activities, substrate/ligand binding, or
other functional activities of a protein.
[0280] The SNPs of the present invention are also useful for
designing RNA interference reagents that specifically target
nucleic acid molecules having particular SNP variants. RNA
interference (RNAi), also referred to as gene silencing, is based
on using double-stranded RNA (dsRNA) molecules to turn genes off.
When introduced into a cell, dsRNAs are processed by the cell into
short fragments (generally about 21, 22, or 23 nucleotides in
length) known as small interfering RNAs (siRNAs) which the cell
uses in a sequence-specific manner to recognize and destroy
complementary RNAs (Thompson, Drug Discovery Today, 7 (17): 912-917
(2002)). Accordingly, an aspect of the present invention
specifically contemplates isolated nucleic acid molecules that are
about 18-26 nucleotides in length, preferably 19-25 nucleotides in
length, and more preferably 20, 21, 22, or 23 nucleotides in
length, and the use of these nucleic acid molecules for RNAi.
Because RNAi molecules, including siRNAs, act in a
sequence-specific manner, the SNPs of the present invention can be
used to design RNAi reagents that recognize and destroy nucleic
acid molecules having specific SNP alleles/nucleotides (such as
deleterious alleles that lead to the production of defective
proteins), while not affecting nucleic acid molecules having
alternative SNP alleles (such as alleles that encode proteins
having normal function). As with antisense reagents, RNAi reagents
may be directly useful as therapeutic agents (e.g., for turning off
defective, disease-causing genes), and are also useful for
characterizing and validating gene function (e.g., in gene
knock-out or knock-down experiments).
[0281] The following references provide a further review of RNAi:
Reynolds et al., "Rational siRNA design for RNA interference", Nat
Biotechnol. 2004 March; 22(3):326-30. Epub 2004 Feb. 1; Chi et al.,
"Genomewide view of gene silencing by small interfering RNAs", PNAS
100(11):6343-6346, 2003; Vickers et al., "Efficient Reduction of
Target RNAs by Small Interfering RNA and RNase H-dependent
Antisense Agents", J. Biol. Chem. 278: 7108-7118, 2003; Agami,
"RNAi and related mechanisms and their potential use for therapy",
Curr Opin Chem Biol. 2002 December; 6(6):829-34; Lavery et al.,
"Antisense and RNAi: powerful tools in drug target discovery and
validation", Curr Opin Drug Discov Devel. 2003 July; 6(4):561-9;
Shi, "Mammalian RNAi for the masses", Trends Genet 2003 January;
19(1):9-12), Shuey et al., "RNAi: gene-silencing in therapeutic
intervention", Drug Discovery Today 2002 October; 7(20):1040-1046;
McManus et al., Nat Rev Genet 2002 October; 3(10):737-47; Xia et
al., Nat Biotechnol 2002 October; 20(10):1006-10; Plasterk et al.,
Curr Opin Genet Dev 2000 October; 10(5):562-7; Bosher et al., Nat
Cell Biol 2000 February; 2(2):E31-6; and Hunter, Curr Biol 1999
June 17; 9(12):R440-2).
[0282] A subject suffering from a pathological condition, such as a
cardiovascular disorder, ascribed to a SNP may be treated so as to
correct the genetic defect (see Kren et al., Proc. Natl. Acad. Sci.
USA 96:10349-10354 (1999)). Such a subject can be identified by any
method that can detect the polymorphism in a biological sample
drawn from the subject. Such a genetic defect may be permanently
corrected by administering to such a subject a nucleic acid
fragment incorporating a repair sequence that supplies the
normal/wild-type nucleotide at the position of the SNP. This
site-specific repair sequence can encompass an RNA/DNA
oligonucleotide that operates to promote endogenous repair of a
subject's genomic DNA. The site-specific repair sequence is
administered in an appropriate vehicle, such as a complex with
polyethylenimine, encapsulated in anionic liposomes, a viral vector
such as an adenovirus, or other pharmaceutical composition that
promotes intracellular uptake of the administered nucleic acid. A
genetic defect leading to an inborn pathology may then be overcome,
as the chimeric oligonucleotides induce incorporation of the normal
sequence into the subject's genome. Upon incorporation, the normal
gene product is expressed, and the replacement is propagated,
thereby engendering a permanent repair and therapeutic enhancement
of the clinical condition of the subject.
[0283] In cases in which a cSNP results in a variant protein that
is ascribed to be the cause of, or a contributing factor to, a
pathological condition, a method of treating such a condition can
include administering to a subject experiencing the pathology the
wild-type/normal cognate of the variant protein. Once administered
in an effective dosing regimen, the wild-type cognate provides
complementation or remediation of the pathological condition.
[0284] The invention further provides a method for identifying a
compound or agent that can be used to treat cardiovascular
disorders. The SNPs disclosed herein are useful as targets for the
identification and/or development of therapeutic agents. A method
for identifying a therapeutic agent or compound typically includes
assaying the ability of the agent or compound to modulate the
activity and/or expression of a SNP-containing nucleic acid or the
encoded product and thus identifying an agent or a compound that
can be used to treat a disorder characterized by undesired activity
or expression of the SNP-containing nucleic acid or the encoded
product. The assays can be performed in cell-based and cell-free
systems. Cell-based assays can include cells naturally expressing
the nucleic acid molecules of interest or recombinant cells
genetically engineered to express certain nucleic acid
molecules.
[0285] Variant gene expression in a patient having a cardiovascular
disorder or undergoing statin treatment can include, for example,
either expression of a SNP-containing nucleic acid sequence (for
instance, a gene that contains a SNP can be transcribed into an
mRNA transcript molecule containing the SNP, which can in turn be
translated into a variant protein) or altered expression of a
normal/wild-type nucleic acid sequence due to one or more SNPs (for
instance, a regulatory/control region can contain a SNP that
affects the level or pattern of expression of a normal
transcript).
[0286] Assays for variant gene expression can involve direct assays
of nucleic acid levels (e.g., mRNA levels), expressed protein
levels, or of collateral compounds involved in a signal pathway.
Further, the expression of genes that are up- or down-regulated in
response to the signal pathway can also be assayed. In this
embodiment, the regulatory regions of these genes can be operably
linked to a reporter gene such as luciferase.
[0287] Modulators of variant gene expression can be identified in a
method wherein, for example, a cell is contacted with a candidate
compound/agent and the expression of mRNA determined. The level of
expression of mRNA in the presence of the candidate compound is
compared to the level of expression of mRNA in the absence of the
candidate compound. The candidate compound can then be identified
as a modulator of variant gene expression based on this comparison
and be used to treat a disorder such as a cardiovascular disorder
that is characterized by variant gene expression (e.g., either
expression of a SNP-containing nucleic acid or altered expression
of a normal/wild-type nucleic acid molecule due to one or more SNPs
that affect expression of the nucleic acid molecule) due to one or
more SNPs of the present invention. When expression of mRNA is
statistically significantly greater in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of nucleic acid expression. When nucleic
acid expression is statistically significantly less in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of nucleic acid
expression.
[0288] The invention further provides methods of treatment, with
the SNP or associated nucleic acid domain (e.g., catalytic domain,
ligand/substrate-binding domain, regulatory/control region, etc.)
or gene, or the encoded mRNA transcript, as a target, using a
compound identified through drug screening as a gene modulator to
modulate variant nucleic acid expression. Modulation can include
either up-regulation (i.e., activation or agonization) or
down-regulation (i.e., suppression or antagonization) of nucleic
acid expression.
[0289] Expression of mRNA transcripts and encoded proteins, either
wild type or variant, may be altered in individuals with a
particular SNP allele in a regulatory/control element, such as a
promoter or transcription factor binding domain, that regulates
expression. In this situation, methods of treatment and compounds
can be identified, as discussed herein, that regulate or overcome
the variant regulatory/control element, thereby generating normal,
or healthy, expression levels of either the wild type or variant
protein.
[0290] The SNP-containing nucleic acid molecules of the present
invention are also useful for monitoring the effectiveness of
modulating compounds on the expression or activity of a variant
gene, or encoded product, in clinical trials or in a treatment
regimen. Thus, the gene expression pattern can serve as an
indicator for the continuing effectiveness of treatment with the
compound, particularly with compounds to which a patient can
develop resistance, as well as an indicator for toxicities. The
gene expression pattern can also serve as a marker indicative of a
physiological response of the affected cells to the compound.
Accordingly, such monitoring would allow either increased
administration of the compound or the administration of alternative
compounds to which the patient has not become resistant. Similarly,
if the level of nucleic acid expression falls below a desirable
level, administration of the compound could be commensurately
decreased.
[0291] In another aspect of the present invention, there is
provided a pharmaceutical pack comprising a therapeutic agent
(e.g., a small molecule drug, antibody, peptide, antisense or RNAi
nucleic acid molecule, etc.) and a set of instructions for
administration of the therapeutic agent to humans diagnostically
tested for one or more SNPs or SNP haplotypes provided by the
present invention.
[0292] The SNPs/haplotypes of the present invention are also useful
for improving many different aspects of the drug development
process. For instance, an aspect of the present invention includes
selecting individuals for clinical trials based on their SNP
genotype. For example, individuals with SNP genotypes that indicate
that they are likely to positively respond to a drug can be
included in the trials, whereas those individuals whose SNP
genotypes indicate that they are less likely to or would not
respond to the drug, or who are at risk for suffering toxic effects
or other adverse reactions, can be excluded from the clinical
trials. This not only can improve the safety of clinical trials,
but also can enhance the chances that the trial will demonstrate
statistically significant efficacy. Furthermore, the SNPs of the
present invention may explain why certain previously developed
drugs performed poorly in clinical trials and may help identify a
subset of the population that would benefit from a drug that had
previously performed poorly in clinical trials, thereby "rescuing"
previously developed drugs, and enabling the drug to be made
available to a particular patient population that can benefit from
it.
[0293] SNPs have many important uses in drug discovery, screening,
and development. A high probability exists that, for any
gene/protein selected as a potential drug target, variants of that
gene/protein will exist in a patient population. Thus, determining
the impact of gene/protein variants on the selection and delivery
of a therapeutic agent should be an integral aspect of the drug
discovery and development process. (Jazwinska, A Trends Guide to
Genetic Variation and Genomic Medicine, 2002 March; S30-S36).
[0294] Knowledge of variants (e.g., SNPs and any corresponding
amino acid polymorphisms) of a particular therapeutic target (e.g.,
a gene, mRNA transcript, or protein) enables parallel screening of
the variants in order to identify therapeutic candidates (e.g.,
small molecule compounds, antibodies, antisense or RNAi nucleic
acid compounds, etc.) that demonstrate efficacy across variants
(Rothberg, Nat Biotechnol 2001 March; 19(3):209-11). Such
therapeutic candidates would be expected to show equal efficacy
across a larger segment of the patient population, thereby leading
to a larger potential market for the therapeutic candidate.
[0295] Furthermore, identifying variants of a potential therapeutic
target enables the most common form of the target to be used for
selection of therapeutic candidates, thereby helping to ensure that
the experimental activity that is observed for the selected
candidates reflects the real activity expected in the largest
proportion of a patient population (Jazwinska, A Trends Guide to
Genetic Variation and Genomic Medicine, 2002 March; S30-S36).
[0296] Additionally, screening therapeutic candidates against all
known variants of a target can enable the early identification of
potential toxicities and adverse reactions relating to particular
variants. For example, variability in drug absorption,
distribution, metabolism and excretion (ADME) caused by, for
example, SNPs in therapeutic targets or drug metabolizing genes,
can be identified, and this information can be utilized during the
drug development process to minimize variability in drug
disposition and develop therapeutic agents that are safer across a
wider range of a patient population. The SNPs of the present
invention, including the variant proteins and encoding polymorphic
nucleic acid molecules provided in Tables 1-2, are useful in
conjunction with a variety of toxicology methods established in the
art, such as those set forth in Current Protocols in Toxicology,
John Wiley & Sons, Inc., N.Y.
[0297] Furthermore, therapeutic agents that target any art-known
proteins (or nucleic acid molecules, either RNA or DNA) may
cross-react with the variant proteins (or polymorphic nucleic acid
molecules) disclosed in Table 1, thereby significantly affecting
the pharmacokinetic properties of the drug. Consequently, the
protein variants and the SNP-containing nucleic acid molecules
disclosed in Tables 1-2 are useful in developing, screening, and
evaluating therapeutic agents that target corresponding art-known
protein forms (or nucleic acid molecules). Additionally, as
discussed above, knowledge of all polymorphic forms of a particular
drug target enables the design of therapeutic agents that are
effective against most or all such polymorphic forms of the drug
target.
[0298] Pharmaceutical Compositions and Administration Thereof
[0299] Any of the cardiovascular disease and/or statin
response-associated proteins, and encoding nucleic acid molecules,
disclosed herein can be used as therapeutic targets (or directly
used themselves as therapeutic compounds) for treating
cardiovascular disorders and related pathologies, and the present
disclosure enables therapeutic compounds (e.g., small molecules,
antibodies, therapeutic proteins, RNAi and antisense molecules,
etc.) to be developed that target (or are comprised of) any of
these therapeutic targets.
[0300] In general, a therapeutic compound will be administered in a
therapeutically effective amount by any of the accepted modes of
administration for agents that serve similar utilities. The actual
amount of the therapeutic compound of this invention, i.e., the
active ingredient, will depend upon numerous factors such as the
severity of the disease to be treated, the age and relative health
of the subject, the potency of the compound used, the route and
form of administration, and other factors.
[0301] Therapeutically effective amounts of therapeutic compounds
may range from, for example, approximately 0.01-50 mg per kilogram
body weight of the recipient per day; preferably about 0.1-20
mg/kg/day. Thus, as an example, for administration to a 70 kg
person, the dosage range would most preferably be about 7 mg to 1.4
g per day.
[0302] In general, therapeutic compounds will be administered as
pharmaceutical compositions by any one of the following routes:
oral, systemic (e.g., transdermal, intranasal, or by suppository),
or parenteral (e.g., intramuscular, intravenous, or subcutaneous)
administration. The preferred manner of administration is oral or
parenteral using a convenient daily dosage regimen, which can be
adjusted according to the degree of affliction. Oral compositions
can take the form of tablets, pills, capsules, semisolids, powders,
sustained release formulations, solutions, suspensions, elixirs,
aerosols, or any other appropriate compositions.
[0303] The choice of formulation depends on various factors such as
the mode of drug administration (e.g., for oral administration,
formulations in the form of tablets, pills, or capsules are
preferred) and the bioavailability of the drug substance. Recently,
pharmaceutical formulations have been developed especially for
drugs that show poor bioavailability based upon the principle that
bioavailability can be increased by increasing the surface area,
i.e., decreasing particle size. For example, U.S. Pat. No.
4,107,288 describes a pharmaceutical formulation having particles
in the size range from 10 to 1,000 nm in which the active material
is supported on a cross-linked matrix of macromolecules. U.S. Pat.
No. 5,145,684 describes the production of a pharmaceutical
formulation in which the drug substance is pulverized to
nanoparticles (average particle size of 400 nm) in the presence of
a surface modifier and then dispersed in a liquid medium to give a
pharmaceutical formulation that exhibits remarkably high
bioavailability.
[0304] Pharmaceutical compositions are comprised of, in general, a
therapeutic compound in combination with at least one
pharmaceutically acceptable excipient. Acceptable excipients are
non-toxic, aid administration, and do not adversely affect the
therapeutic benefit of the therapeutic compound. Such excipients
may be any solid, liquid, semi-solid or, in the case of an aerosol
composition, gaseous excipient that is generally available to one
skilled in the art.
[0305] Solid pharmaceutical excipients include starch, cellulose,
talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, magnesium stearate, sodium stearate, glycerol
monostearate, sodium chloride, dried skim milk and the like. Liquid
and semisolid excipients may be selected from glycerol, propylene
glycol, water, ethanol and various oils, including those of
petroleum, animal, vegetable or synthetic origin, e.g., peanut oil,
soybean oil, mineral oil, sesame oil, etc. Preferred liquid
carriers, particularly for injectable solutions, include water,
saline, aqueous dextrose, and glycols.
[0306] Compressed gases may be used to disperse a compound of this
invention in aerosol form. Inert gases suitable for this purpose
are nitrogen, carbon dioxide, etc.
[0307] Other suitable pharmaceutical excipients and their
formulations are described in Remington's Pharmaceutical Sciences,
edited by E. W. Martin (Mack Publishing Company, 18th ed.,
1990).
[0308] The amount of the therapeutic compound in a formulation can
vary within the full range employed by those skilled in the art.
Typically, the formulation will contain, on a weight percent (wt %)
basis, from about 0.01-99.99 wt % of the therapeutic compound based
on the total formulation, with the balance being one or more
suitable pharmaceutical excipients. Preferably, the compound is
present at a level of about 1-80 wt %.
[0309] Therapeutic compounds can be administered alone or in
combination with other therapeutic compounds or in combination with
one or more other active ingredient(s). For example, an inhibitor
or stimulator of a cardiovascular disorder-associated protein can
be administered in combination with another agent that inhibits or
stimulates the activity of the same or a different cardiovascular
disorder-associated protein to thereby counteract the affects of a
cardiovascular disorder.
[0310] For further information regarding pharmacology, see Current
Protocols in Pharmacology, John Wiley & Sons, Inc., N.Y.
[0311] Human Identification Applications
[0312] In addition to their diagnostic and therapeutic uses in
cardiovascular disorders and statin treatment of cardiovascular
disorders, the SNPs provided by the present invention are also
useful as human identification markers for such applications as
forensics, paternity testing, and biometrics (see, e.g., Gill, "An
assessment of the utility of single nucleotide polymorphisms (SNPs)
for forensic purposes", Int J Legal Med. 2001; 114(4-5):204-10).
Genetic variations in the nucleic acid sequences between
individuals can be used as genetic markers to identify individuals
and to associate a biological sample with an individual.
Determination of which nucleotides occupy a set of SNP positions in
an individual identifies a set of SNP markers that distinguishes
the individual. The more SNP positions that are analyzed, the lower
the probability that the set of SNPs in one individual is the same
as that in an unrelated individual. Preferably, if multiple sites
are analyzed, the sites are unlinked (i.e., inherited
independently). Thus, preferred sets of SNPs can be selected from
among the SNPs disclosed herein, which may include SNPs on
different chromosomes, SNPs on different chromosome arms, and/or
SNPs that are dispersed over substantial distances along the same
chromosome arm.
[0313] Furthermore, among the SNPs disclosed herein, preferred SNPs
for use in certain forensic/human identification applications
include SNPs located at degenerate codon positions (i.e., the third
position in certain codons which can be one of two or more
alternative nucleotides and still encode the same amino acid),
since these SNPs do not affect the encoded protein. SNPs that do
not affect the encoded protein are expected to be under less
selective pressure and are therefore expected to be more
polymorphic in a population, which is typically an advantage for
forensic/human identification applications. However, for certain
forensics/human identification applications, such as predicting
phenotypic characteristics (e.g., inferring ancestry or inferring
one or more physical characteristics of an individual) from a DNA
sample, it may be desirable to utilize SNPs that affect the encoded
protein.
[0314] For many of the SNPs disclosed in Tables 1-2 (which are
identified as "Applera" SNP source), Tables 1-2 provide SNP allele
frequencies obtained by re-sequencing the DNA of chromosomes from
39 individuals (Tables 1-2 also provide allele frequency
information for "Celera" source SNPs and, where available, public
SNPs from dbEST, HGBASE, and/or HGMD). The allele frequencies
provided in Tables 1-2 enable these SNPs to be readily used for
human identification applications. Although any SNP disclosed in
Table 1 and/or Table 2 could be used for human identification, the
closer that the frequency of the minor allele at a particular SNP
site is to 50%, the greater the ability of that SNP to discriminate
between different individuals in a population since it becomes
increasingly likely that two randomly selected individuals would
have different alleles at that SNP site. Using the SNP allele
frequencies provided in Tables 1-2, one of ordinary skill in the
art could readily select a subset of SNPs for which the frequency
of the minor allele is, for example, at least 1%, 2%, 5%, 10%, 20%,
25%, 30%, 40%, 45%, or 50%, or any other frequency in-between.
Thus, since Tables 1-2 provide allele frequencies based on the
re-sequencing of the chromosomes from 39 individuals, a subset of
SNPs could readily be selected for human identification in which
the total allele count of the minor allele at a particular SNP site
is, for example, at least 1, 2, 4, 8, 10, 16, 20, 24, 30, 32, 36,
38, 39, 40, or any other number in-between.
[0315] Furthermore, Tables 1-2 also provide population group
(interchangeably referred to herein as ethnic or racial groups)
information coupled with the extensive allele frequency
information. For example, the group of 39 individuals whose DNA was
re-sequenced was made-up of 20 Caucasians and 19 African-Americans.
This population group information enables further refinement of SNP
selection for human identification. For example, preferred SNPs for
human identification can be selected from Tables 1-2 that have
similar allele frequencies in both the Caucasian and
African-American populations; thus, for example, SNPs can be
selected that have equally high discriminatory power in both
populations. Alternatively, SNPs can be selected for which there is
a statistically significant difference in allele frequencies
between the Caucasian and African-American populations (as an
extreme example, a particular allele may be observed only in either
the Caucasian or the African-American population group but not
observed in the other population group); such SNPs are useful, for
example, for predicting the race/ethnicity of an unknown
perpetrator from a biological sample such as a hair or blood stain
recovered at a crime scene. For a discussion of using SNPs to
predict ancestry from a DNA sample, including statistical methods,
see Frudakis et al., "A Classifier for the SNP-Based Inference of
Ancestry", Journal of Forensic Sciences 2003; 48(4):771-782.
[0316] SNPs have numerous advantages over other types of
polymorphic markers, such as short tandem repeats (STRs). For
example, SNPs can be easily scored and are amenable to automation,
making SNPs the markers of choice for large-scale forensic
databases. SNPs are found in much greater abundance throughout the
genome than repeat polymorphisms. Population frequencies of two
polymorphic forms can usually be determined with greater accuracy
than those of multiple polymorphic forms at multi-allelic loci.
SNPs are mutationaly more stable than repeat polymorphisms. SNPs
are not susceptible to artefacts such as stutter bands that can
hinder analysis. Stutter bands are frequently encountered when
analyzing repeat polymorphisms, and are particularly troublesome
when analyzing samples such as crime scene samples that may contain
mixtures of DNA from multiple sources. Another significant
advantage of SNP markers over STR markers is the much shorter
length of nucleic acid needed to score a SNP. For example, STR
markers are generally several hundred base pairs in length. A SNP,
on the other hand, comprises a single nucleotide, and generally a
short conserved region on either side of the SNP position for
primer and/or probe binding. This makes SNPs more amenable to
typing in highly degraded or aged biological samples that are
frequently encountered in forensic casework in which DNA may be
fragmented into short pieces.
[0317] SNPs also are not subject to microvariant and "off-ladder"
alleles frequently encountered when analyzing STR loci.
Microvariants are deletions or insertions within a repeat unit that
change the size of the amplified DNA product so that the amplified
product does not migrate at the same rate as reference alleles with
normal sized repeat units. When separated by size, such as by
electrophoresis on a polyacrylamide gel, microvariants do not align
with a reference allelic ladder of standard sized repeat units, but
rather migrate between the reference alleles. The reference allelic
ladder is used for precise sizing of alleles for allele
classification; therefore alleles that do not align with the
reference allelic ladder lead to substantial analysis problems.
Furthermore, when analyzing multi-allelic repeat polymorphisms,
occasionally an allele is found that consists of more or less
repeat units than has been previously seen in the population, or
more or less repeat alleles than are included in a reference
allelic ladder. These alleles will migrate outside the size range
of known alleles in a reference allelic ladder, and therefore are
referred to as "off-ladder" alleles. In extreme cases, the allele
may contain so few or so many repeats that it migrates well out of
the range of the reference allelic ladder. In this situation, the
allele may not even be observed, or, with multiplex analysis, it
may migrate within or close to the size range for another locus,
further confounding analysis.
[0318] SNP analysis avoids the problems of microvariants and
off-ladder alleles encountered in STR analysis. Importantly,
microvariants and off-ladder alleles may provide significant
problems, and may be completely missed, when using analysis methods
such as oligonucleotide hybridization arrays, which utilize
oligonucleotide probes specific for certain known alleles.
Furthermore, off-ladder alleles and microvariants encountered with
STR analysis, even when correctly typed, may lead to improper
statistical analysis, since their frequencies in the population are
generally unknown or poorly characterized, and therefore the
statistical significance of a matching genotype may be
questionable. All these advantages of SNP analysis are considerable
in light of the consequences of most DNA identification cases,
which may lead to life imprisonment for an individual, or
re-association of remains to the family of a deceased
individual.
[0319] DNA can be isolated from biological samples such as blood,
bone, hair, saliva, or semen, and compared with the DNA from a
reference source at particular SNP positions. Multiple SNP markers
can be assayed simultaneously in order to increase the power of
discrimination and the statistical significance of a matching
genotype. For example, oligonucleotide arrays can be used to
genotype a large number of SNPs simultaneously. The SNPs provided
by the present invention can be assayed in combination with other
polymorphic genetic markers, such as other SNPs known in the art or
STRs, in order to identify an individual or to associate an
individual with a particular biological sample.
[0320] Furthermore, the SNPs provided by the present invention can
be genotyped for inclusion in a database of DNA genotypes, for
example, a criminal DNA databank such as the FBI's Combined DNA
Index System (CODIS) database. A genotype obtained from a
biological sample of unknown source can then be queried against the
database to find a matching genotype, with the SNPs of the present
invention providing nucleotide positions at which to compare the
known and unknown DNA sequences for identity. Accordingly, the
present invention provides a database comprising novel SNPs or SNP
alleles of the present invention (e.g., the database can comprise
information indicating which alleles are possessed by individual
members of a population at one or more novel SNP sites of the
present invention), such as for use in forensics, biometrics, or
other human identification applications. Such a database typically
comprises a computer-based system in which the SNPs or SNP alleles
of the present invention are recorded on a computer readable medium
(see the section of the present specification entitled
"Computer-Related Embodiments").
[0321] The SNPs of the present invention can also be assayed for
use in paternity testing. The object of paternity testing is
usually to determine whether a male is the father of a child. In
most cases, the mother of the child is known and thus, the mother's
contribution to the child's genotype can be traced. Paternity
testing investigates whether the part of the child's genotype not
attributable to the mother is consistent with that of the putative
father. Paternity testing can be performed by analyzing sets of
polymorphisms in the putative father and the child, with the SNPs
of the present invention providing nucleotide positions at which to
compare the putative father's and child's DNA sequences for
identity. If the set of polymorphisms in the child attributable to
the father does not match the set of polymorphisms of the putative
father, it can be concluded, barring experimental error, that the
putative father is not the father of the child. If the set of
polymorphisms in the child attributable to the father match the set
of polymorphisms of the putative father, a statistical calculation
can be performed to determine the probability of coincidental
match, and a conclusion drawn as to the likelihood that the
putative father is the true biological father of the child.
[0322] In addition to paternity testing, SNPs are also useful for
other types of kinship testing, such as for verifying familial
relationships for immigration purposes, or for cases in which an
individual alleges to be related to a deceased individual in order
to claim an inheritance from the deceased individual, etc. For
further information regarding the utility of SNPs for paternity
testing and other types of kinship testing, including methods for
statistical analysis, see Krawczak, "Informativity assessment for
biallelic single nucleotide polymorphisms", Electrophoresis 1999
June; 20(8):1676-81.
[0323] The use of the SNPs of the present invention for human
identification further extends to various authentication systems,
commonly referred to as biometric systems, which typically convert
physical characteristics of humans (or other organisms) into
digital data. Biometric systems include various technological
devices that measure such unique anatomical or physiological
characteristics as finger, thumb, or palm prints; hand geometry;
vein patterning on the back of the hand; blood vessel patterning of
the retina and color and texture of the iris; facial
characteristics; voice patterns; signature and typing dynamics; and
DNA. Such physiological measurements can be used to verify identity
and, for example, restrict or allow access based on the
identification. Examples of applications for biometrics include
physical area security, computer and network security, aircraft
passenger check-in and boarding, financial transactions, medical
records access, government benefit distribution, voting, law
enforcement, passports, visas and immigration, prisons, various
military applications, and for restricting access to expensive or
dangerous items, such as automobiles or guns (see, for example,
O'Connor, Stanford Technology Law Review and U.S. Pat. No.
6,119,096).
[0324] Groups of SNPs, particularly the SNPs provided by the
present invention, can be typed to uniquely identify an individual
for biometric applications such as those described above. Such SNP
typing can readily be accomplished using, for example, DNA
chips/arrays. Preferably, a minimally invasive means for obtaining
a DNA sample is utilized. For example, PCR amplification enables
sufficient quantities of DNA for analysis to be obtained from
buccal swabs or fingerprints, which contain DNA-containing skin
cells and oils that are naturally transferred during contact.
[0325] Further information regarding techniques for using SNPs in
forensic/human identification applications can be found in, for
example, Current Protocols in Human Genetics, John Wiley &
Sons, N.Y. (2002), 14.1-14.7.
[0326] Variant Proteins, Antibodies, Vectors & Host Cells,
& Uses Thereof
[0327] Variant Proteins Encoded by SNP-Containing Nucleic Acid
Molecules
[0328] The present invention provides SNP-containing nucleic acid
molecules, many of which encode proteins having variant amino acid
sequences as compared to the art-known (i.e., wild-type) proteins.
Amino acid sequences encoded by the polymorphic nucleic acid
molecules of the present invention are provided as SEQ ID
NOS:61-120 in Table 1 and the Sequence Listing. These variants will
generally be referred to herein as variant
proteins/peptides/polypeptides, or polymorphic
proteins/peptides/polypeptides of the present invention. The terms
"protein", "peptide", and "polypeptide" are used herein
interchangeably.
[0329] A variant protein of the present invention may be encoded
by, for example, a nonsynonymous nucleotide substitution at any one
of the cSNP positions disclosed herein. In addition, variant
proteins may also include proteins whose expression, structure,
and/or function is altered by a SNP disclosed herein, such as a SNP
that creates or destroys a stop codon, a SNP that affects splicing,
and a SNP in control/regulatory elements, e.g. promoters,
enhancers, or transcription factor binding domains.
[0330] As used herein, a protein or peptide is said to be
"isolated" or "purified" when it is substantially free of cellular
material or chemical precursors or other chemicals. The variant
proteins of the present invention can be purified to homogeneity or
other lower degrees of purity. The level of purification will be
based on the intended use. The key feature is that the preparation
allows for the desired function of the variant protein, even if in
the presence of considerable amounts of other components.
[0331] As used herein, "substantially free of cellular material"
includes preparations of the variant protein having less than about
30% (by dry weight) other proteins (i.e., contaminating protein),
less than about 20% other proteins, less than about 10% other
proteins, or less than about 5% other proteins. When the variant
protein is recombinantly produced, it can also be substantially
free of culture medium, i.e., culture medium represents less than
about 20% of the volume of the protein preparation.
[0332] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the variant protein in
which it is separated from chemical precursors or other chemicals
that are involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the variant protein having less than about
30% (by dry weight) chemical precursors or other chemicals, less
than about 20% chemical precursors or other chemicals, less than
about 10% chemical precursors or other chemicals, or less than
about 5% chemical precursors or other chemicals.
[0333] An isolated variant protein may be purified from cells that
naturally express it, purified from cells that have been altered to
express it (recombinant host cells), or synthesized using known
protein synthesis methods. For example, a nucleic acid molecule
containing SNP(s) encoding the variant protein can be cloned into
an expression vector, the expression vector introduced into a host
cell, and the variant protein expressed in the host cell. The
variant protein can then be isolated from the cells by any
appropriate purification scheme using standard protein purification
techniques. Examples of these techniques are described in detail
below (Sambrook and Russell, 2000, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.).
[0334] The present invention provides isolated variant proteins
that comprise, consist of or consist essentially of amino acid
sequences that contain one or more variant amino acids encoded by
one or more codons which contain a SNP of the present
invention.
[0335] Accordingly, the present invention provides variant proteins
that consist of amino acid sequences that contain one or more amino
acid polymorphisms (or truncations or extensions due to creation or
destruction of a stop codon, respectively) encoded by the SNPs
provided in Table 1 and/or Table 2. A protein consists of an amino
acid sequence when the amino acid sequence is the entire amino acid
sequence of the protein.
[0336] The present invention further provides variant proteins that
consist essentially of amino acid sequences that contain one or
more amino acid polymorphisms (or truncations or extensions due to
creation or destruction of a stop codon, respectively) encoded by
the SNPs provided in Table 1 and/or Table 2. A protein consists
essentially of an amino acid sequence when such an amino acid
sequence is present with only a few additional amino acid residues
in the final protein.
[0337] The present invention further provides variant proteins that
comprise amino acid sequences that contain one or more amino acid
polymorphisms (or truncations or extensions due to creation or
destruction of a stop codon, respectively) encoded by the SNPs
provided in Table 1 and/or Table 2. A protein comprises an amino
acid sequence when the amino acid sequence is at least part of the
final amino acid sequence of the protein. In such a fashion, the
protein may contain only the variant amino acid sequence or have
additional amino acid residues, such as a contiguous encoded
sequence that is naturally associated with it or heterologous amino
acid residues. Such a protein can have a few additional amino acid
residues or can comprise many more additional amino acids. A brief
description of how various types of these proteins can be made and
isolated is provided below.
[0338] The variant proteins of the present invention can be
attached to heterologous sequences to form chimeric or fusion
proteins. Such chimeric and fusion proteins comprise a variant
protein operatively linked to a heterologous protein having an
amino acid sequence not substantially homologous to the variant
protein. "Operatively linked" indicates that the coding sequences
for the variant protein and the heterologous protein are ligated
in-frame. The heterologous protein can be fused to the N-terminus
or C-terminus of the variant protein. In another embodiment, the
fusion protein is encoded by a fusion polynucleotide that is
synthesized by conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification of gene fragments
can be carried out using anchor primers which give rise to
complementary overhangs between two consecutive gene fragments
which can subsequently be annealed and re-amplified to generate a
chimeric gene sequence (see Ausubel et al., Current Protocols in
Molecular Biology, 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST protein). A variant protein-encoding nucleic acid can be cloned
into such an expression vector such that the fusion moiety is
linked in-frame to the variant protein.
[0339] In many uses, the fusion protein does not affect the
activity of the variant protein. The fusion protein can include,
but is not limited to, enzymatic fusion proteins, for example,
beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His
fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion
proteins, particularly poly-His fusions, can facilitate their
purification following recombinant expression. In certain host
cells (e.g., mammalian host cells), expression and/or secretion of
a protein can be increased by using a heterologous signal sequence.
Fusion proteins are further described in, for example, Terpe,
"Overview of tag protein fusions: from molecular and biochemical
fundamentals to commercial systems", Appl Microbiol Biotechnol.
2003 January; 60(5):523-33. Epub 2002 Nov. 7; Graddis et al.,
"Designing proteins that work using recombinant technologies", Curr
Pharm Biotechnol. 2002 December; 3(4):285-97; and Nilsson et al.,
"Affinity fusion strategies for detection, purification, and
immobilization of recombinant proteins", Protein Expr Punf 1997
October; 11(1):1-16.
[0340] The present invention also relates to further obvious
variants of the variant polypeptides of the present invention, such
as naturally-occurring mature forms (e.g., alleleic variants),
non-naturally occurring recombinantly-derived variants, and
orthologs and paralogs of such proteins that share sequence
homology. Such variants can readily be generated using art-known
techniques in the fields of recombinant nucleic acid technology and
protein biochemistry. It is understood, however, that variants
exclude those known in the prior art before the present
invention.
[0341] Further variants of the variant polypeptides disclosed in
Table 1 can comprise an amino acid sequence that shares at least
70-80%, 80-85%, 85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% sequence identity with an amino acid sequence disclosed in
Table 1 (or a fragment thereof) and that includes a novel amino
acid residue (allele) disclosed in Table 1 (which is encoded by a
novel SNP allele). Thus, an aspect of the present invention that is
specifically contemplated are polypeptides that have a certain
degree of sequence variation compared with the polypeptide
sequences shown in Table 1, but that contain a novel amino acid
residue (allele) encoded by a novel SNP allele disclosed herein. In
other words, as long as a polypeptide contains a novel amino acid
residue disclosed herein, other portions of the polypeptide that
flank the novel amino acid residue can vary to some degree from the
polypeptide sequences shown in Table 1.
[0342] Full-length pre-processed forms, as well as mature processed
forms, of proteins that comprise one of the amino acid sequences
disclosed herein can readily be identified as having complete
sequence identity to one of the variant proteins of the present
invention as well as being encoded by the same genetic locus as the
variant proteins provided herein.
[0343] Orthologs of a variant peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of a variant peptide as well as being encoded by a
gene from another organism. Preferred orthologs will be isolated
from non-human mammals, preferably primates, for the development of
human therapeutic targets and agents. Such orthologs can be encoded
by a nucleic acid sequence that hybridizes to a variant
peptide-encoding nucleic acid molecule under moderate to stringent
conditions depending on the degree of relatedness of the two
organisms yielding the homologous proteins.
[0344] Variant proteins include, but are not limited to, proteins
containing deletions, additions and substitutions in the amino acid
sequence caused by the SNPs of the present invention. One class of
substitutions is conserved amino acid substitutions in which a
given amino acid in a polypeptide is substituted for another amino
acid of like characteristics. Typical conservative substitutions
are replacements, one for another, among the aliphatic amino acids
Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser
and Thr; exchange of the acidic residues Asp and Glu; substitution
between the amide residues Asn and Gln; exchange of the basic
residues Lys and Arg; and replacements among the aromatic residues
Phe and Tyr. Guidance concerning which amino acid changes are
likely to be phenotypically silent are found in, for example, Bowie
et al., Science 247:1306-1310 (1990).
[0345] Variant proteins can be fully functional or can lack
function in one or more activities, e.g. ability to bind another
molecule, ability to catalyze a substrate, ability to mediate
signaling, etc. Fully functional variants typically contain only
conservative variations or variations in non-critical residues or
in non-critical regions. Functional variants can also contain
substitution of similar amino acids that result in no change or an
insignificant change in function. Alternatively, such substitutions
may positively or negatively affect function to some degree.
Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, truncations or extensions, or a substitution,
insertion, inversion, or deletion of a critical residue or in a
critical region.
[0346] Amino acids that are essential for function of a protein can
be identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)), particularly using the amino acid
sequence and polymorphism information provided in Table 1. The
latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as enzyme activity or in assays
such as an in vitro proliferative activity. Sites that are critical
for binding partner/substrate binding can also be determined by
structural analysis such as crystallization, nuclear magnetic
resonance or photoaffinity labeling (Smith et al., J. Mol. Biol.
224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).
[0347] Polypeptides can contain amino acids other than the 20 amino
acids commonly referred to as the 20 naturally occurring amino
acids. Further, many amino acids, including the terminal amino
acids, may be modified by natural processes, such as processing and
other post-translational modifications, or by chemical modification
techniques well known in the art. Accordingly, the variant proteins
of the present invention also encompass derivatives or analogs in
which a substituted amino acid residue is not one encoded by the
genetic code, in which a substituent group is included, in which
the mature polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (e.g.,
polyethylene glycol), or in which additional amino acids are fused
to the mature polypeptide, such as a leader or secretory sequence
or a sequence for purification of the mature polypeptide or a
pro-protein sequence.
[0348] Known protein modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0349] Such protein modifications are well known to those of skill
in the art and have been described in great detail in the
scientific literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993); Wold, F.,
Posttranslational Covalent Modification of Proteins, B. C. Johnson,
Ed., Academic Press, New York 1-12 (1983); Seifter et al., Meth.
Enzymol. 182: 626-646 (1990); and Rattan et al., Ann. N.Y. Acad.
Sci. 663:48-62 (1992).
[0350] The present invention further provides fragments of the
variant proteins in which the fragments contain one or more amino
acid sequence variations (e.g., substitutions, or truncations or
extensions due to creation or destruction of a stop codon) encoded
by one or more SNPs disclosed herein. The fragments to which the
invention pertains, however, are not to be construed as
encompassing fragments that have been disclosed in the prior art
before the present invention.
[0351] As used herein, a fragment may comprise at least about 4, 8,
10, 12, 14, 16, 18, 20, 25, 30, 50, 100 (or any other number
in-between) or more contiguous amino acid residues from a variant
protein, wherein at least one amino acid residue is affected by a
SNP of the present invention, e.g., a variant amino acid residue
encoded by a nonsynonymous nucleotide substitution at a cSNP
position provided by the present invention. The variant amino acid
encoded by a cSNP may occupy any residue position along the
sequence of the fragment. Such fragments can be chosen based on the
ability to retain one or more of the biological activities of the
variant protein or the ability to perform a function, e.g., act as
an immunogen. Particularly important fragments are biologically
active fragments. Such fragments will typically comprise a domain
or motif of a variant protein of the present invention, e.g.,
active site, transmembrane domain, or ligand/substrate binding
domain. Other fragments include, but are not limited to, domain or
motif-containing fragments, soluble peptide fragments, and
fragments containing immunogenic structures. Predicted domains and
functional sites are readily identifiable by computer programs well
known to those of skill in the art (e.g., PROSITE analysis)
(Current Protocols in Protein Science, John Wiley & Sons, N.Y.
(2002)).
[0352] Uses of Variant Proteins
[0353] The variant proteins of the present invention can be used in
a variety of ways, including but not limited to, in assays to
determine the biological activity of a variant protein, such as in
a panel of multiple proteins for high-throughput screening; to
raise antibodies or to elicit another type of immune response; as a
reagent (including the labeled reagent) in assays designed to
quantitatively determine levels of the variant protein (or its
binding partner) in biological fluids; as a marker for cells or
tissues in which it is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation
or development or in a disease state); as a target for screening
for a therapeutic agent; and as a direct therapeutic agent to be
administered into a human subject. Any of the variant proteins
disclosed herein may be developed into reagent grade or kit format
for commercialization as research products. Methods for performing
the uses listed above are well known to those skilled in the art
(see, e.g., Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Sambrook and Russell, 2000, and Methods in
Enzymology: Guide to Molecular Cloning Techniques, Academic Press,
Berger, S. L. and A. R. Kimmel eds., 1987).
[0354] In a specific embodiment of the invention, the methods of
the present invention include detection of one or more variant
proteins disclosed herein. Variant proteins are disclosed in Table
1 and in the Sequence Listing as SEQ ID NOS: 61-120. Detection of
such proteins can be accomplished using, for example, antibodies,
small molecule compounds, aptamers, ligands/substrates, other
proteins or protein fragments, or other protein-binding agents.
Preferably, protein detection agents are specific for a variant
protein of the present invention and can therefore discriminate
between a variant protein of the present invention and the
wild-type protein or another variant form. This can generally be
accomplished by, for example, selecting or designing detection
agents that bind to the region of a protein that differs between
the variant and wild-type protein, such as a region of a protein
that contains one or more amino acid substitutions that is/are
encoded by a non-synonymous cSNP of the present invention, or a
region of a protein that follows a nonsense mutation-type SNP that
creates a stop codon thereby leading to a shorter polypeptide, or a
region of a protein that follows a read-through mutation-type SNP
that destroys a stop codon thereby leading to a longer polypeptide
in which a portion of the polypeptide is present in one version of
the polypeptide but not the other.
[0355] In another specific aspect of the invention, the variant
proteins of the present invention are used as targets for
evaluating an individual's predisposition to developing a
cardiovascular disorder, particularly an acute coronary event such
as myocardial infarction, or stroke, for treating and/or preventing
cardiovascular disorders, of for predicting an individuals response
to statin treatment of cardiovascular disorders, etc. Accordingly,
the invention provides methods for detecting the presence of, or
levels of, one or more variant proteins of the present invention in
a cell, tissue, or organism. Such methods typically involve
contacting a test sample with an agent (e.g., an antibody, small
molecule compound, or peptide) capable of interacting with the
variant protein such that specific binding of the agent to the
variant protein can be detected. Such an assay can be provided in a
single detection format or a multi-detection format such as an
array, for example, an antibody or aptamer array (arrays for
protein detection may also be referred to as "protein chips"). The
variant protein of interest can be isolated from a test sample and
assayed for the presence of a variant amino acid sequence encoded
by one or more SNPs disclosed by the present invention. The SNPs
may cause changes to the protein and the corresponding protein
function/activity, such as through non-synonymous substitutions in
protein coding regions that can lead to amino acid substitutions,
deletions, insertions, and/or rearrangements; formation or
destruction of stop codons; or alteration of control elements such
as promoters. SNPs may also cause inappropriate post-translational
modifications.
[0356] One preferred agent for detecting a variant protein in a
sample is an antibody capable of selectively binding to a variant
form of the protein (antibodies are described in greater detail in
the next section). Such samples include, for example, tissues,
cells, and biological fluids isolated from a subject, as well as
tissues, cells and fluids present within a subject.
[0357] In vitro methods for detection of the variant proteins
associated with cardiovascular disorders and/or statin response
that are disclosed herein and fragments thereof include, but are
not limited to, enzyme linked immunosorbent assays (ELISAs),
radioimmunoassays (RIA), Western blots, immunoprecipitations,
immunofluorescence, and protein arrays/chips (e.g., arrays of
antibodies or aptamers). For further information regarding
immunoassays and related protein detection methods, see Current
Protocols in Immunology, John Wiley & Sons, N.Y., and Hage,
"Immunoassays", Anal Chem. 1999 June 15; 71(12):294R-304R.
[0358] Additional analytic methods of detecting amino acid variants
include, but are not limited to, altered electrophoretic mobility,
altered tryptic peptide digest, altered protein activity in
cell-based or cell-free assay, alteration in ligand or
antibody-binding pattern, altered isoelectric point, and direct
amino acid sequencing.
[0359] Alternatively, variant proteins can be detected in vivo in a
subject by introducing into the subject a labeled antibody (or
other type of detection reagent) specific for a variant protein.
For example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
[0360] Other uses of the variant peptides of the present invention
are based on the class or action of the protein. For example,
proteins isolated from humans and their mammalian orthologs serve
as targets for identifying agents (e.g., small molecule drugs or
antibodies) for use in therapeutic applications, particularly for
modulating a biological or pathological response in a cell or
tissue that expresses the protein. Pharmaceutical agents can be
developed that modulate protein activity.
[0361] As an alternative to modulating gene expression, therapeutic
compounds can be developed that modulate protein function. For
example, many SNPs disclosed herein affect the amino acid sequence
of the encoded protein (e.g., non-synonymous cSNPs and nonsense
mutation-type SNPs). Such alterations in the encoded amino acid
sequence may affect protein function, particularly if such amino
acid sequence variations occur in functional protein domains, such
as catalytic domains, ATP-binding domains, or ligand/substrate
binding domains. It is well established in the art that variant
proteins having amino acid sequence variations in functional
domains can cause or influence pathological conditions. In such
instances, compounds (e.g., small molecule drugs or antibodies) can
be developed that target the variant protein and modulate (e.g.,
up- or down-regulate) protein function/activity.
[0362] The therapeutic methods of the present invention further
include methods that target one or more variant proteins of the
present invention. Variant proteins can be targeted using, for
example, small molecule compounds, antibodies, aptamers,
ligands/substrates, other proteins, or other protein-binding
agents. Additionally, the skilled artisan will recognize that the
novel protein variants (and polymorphic nucleic acid molecules)
disclosed in Table 1 may themselves be directly used as therapeutic
agents by acting as competitive inhibitors of corresponding
art-known proteins (or nucleic acid molecules such as mRNA
molecules).
[0363] The variant proteins of the present invention are
particularly useful in drug screening assays, in cell-based or
cell-free systems. Cell-based systems can utilize cells that
naturally express the protein, a biopsy specimen, or cell cultures.
In one embodiment, cell-based assays involve recombinant host cells
expressing the variant protein. Cell-free assays can be used to
detect the ability of a compound to directly bind to a variant
protein or to the corresponding SNP-containing nucleic acid
fragment that encodes the variant protein.
[0364] A variant protein of the present invention, as well as
appropriate fragments thereof, can be used in high-throughput
screening assays to test candidate compounds for the ability to
bind and/or modulate the activity of the variant protein. These
candidate compounds can be further screened against a protein
having normal function (e.g., a wild-type/non-variant protein) to
further determine the effect of the compound on the protein
activity. Furthermore, these compounds can be tested in animal or
invertebrate systems to determine in vivo activity/effectiveness.
Compounds can be identified that activate (agonists) or inactivate
(antagonists) the variant protein, and different compounds can be
identified that cause various degrees of activation or inactivation
of the variant protein.
[0365] Further, the variant proteins can be used to screen a
compound for the ability to stimulate or inhibit interaction
between the variant protein and a target molecule that normally
interacts with the protein. The target can be a ligand, a substrate
or a binding partner that the protein normally interacts with (for
example, epinephrine or norepinephrine). Such assays typically
include the steps of combining the variant protein with a candidate
compound under conditions that allow the variant protein, or
fragment thereof, to interact with the target molecule, and to
detect the formation of a complex between the protein and the
target or to detect the biochemical consequence of the interaction
with the variant protein and the target, such as any of the
associated effects of signal transduction.
[0366] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al., Nature
354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0367] One candidate compound is a soluble fragment of the variant
protein that competes for ligand binding. Other candidate compounds
include mutant proteins or appropriate fragments containing
mutations that affect variant protein function and thus compete for
ligand. Accordingly, a fragment that competes for ligand, for
example with a higher affinity, or a fragment that binds ligand but
does not allow release, is encompassed by the invention.
[0368] The invention further includes other end point assays to
identify compounds that modulate (stimulate or inhibit) variant
protein activity. The assays typically involve an assay of events
in the signal transduction pathway that indicate protein activity.
Thus, the expression of genes that are up or down-regulated in
response to the variant protein dependent signal cascade can be
assayed. In one embodiment, the regulatory region of such genes can
be operably linked to a marker that is easily detectable, such as
luciferase. Alternatively, phosphorylation of the variant protein,
or a variant protein target, could also be measured. Any of the
biological or biochemical functions mediated by the variant protein
can be used as an endpoint assay. These include all of the
biochemical or biological events described herein, in the
references cited herein, incorporated by reference for these
endpoint assay targets, and other functions known to those of
ordinary skill in the art.
[0369] Binding and/or activating compounds can also be screened by
using chimeric variant proteins in which an amino terminal
extracellular domain or parts thereof, an entire transmembrane
domain or subregions, and/or the carboxyl terminal intracellular
domain or parts thereof, can be replaced by heterologous domains or
subregions. For example, a substrate-binding region can be used
that interacts with a different substrate than that which is
normally recognized by a variant protein. Accordingly, a different
set of signal transduction components is available as an end-point
assay for activation. This allows for assays to be performed in
other than the specific host cell from which the variant protein is
derived.
[0370] The variant proteins are also useful in competition binding
assays in methods designed to discover compounds that interact with
the variant protein. Thus, a compound can be exposed to a variant
protein under conditions that allow the compound to bind or to
otherwise interact with the variant protein. A binding partner,
such as ligand, that normally interacts with the variant protein is
also added to the mixture. If the test compound interacts with the
variant protein or its binding partner, it decreases the amount of
complex formed or activity from the variant protein. This type of
assay is particularly useful in screening for compounds that
interact with specific regions of the variant protein (Hodgson,
Bio/technology, 1992, September 10(9), 973-80).
[0371] To perform cell-free drug screening assays, it is sometimes
desirable to immobilize either the variant protein or a fragment
thereof, or its target molecule, to facilitate separation of
complexes from uncomplexed forms of one or both of the proteins, as
well as to accommodate automation of the assay. Any method for
immobilizing proteins on matrices can be used in drug screening
assays. In one embodiment, a fusion protein containing an added
domain allows the protein to be bound to a matrix. For example,
glutathione-S-transferase/.sup.125I fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the cell lysates (e.g., .sup.35S-labeled) and a
candidate compound, such as a drug candidate, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads can be washed to remove any unbound label, and the matrix
immobilized and radiolabel determined directly, or in the
supernatant after the complexes are dissociated. Alternatively, the
complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of bound material found in the bead
fraction quantitated from the gel using standard electrophoretic
techniques.
[0372] Either the variant protein or its target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Alternatively, antibodies reactive with the variant protein but
which do not interfere with binding of the variant protein to its
target molecule can be derivatized to the wells of the plate, and
the variant protein trapped in the wells by antibody conjugation.
Preparations of the target molecule and a candidate compound are
incubated in the variant protein-presenting wells and the amount of
complex trapped in the well can be quantitated. Methods for
detecting such complexes, in addition to those described above for
the GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the protein target molecule, or
which are reactive with variant protein and compete with the target
molecule, and enzyme-linked assays that rely on detecting an
enzymatic activity associated with the target molecule.
[0373] Modulators of variant protein activity identified according
to these drug screening assays can be used to treat a subject with
a disorder mediated by the protein pathway, such as cardiovascular
disease. These methods of treatment typically include the steps of
administering the modulators of protein activity in a
pharmaceutical composition to a subject in need of such
treatment.
[0374] The variant proteins, or fragments thereof, disclosed herein
can themselves be directly used to treat a disorder characterized
by an absence of, inappropriate, or unwanted expression or activity
of the variant protein. Accordingly, methods for treatment include
the use of a variant protein disclosed herein or fragments
thereof.
[0375] In yet another aspect of the invention, variant proteins can
be used as "bait proteins" in a two-hybrid assay or three-hybrid
assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993)
Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300)
to identify other proteins that bind to or interact with the
variant protein and are involved in variant protein activity. Such
variant protein-binding proteins are also likely to be involved in
the propagation of signals by the variant proteins or variant
protein targets as, for example, elements of a protein-mediated
signaling pathway. Alternatively, such variant protein-binding
proteins are inhibitors of the variant protein.
[0376] The two-hybrid system is based on the modular nature of most
transcription factors, which typically consist of separable
DNA-binding and activation domains. Briefly, the assay typically
utilizes two different DNA constructs. In one construct, the gene
that codes for a variant protein is fused to a gene encoding the
DNA binding domain of a known transcription factor (e.g., GAL-4).
In the other construct, a DNA sequence, from a library of DNA
sequences, that encodes an unidentified protein ("prey" or
"sample") is fused to a gene that codes for the activation domain
of the known transcription factor. If the "bait" and the "prey"
proteins are able to interact, in vivo, forming a variant
protein-dependent complex, the DNA-binding and activation domains
of the transcription factor are brought into close proximity. This
proximity allows transcription of a reporter gene (e.g., LacZ) that
is operably linked to a transcriptional regulatory site responsive
to the transcription factor. Expression of the reporter gene can be
detected, and cell colonies containing the functional transcription
factor can be isolated and used to obtain the cloned gene that
encodes the protein that interacts with the variant protein.
[0377] Antibodies Directed to Variant Proteins
[0378] The present invention also provides antibodies that
selectively bind to the variant proteins disclosed herein and
fragments thereof. Such antibodies may be used to quantitatively or
qualitatively detect the variant proteins of the present invention.
As used herein, an antibody selectively binds a target variant
protein when it binds the variant protein and does not
significantly bind to non-variant proteins, i.e., the antibody does
not significantly bind to normal, wild-type, or art-known proteins
that do not contain a variant amino acid sequence due to one or
more SNPs of the present invention (variant amino acid sequences
may be due to, for example, nonsynonymous cSNPs, nonsense SNPs that
create a stop codon, thereby causing a truncation of a polypeptide
or SNPs that cause read-through mutations resulting in an extension
of a polypeptide).
[0379] As used herein, an antibody is defined in terms consistent
with that recognized in the art: they are multi-subunit proteins
produced by an organism in response to an antigen challenge. The
antibodies of the present invention include both monoclonal
antibodies and polyclonal antibodies, as well as antigen-reactive
proteolytic fragments of such antibodies, such as Fab,
F(ab)'.sub.2, and Fv fragments. In addition, an antibody of the
present invention further includes any of a variety of engineered
antigen-binding molecules such as a chimeric antibody (U.S. Pat.
Nos. 4,816,567 and 4,816,397; Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851, 1984; Neuberger et al., Nature 312:604, 1984), a
humanized antibody (U.S. Pat. Nos. 5,693,762; 5,585,089; and
5,565,332), a single-chain Fv (U.S. Pat. No. 4,946,778; Ward et
al., Nature 334:544, 1989), a bispecific antibody with two binding
specificities (Segal et al., J. Immunol. Methods 248:1, 2001;
Carter, J. Immunol. Methods 248:7, 2001), a diabody, a triabody,
and a tetrabody (Todorovska et al., J. Immunol. Methods, 248:47,
2001), as well as a Fab conjugate (dimer or trimer), and a
minibody.
[0380] Many methods are known in the art for generating and/or
identifying antibodies to a given target antigen (Harlow,
Antibodies, Cold Spring Harbor Press, (1989)). In general, an
isolated peptide (e.g., a variant protein of the present invention)
is used as an immunogen and is administered to a mammalian
organism, such as a rat, rabbit, hamster or mouse. Either a
full-length protein, an antigenic peptide fragment (e.g., a peptide
fragment containing a region that varies between a variant protein
and a corresponding wild-type protein), or a fusion protein can be
used. A protein used as an immunogen may be naturally-occurring,
synthetic or recombinantly produced, and may be administered in
combination with an adjuvant, including but not limited to,
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substance such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanin, dinitrophenol, and the like.
[0381] Monoclonal antibodies can be produced by hybridoma
technology (Kohler and Milstein, Nature, 256:495, 1975), which
immortalizes cells secreting a specific monoclonal antibody. The
immortalized cell lines can be created in vitro by fusing two
different cell types, typically lymphocytes, and tumor cells. The
hybridoma cells may be cultivated in vitro or in vivo.
Additionally, fully human antibodies can be generated by transgenic
animals (He et al., J. Immunol., 169:595, 2002). Fd phage and Fd
phagemid technologies may be used to generate and select
recombinant antibodies in vitro (Hoogenboom and Chames, Immunol.
Today 21:371, 2000; Liu et al., J. Mol. Biol. 315:1063, 2002). The
complementarity-determining regions of an antibody can be
identified, and synthetic peptides corresponding to such regions
may be used to mediate antigen binding (U.S. Pat. No.
5,637,677).
[0382] Antibodies are preferably prepared against regions or
discrete fragments of a variant protein containing a variant amino
acid sequence as compared to the corresponding wild-type protein
(e.g., a region of a variant protein that includes an amino acid
encoded by a nonsynonymous cSNP, a region affected by truncation
caused by a nonsense SNP that creates a stop codon, or a region
resulting from the destruction of a stop codon due to read-through
mutation caused by a SNP). Furthermore, preferred regions will
include those involved in function/activity and/or protein/binding
partner interaction. Such fragments can be selected on a physical
property, such as fragments corresponding to regions that are
located on the surface of the protein, e.g., hydrophilic regions,
or can be selected based on sequence uniqueness, or based on the
position of the variant amino acid residue(s) encoded by the SNPs
provided by the present invention. An antigenic fragment will
typically comprise at least about 8-10 contiguous amino acid
residues in which at least one of the amino acid residues is an
amino acid affected by a SNP disclosed herein. The antigenic
peptide can comprise, however, at least 12, 14, 16, 20, 25, 50, 100
(or any other number in-between) or more amino acid residues,
provided that at least one amino acid is affected by a SNP
disclosed herein.
[0383] Detection of an antibody of the present invention can be
facilitated by coupling (i.e., physically linking) the antibody or
an antigen-reactive fragment thereof to a detectable substance.
Detectable substances include, but are not limited to, various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0384] Antibodies, particularly the use of antibodies as
therapeutic agents, are reviewed in: Morgan, "Antibody therapy for
Alzheimer's disease", Expert Rev Vaccines. 2003 February;
2(1):53-9; Ross et al., "Anticancer antibodies", Am J Clin Pathol.
2003 April; 119(4):472-85; Goldenberg, "Advancing role of
radiolabeled antibodies in the therapy of cancer", Cancer Immunol
Immunother. 2003 May; 52(5):281-96. Epub 2003 Mar. 11; Ross et al.,
"Antibody-based therapeutics in oncology", Expert Rev Anticancer
Ther. 2003 February; 3(1):107-21; Cao et al., "Bispecific antibody
conjugates in therapeutics", Adv Drug Deliv Rev. 2003 Feb. 10;
55(2):171-97; von Mehren et al., "Monoclonal antibody therapy for
cancer", Annu Rev Med. 2003; 54:343-69. Epub 2001 Dec. 3; Hudson et
al., "Engineered antibodies", Nat Med. 2003 January; 9(1):129-34;
Brekke et al., "Therapeutic antibodies for human diseases at the
dawn of the twenty-first century", Nat Rev Drug Discov. 2003
January; 2(1):52-62 (Erratum in: Nat Rev Drug Discov. 2003 March;
2(3):240); Houdebine, "Antibody manufacture in transgenic animals
and comparisons with other systems", Curr Opin Biotechnol. 2002
December; 13(6):625-9; Andreakos et al., "Monoclonal antibodies in
immune and inflammatory diseases", Curr Opin Biotechnol. 2002
December; 13(6):615-20; Kellermann et al., "Antibody discovery: the
use of transgenic mice to generate human monoclonal antibodies for
therapeutics", Curr Opin Biotechnol. 2002 December; 13(6):593-7;
Pini et al., "Phage display and colony filter screening for
high-throughput selection of antibody libraries", Comb Chem High
Throughput Screen. 2002 November; 5(7):503-10; Batra et al.,
"Pharmacokinetics and biodistribution of genetically engineered
antibodies", Curr Opin Biotechnol. 2002 December; 13(6):603-8; and
Tangri et al., "Rationally engineered proteins or antibodies with
absent or reduced immunogenicity", Curr Med Chem. 2002 December;
9(24):2191-9.
[0385] Uses of Antibodies
[0386] Antibodies can be used to isolate the variant proteins of
the present invention from a natural cell source or from
recombinant host cells by standard techniques, such as affinity
chromatography or immunoprecipitation. In addition, antibodies are
useful for detecting the presence of a variant protein of the
present invention in cells or tissues to determine the pattern of
expression of the variant protein among various tissues in an
organism and over the course of normal development or disease
progression. Further, antibodies can be used to detect variant
protein in situ, in vitro, in a bodily fluid, or in a cell lysate
or supernatant in order to evaluate the amount and pattern of
expression. Also, antibodies can be used to assess abnormal tissue
distribution, abnormal expression during development, or expression
in an abnormal condition, such as in a cardiovascular disorder or
during statin treatment. Additionally, antibody detection of
circulating fragments of the full-length variant protein can be
used to identify turnover.
[0387] Antibodies to the variant proteins of the present invention
are also useful in pharmacogenomic analysis. Thus, antibodies
against variant proteins encoded by alternative SNP alleles can be
used to identify individuals that require modified treatment
modalities.
[0388] Further, antibodies can be used to assess expression of the
variant protein in disease states such as in active stages of the
disease or in an individual with a predisposition to a disease
related to the protein's function, such as a cardiovascular
disorder, or during the course of a treatment regime, such as
during statin treatment. Antibodies specific for a variant protein
encoded by a SNP-containing nucleic acid molecule of the present
invention can be used to assay for the presence of the variant
protein, such as to predict an individual's response to statin
treatment or predisposition/susceptibility to an acute coronary
event, as indicated by the presence of the variant protein.
[0389] Antibodies are also useful as diagnostic tools for
evaluating the variant proteins in conjunction with analysis by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays well known in the art.
[0390] Antibodies are also useful for tissue typing. Thus, where a
specific variant protein has been correlated with expression in a
specific tissue, antibodies that are specific for this protein can
be used to identify a tissue type.
[0391] Antibodies can also be used to assess aberrant subcellular
localization of a variant protein in cells in various tissues. The
diagnostic uses can be applied, not only in genetic testing, but
also in monitoring a treatment modality. Accordingly, where
treatment is ultimately aimed at correcting the expression level or
the presence of variant protein or aberrant tissue distribution or
developmental expression of a variant protein, antibodies directed
against the variant protein or relevant fragments can be used to
monitor therapeutic efficacy.
[0392] The antibodies are also useful for inhibiting variant
protein function, for example, by blocking the binding of a variant
protein to a binding partner. These uses can also be applied in a
therapeutic context in which treatment involves inhibiting a
variant protein's function. An antibody can be used, for example,
to block or competitively inhibit binding, thus modulating
(agonizing or antagonizing) the activity of a variant protein.
Antibodies can be prepared against specific variant protein
fragments containing sites required for function or against an
intact variant protein that is associated with a cell or cell
membrane. For in vivo administration, an antibody may be linked
with an additional therapeutic payload such as a radionuclide, an
enzyme, an immunogenic epitope, or a cytotoxic agent. Suitable
cytotoxic agents include, but are not limited to, bacterial toxin
such as diphtheria, and plant toxin such as ricin. The in vivo
half-life of an antibody or a fragment thereof may be lengthened by
pegylation through conjugation to polyethylene glycol (Leong et
al., Cytokine 16:106, 2001).
[0393] The invention also encompasses kits for using antibodies,
such as kits for detecting the presence of a variant protein in a
test sample. An exemplary kit can comprise antibodies such as a
labeled or labelable antibody and a compound or agent for detecting
variant proteins in a biological sample; means for determining the
amount, or presence/absence of variant protein in the sample; means
for comparing the amount of variant protein in the sample with a
standard; and instructions for use.
[0394] Vectors and Host Cells
[0395] The present invention also provides vectors containing the
SNP-containing nucleic acid molecules described herein. The term
"vector" refers to a vehicle, preferably a nucleic acid molecule,
which can transport a SNP-containing nucleic acid molecule. When
the vector is a nucleic acid molecule, the SNP-containing nucleic
acid molecule can be covalently linked to the vector nucleic acid.
Such vectors include, but are not limited to, a plasmid, single or
double stranded phage, a single or double stranded RNA or DNA viral
vector, or artificial chromosome, such as a BAC, PAC, YAC, or
MAC.
[0396] A vector can be maintained in a host cell as an
extrachromosomal element where it replicates and produces
additional copies of the SNP-containing nucleic acid molecules.
Alternatively, the vector may integrate into the host cell genome
and produce additional copies of the SNP-containing nucleic acid
molecules when the host cell replicates.
[0397] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
SNP-containing nucleic acid molecules. The vectors can function in
prokaryotic or eukaryotic cells or in both (shuttle vectors).
[0398] Expression vectors typically contain cis-acting regulatory
regions that are operably linked in the vector to the
SNP-containing nucleic acid molecules such that transcription of
the SNP-containing nucleic acid molecules is allowed in a host
cell. The SNP-containing nucleic acid molecules can also be
introduced into the host cell with a separate nucleic acid molecule
capable of affecting transcription. Thus, the second nucleic acid
molecule may provide a trans-acting factor interacting with the
cis-regulatory control region to allow transcription of the
SNP-containing nucleic acid molecules from the vector.
Alternatively, a trans-acting factor may be supplied by the host
cell. Finally, a trans-acting factor can be produced from the
vector itself. It is understood, however, that in some embodiments,
transcription and/or translation of the nucleic acid molecules can
occur in a cell-free system.
[0399] The regulatory sequences to which the SNP-containing nucleic
acid molecules described herein can be operably linked include
promoters for directing mRNA transcription. These include, but are
not limited to, the left promoter from bacteriophage X, the lac,
TRP, and TAC promoters from E. coli, the early and late promoters
from SV40, the CMV immediate early promoter, the adenovirus early
and late promoters, and retrovirus long-terminal repeats.
[0400] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0401] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region, a ribosome-binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. A person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors (see, e.g.,
Sambrook and Russell, 2000, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
[0402] A variety of expression vectors can be used to express a
SNP-containing nucleic acid molecule. Such vectors include
chromosomal, episomal, and virus-derived vectors, for example,
vectors derived from bacterial plasmids, from bacteriophage, from
yeast episomes, from yeast chromosomal elements, including yeast
artificial chromosomes, from viruses such as baculoviruses,
papovaviruses such as SV40, Vaccinia viruses, adenoviruses,
poxviruses, pseudorabies viruses, and retroviruses. Vectors can
also be derived from combinations of these sources such as those
derived from plasmid and bacteriophage genetic elements, e.g.,
cosmids and phagemids. Appropriate cloning and expression vectors
for prokaryotic and eukaryotic hosts are described in Sambrook and
Russell, 2000, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0403] The regulatory sequence in a vector may provide constitutive
expression in one or more host cells (e.g., tissue specific
expression) or may provide for inducible expression in one or more
cell types such as by temperature, nutrient additive, or exogenous
factor, e.g., a hormone or other ligand. A variety of vectors that
provide constitutive or inducible expression of a nucleic acid
sequence in prokaryotic and eukaryotic host cells are well known to
those of ordinary skill in the art.
[0404] A SNP-containing nucleic acid molecule can be inserted into
the vector by methodology well-known in the art. Generally, the
SNP-containing nucleic acid molecule that will ultimately be
expressed is joined to an expression vector by cleaving the
SNP-containing nucleic acid molecule and the expression vector with
one or more restriction enzymes and then ligating the fragments
together. Procedures for restriction enzyme digestion and ligation
are well known to those of ordinary skill in the art.
[0405] The vector containing the appropriate nucleic acid molecule
can be introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial host cells
include, but are not limited to, E. coli, Streptomyces, and
Salmonella typhimurium. Eukaryotic host cells include, but are not
limited to, yeast, insect cells such as Drosophila, animal cells
such as COS and CHO cells, and plant cells.
[0406] As described herein, it may be desirable to express the
variant peptide as a fusion protein. Accordingly, the invention
provides fusion vectors that allow for the production of the
variant peptides. Fusion vectors can, for example, increase the
expression of a recombinant protein, increase the solubility of the
recombinant protein, and aid in the purification of the protein by
acting, for example, as a ligand for affinity purification. A
proteolytic cleavage site may be introduced at the junction of the
fusion moiety so that the desired variant peptide can ultimately be
separated from the fusion moiety. Proteolytic enzymes suitable for
such use include, but are not limited to, factor Xa, thrombin, and
enterokinase. Typical fusion expression vectors include pGEX (Smith
et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly,
Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185:60-89 (1990)).
[0407] Recombinant protein expression can be maximized in a
bacterial host by providing a genetic background wherein the host
cell has an impaired capacity to proteolytically cleave the
recombinant protein (Gottesman, S., Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)
119-128). Alternatively, the sequence of the SNP-containing nucleic
acid molecule of interest can be altered to provide preferential
codon usage for a specific host cell, for example, E. coli (Wada et
al., Nucleic Acids Res. 20:2111-2118 (1992)).
[0408] The SNP-containing nucleic acid molecules can also be
expressed by expression vectors that are operative in yeast.
Examples of vectors for expression in yeast (e.g., S. cerevisiae)
include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa
(Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al.,
Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San
Diego, Calif.).
[0409] The SNP-containing nucleic acid molecules can also be
expressed in insect cells using, for example, baculovirus
expression vectors. Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and
the pVL series (Lucklow et al., Virology 170:31-39 (1989)).
[0410] In certain embodiments of the invention, the SNP-containing
nucleic acid molecules described herein are expressed in mammalian
cells using mammalian expression vectors. Examples of mammalian
expression vectors include pCDM8 (Seed, B. Nature 329:840(1987))
and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).
[0411] The invention also encompasses vectors in which the
SNP-containing nucleic acid molecules described herein are cloned
into the vector in reverse orientation, but operably linked to a
regulatory sequence that permits transcription of antisense RNA.
Thus, an antisense transcript can be produced to the SNP-containing
nucleic acid sequences described herein, including both coding and
non-coding regions. Expression of this antisense RNA is subject to
each of the parameters described above in relation to expression of
the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0412] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include, for example, prokaryotic cells, lower eukaryotic cells
such as yeast, other eukaryotic cells such as insect cells, and
higher eukaryotic cells such as mammalian cells.
[0413] The recombinant host cells can be prepared by introducing
the vector constructs described herein into the cells by techniques
readily available to persons of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those described in
Sambrook and Russell, 2000, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.).
[0414] Host cells can contain more than one vector. Thus, different
SNP-containing nucleotide sequences can be introduced in different
vectors into the same cell. Similarly, the SNP-containing nucleic
acid molecules can be introduced either alone or with other nucleic
acid molecules that are not related to the SNP-containing nucleic
acid molecules, such as those providing trans-acting factors for
expression vectors. When more than one vector is introduced into a
cell, the vectors can be introduced independently, co-introduced,
or joined to the nucleic acid molecule vector.
[0415] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication can occur in host
cells that provide functions that complement the defects.
[0416] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be inserted in the
same vector that contains the SNP-containing nucleic acid molecules
described herein or may be in a separate vector. Markers include,
for example, tetracycline or ampicillin-resistance genes for
prokaryotic host cells, and dihydrofolate reductase or neomycin
resistance genes for eukaryotic host cells. However, any marker
that provides selection for a phenotypic trait can be
effective.
[0417] While the mature variant proteins can be produced in
bacteria, yeast, mammalian cells, and other cells under the control
of the appropriate regulatory sequences, cell-free transcription
and translation systems can also be used to produce these variant
proteins using RNA derived from the DNA constructs described
herein.
[0418] Where secretion of the variant protein is desired, which is
difficult to achieve with multi-transmembrane domain containing
proteins such as G-protein-coupled receptors (GPCRs), appropriate
secretion signals can be incorporated into the vector. The signal
sequence can be endogenous to the peptides or heterologous to these
peptides.
[0419] Where the variant protein is not secreted into the medium,
the protein can be isolated from the host cell by standard
disruption procedures, including freeze/thaw, sonication,
mechanical disruption, use of lysing agents, and the like. The
variant protein can then be recovered and purified by well-known
purification methods including, for example, ammonium sulfate
precipitation, acid extraction, anion or cationic exchange
chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0420] It is also understood that, depending upon the host cell in
which recombinant production of the variant proteins described
herein occurs, they can have various glycosylation patterns, or may
be non-glycosylated, as when produced in bacteria. In addition, the
variant proteins may include an initial modified methionine in some
cases as a result of a host-mediated process.
[0421] For further information regarding vectors and host cells,
see Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y.
[0422] Uses of Vectors and Host Cells, and Transgenic Animals
[0423] Recombinant host cells that express the variant proteins
described herein have a variety of uses. For example, the cells are
useful for producing a variant protein that can be further purified
into a preparation of desired amounts of the variant protein or
fragments thereof. Thus, host cells containing expression vectors
are useful for variant protein production.
[0424] Host cells are also useful for conducting cell-based assays
involving the variant protein or variant protein fragments, such as
those described above as well as other formats known in the art.
Thus, a recombinant host cell expressing a variant protein is
useful for assaying compounds that stimulate or inhibit variant
protein function. Such an ability of a compound to modulate variant
protein function may not be apparent from assays of the compound on
the native/wild-type protein, or from cell-free assays of the
compound. Recombinant host cells are also useful for assaying
functional alterations in the variant proteins as compared with a
known function.
[0425] Genetically-engineered host cells can be further used to
produce non-human transgenic animals. A transgenic animal is
preferably a non-human mammal, for example, a rodent, such as a rat
or mouse, in which one or more of the cells of the animal include a
transgene. A transgene is exogenous DNA containing a SNP of the
present invention which is integrated into the genome of a cell
from which a transgenic animal develops and which remains in the
genome of the mature animal in one or more of its cell types or
tissues. Such animals are useful for studying the function of a
variant protein in vivo, and identifying and evaluating modulators
of variant protein activity. Other examples of transgenic animals
include, but are not limited to, non-human primates, sheep, dogs,
cows, goats, chickens, and amphibians. Transgenic non-human mammals
such as cows and goats can be used to produce variant proteins
which can be secreted in the animal's milk and then recovered.
[0426] A transgenic animal can be produced by introducing a
SNP-containing nucleic acid molecule into the male pronuclei of a
fertilized oocyte, e.g., by microinjection or retroviral infection,
and allowing the oocyte to develop in a pseudopregnant female
foster animal. Any nucleic acid molecules that contain one or more
SNPs of the present invention can potentially be introduced as a
transgene into the genome of a non-human animal.
[0427] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the
variant protein in particular cells or tissues.
[0428] Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described in, for
example, U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et
al., U.S. Pat. No. 4,873,191 by Wagner et al., and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the transgene
in its genome and/or expression of transgenic mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes a non-human animal in which the entire animal
or tissues in the animal have been produced using the homologously
recombinant host cells described herein.
[0429] In another embodiment, transgenic non-human animals can be
produced which contain selected systems that allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1 (Lakso et al. PNAS
89:6232-6236 (1992)). Another example of a recombinase system is
the FLP recombinase system of S. cerevisiae (O'Gorman et al.
Science 251:1351-1355 (1991)). If a cre/loxP recombinase system is
used to regulate expression of the transgene, animals containing
transgenes encoding both the Cre recombinase and a selected protein
are generally needed. Such animals can be provided through the
construction of "double" transgenic animals, e.g., by mating two
transgenic animals, one containing a transgene encoding a selected
variant protein and the other containing a transgene encoding a
recombinase.
[0430] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in, for
example, Wilmut, I. et al. Nature 385:810-813 (1997) and PCT
International Publication Nos. WO 97/07668 and WO 97/07669. In
brief, a cell (e.g., a somatic cell) from the transgenic animal can
be isolated and induced to exit the growth cycle and enter G.sub.o
phase. The quiescent cell can then be fused, e.g., through the use
of electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyst and then transferred to pseudopregnant female
foster animal. The offspring born of this female foster animal will
be a clone of the animal from which the cell (e.g., a somatic cell)
is isolated.
[0431] Transgenic animals containing recombinant cells that express
the variant proteins described herein are useful for conducting the
assays described herein in an in vivo context. Accordingly, the
various physiological factors that are present in vivo and that
could influence ligand or substrate binding, variant protein
activation, signal transduction, or other processes or
interactions, may not be evident from in vitro cell-free or
cell-based assays. Thus, non-human transgenic animals of the
present invention may be used to assay in vivo variant protein
function as well as the activities of a therapeutic agent or
compound that modulates variant protein function/activity or
expression. Such animals are also suitable for assessing the
effects of null mutations (i.e., mutations that substantially or
completely eliminate one or more variant protein functions).
[0432] For further information regarding transgenic animals, see
Houdebine, "Antibody manufacture in transgenic animals and
comparisons with other systems", Curr Opin Biotechnol. 2002
December; 13(6):625-9; Petters et al., "Transgenic animals as
models for human disease", Transgenic Res. 2000; 9(4-5):347-51;
discussion 345-6; Wolf et al., "Use of transgenic animals in
understanding molecular mechanisms of toxicity", J Pharm Pharmacol.
1998 June; 50(6):567-74; Echelard, "Recombinant protein production
in transgenic animals", Curr Opin Biotechnol. 1996 October;
7(5):536-40; Houdebine, "Transgenic animal bioreactors", Transgenic
Res. 2000; 9(4-5):305-20; Pirity et al., "Embryonic stem cells,
creating transgenic animals", Methods Cell Biol. 1998; 57:279-93;
and Robl et al., "Artificial chromosome vectors and expression of
complex proteins in transgenic animals", Theriogenology. 2003 Jan.
1; 59(1):107-13.
Computer-Related Embodiments
[0433] The SNPs provided in the present invention may be "provided"
in a variety of mediums to facilitate use thereof. As used in this
section, "provided" refers to a manufacture, other than an isolated
nucleic acid molecule, that contains SNP information of the present
invention. Such a manufacture provides the SNP information in a
form that allows a skilled artisan to examine the manufacture using
means not directly applicable to examining the SNPs or a subset
thereof as they exist in nature or in purified form. The SNP
information that may be provided in such a form includes any of the
SNP information provided by the present invention such as, for
example, polymorphic nucleic acid and/or amino acid sequence
information such as SEQ ID NOS:1-60, SEQ ID NOS:61-120, SEQ ID
NOS:201-250, SEQ ID NOS:121-200, and SEQ ID NOS:251-410;
information about observed SNP alleles, alternative codons,
populations, allele frequencies, SNP types, and/or affected
proteins; or any other information provided by the present
invention in Tables 1-2 and/or the Sequence Listing.
[0434] In one application of this embodiment, the SNPs of the
present invention can be recorded on a computer readable medium. As
used herein, "computer readable medium" refers to any medium that
can be read and accessed directly by a computer. Such media
include, but are not limited to: magnetic storage media, such as
floppy discs, hard disc storage medium, and magnetic tape; optical
storage media such as CD-ROM; electrical storage media such as RAM
and ROM; and hybrids of these categories such as magnetic/optical
storage media. A skilled artisan can readily appreciate how any of
the presently known computer readable media can be used to create a
manufacture comprising computer readable medium having recorded
thereon a nucleotide sequence of the present invention. One such
medium is provided with the present application, namely, the
present application contains computer readable medium (CD-R) that
has nucleic acid sequences (and encoded protein sequences)
containing SNPs provided/recorded thereon in ASCII text format in a
Sequence Listing along with accompanying Tables that contain
detailed SNP and sequence information (transcript sequences are
provided as SEQ ID NOS:1-60, protein sequences are provided as SEQ
ID NOS:61-120, genomic sequences are provided as SEQ ID
NOS:201-250, transcript-based context sequences are provided as SEQ
ID NOS:121-200, and genomic-based context sequences are provided as
SEQ ID NOS:251-410).
[0435] As used herein, "recorded" refers to a process for storing
information on computer readable medium. A skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the SNP information of the present invention.
[0436] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide/amino acid sequence information of the
present invention on computer readable medium. For example, the
sequence information can be represented in a word processing text
file, formatted in commercially-available software such as
WordPerfect and Microsoft Word, represented in the form of an ASCII
file, or stored in a database application, such as OB2, Sybase,
Oracle, or the like. A skilled artisan can readily adapt any number
of data processor structuring formats (e.g., text file or database)
in order to obtain computer readable medium having recorded thereon
the SNP information of the present invention.
[0437] By providing the SNPs of the present invention in computer
readable form, a skilled artisan can routinely access the SNP
information for a variety of purposes. Computer software is
publicly available which allows a skilled artisan to access
sequence information provided in a computer readable medium.
Examples of publicly available computer software include BLAST
(Altschul et at, J. Mol. Biol. 215:403-410 (1990)) and BLAZE
(Brutlag et at, Comp. Chem. 17:203-207 (1993)) search
algorithms.
[0438] The present invention further provides systems, particularly
computer-based systems, which contain the SNP information described
herein. Such systems may be designed to store and/or analyze
information on, for example, a large number of SNP positions, or
information on SNP genotypes from a large number of individuals.
The SNP information of the present invention represents a valuable
information source. The SNP information of the present invention
stored/analyzed in a computer-based system may be used for such
computer-intensive applications as determining or analyzing SNP
allele frequencies in a population, mapping disease genes,
genotype-phenotype association studies, grouping SNPs into
haplotypes, correlating SNP haplotypes with response to particular
drugs, or for various other bioinformatic, pharmacogenomic, drug
development, or human identification/forensic applications.
[0439] As used herein, "a computer-based system" refers to the
hardware means, software means, and data storage means used to
analyze the SNP information of the present invention. The minimum
hardware means of the computer-based systems of the present
invention typically comprises a central processing unit (CPU),
input means, output means, and data storage means. A skilled
artisan can readily appreciate that any one of the currently
available computer-based systems are suitable for use in the
present invention. Such a system can be changed into a system of
the present invention by utilizing the SNP information provided on
the CD-R, or a subset thereof, without any experimentation.
[0440] As stated above, the computer-based systems of the present
invention comprise a data storage means having stored therein SNPs
of the present invention and the necessary hardware means and
software means for supporting and implementing a search means. As
used herein, "data storage means" refers to memory which can store
SNP information of the present invention, or a memory access means
which can access manufactures having recorded thereon the SNP
information of the present invention.
[0441] As used herein, "search means" refers to one or more
programs or algorithms that are implemented on the computer-based
system to identify or analyze SNPs in a target sequence based on
the SNP information stored within the data storage means. Search
means can be used to determine which nucleotide is present at a
particular SNP position in the target sequence. As used herein, a
"target sequence" can be any DNA sequence containing the SNP
position(s) to be searched or queried.
[0442] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences containing a SNP position in which the sequence(s) is
chosen based on a three-dimensional configuration that is formed
upon the folding of the target motif. There are a variety of target
motifs known in the art. Protein target motifs include, but are not
limited to, enzymatic active sites and signal sequences. Nucleic
acid target motifs include, but are not limited to, promoter
sequences, hairpin structures, and inducible expression elements
(protein binding sequences).
[0443] A variety of structural formats for the input and output
means can be used to input and output the information in the
computer-based systems of the present invention. An exemplary
format for an output means is a display that depicts the presence
or absence of specified nucleotides (alleles) at particular SNP
positions of interest. Such presentation can provide a rapid,
binary scoring system for many SNPs simultaneously.
[0444] One exemplary embodiment of a computer-based system
comprising SNP information of the present invention is provided in
FIG. 1. FIG. 1 provides a block diagram of a computer system 102
that can be used to implement the present invention. The computer
system 102 includes a processor 106 connected to a bus 104. Also
connected to the bus 104 are a main memory 108 (preferably
implemented as random access memory, RAM) and a variety of
secondary storage devices 110, such as a hard drive 112 and a
removable medium storage device 114. The removable medium storage
device 114 may represent, for example, a floppy disk drive, a
CD-ROM drive, a magnetic tape drive, etc. A removable storage
medium 116 (such as a floppy disk, a compact disk, a magnetic tape,
etc.) containing control logic and/or data recorded therein may be
inserted into the removable medium storage device 114. The computer
system 102 includes appropriate software for reading the control
logic and/or the data from the removable storage medium 116 once
inserted in the removable medium storage device 114.
[0445] The SNP information of the present invention may be stored
in a well-known manner in the main memory 108, any of the secondary
storage devices 110, and/or a removable storage medium 116.
Software for accessing and processing the SNP information (such as
SNP scoring tools, search tools, comparing tools, etc.) preferably
resides in main memory 108 during execution.
EXAMPLES
Genetic Polymorphisms Associated with MI and Statin Response
[0446] The following examples are offered to illustrate, but not to
limit the claimed invention.
[0447] Study Designs
[0448] In order to identify genetic markers associated with acute
coronary events (e.g. MI, stroke, unstable angina, congestive heart
failure, etc.) or response to statin treatment for the prevention
of coronary events, samples were genotyped in two clinical trials:
the Cholesterol and Recurrent Events (CARE) study (a randomized
multicentral double-blinded trial on secondary prevention of acute
coronary events with pravastatin; Sacks et al., Am. J. Cardiol. 68:
1436-1446 [1991]), and the West of Scotland Coronary Prevention
Study (WOSCOPS) study (Shepherd et al., N Eng J Med 333 (20), pp.
1301-7, Nov. 16, 1995; Packard et al., N Eng J Med 343 (16), pp.
1148-55, Oct. 19, 2000).
[0449] A well-documented myocardial infarction (MI) was one of the
enrollment criteria for entry into the CARE study. Patients were
enrolled in the CARE trial from 80 participating study centers. Men
and post-menopausal women were eligible for the trial if they had
had an acute MI between 3 and 20 months prior to randomization,
were 21 to 75 years of age, and had plasma total cholesterol levels
of less than 240 mg/deciliter, LDL cholesterol levels of 115 to 174
mgs/deciliter, fasting triglyceride levels of less than 350
mgs/deciliter, fasting glucose levels of no more than 220
mgs/deciliter, left ventricular ejection fractions of no less than
25%, and no symptomatic congestive heart failure. Patients were
randomized to receive either 40 mg of pravastatin once daily or a
matching placebo. The primary endpoint of the trial was death from
coronary heart disease and the median duration of follow-up was 5.0
years (range, 4.0 to 6.2 years). All individuals included in the
study had signed informed consent forms and the study protocol was
approved by the respective Institutional Review Boards (IRBs).
[0450] For genotyping SNPs in CARE patient samples, DNA was
extracted from blood samples using conventional DNA extraction
methods such as the QIAamp kit from Qiagen. Genotypes were obtained
by a method similar to that described by Lannone (M. A. Lannone et
al., Cytometry 39(2):131-140 [Feb. 1, 2000]; also described in
section "SNP DETECTION REAGENTS," supra). Briefly, target DNA was
amplified by multiplex PCR, the amplified product was submitted to
a multiplex oligo ligation assay (OLA), the specific ligated
products were hybridized to unique universal "ZIP code" oligomer
sequences covalently attached to Luminex xMAP.RTM. Multi-Analyte
COOH Microspheres, and these hybridization products/microspheres
were detected in a rapid automated multiplex system using the
Luminex 100 instrument. Alternatively, allele specific primers were
designed for detecting each SNP and genotypes were obtained on an
ABI PRISM.RTM. 7900HT Sequence Detection PCR system (Applied
Biosystems) by allele-specific PCR, similar to the method described
by Germer et al (Germer S., Holland M. J., Higuchi R. 2000, Genome
Res. 10: 258-266).
[0451] Genetic markers identified as associated with acute coronary
events or response to statin treatment for the prevention of
coronary events in the CARE samples were also genotyped in a second
sample set, the West of Scotland Coronary Prevention Study
(WOSCOPS). The design of the original WOSCOPS cohort and the nested
case-control study have been described (Shepherd et al., N Eng J
Med 333 (20), pp. 1301-7, Nov. 16, 1995; Packard et al., N Eng J
Med 343 (16), pp. 1148-55, Oct. 19, 2000). The objective of the
WOSCOPS trial was to assess pravastatin efficacy at reducing risk
of primary MI or coronary death among Scottish men with
hypercholesterolemia (fasting LDL cholesterol >155 mg/dl).
Participants in the WOSCOPS study were 45-64 years of age and
followed for an average of 4.9 years for coronary events. The
nested case-control study included as cases all WOSCOPS patients
who experienced a coronary event (confirmed nonfatal MI, death from
coronary heart disease, or a revascularization procedure; N=580).
Controls were age- and smoking-matched unaffected patients. All
individuals included in the study had signed informed consent forms
and the study protocol was approved by the IRBs. DNA was extracted
and genotyped as described above.
Example 1
Genetic Polymorphism hCV3054799 Associated with Risk of MI/RMI and
Response to Statin Treatment
[0452] The association of hCV3054799 with risk of MI in patients
was tested in two clinical trials, CARE and WOSCOPS, with patients
receiving treatment with either pravastatin or placebo. CARE was a
secondary prevention trial (i.e., recurrent MI as endpoint) and
WOSCOPS was a primary prevention trial. SNP genotype frequencies in
a group of 244 patients who had a second MI during the five years
of CARE follow-up (cases) were compared with the frequencies in the
group of 2471 CARE patients who did not experience a second MI
(controls). To replicate this finding, SNP genotype frequencies in
a group of 483 patients who had an MI within the five years of the
WOSCOPS follow-up (cases) were compared with the frequencies in the
group of 1094 WOSCOPS patients who did not experience MI
(controls). The analysis in WOSCOPS (a nested case-control study)
was performed using the MI as an endpoint.
[0453] Tables 5-7 list the counts of total patients studied, as
well as the number of patients in each treatment arm (study
strata). Also presented are the counts of specific genotypes
observed following the genotyping assays, their frequencies and the
frequencies of the two alleles for this SNP. The counts represent
the total number of valid genotyping assay results obtained, which
in some instances is slightly less than the total number of
patients enrolled in the study.
[0454] The statistics in Table 8 demonstrate the association of
hCV3054799 with MI/RMI. In both the CARE and WOSCOPS studies, an
association analysis was performed for each of the two modes of
inheritance, dominant and recessive. Additionally, a genotypic test
(heterozygote v. major homozygote) and an allelic test of
association were performed using Fisher's exact method. Estimates
of odds ratios and corresponding ninety-five percent confidence
intervals were calculated. The significance of the disease
association for hCV3054799 with the overall (unstratified)
population, and with the placebo- or pravastatin-treated patients
are indicated in Table 8. Analysis of these statistics shows that
the association of hCV3054799 with MI risk is especially
significant, and is replicated in both CARE and WOSCOPS studies. In
patients that are heterozygous carriers of the minor allele, the
odds ratio for risk of MI relative to patients with no minor allele
is 1.7, with a P value below 0.01 both studies. The odds ratio is
approximately the same (1.6-1.7) when calculated for the dominant
mode of inheritance (a genotype with one or two minor alleles vs.
none), with a P value less than or equal to 0.01 in both
studies.
[0455] A logistic regression method was used to adjust for major
epidemiologic risk factors in the CARE and WOSCOPS study samples
(Table 8). In the CARE samples, this method adjusted for the risk
factors associated with sex, family history, smoking status, body
mass index and hypertension. In the WOSCOPS set, logistic
regression was performed to adjust for the risk factors of family
history and hypertension. No adjustments were made for sex or
smoker status in the WOSCOPS set because all patients were male,
and the cases and controls were matched as to smoker status. The
odds ratios shown in Table 9 for heterozygous samples clearly
confirm the association of hCV3054799 with MI risk, independent of
and uninfluenced by other conventional risk factors. As in Table 8,
the OR for risk of MI in a heterozygous carrier is approximately
1.7, with P values below 0.01 in both studies.
[0456] Also analyzed was whether hCV3054799 is associated with
response to statin treatment (specifically, a decreased risk of MI
as a result of treatment with pravastatin). Table 10 presents these
risk association results. The number of heterozygous patients in
the pravastatin treatment group who had a second MI during the
five-year followup (cases) were compared with the number of
heterozygous individuals who did not experience a second MI
(controls). Heterozygous carriers in the placebo treatment group
were used as a reference in determining the odds ratios. Results in
the columns under the heading "Risk Reduction by Statin"
demonstrate the strong association of hCV3054799 with response to
pravastatin treatment: an odds ratio of approximately 0.5 for
carriers treated with pravastatin, with P values in both studies
well below 0.01. These data indicate that carriers of this allele
had a reduced risk of having an MI if they received statin
treatment and particularly pravistatin treatment. For comparison,
the odds ratio for association of this SNP with MI risk in
placebo-treated carriers is also presented, under the heading
"MI/RMI risk estimation" (data from Table 8; again, heterozygous
carriers).
[0457] See also Tables 17 and 18 for additional data supporting the
conclusion that individuals carrying certain hCV3054799 alleles had
an increased risk of developing MI/RMI, and that they responded
well to statin treatment. Data in Tables 17 and 18 were derived as
follows:
[0458] In analyzing the CARE patients, statistical analysis was
performed with SAS version 9. Cox proportional hazard models (Wald
tests) were used in CARE to assess the association of genotype with
both the risk of incident MI in the placebo arm and the effect of
on MI pravastatin compared to placebo in subgroups defined by
genotypes. In analyzing the WOSCOPS patients, a conditional
logistic regression model was used in WOSCOPS given that the
controls had been previously matched to the cases. The column
entitled "On-trial MI" lists the number of patients who suffered an
RMI during the clinical trial period.
[0459] In Table 18, p interaction values were calculated. An
interaction (or effect modification) is formed when a third
variable modifies the relation between an exposure and outcome. A p
interaction <0.05 indicates that a third variable (genotype)
modifies the relation between an exposure (statin treatment) and
outcome (MI). Genotype and drug interaction is present when the
incidence rate of disease in the presence of different genotypes
under placebo and statin treatment differs from the incidence rate
expected to result from their individual effects. HR stands for
Hazard Ratio, which is a concept similar to Odds Ratio (OR). The
hazard ratio in survival analysis is the effect of an explanatory
variable on the hazard or risk of an event. It describes the
likelihood of developing MI based on comparison of event rate
between patient treated with pravastatin and placebo. The
hCV3054799 risk allele predicted risk of MI in CARE and was
associated with odds of CHD in WOSCOPS. In both trials, carriers of
this risk allele (about 60% of population) benefited from
pravastatin treatment.
Example 2
Other Genetic Polymorphisms Associated with Risk of MI/RMI and
Response to Statin Treatment
[0460] An additional 175 markers were analyzed in samples from the
CARE study (secondary MI prevention trial). All patients in CARE
had an MI within ten months prior to enrollment in the trial.
During the five years of follow-up, 264 patients experienced a
recurrent MI (cases) and 2649 patients did not (controls). Of the
cases, 150 received placebo treatment and 114 received pravastatin;
of the controls, 1290 received placebo and 1359 received
pravastatin.
[0461] Tables 11-13 show the total counts of case and control
samples for each SNP, and a breakdown of cases and controls by
genotype: major and minor homozygous individuals, and heterozygous
individuals. Table 11 shows the data for the placebo-treatment arm
of the study, Table 12, the pravastatin-treatment arm, and Table
13, data for all samples unstratified by treatment.
[0462] The genotypes for each SNP were tested for association with
RMI. This genotypic test of association (i.e., minor homozygote
genotype vs. major, and heterozygote vs. major) was performed
separately for placebo-treated patients and for all patients
unstratified by treatment group, using the Fisher's exact method.
Table 14 shows the odds ratios obtained from the genotypic analysis
of the placebo-treatment arm (under the headings "Homozygous Minor"
and "Heterozygous Carriers"). The reliability of the OR for each
particular SNP is confirmed by its respective 95% confidence
interval (CI) and P value, also shown. From Table 14 one can see
the association of several SNPs with MI risk when they are present
in a Homozygous Minor genotype (association based on an OR >1,
the lower limit of the 95% CI >1 and a P value <0.05). These
SNPs include but are not limited to: hCV11433557, hCV16054991,
hCV25472673, and hCV7514870. In the Heterozygous Carriers genotype,
SNPs associated with an increased risk of RMI in Table 14 are:
hCV11450617, hCV16189747, hCV25963691, hCV3054799 and
hCV465412.
[0463] The Dominant and Recessive modes of inheritance for each SNP
were also tested for association with MI risk (i.e., Dominant=one
or two minor alleles vs. none; Recessive=two minor alleles vs. one
or none), in placebo-treated and unstratified sample groups. Table
14 shows modal results for the placebo-treated patients. According
to the OR's, P values and confidence intervals listed, all of the
SNPs mentioned above as associated with MI risk in Heterozygous
Carriers are also associated with increased risk for MI in the
Dominant inheritance mode, excepting hCV25963691 and with the
addition of hCV1801149 (OR 2.14, P value 0.04) Likewise, all of the
SNPs listed above as associated with risk in Homozygous Minor
patients are also associated with increased risk for MI in the
Recessive inheritance mode.
[0464] Table 15 shows results of the genotypic and modal tests of
association performed on all patients unstratified by treatment
arm; i.e., including statin-treated patients. In this analysis
(again according to OR, 95% CI and P value), SNP's significantly
associated with MI when present in the Homozygous Minor genotype
include: hCV16203383, hCV2146578 and hCV25936375. In the
Heterozygous Carriers genotype, MI-risk SNP's include: hCV11450617,
hCV16189747, hCV25624196, hCV25963691 and hCV3054799.
[0465] The Dominant and Recessive modal results of Table 15
(patients unstratified by treatment arm) indicate that in the
Dominant mode, SNP's associated with an increased risk of RMI
include: hCV11450617, hCV16189747, hCV25624196, hCV25741584 and
hCV3054799. In the Recessive mode, SNP's associated with an
increases risk of RMI include: hCV16054991, hCV16203383 and
hCV25936375.
[0466] The statistical results in Table 16 demonstrate the SNPs
predictive of response to statin treatment in the prevention of
RMI. All of these SNPs show a significant difference in risk
association between their respective placebo- and statin-treated
patient groups, as evidenced by Breslow Day P value for each SNP
(<0.05). For all SNPs in Table 16, the minor homozygous
individuals showed statistically different associations with statin
response (OR of placebo vs. pravastatin treatment) when compared to
heterozygous and major homozygous individuals. That is, these SNPs,
when present in a genotype of one and/or two alleles, are
associated with a response to statin treatment such that risk of MI
is reduced. Here the Breslow-Day statistics tests whether the odds
ratios for MI are different between carriers and non-carriers. A
Breslow-Day p value <0.05 means that the association between
treatment group and MI is not homogenous across genotypes,
therefore, this SNP may be associated with variability in statin
response.
[0467] As shown in Tables 19 and 20, certain individuals carrying
SNPs who were identified in Table 14 as being associated with an
increased risk of developing MI/RMI can benefit from being treated
with statin because statin reduced their risk of developing MI/RMI,
as compared those who did not undergo statin treatment. For
example, in the case of hCV11450617, individuals with Heterozygous
genotype and the Dominant genotype have their risk of developing MI
reduced by statin treatment.
Example 3
Additional SNPS in LD with SNPS that are Associated with Risk of
MI/RMI and Response to Statin Treatment
[0468] An investigation was conducted to identify SNP markers in
linkage disequilibrium (LD) with SNPs which have been found to be
associated with risk of MI/RMI and response to statin treatment,
such as those shown in Tables 1-20. Briefly, the power threshold
(T) was set at 51% for detecting disease association using LD
markers. This power threshold is based on equation (31) above,
which incorporates allele frequency data from previous disease
association studies, the predicted error rate for not detecting
truly disease-associated markers, and a significance level of 0.05.
Using this power calculation and the sample size, for each
interrogated SNP a threshold level of LD, or r.sup.2 value, was
derived (r.sub.T.sup.2, equations (32) and (33)). The threshold
value r.sub.T.sup.2 is the minimum value of linkage disequilibrium
between the interrogated SNP and its LD SNPs possible such that the
non-interrogated SNP still retains a power greater or equal to T
for detecting disease-association.
[0469] Based on the methodology described above, LD SNPs were
discovered for the interrogated SNPs shown in Tables 5-20. Table 21
shows a listing of some of the LD SNPs, each of which is associated
with its respective interrogated SNP. Also shown are the public SNP
IDs (rs numbers) for interrogated and LD SNPs, the threshold
r.sup.2 value and the power used to determine this, and the r.sup.2
value of linkage disequilibrium between the interrogated SNP and
its matching LD SNP. As an example in Table LD SNP,
MI/RMI-associated SNP hCV11450617 was calculated to be in LD with
hCV27846382 at an r.sup.2 value of 0.73586, based on a 51% power
calculation. Thus, hCV27846382 would also be a MI/RMI-associated
SNP due to its being in LD with the interrogated SNP
hCV11450617.
[0470] All publications and patents cited in this specification are
herein incorporated by reference in their entirety. Various
modifications and variations of the described compositions, methods
and systems of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection
with specific preferred embodiments and certain working examples,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the above-described modes for carrying out the
invention that are obvious to those skilled in the field of
molecular biology, genetics and related fields are intended to be
within the scope of the following claims.
TABLE-US-00004 TABLE 5 Allelic counts for hCV3054799. Major Allele
Minor Allele Case Cont Cont Cont Study Stratum All Count Count
Count Allele Case Freq Freq Allele Case Freq Freq CARE Unstratified
2714 244 2470 A 0.60 0.64 G 0.40 0.36 Placebo 1334 142 1192 A 0.60
0.65 G 0.40 0.35 Pravastatin 1380 102 1278 A 0.60 0.64 G 0.40 0.36
WOSCOPS Unstratified 1548 478 1070 A 0.63 0.66 G 0.37 0.34 Placebo
808 286 522 A 0.62 0.69 G 0.38 0.31 Pravastatin 740 192 548 A 0.65
0.63 G 0.35 0.37
TABLE-US-00005 TABLE 6 Genotypic counts for hCV3054799. Major
homozygous Minor homozygous Heterozygous Case Cont Case Cont Case
Cont Case Cont Case Cont Case Cont Study Genot Count Count Freq
Freq Genot Count Count Freq Freq Genot Count Count Freq Freq CARE
AA 82 1030 0.34 0.42 GG 35 314 0.14 0.13 GA 127 1126 0.52 0.46 AA
44 502 0.31 0.42 GG 17 143 0.12 0.12 GA 81 547 0.57 0.46 AA 38 528
0.37 0.41 GG 18 171 0.18 0.13 GA 46 579 0.45 0.45 WOSCOPS AA 189
475 0.40 0.44 GG 67 132 0.14 0.12 GA 222 463 0.46 0.43 AA 105 259
0.37 0.50 GG 39 58 0.14 0.11 GA 142 205 0.50 0.39 AA 84 216 0.44
0.39 GG 28 74 0.15 0.14 GA 80 258 0.42 0.47
TABLE-US-00006 TABLE 7 Combined counts for hCV3054799 Maj hom + Het
Het + MM hom Case Cont Case Cont Study Count Count Count Count CARE
209 2156 162 1440 125 1049 98 690 84 1107 64 750 WOSCOPS 411 938
289 595 247 464 181 263 164 474 108 332
TABLE-US-00007 TABLE 8 Association of genetic polymorphism
hCV3054799 with MI/RMI risk - estimates unadjusted. Heterozygous
Carriers Dominant ([Het + Recessive (Min hom vs. Allelic (Het vs.
Maj hom) Min hom] vs. Maj hom) [Maj hom + Het]) P Study Stratum OR
95% CI P value* OR 95% CI P value* OR 95% CI P value* OR 95% CI
value* CARE Unstratified 1.42 1.06-1.89 0.0201 1.41 1.07-1.86
0.0141 1.15 0.79-1.68 0.4825 1.23 1.02-1.49 0.0336 Placebo 1.69
1.15-2.49 0.0078 1.62 1.12-2.35 0.0114 1.00 0.58-1.71 1.0000 1.27
0.98-1.63 0.0666 Pravastatin 1.10 0.71-1.72 0.7342 1.19 0.78-1.80
0.4647 1.39 0.81-2.37 0.2314 1.19 0.89-1.60 0.2570 WOSCOPS
Unstratified 1.21 0.96-1.52 0.1241 1.22 0.98-1.52 0.0756 1.16
0.84-1.59 0.3667 1.15 0.98-1.35 0.0796 Placebo 1.71 1.25-2.33
0.0009 1.70 1.26-2.28 0.0005 1.26 0.82-1.95 0.3091 1.41 1.14-1.74
0.0020 Pravastatin 0.80 0.56-1.14 0.2381 0.84 0.60-1.17 0.3061 1.09
0.68-1.75 0.7159 0.93 0.73-1.19 0.5800 *Fisher's exact P value.
TABLE-US-00008 TABLE 9 Association of genetic polymorphism
hCV3054799 with MURMI risk-estimates adjusted by logistic
regression. Study Genotype Risk Freq* OR** 95% Cl P value*** CARE
Min hom 0.12 1.36 0.75-2.44 0.321 Het 0.47 1.69 1.14-2.49 0.008
WOSCOPS MM hom 0.12 1.62 1.01-2.60 0.043 Het 0.43 1.65 1.21-2.25
0.002 *Chi-square test for frequency of risk genotype. **Odds ratio
derived from patients in Placebo treatment arm. ***Fisher's exact P
value.
TABLE-US-00009 TABLE 10 Association of genetic polymorphism
hCV3054799 with MI/RMI risk and response to statin treatment.
MI/RMI risk estimation (Het vs. Maj hom)* Risk Reduction by Statin
Placebo stratum (Pravastatin vs. Placebo) P Heterozygous carriers
Study OR 95% CI value** OR 95% Cl P value** CARE 1.69 1.15-2.49
0.0078 0.54 0.36-0.78 0.00139 WOSCOPS 1.71 1.25-2.33 0.0009 0.45
0.32-0.62 0.0000014 *Data also presented in Table 9, Heterozygous
carriers. **Fisher's exact P value.
TABLE-US-00010 TABLE 11 Genotypic counts for genetic polymorphisms
in CARE study - Placebo arm. Major homozygous Minor homozygous
Heterozygous Cont Case Cont Case Cont Case Cont Marker Case Count
Count Genotype Count Count Genotype Count Count Genotype Count
Count hCV11433557 102 943 CC 57 592 GG 9 41 GC 36 310 hCV11450617
145 1220 GG 61 633 AA 13 101 AG 71 486 hCV12107274 104 942 CC 54
423 TT 8 122 TC 42 397 hCV15965459 133 1157 TT 95 843 AA 1 20 AT 37
294 hCV15973230 124 1023 GG 91 657 AA 3 61 AG 30 305 hCV15974589
101 938 CC 31 238 TT 21 220 TC 49 480 hCV1603656 104 947 CC 82 818
TT 2 6 TC 20 123 hCV16054991 132 1145 GG 60 599 AA 17 81 AG 55 465
hCV16189747 138 1174 CC 61 641 AA 9 75 AC 68 458 hCV1801149 50 403
GG 36 341 AA 1 2 AG 13 60 hCV22275215 103 947 CC 79 632 TT 2 39 TC
22 276 hCV2476746 123 1016 CC 55 405 TT 9 138 TC 59 473 hCV25472673
104 947 TT 29 338 CC 22 133 CT 53 476 hCV25623506 104 947 CC 101
894 TT 0 2 TC 3 51 hCV25745415 145 1217 GG 91 718 TT 2 63 TG 52 436
hCV25749177 145 1217 GG 89 704 AA 4 75 AG 52 438 hCV25963691 145
1217 CC 84 793 GG 3 52 GC 58 372 hCV2741051 104 947 TT 66 494 CC 3
67 CT 35 386 hCV3054799 104 945 AA 29 398 GG 13 116 GA 62 431
hCV465412 133 1157 CC 88 881 AA 2 27 AC 43 249 hCV7514870 103 936
CC 41 436 AA 17 90 AC 45 410 hCV881283 145 1219 GG 130 1129 CC 0 2
CG 15 88
TABLE-US-00011 TABLE 12 Genotypic counts for genetic polymorphisms
in CARE study - Pravastatin treatment arm. Case Cont Case Cont Case
Cont Case Cont Marker Count Count Genotype Count Count Genotype
Count Count Genotype Count Count hCV12107274 84 1040 CC 34 481 TC
37 446 TT 13 113 hCV15965459 99 1231 TT 85 918 AT 13 292 AA 1 21
hCV15974589 85 1036 CC 16 286 TC 35 514 TT 34 236 hCV1603656 85
1047 CC 76 873 TC 9 166 TT 0 8 hCV16054991 95 1216 GG 58 661 AG 29
467 AA 8 88 hCV25472673 85 1046 TT 38 392 CT 33 482 CC 14 172
hCV25623506 85 1046 CC 72 984 TC 12 62 TT 1 0 hCV25745415 107 1302
GG 57 798 TG 39 445 TT 11 59 hCV25749177 107 1300 GG 56 776 AG 39
444 AA 12 80 hCV2741051 85 1046 TT 39 533 CT 40 433 CC 6 80
hCV881283 107 1303 GG 103 1179 CG 4 122 CC 0 2
TABLE-US-00012 TABLE 13 Genotypic counts for genetic polymorphisms
in CARE study - Unstratified by treatment arm. Case Case Cont Case
Cont Case Cont Marker Count Cont Count Genot Count Count Genot
Count Count Genot Count Count hCV11450617 252 2523 AA 22 204 AG 120
1034 GG 110 1285 hCV15973230 216 2135 AA 5 118 AG 62 664 GG 149
1353 hCV16054991 227 2361 AA 25 169 AG 84 932 GG 118 1260
hCV16189747 241 2428 AA 15 153 AC 120 909 CC 106 1366 hCV16203383
248 2513 TT 35 253 TC 114 1077 CC 99 1183 hCV1690777 213 2104 GG 0
4 GA 7 147 AA 206 1953 hCV2146578 215 2127 TT 49 372 TC 103 1033 CC
63 722 hCV22275215 188 1994 TT 4 76 TC 41 592 CC 143 1326
hCV2476746 215 2117 TT 18 283 TC 96 967 CC 101 867 hCV25624196 213
2101 AA 5 52 AG 62 471 GG 146 1578 hCV25741584 232 2390 CC 0 0 CG 8
35 GG 224 2355 hCV25936375 233 2402 AA 12 64 AG 62 649 GG 159 1689
hCV25963691 251 2521 GG 5 100 GC 99 793 CC 147 1628 hCV3054799 189
1990 GG 29 259 GA 100 898 AA 60 833 hCV3200162 189 1993 GG 16 234
GA 75 885 AA 98 874 hCV945276 243 2421 TT 34 459 TG 121 1181 GG 88
781 hCV9879153 188 1992 TT 36 436 TC 85 986 CC 67 570
TABLE-US-00013 TABLE 14 RMI risk association of genetic
polymorphisms in CARE study - Placebo treatment arm. Minor
Homozygous Heterozygous Carriers Dominant ([Het + Recessive (Min
hom vs. Maj hom) (Het vs. Maj hom) Min hom]) vs. Maj hom Min hom
vs. ([Het + Maj hom]) Marker OR 95% Cl P value* OR 95% Cl P value*
OR 95% Cl P value* OR 95% Cl P value* hCV11433557 2.28 1.05-4.93
0.0425 1.21 0.78-1.87 0.4242 1.33 0.88-2.01 0.1972 2.13 1.00-4.52
0.0519 hCV11450617 1.34 0.71-2.52 0.3807 1.52 1.06-2.18 0.0262 1.48
1.05-2.10 0.0280 1.09 0.60-2.00 0.7512 hCV15973230 0.36 0.11-1.16
0.0999 0.71 0.46-1.10 0.1437 0.65 0.43-0.99 0.0459 0.39 0.12-1.27
0.1437 hCV16054991 2.10 1.17-3.77 0.0186 1.18 0.80-1.74 0.4295 1.32
0.92-1.89 0.1419 1.94 1.11-3.39 0.0240 hCV16189747 1.26 0.60-2.64
0.5424 1.56 1.08-2.25 0.0186 1.52 1.06-2.17 0.0239 1.02 0.50-2.09
0.8559 hCV1801149 4.74 0.42-53.53 0.2652 2.05 1.03-4.10 0.0616 2.14
1.09-4.20 0.0422 4.09 0.36-45.96 0.2965 hCV22275215 0.41 0.10-1.73
0.3005 0.64 0.39-1.04 0.0842 0.61 0.38-0.98 0.0454 0.46 0.11-1.94
0.4208 hCV2476746 0.48 0.23-1.00 0.0456 0.92 0.62-1.36 0.6905 0.82
0.56-1.19 0.3307 0.50 0.25-1.01 0.0627 hCV25472673 1.93 1.07-3.48
0.0352 1.30 0.81-2.08 0.2918 1.44 0.92-2.25 0.1292 1.64 0.99-2.72
0.0584 hCV25745415 0.25 0.06-1.04 0.0362 0.94 0.66-1.35 0.7841 0.85
0.60-1.22 0.4211 0.26 0.06-1.06 0.0389 hCV25963691 0.54 0.17-1.78
0.4709 1.47 1.03-2.10 0.0373 1.36 0.96-1.93 0.0984 0.47 0.15-1.54
0.2657 hCV2741051 0.34 0.10-1.10 0.0664 0.68 0.44-1.04 0.0892 0.63
0.41-0.95 0.0299 0.39 0.12-1.26 0.1435 hCV3054799 1.54 0.77-3.05
0.2528 1.97 1.24-3.13 0.0038 1.88 1.20-2.94 0.0060 1.02 0.55-1.88
0.8762 hCV465412 0.74 0.17-3.17 1.0000 1.73 1.17-2.56 0.0083 1.63
1.11-2.40 0.0147 0.64 0.15-2.72 0.7608 hCV7514870 2.01 1.09-3.69
0.0308 1.17 0.75-1.82 0.4996 1.32 0.87-2.00 0.2117 1.86 1.06-3.27
0.0389 For hCV15973230, hCV22275215 and hCV2741051, major
homozygotes were associated with RMI using heterozygotes and minor
homozygotes as reference. For hCV2476746 and hCV25745415, major
homozygotes were associated with RMI using minor homozygotes as
reference.
TABLE-US-00014 TABLE 15 RMI risk association of genetic
polymorphisms in CARE study - Unstratified by treatment arm.
Dominant Recessive Minor Homozygous Heterozygous carriers ([Het +
Min hom]) Min hom vs. (Min hom vs. Maj hom) (Het vs. Maj hom) vs.
Maj hom ([Het + Maj hom]) P P P P Marker OR 95% Cl value* OR 95% Cl
value* OR 95% Cl value* OR 95% Cl value* hCV11450617 1.26 0.78-2.04
0.3583 1.36 1.03-1.78 0.0312 1.34 1.03-1.74 0.0292 1.09 0.69-1.72
0.7169 hCV15973230 0.38 0.15-0.96 0.0357 0.85 0.62-1.16 0.3162 0.78
0.58-1.05 0.1183 0.41 0.16-1.00 0.0520 hCV16054991 1.58 1.00-2.50
0.0609 0.96 0.72-1.29 0.8237 1.06 0.80-1.39 0.7278 1.61 1.03-2.50
0.0464 hCV16189747 1.26 0.72-2.22 0.4347 1.70 1.29-2.24 0.0002 1.64
1.25-2.14 0.0003 0.99 0.57-1.71 1.0000 hCV16203383 1.65 1.10-2.49
0.0192 1.26 0.95-1.68 0.1144 1.34 1.03-1.75 0.0327 1.47 1.00-2.15
0.0502 hCV1690777 0.00 0.00-0.00 1.0000 0.45 0.21-0.98 0.0420 0.44
0.20-0.95 0.0314 0.00 0.00-0.00 1.0000 hCV2146578 1.51 1.02-2.24
0.0476 1.14 0.82-1.59 0.4577 1.24 0.91-1.69 0.1734 1.39 0.99-1.95
0.0619 hCV22275215 0.49 0.18-1.35 0.2361 0.64 0.45-0.92 0.0147 0.62
0.44-0.88 0.0072 0.55 0.20-1.52 0.3109 hCV2476746 0.55 0.32-0.92
0.0230 0.85 0.64-1.14 0.2941 0.78 0.59-1.04 0.0948 0.59 0.36-0.97
0.0418 hCV25624196 1.04 0.41-2.64 0.8114 1.42 1.04-1.95 0.0320 1.38
1.02-1.88 0.0392 0.95 0.37-2.40 1.0000 hCV25741584 0.00 0.00-0.00
1.0000 2.40 1.10-5.24 0.0498 2.40 1.10-5.24 0.0498 0.00 0.00-0.00
1.0000 hCV25936375 1.99 1.05-3.77 0.0390 1.01 0.75-1.38 0.9374 1.10
0.83-1.47 0.5008 1.98 1.05-3.73 0.0391 hCV25963691 0.55 0.22-1.38
0.2671 1.38 1.06-1.81 0.0193 1.29 0.99-1.68 0.0626 0.49 0.20-1.22
0.1623 hCV3054799 1.55 0.98-2.47 0.0718 1.55 1.11-2.16 0.0103 1.55
1.13-2.13 0.0067 1.21 0.80-1.84 0.3687 hCV3200162 0.61 0.35-1.05
0.0872 0.76 0.55-1.04 0.0940 0.73 0.54-0.98 0.0386 0.70 0.41-1.18
0.1904 hCV945276 0.66 0.44-0.99 0.0483 0.91 0.68-1.21 0.5526 0.84
0.64-1.10 0.2225 0.70 0.48-1.01 0.0570 hCV9879153 0.70 0.46-1.07
0.1164 0.73 0.52-1.03 0.0787 0.72 0.53-0.99 0.0445 0.85 0.58-1.23
0.4061
TABLE-US-00015 TABLE 16 Association of genetic polymorphisms with
response to statin treatment in CARE study. Risk of RMI in
placebo-vs. pravastatin-treated patients. Major homozygous Minor
homozygous Heterozygous Marker OR 95% Cl P value* OR 95% Cl P
value* OR 95% Cl P value* BD P value** hCV12107274 0.55 0.35-0.87
0.0100 1.75 0.70-4.39 0.2598 0.78 0.49-1.24 0.3462 0.0255
hCV15965459 0.82 0.60-1.12 0.2115 0.95 0.06-16.28 1.0000 0.35
0.18-0.68 0.0011 0.0408 hCV15974589 0.43 0.23-0.80 0.0090 1.51
0.85-2.68 0.1979 0.67 0.42-1.05 0.0881 0.0018 hCV1603656 0.87
0.63-1.20 0.4064 0.00 0.00-0.00 0.4667 0.33 0.15-0.76 0.0099 0.0097
hCV16054991 0.88 0.60-1.28 0.5015 0.43 0.18-1.06 0.0854 0.53
0.33-0.84 0.0063 0.0463 hCV25472673 1.13 0.68-1.87 0.7014 0.49
0.24-1.00 0.0524 0.61 0.39-0.97 0.0422 0.0382 hCV25623506 0.65
0.47-0.89 0.0069 0.00 0.00-0.00 0.3333 3.29 0.88-12.30 0.0939
0.0035 hCV25745415 0.56 0.40-0.80 0.0010 5.87 1.25-27.61 0.0176
0.73 0.48-1.14 0.1865 0.0073 hCV25749177 0.57 0.40-0.81 0.0017 2.81
0.87-9.10 0.1124 0.74 0.48-1.14 0.1873 0.0169 hCV2741051 0.55
0.36-0.83 0.0041 1.68 0.40-6.95 0.7321 1.02 0.63-1.64 1.0000 0.0307
hCV881283 0.76 0.58-0.99 0.0464 0.00 0.00-0.00 1.0000 0.19
0.06-0.60 0.0029 0.0185 *Fisher's exact P value. **Breslow-Day P
value.
TABLE-US-00016 TABLE 17 Association of KIF6 hCV3054799 with MI and
CHD in the placebo arm of CARE and WOSCOPS On-trial Unadjusted
Adjusted.sup..dagger. Study Genotype MI* Total* HR 95% Cl p Value
HR 95% Cl p Value CARE GG 16 155 1.33 0.75-2.35 0.33 1.33 0.75-2.36
0.33 GA 82 636 1.63 1.13-2.35 0.009 1.54 1.07-2.23 0.02 GG + GA 98
791 1.57 1.10-2.25 0.013 1.50 1.05-2.15 0.03 AA 44 542 1.00 ref
1.00 ref Matched.sup..dagger-dbl. Adjusted.sup..dagger..dagger-dbl.
Study Genotype Case* Control* OR 95% Cl p Value OR 95% Cl p Value
WOSCOPS GG 35 59 1.49 0.92-2.40 0.10 1.48 0.91-2.41 0.11 GA 137 204
1.61 1.18-2.21 0.003 1.56 1.14-2.15 0.006 GG + GA 172 263 1.59
1.18-2.14 0.003 1.55 1.14-2.09 0.005 AA 104 256 1.00 ref 1.00 ref
*Data for patients presented as number of patients
.sup..dagger.Adjusted for age (continuous), sex, smoking (current
versus non-current) (except in WOSCOPS, where no adjustments for
age and smoking were made because cases and controls were matched),
history of hypertension, history of diabetes, BMI (continuous),
baseline LDL-C level (continuous), and baseline HDL-C level
(continuous). .sup..dagger-dbl.Cases and controls were matched for
age (in two-year age groups) and smoking (current versus
non-current), all were men.
TABLE-US-00017 TABLE 18 Effect of pravastatin on MI and CHD in KIF6
hCV3054799 subgroups: CARE and WOSCOPS On-trial Unadjusted
Adjusted.sup..dagger. Study Genotype Treatment MI* Total* HR 95% CI
p Value HR 95% CI p Value CARE GG + GA Pravastatin 64 810 0.63
0.46-0.86 0.004 0.63 0.46-0.87 0.005 Placebo 98 791 1.00 ref 1.00
ref AA Pravastatin 39 554 0.86 0.56-1.33 0.50 0.80 0.52-1.24 0.32
Placebo 44 542 1.00 ref 1.00 ref p interaction 0.25.sup..sctn. p
interaction 0.39.sup..sctn. Matched.sup..dagger-dbl.
Adjusted.sup..dagger..dagger-dbl. Study Genotype Treatment Case*
Control* OR.sup..dagger. 95% CI.sup..dagger. p Value
OR.sup..dagger. 95% CI p Value WOSCOPS GG + GA Pravastatin 108 330
0.50 0.38-0.67 <0.0001 0.50 0.38-0.67 <0.0001 Placebo 172 263
1.00 ref 1.00 ref AA Pravastatin 81 213 0.94 0.67-1.33 0.73 0.91
0.64-1.28 0.58 Placebo 104 256 1.00 ref 1.00 ref p interaction
0.006.sup..sctn. p interaction 0.011.sup..sctn. *Data for patients
presented as number of patients .sup..dagger.Adjusted for age
(continuous), smoking (current versus non-current) (except in
WOSCOPS, where no adjustment for age and smoking were made because
cases and controls were matched), history of hypertension, history
of diabetes, BMI (continuous), baseline LDL-C level (continuous),
and baseline HDL-C level (continuous). .sup..dagger-dbl.Cases and
controls were matched for age (in two-year age groups) and smoking
(current versus non-current), all were men. .sup..sctn.Likelihood
ratio test of interaction between KIF6 hCV3054799 genotype and
treatment.
TABLE-US-00018 TABLE 19 Genotypic counts for genetic polymorphisms
in CARE study - Placebo arm and statin arm Marker Treatment Genot
case cnt control cnt Genot case cnt control cnt Genot case cnt
control cnt hCV11450617 placebo A A 13 101 A G 71 486 G G 61 633
hCV11450617 statin A A 9 103 A G 49 548 G G 49 652 hCV15973230
placebo A A 3 61 A G 30 305 G G 91 657 hCV15973230 statin A A 2 57
A G 32 359 G G 58 696 hCV25745415 placebo T T 2 63 T G 52 436 G G
91 718 hCV25745415 statin T T 11 59 T G 39 445 G G 57 798
hCV25963691 placebo G G 3 52 G C 58 372 C C 84 793 hCV25963691
statin G G 2 48 G C 41 421 C C 63 835 hCV2741051 placebo C C 3 67 C
T 35 386 T T 66 494 hCV2741051 statin C C 6 80 C T 40 433 T T 39
533 hCV3054799 placebo G G 13 116 G A 62 431 A A 29 398 hCV3054799
statin G G 16 143 G A 38 467 A A 31 435 hCV465412 placebo A A 2 27
A C 43 249 C C 88 881 hCV465412 statin A A 2 20 A C 24 298 C C 73
910 hCV7514870 placebo A A 17 90 A C 45 410 C C 41 436 hCV7514870
statin A A 9 130 A C 40 439 C C 35 466
TABLE-US-00019 TABLE 20 Risk reduction by pravastatin in CARE
patients stratified by genotypes of the SNPs associated with MI
(table 14) Risk of RMI in placebo- vs. pravastatin-treated
patients. Minor Homozygous Heterozygous Major Homozygous Dominant P
P P P Marker OR_min 95% CI value* OR_het 95% CI value* OR_maj 95%
CI value* OR_dom 95% CI value* hCV11450617 0.68 0.28-1.66 0.502
0.61 0.42-0.9 0.012 0.78 0.53-1.16 0.234 0.62 0.44-0.89 0.010
hCV11450617 hCV15973230 0.71 0.12-4.43 1.000 0.91 0.54-1.53 0.790
0.60 0.43-0.86 0.004 0.91 0.56-1.5 0.704 hCV15973230 hCV25745415
5.87 1.25-27.62 0.018 0.73 0.48-1.14 0.187 0.56 0.4-0.8 0.001 0.92
0.62-1.38 0.682 hCV25745415 hCV25963691 0.72 0.12-4.52 1.000 0.62
0.41-0.96 0.033 0.71 0.51-1.01 0.058 0.64 0.43-0.97 0.038
hCV25963691 hCV2741051 1.68 0.41-6.96 0.732 1.02 0.64-1.64 1.000
0.55 0.37-0.83 0.004 1.07 0.69-1.68 0.820 hCV2741051 hCV3054799
1.00 0.47-2.17 1.000 0.57 0.37-0.87 0.008 0.98 0.58-1.66 1.000 0.65
0.45-0.94 0.020 hCV3054799 hCV465412 1.35 0.18-10.42 1.000 0.47
0.28-0.79 0.004 0.80 0.59-1.12 0.189 0.50 0.31-0.84 0.008 hCV465412
hCV7514870 0.37 0.16-0.86 0.021 0.83 0.54-1.3 0.428 0.80 0.5-1.28
0.403 0.69 0.47-1.03 0.073 hCV7514870 *Fisher's exact P value.
TABLE-US-00020 TABLE 21 LD SNPs associated with MI/RMI risks and
statin response Interrogated SNP Interrogated SNP RS LD SNP LD SNP
RS Power Threshold r.sub.T.sup.2 r.sup.2 hCV11450617 rs1944270
hCV27846382 rs7234924 0.51 0.58 0.73586 hCV15973230 rs2304024
hCV25639371 rs3734051 0.51 0.39 0.60534 hCV15973230 rs2304024
hCV25951598 rs3734049 0.51 0.39 0.8599 hCV15973230 rs2304024
hCV25953895 rs3734047 0.51 0.39 0.86466 hCV15973230 rs2304024
hCV31985917 rs6870052 0.51 0.39 0.86466 hCV15974589 rs4875
hCV11238304 rs8455 0.51 0.93 0.9329 hCV15974589 rs4875 hCV2004219
rs891464 0.51 0.93 1 hCV15974589 rs4875 hCV29114176 rs6804259 0.51
0.93 0.96148 hCV15974589 rs4875 hCV29114183 rs7621897 0.51 0.93 1
hCV15974589 rs4875 hCV29114184 rs6772483 0.51 0.93 1 hCV15974589
rs4875 hCV29593475 rs9819567 0.51 0.93 1 hCV15974589 rs4875
hCV29864539 rs6803047 0.51 0.93 1 hCV15974589 rs4875 hCV3175476
rs6800914 0.51 0.93 0.96448 hCV16189747 rs2298566 hCV1067003
rs735094 0.51 0.24 0.32038 hCV16189747 rs2298566 hCV11345437
rs7937411 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV11345444
rs7937166 0.51 0.24 0.84361 hCV16189747 rs2298566 hCV25626063
rs3751039 0.51 0.24 0.3836 hCV16189747 rs2298566 hCV25626077
rs4414223 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV25626089
rs4586174 0.51 0.24 0.9631 hCV16189747 rs2298566 hCV25626101
rs6590537 0.51 0.24 0.84541 hCV16189747 rs2298566 hCV25759522
rs3751037 0.51 0.24 0.96383 hCV16189747 rs2298566 hCV25767057
rs12421950 0.51 0.24 0.24257 hCV16189747 rs2298566 hCV26490015
rs7942621 0.51 0.24 0.41952 hCV16189747 rs2298566 hCV26490026
rs7924461 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV26490027
rs7937782 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV26490028
rs7924736 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV26490038
rs6590523 0.51 0.24 0.85859 hCV16189747 rs2298566 hCV26490043
rs6421609 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV26490055
rs3794143 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV26490056
rs3794140 0.51 0.24 0.67868 hCV16189747 rs2298566 hCV26490057
rs7925664 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV26490064
rs3829270 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV26490066
rs3794132 0.51 0.24 0.89643 hCV16189747 rs2298566 hCV26490121
rs2875322 0.51 0.24 0.47739 hCV16189747 rs2298566 hCV27467549
rs3190331 0.51 0.24 0.41054 hCV16189747 rs2298566 hCV27497392
rs3829271 0.51 0.24 0.34599 hCV16189747 rs2298566 hCV27502173
rs3794139 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV27868997
rs4622274 0.51 0.24 0.29087 hCV16189747 rs2298566 hCV27868998
rs4936121 0.51 0.24 0.38654 hCV16189747 rs2298566 hCV27869000
rs4436551 0.51 0.24 0.41054 hCV16189747 rs2298566 hCV27869001
rs4264159 0.51 0.24 0.40152 hCV16189747 rs2298566 hCV27931232
rs4456262 0.51 0.24 0.41054 hCV16189747 rs2298566 hCV27996234
rs4457753 0.51 0.24 0.41296 hCV16189747 rs2298566 hCV29138817
rs4936120 0.51 0.24 0.31397 hCV16189747 rs2298566 hCV29138819
rs7116068 0.51 0.24 0.36418 hCV16189747 rs2298566 hCV29138820
rs7119425 0.51 0.24 0.39687 hCV16189747 rs2298566 hCV29138822
rs6590518 0.51 0.24 0.39687 hCV16189747 rs2298566 hCV29138826
rs4936123 0.51 0.24 0.41054 hCV16189747 rs2298566 hCV29138827
rs6590520 0.51 0.24 0.40531 hCV16189747 rs2298566 hCV29138833
rs6590532 0.51 0.24 0.75456 hCV16189747 rs2298566 hCV29138834
rs6590533 0.51 0.24 0.84765 hCV16189747 rs2298566 hCV29138837
rs7110968 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV29138841
rs7933747 0.51 0.24 0.84826 hCV16189747 rs2298566 hCV29138842
rs7933767 0.51 0.24 0.86378 hCV16189747 rs2298566 hCV29138843
rs7930661 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV29138844
rs7943935 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV29138848
rs7110591 0.51 0.24 0.86498 hCV16189747 rs2298566 hCV29138854
rs7949722 0.51 0.24 0.85299 hCV16189747 rs2298566 hCV3108695
rs4937582 0.51 0.24 1 hCV16189747 rs2298566 hCV3108709 rs948085
0.51 0.24 1 hCV16189747 rs2298566 hCV3108767 rs3198419 0.51 0.24
0.86498 hCV16189747 rs2298566 hCV31258077 rs6590515 0.51 0.24
0.39687 hCV16189747 rs2298566 hCV31258078 rs6590517 0.51 0.24
0.39687 hCV16189747 rs2298566 hCV31258101 rs4459316 0.51 0.24
0.41952 hCV16189747 rs2298566 hCV31258105 rs7106973 0.51 0.24
0.36538 hCV16189747 rs2298566 hCV31258108 rs7107595 0.51 0.24
0.41054 hCV16189747 rs2298566 hCV31258111 rs7937514 0.51 0.24
0.86498 hCV16189747 rs2298566 hCV31258124 rs3794136 0.51 0.24
0.86252 hCV16189747 rs2298566 hCV3251044 rs7114655 0.51 0.24 0.3931
hCV16189747 rs2298566 hCV3251060 rs7943757 0.51 0.24 0.29892
hCV16189747 rs2298566 hCV3251061 rs4366492 0.51 0.24 0.43166
hCV16189747 rs2298566 hCV3251063 rs6590512 0.51 0.24 0.3731
hCV16189747 rs2298566 hCV3251074 rs7106373 0.51 0.24 0.96341
hCV16189747 rs2298566 hCV3251078 rs4601795 0.51 0.24 0.42463
hCV16189747 rs2298566 hDV71141362 rs876641 0.51 0.24 0.40253
hCV16189747 rs2298566 hDV71218734 rs7110949 0.51 0.24 0.86498
hCV1801149 rs4750685 hCV11276365 rs4750935 0.51 0.73 0.88081
hCV1801149 rs4750685 hCV1801142 rs9787436 0.51 0.73 1 hCV1801149
rs4750685 hCV1801143 rs7100645 0.51 0.73 1 hCV1801149 rs4750685
hCV1801150 rs7918199 0.51 0.73 1 hCV1801149 rs4750685 hCV1801168
rs3781301 0.51 0.73 0.88095 hCV1801149 rs4750685 hCV218623
rs12261926 0.51 0.73 0.75008 hCV1801149 rs4750685 hCV31968274
rs11016063 0.51 0.73 0.75008 hCV1801149 rs4750685 hCV31968275
rs11016062 0.51 0.73 0.74272 hCV1801149 rs4750685 hCV374877
rs12252890 0.51 0.73 0.75008 hCV1801149 rs4750685 hCV8903188
rs997983 0.51 0.73 1 hCV22275215 rs3826543 hCV25610955 rs7221716
0.51 0.24 0.64584 hCV22275215 rs3826543 hCV25610955 rs7221716 0.51
0.560671413 0.64584 hCV22275215 rs3826543 hCV26960050 rs7221547
0.51 0.24 0.64511 hCV22275215 rs3826543 hCV26960050 rs7221547 0.51
0.560671413 0.64511 hCV22275215 rs3826543 hCV27915850 rs4792722
0.51 0.24 0.50871 hCV22275215 rs3826543 hCV29573094 rs8079361 0.51
0.24 0.4 hCV22275215 rs3826543 hCV30294410 rs9899432 0.51 0.24
0.61158 hCV22275215 rs3826543 hCV30294410 rs9899432 0.51
0.560671413 0.61158 hCV22275215 rs3826543 hCV30957093 rs7225429
0.51 0.24 0.81833 hCV22275215 rs3826543 hCV30957093 rs7225429 0.51
0.560671413 0.81833 hCV22275215 rs3826543 hCV30957101 rs6503096
0.51 0.24 0.81833 hCV22275215 rs3826543 hCV30957101 rs6503096 0.51
0.560671413 0.81833 hCV22275215 rs3826543 hCV30957112 rs7208297
0.51 0.24 0.48325 hCV22275215 rs3826543 hCV31793593 rs4792590 0.51
0.24 0.59064 hCV22275215 rs3826543 hCV31793593 rs4792590 0.51
0.560671413 0.59064 hCV22275215 rs3826543 hCV7453127 rs8531 0.51
0.24 0.25349 hCV22275215 rs3826543 hDV71102112 rs869773 0.51 0.24
0.26288 hCV3200162 rs10455 hCV27053832 rs2542939 0.51 0.67 0.95761
hCV3200162 rs10455 hCV31898499 rs11675373 0.51 0.67 0.95787
hCV3200162 rs10455 hCV3200161 rs2542941 0.51 0.67 1 hCV3200162
rs10455 hDV71152540 rs950163 0.51 0.67 1
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150111775A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150111775A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References