U.S. patent application number 12/705754 was filed with the patent office on 2010-07-22 for methods to predict cholesterol elevations during immunosuppressant therapy.
Invention is credited to Sridhar Kudaravalli, Mihael Hristos Polymeropoulos, Rosarelis Torres, Curt Douglas Wolfgang.
Application Number | 20100184798 12/705754 |
Document ID | / |
Family ID | 32043422 |
Filed Date | 2010-07-22 |
United States Patent
Application |
20100184798 |
Kind Code |
A1 |
Kudaravalli; Sridhar ; et
al. |
July 22, 2010 |
Methods to Predict Cholesterol Elevations during Immunosuppressant
Therapy
Abstract
This invention provides methods to predict the degree of
elevation of serum cholesterol levels in patients treated with
immunosuppressive medication. This invention also provides
treatment strategies based on these predictions and kits to carry
out these methods.
Inventors: |
Kudaravalli; Sridhar;
(Chicago, IL) ; Polymeropoulos; Mihael Hristos;
(Potomac, MD) ; Torres; Rosarelis; (Bethesda,
MD) ; Wolfgang; Curt Douglas; (Germantown,
MD) |
Correspondence
Address: |
NOVARTIS;CORPORATE INTELLECTUAL PROPERTY
ONE HEALTH PLAZA 104/3
EAST HANOVER
NJ
07936-1080
US
|
Family ID: |
32043422 |
Appl. No.: |
12/705754 |
Filed: |
February 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10529613 |
Jun 8, 2005 |
7732134 |
|
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PCT/EP2003/010798 |
Sep 29, 2003 |
|
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12705754 |
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60415123 |
Sep 30, 2002 |
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Current U.S.
Class: |
514/291 ;
435/6.16 |
Current CPC
Class: |
C12Q 2600/156 20130101;
A61P 3/06 20180101; A61P 37/06 20180101; C12Q 1/6883 20130101 |
Class at
Publication: |
514/291 ;
435/6 |
International
Class: |
A61K 31/436 20060101
A61K031/436; C12Q 1/68 20060101 C12Q001/68; A61P 37/06 20060101
A61P037/06 |
Claims
1.-19. (canceled)
20. A method of determining predisposition to serum cholesterol
elevation of greater than 239 mg/dL in a human patient upon
treatment with everolimus comprising: a) assaying a blood sample
from the patient for the presence of a Thymine (T) at polymorphic
site -511 of the IL-1.beta. gene, which is position 1423 of SEQ ID
NO 11; and b) determining that the patient has a predisposition to
serum cholesterol elevation of greater than 239 mg/dL upon
treatment with everolimus by the presence of a T at the polymorphic
site -511 of the human IL-1.beta. gene.
21. A method of treating a human patient with everolimus
comprising: a) assaying a blood sample from the patient for a
thymine (T) or cytosine (C) at polymorphic site -511 of the human
IL-1.beta. gene, which is position 1423 of SEQ ID NO:11; and b)
treating the patient with everolimus if the patient has a C for
both alleles at the polymorphic site -511 of the human IL-1.beta.
gene or treating the patient with everolimus and an alternative
treatment if the patient has a T allele at the polymorphic site
-511 of the human IL-1.beta. gene.
22. The method of claim 2, wherein the alternative treatment
comprises a cholesterol lowering medication selected from the group
consisting of a bile acid sequestrant, a fibric acid derivative, an
HMG-CoA reductase inhibitor, and nicotinic acid.
Description
[0001] This is a Divisional Application of 10/529,613 filed Mar.
30, 2005, which is a 35 U.S.C. 371 of PCT/EP30/10798 filed Sep. 29,
2003, which claims the benefit of U.S. Provisional Application No.
60/415,123 filed Sep. 30, 2002, which in their entirety are herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention belongs to the fields of pharmacology
and medicine and provides methods to determine which patients will
develop elevated serum cholesterol levels during treatment with an
immunosuppressant drug. In particular, this invention relates to
the use of genomic analysis to identify patients at risk for
developing increased cholesterol levels during immunosuppressant
drug therapy and to methods to determine optimal treatment
strategies for these patients.
[0004] 2. Description of the Related Art
[0005] Immunosuppressant drugs have many important applications in
modern medicine. These drugs are used to suppress the rejection of
transplanted organs, including hearts, lungs and kidneys to prolong
the useful life of the transplanted organ. In addition,
immunosuppressant drugs are used to treat a wide variety of other
diseases such as autoimmune diseases, myocarditis and rheumatoid
arthritis. However, the immunosuppressant drugs have numerous, and
sometimes severe, side effects which include causing cancer and
lymphomas and producing a variety of toxic effects on internal
organs, such as the kidney.
[0006] Because of the toxic effect of the immunosuppressant drugs,
there has been a great deal of effort to develop less toxic
alternatives and to find drugs whose mechanism of action differs
from that of the other immunosuppressant drugs so that synergistic
combinations can be used with fewer overall side effects.
[0007] Recently an anti-fungal, anti-tumor and immunosuppressive
antibiotic called rapamycin (also known as sirolimus and
RAPAMUNE.TM.) has been found to be effective at inhibiting
allograft rejection. Rapamycin has a mechanism of action that is
unique and markedly different from that of other immunosuppressant
drugs. Rapamycin and its derivatives, such as everolimus
(CERTICAN.TM.) (RAD) act by inhibiting the biochemical pathways
involved in the G1-S phase progression of activated T cells in a
Ca.sup.2+ independent manner. See Schuler et al., Transplantation,
Vol. 64, pp. 36-42 (1997). In this way, rapamycin derivatives such
as everolimus block cytokine signal transduction rather than
blocking the production of cytokines as in the case of other
immunosuppressant drugs, such as cyclosporine.
[0008] Rapamycin and its derivatives and mycophenolic acid are
effective immunosuppressant drugs, however, in some patients the
administration of these drugs has been found to cause elevations in
serum cholesterol and triglycerides, i.e., hypercholesterolemia and
hyperlipidemia. Both of these conditions are risk factors for
coronary artery disease (CAD) and atherosclerosis in general,
especially in diabetic patients.
[0009] Hypercholesterolemia itself, is a common condition and can
be treated with several major classes of drugs. These include the
HMG-CoA reductase inhibitors or the so-called statins, the bile
acid-binding resins and nicotinic acid.
[0010] Increased serum cholesterol levels during treatment with an
immunosuppressant drug, such as rapamycin or its derivatives
including, but not limited to, everolimus (CERTICAN.TM.) (RAD) or
with mycophenolic acid, is a serious adverse side effect. This is
especially true for organ transplant patients since these patients
require long-term (generally life long) treatment. The increase in
serum cholesterol levels varies widely from patient to patient and
prior to the present invention it was not possible to predict which
patients would develop these increases. Thus, there is a need for
methods to predict which patients will experience elevations in
serum cholesterol when immunosuppressant drugs, such as rapamycin
and its derivatives or mycophenolic acid are administered to
patients, especially for long-term use.
SUMMARY OF THE INVENTION
[0011] The present invention overcomes this problem by providing a
method to determine the degree of serum cholesterol elevation which
will occur in a patient during treatment with an immunosuppressant
medication comprising: determining for the two copies of the
IL-1.beta. gene present in the patient the identity of the
nucleotide pair at the polymorphic site -511 C.fwdarw.T (position
1423 of sequence X04500, which is incorporated by reference and is
SEQ ID NO:11 of the sequence listing), of the IL-1.beta. gene; and
assigning the patient to a high cholesterol elevation group if both
pairs are AT, assigning the patient to an intermediate cholesterol
elevation group if one pair is AT and one pair is GC and assigning
the patient to a low cholesterol elevation group if both pairs are
GC.
[0012] In a further embodiment this invention provides another
method to treat a patient with an immunosuppressive medication
comprising: determining for the two copies of the IL-1.beta. gene
present in the patient the identity of the nucleotide pair at the
polymorphic site -511 C.fwdarw.J (position 1423 of sequence X04500,
which is incorporated by reference and is SEQ ID NO:11 of the
sequence listing) of the IL-1.beta. gene; and treating the patient
with the immunosuppression medication if both pairs are GC and
using alternative treatment if one pair is AT and one pair is GC or
if both pairs are AT. The immunosuppressive medication may be
selected from the list in Table 2 and may be everolimus. In
addition this invention provides that the alternative treatment
comprises the addition of a cholesterol-lowering medication chosen
from those listed in Table 1.
[0013] In a further embodiment this invention provides a method to
determine the degree of serum cholesterol elevation which will
occur in a patient during treatment with an immunosuppressant
medication comprising: determining for the two copies of the
IL-1.beta. gene present in the patient the identity of the
nucleotide pair at the polymorphic site -31 T.fwdarw.C (position
1903 of sequence X04500, which is incorporated by reference and is
SEQ ID NO:11 of the sequence listing) of the IL-1.beta. gene; and
assigning the patient to a high cholesterol elevation group if both
pairs are CG, assigning the patient to an intermediate cholesterol
elevation group if one pair is AT and one pair is GC and assigning
the patient to a low cholesterol elevation group if both pairs are
AT.
[0014] In a still further embodiment this invention provides a
method to treat a patient with an immunosuppressive medication
comprising: determining for the two copies of the IL-1.beta. gene
present in the patient the identity of the nucleotide pair at the
polymorphic site -31 T.fwdarw.C (position 1903 of sequence X04500,
which is incorporated by reference and is SEQ ID NO:11 of the
sequence listing) of the IL-1.beta. gene; and treating the patient
with the immunosuppression medication if both pairs are AT and
using alternative treatment if one pair is AT and one pair is GC or
if both pairs are CG. The immunosuppressive medication may be
selected from the list in Table 2 and may be everolimus. In
addition the alternative treatment may comprise the addition of a
cholesterol-lowering medication chosen from those listed in Table
1.
[0015] In a still further embodiment this invention provides a kit
for determining the nucleotide pair at the polymorphic site -511 in
the IL-1.beta. gene in a patient, comprising: a container
containing at least one reagent specific for detecting the nature
of the nucleotide pair at the polymorphic site -511 of the
IL-1.beta. gene; and instructions for recommended treatment options
based on the nature of the said nucleotide pair.
[0016] In a still further embodiment this invention provides a kit
for determining the nucleotide pair at the polymorphic site -31 in
the IL-1.beta. gene in a patient, comprising: a container
containing at least one reagent specific for detecting the nature
of the nucleotide pair at the polymorphic site -31 of the
IL-1.beta. gene; and instructions for recommended treatment options
based on the nature of the said nucleotide pair.
[0017] In a further embodiment this invention provides a method to
determine the degree of serum cholesterol elevation which will
occur in a patient during treatment with an immunosuppressant
medication comprising determining, for the two copies, containing
the IL-1.beta. gene, present in the patient, the haplotype with
regard to the IL-1.beta. gene. The term "haplotype with regard to
the IL-1.beta. gene" shall refer to the haplotype consisting of the
combination of the polymorphisms at the -511 and the -31 position
of the IL-1.beta. gene. The patient would be assigned to a high
cholesterol elevation group if both chromosomes contain the "high
cholesterol" haplotype i.e., T for C at site -511 and C for T at
site -31 of the IL-1.beta. gene, and the patient would be assigned
to an intermediate cholesterol elevation group if one chromosome
contains the "high cholesterol" haplotype and one contains the "low
cholesterol" haplotype and the patient would be assigned to a low
cholesterol elevation group if both chromosomes contain the "low
cholesterol" haplotype, i.e. C at site -511 and T at site -31.
[0018] In a still further embodiment this invention provides a
method to treat a patient with an immunosuppressive medication
comprising determining, for the two chromosomes containing the
IL-1B gene, present in the patient, the haplotype with regard to
the IL-1.beta. gene, and treating the patient with the
immunosuppression medication if both chromosomes contain the "low
cholesterol" haplotype or if one chromosome contains the "low
cholesterol" haplotype and one contains the "high cholesterol"
haplotype and using alternative treatment if both chromosomes
contain the "high cholesterol" haplotype. The immunosuppressive
medication may be selected from the list in Table 2 and may be
everolimus. The alternative treatment may comprise the addition of
a cholesterol-lowering medication chosen from those listed in Table
1.
[0019] In a further embodiment this invention provides methods of
determining the identity of the nucleotide pair at the site -511
and -31 of the IL-1.beta. gene in a patient or the haplotype of the
IL-1.beta. gene in a patient by finding SNPs anywhere in the
chromosome which are in linkage disequilibrium with the -511
polymorphism or the .+-.31 polymorphism in the IL-1.beta. gene and
using the relationship of the said SNP or SNPs to determine the
nature of the nucleotide pair or haplotype of interest and using
this information to estimate cholesterol elevation during IM
therapy and to make treatment decisions.
[0020] A further embodiment of this invention is a kit for
determining the nature of the haplotype of the IL-1.beta. gene
which includes; a container containing at least one reagent
specific for detecting the nature of the nucleotide pair at the
polymorphic site -511 of the IL-1.beta. gene; and a container
containing at least one reagent specific for detecting the nature
of the nucleotide pair at the polymorphic site -31 of the
IL-1.beta. gene; and instructions for determining the haplotype
from the results of the above and instructions for recommended
treatment options based on the nature of the indicated
haplotype.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1: LS mean total cholesterol levels compared to the
(-511) IL-1.beta. CC, CT or TT genotypes in all treatment groups
combined within the RAD B251 clinical trial.
[0022] FIG. 2: LS mean total cholesterol levels compared to the
(-31) IL-1.beta. CC, CT or TT genotypes in all treatment groups
combined within the RAD B251 clinical trial.
[0023] FIG. 3: LS mean HDL cholesterol levels compared to the
(-511) IL-1.beta. CC, CT or TT genotypes in all treatment groups
combined within the RAD B251 clinical trial.
[0024] FIG. 4: LS mean HDL cholesterol levels compared to the (-31)
IL-1.beta. CC, CT or TT genotypes in all treatment groups combined
within the RAD B251 clinical trial.
[0025] FIG. 5: LS mean LDL cholesterol levels compared to the
(-511) IL-1.beta. CC, CT or TT genotypes in all treatment groups
within the RAD B251 clinical trial.
[0026] FIG. 6: LS mean LDL cholesterol levels compared to the (-31)
IL-1.beta. CC, CT or TT genotypes in all treatments groups within
the RAD B251 clinical trial.
[0027] FIG. 7: Study Schematic for the RAD B251 clinical trial.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention provides methods to determine the
degree of serum cholesterol elevation that will occur in a patient
during treatment with an immunosuppressant medication (IM), such as
rapamycin or its derivatives.
[0029] In one embodiment, a patient who is a potential candidate
for treatment with an IM would have blood drawn for a determination
of the presence of a polymorphism, i.e., cytosine
(C).fwdarw.thymine (T) at nucleotide position -511 (in the promoter
region with no amino acid change); this is a C .fwdarw.T change at
nucleotide position 1423 of sequence X04500, which is incorporated
by reference and is SEQ ID NO:11 of the sequence listing, in the
two copies of the interleukin-1-beta (IL-1.beta.) gene present in
the patient. If the nucleotide pair at position -511 is AT in both
copies of the gene, then the patient will experience a high
elevation in serum cholesterol levels during treatment with an
IM.
[0030] If the nucleotide pair at position -511 is AT in one copy
and CG in the other copy, then the patient will have an
intermediate elevation in their cholesterol levels during treatment
with an immunosuppressant medication.
[0031] If the nucleotide pair is GC at both copies at position
-511, then the patient will have a low elevation in serum
cholesterol levels during treatment with an IM.
[0032] In another embodiment, a determination of which medications
to use to treat a patient in need of treatment with an IM would be
based on the results of the determination of the nature of the
nucleotide pairs at position -511 in the IL-1.beta. gene present in
the patient.
[0033] If both nucleotide pairs are GC then the patient would be
treated with an IM. This immunosuppressant medication could be any
of those shown in Table 2, including rapamycin or one of its
derivatives, including, but not limited to, everolimus
(Certican.TM.) (RAD).
[0034] If both nucleotide pairs are AT, or if one pair is AT and
one pair is GC, then the patient would be treated with an
alternative medication that did not raise cholesterol levels or
alternatively the patient would be treated with a
cholesterol-lowering drug in addition to the IM and the patients'
cholesterol levels would be monitored during treatment. This
cholesterol-lowering drug, which would be used in combination with
IM, could be one or more of the medications chosen from the list in
Table 1 below.
[0035] In a further embodiment, a patient who is a potential
candidate for treatment with an IM would have blood drawn for a
determination of the presence of a polymorphism T.fwdarw.C at
nucleotide position -31 (in the promoter region with no amino acid
change); this is a T.fwdarw.C change at nucleotide position 1903 of
sequence X04500, which is incorporated by reference and is SEQ ID
NO:11 of the sequence listing, in the two copies of the IL-1.beta.
gene present in the patient. If the nucleotide pair at position -31
is a CG in both copies of the gene, then the patient will
experience a high elevation in serum cholesterol levels during
treatment with an IM. If the nucleotide pair at position -31 is AT
in one copy and CG in the other copy, then the patient will have an
intermediate elevation in their cholesterol levels during treatment
with an IM. If the nucleotide pair is AT at both copies at position
-31, then the patient will have a low elevation in serum
cholesterol levels during treatment with an IM.
[0036] In another embodiment, a determination of which medications
to use to treat a patient in need of treatment with an IM would be
based on the results of the determination of the nature of the
nucleotide pairs at position -31 in the IL-1.beta. gene present in
the patient. If both nucleotide pairs are AT then the patient would
be treated with an IM.
[0037] This IM could any of those shown in Table 2 including, but
not limited to, rapamycin or one of its derivatives including, but
not limited to, everolimus (CERTICAN.TM.) (RAD).
[0038] If both nucleotide pairs are GC or if the nucleotide pair is
AT in one copy and CG in the other copy, then the patient would be
treated with an alternative medication that did not raise
cholesterol levels or alternatively the patient would be treated
with a cholesterol-lowering drug in addition to the IM and the
patients' cholesterol levels would be monitored during treatment.
This cholesterol-lowering drug, which would be used in combination
with IM, could be one or more of the medications chosen from the
list in Table 1 below.
[0039] Suitable rapamycins for use in the methods of this invention
are, e.g., as described in U.S. Pat. Nos. 3,929,992 and 5,258,389
and in WO 94/09010 and WO 01/60345, all of which are hereby
incorporated by reference in their entirety and for all
purposes.
TABLE-US-00001 TABLE 1 Antilipemic Agents (Cholesterol-Lowering
Drugs) Bile Acid Sequestrants Colestipol (COLESTID .TM. Pharmacia
& Upjohn) Fibric Acid Derivatives Clofibrate (ATROMID-S .TM.
Wyeth-Ayerst) Gemfibrozil (LOPID .TM. Park-Davis) Fenofibrate
(TRICOR .TM. Abbott) HMG-CoA Reductase Inhibitors Fluvastatin
(LESCOL .TM. Novartis) Atorvastatin (LIPITOR .TM. Parke-Davis)
Lovastatin (MEVACOR .TM. Merck) Pravastatin (PRAVACOL .TM.
Bristol-Myers Squibb) Simvastatin (ZOCOR .TM. Merck) Nicotinic Acid
Niacin (NIASPAN .TM. Kos)
TABLE-US-00002 TABLE 2 Immunosuppressant Drugs Rapamycin
(sirolimus, RAPAMUNE .TM.) Everolimus (CERTICAN .TM.) (RAD)
Mycophenolic acid and Mycophenolate Mofetil (CELLCEPT .TM.) (MMF)
Azathioprine (IMURAN .TM.) Cyclosporine (NEORAL .TM.) Tacrolimus
(PROGRAF .TM.)
[0040] The following example is provided for the purpose of further
illustration only and is not intended to be a limitation on the
disclosed invention.
Example 1
The RAD B251 Study
Overall Study Design
[0041] The RAD B251 study was a randomized, multicenter,
double-blind, parallel group study of the efficacy and safety of
everolimus (Certican.TM.) (RAD) versus mycophenolate mofetil (MMF)
used in combination with cyclosporine (CsA) (NEORAL.RTM.) and
prednisone. The study consisted of three periods: a Screening
period, a Baseline period and a Double-Blind Treatment period.
Following the Baseline assessments, patients who meet the
inclusion/exclusion criteria were randomized into one of the three
treatment groups (1:1:1) once it has been ascertained that the
allograft is functional and that oral medication can be tolerated
(within 48 hours post-transplantation). Determination of allograft
function was by the investigator's judgement and was based upon
adequate urine output and evidence of falling creatinine levels.
The day of randomization and administration of the first dose of
study medication was recorded as Day 1 of the study. The three
treatment groups are described below:
[0042] Dose Level 1: 0.75 mg RAD bid+NEORAL.RTM.+prednisone
[0043] Dose Level 2: 1.5 mg RAD bid+NEORAL.RTM.+prednisone
[0044] Comparator: 1 g MMF bid+NEORAL.RTM.+prednisone
Concomitant Therapy
Initiation and Maintenance of NEORAL.RTM.
[0045] Oral CsA (NEORAL.RTM.) administration was begun at 6-12
mg/kg/day p.o. and was adjusted to maintain a 12-hour trough level
reflecting a standard target assay range. Intravenous (i.v.)
administration of CsA was avoided unless mandated by the clinical
situation. Whole blood levels were brought into the therapeutic
ranges listed below as rapidly as possible. Once this was achieved,
doses of CsA were only adjusted for maintaining trough blood levels
within the target ranges.
[0046] Weeks 1-4: 200-350 ng/mL
[0047] Months 2-36: 100-300 ng/mL
[0048] On the days when blood for CsA or RAD measurements was to be
drawn, the patient received the prior dose of NEORAL.RTM. and study
medication 12.+-.1 hour prior to the blood draw. The patients were
instructed to adjust their medication schedule on the day previous
to the blood draw to achieve proper timing and the exact time of
administration of the evening dose was recorded. Study medication
and NEORAL.RTM. due on the day of the blood draw were not to be
taken by the patient, but were brought to the clinic and taken
after the blood draw was completed.
Prednisone
[0049] Immediately prior to transplant, patients could receive up
to 1 g methylprednisolone i.v. and then up to 500 mg
methylprednisolone i.v. 12 hours later. As soon as possible
post-transplantation, oral prednisone was initiated at 0.35-2.0
mg/kg/day and tapered in order to achieve a dose of 20 mg/day, or
0.25 mg/kg/day, by Day 30 and of no less than 5 mg/day for the
first six months.
Cytomegalovirus (CMV) Prophylaxis
[0050] CMV prophylaxis was mandatory for all cases in which the
donor tests positive and the recipient tests negative for CMV.
Treatment with ganciclovir, CMV hyperimmune globulin or acyclovir
was permitted and was administered according to local practice. All
cases other than CMV positive donors to CMV negative recipients
were treated according to local practice. CMV prophylaxis was also
recommended following any antibody treatment of acute rejection
episodes.
PCP Prophylaxis
[0051] All patients were also started on
trimethoprim-sulfamethoxazole, one single strength tablet per day,
starting when oral medication could be tolerated and continuing for
the first six months after transplantation. Dosage was decreased at
the investigator's discretion to one single strength tablet 3 times
a week for the second six months. Treatment after one year was
according to local practice. Aerosolized pentamidine or dapsone was
administered to patients unable to tolerate
trimethoprim-sulfamethoxazole.
Other Concomitant Therapy
[0052] No medication other than the study drugs, study prophylaxis
and the usual medications of the patients were given during the
full treatment period of the study, i.e., from the initial day of
screening until all of the final study evaluations have been
completed. Exceptions to this rule applied only to medications that
were needed to treat adverse events (AEs). The administration of
any additional medication (including over-the-counter medications
and vitamins) were clearly documented on the Prior and Concomitant
Medications Case Report Form (CRF). If required for an AE,
concomitant medications were clearly documented and
cross-referenced on the AEs CRF.
[0053] All immunosuppressive drugs other than those specified by
protocol were disallowed. Permissible anti-rejection therapy
includes methylprednisolone and anti-lymphocyte antibody therapy
according to the guidelines in "Treatment of Acute Rejection
Episodes". Patients requiring tacrolimus or MMF for rescue therapy
were discontinued from study drug. Terfenadine, astemizole and
cisapride were prohibited while the patient is on study medication.
The use of phenobarbital, phenyloin, carbamazepine, or ketoconazole
was strongly discouraged.
[0054] A treatment period of three years followed transplantation.
During the Double-Blind Treatment period, the patients were seen at
Days 7, 14 and 28 and at Months 2, 3, 6, 9, 12, 18, 24, 30 and 36.
Renal biopsies were required at Baseline (may be intra-operative)
and at the time of any suspected rejection. Blinded study drug
administration ceased at 3 years.
[0055] Male and female patients, 16-65 years of age who are
scheduled to undergo primary cadaveric, living unrelated or non-HLA
identical related donor kidney transplantation, were allowed to
enter the study. Patients who discontinued the study prematurely
were not replaced. Each patient had to meet all of the
inclusion/exclusion criteria to be eligible for entry into the
study.
[0056] The RAD 8251 study was designed to assess the safety and
efficacy of two oral doses of RAD compared to MMF in de novo renal
transplant recipients as measured by the incidence of biopsy proven
acute allograft rejection episodes, graft loss or death. MMF was
chosen as the comparator agent because of its widespread use in
renal transplantation.
Pharmacogenetics Analysis
[0057] In an effort to identify genetic factors that are associated
with increased cholesterol and lipid levels observed in patients
treated with everolimus (RAD), 47 single nucleotide polymorphisms
(SNPs) from 24 genes within genomic polydeoxyribonucleotide (DNA)
of patients participating in the RAD B251 clinical trials were
examined. Of the 47 SNPs that were examined, 21 were experimentally
determined to be not polymorphic. Of the 26 that were polymorphic,
two SNPs in the IL-1.beta. gene promoter at positions (-511) and
(-31) showed statistical significance in relation to changes in
cholesterol levels in patients participating in the RAD B251
clinical trial.
[0058] Patients who were homozygous for the IL-1.beta. (-511)
C.fwdarw.T base transition (T-T) or the IL-1.beta. (-31) T.fwdarw.C
base transition (C-C) had the highest least mean levels of total
cholesterol at their last visit regardless of treatment received
during the study (p=0.0018 and p=0.0013 respectively). (The values
on the figures, etc. refer to the absolute serum cholesterol levels
at the last visit, however during the statistical analysis this
value was defined as the dependent variable and the cholesterol
level at baseline as the independent variable thus automatically
taking into consideration the baseline level).
[0059] The increase in total cholesterol levels was due to both
increased levels of HDL and LDL: patients homozygous for the T
allele at the (-511) position or the C allele at the (-31) position
had the highest least square mean levels of HDL (p=0.0214 and
p=0.0514 respectively) and LDL (p=0.0159 and p=0.0091 respectively)
at their last visit. Importantly, however, the HDL to LDL ratios
remained the same regardless of genotype.
[0060] Therefore, our findings suggest that individuals homozygous
for the T allele at position (-511) and homozygous for the C allele
at position (-31) of the IL-1.beta. gene promoter may be
predisposed to larger increases in total blood cholesterol levels
upon treatment with either the RAD/NEORAL.RTM. or MMF/NEORAL.RTM.
regimens.
Methods
Samples
[0061] A total of 82 unique samples from the RAD B251 clinical
trial were genotyped upon their consent to participate in the
pharmacogenetic evaluation. This represents about 15% of the total
population that participated in the RAD B251 clinical trial. Blood
samples from each patient were collected at the individual trial
sites and then shipped to Covance (Geneva, Switzerland). The
genomic DNA of each patient was extracted from the blood by Covance
using the PUREGENE.TM. DNA Isolation Kit (D-50K) (Gentra,
Minneapolis, Minn.).
Genotyping
[0062] A total of 47 unique polymorphisms corresponding to 24 genes
were analyzed for each clinical trial. Candidate genes involved in
metabolism of the drug, hypercholesterolemia, hyperlipidemia,
immunosuppression and inflammation were chosen for this study. SNP
assays were designed using information from public databases, such
as OMIM, the SNP Consortium, Locus Link and dbSNP, and the Third
Wave Technologies, Inc. (TWT, Madison, Wis.) website. The resulting
probe sets for the genotyping assay were generated by TWT.
Genotyping was performed with 60 ng of genomic DNA using the
INVADER.RTM. assay developed by TWT (9-10) according to the
manufacturer's instructions. See Lyamichev et al., Nat.
Biotechnol., Vol. 17, pp. 292-296 (1999); and Ryan, Mol. Diagn.,
Vol. 4, pp. 135-144 (1999).
[0063] Polymerase Chain Reaction (PCR) for the (-511) IL-1.beta.
SNP was performed in a 20 .mu.L reaction containing: 10-70 ng
genomic DNA, 160 .mu.M dNTPs, 10 mM Tris-HCl [pH 8.3], 50 mM KCl,
1.5 mM MgCl.sub.2, 0.6 .mu.M IL-1.beta.(-511)-forward primer, 0.6
.mu.M IL-1.beta. (-511)-reverse primer and 0.03 U Taq DNA
polymerase (Applied Biosystems, Foster City, Calif.). Thirty-six
(36) rounds of amplification were performed using the following
conditions: 94.degree. C., 30 seconds; 55.degree. C., 30 seconds;
72.degree. C., 30 seconds. Nine samples were then fractionated (5
.mu.L) on a 2% agarose gel and visualized by ethidium bromide
staining to confirm amplification. A 1:7 dilution of the PCR
product was run against TWT SNP#128069 using a 384-well biplex
plate for amplified DNA.
[0064] Primer sequences are as follows:
TABLE-US-00003 IL-1.beta.(-511)-forward 5'-GCAGAGCTCATCTGGCATTG-3';
(SEQ ID No. 1) IL-1.beta.(-511)-reverse 5'-TATGTGGGACAAAG
TGGAAG-3'. (SEQ ID No. 2)
[0065] PCR for the (-31) IL-1.beta. SNP was performed in a 25 .mu.L
reaction containing: 1 ng genomic DNA, 40 .mu.M dNTPs, 10 mM
Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl.sub.2, 0.75 .mu.M
IL-1.beta. (-31)-forward primer, 0.75 .mu.M IL-1.beta.
(-31)-reverse primer and 0.15 U Gold Tag DNA polymerase (Applied
Biosystems, Foster City, Calif.). Thirty-eight (38) rounds of
amplification were performed using the following conditions:
94.degree. C., 30 seconds; 58.degree. C., 30 seconds; 72.degree.
C., 30 seconds. All samples were then fractionated (5 .mu.L) on a
2% agarose gel and visualized by ethidium bromide staining to
confirm amplification.
[0066] Primer sequences are as follows:
TABLE-US-00004 IL-1.beta.(-31)-forward
5'-GCACAACGATTGTCAGGAAAAC-3'; (SEQ ID No. 3)
IL-1.beta.(-31)-reverse (5'-ATGCATACA CACAAAGAGGCAG-3'. (SEQ ID No.
4)
[0067] A 1:10 dilution of the PCR product was run against TWT SNP#
274339 for RAD 13251 using a 384-well biplex plate for amplified
DNA. RFLP analysis was used to determine genotypes using the Alu-I
restriction enzyme (New England Biolabs, Beverly, Mass.). RFLP
digests were performed in a 20 .mu.L reaction containing: 50 mM
NaCl, 10 mM Tris-HCl, 10 mM MgCl.sub.2, 1 mM DTT [pH 7.9], 8 ng
amplified genomic DNA and 0.5 mM Alu-1 enzyme. All samples were
incubated for 17 hours at 37.degree. C. and then fractionated (19
.mu.L) on a 3% agarose gel and visualized by ethidium bromide
staining to determine band size.
Nucleotide Sequence Surrounding the (-511) IL-1.beta.
Polymorphism
TABLE-US-00005 [0068] Allele Allele Gene Position 1 2 Surrounding
Sequence IL-1.beta. -511 C T CTGCAATTGACAGAGAGCTCC
[C,T]GAGGCAGAGAACAGCA CCCAAGGTAGAGACCCA (SEQ ID No. 9) Allele 1
(SEQ ID No. 5); CTGCAATTGACAGAGAGCTCC[C]GAGGCAGAGAACAGCACCCAAGGTAG
AGACCCA Allele 2 (SEQ ID No. 6);
CTGCAATTGACAGAGAGCTCC[T]GAGGCAGAGAACAGCACCCAAGGTAG AGACCCA
Nucleotide Sequence Surrounding the (-31) IL-1.beta.
Polymorphism
TABLE-US-00006 [0069] Allele Allele Gene Position 1 2 Surrounding
Sequence IL-1.beta. -31 C T TCCTACTTCTGCTTTTGAAAGC
[T,C]ATAAAAACAGCGAGGGA GAAACTGGCAGATACCAAACCT C (SEQ ID No. 10)
Allele 1 (SEQ ID No. 7)
TCCTACTTCTGCTTTTGAAAGC[C]ATAAAAACAGcGAGGGAGAAACTGG CAGATACCAAACCTC
Allele 2 (SEQ ID No. 8)
TCCTACTTCTGCTTTTGAAAGC[T]ATAAAAACAGCGAGGGAGAAACTGG
CAGATACCAAACCTC
Statistical Analysis
[0070] An analysis of covariance model was used for the analysis of
the effect of genotype and treatment on cholesterol levels using
the 24-month lab_b.sd2 RADB 251 clinical data set. Terms in the
model include the final cholesterol level, the initial cholesterol
level as the covariant, and the genotype and treatment as the main
effectors. The odds ratios, 95% confidence limits, and Chi-square
analysis were calculated where applicable. All statistical analyses
were performed using the SAS 8.02 software. To correct for multiple
testing, the Bonferroni correction method was performed.
Results
[0071] In the RAD B251 study, 47 unique SNPs corresponding to 24
genes were genotyped for each patient consenting to
pharmacogenetics analysis participating in the RAD B251 clinical
trial. A comparison of the patients that consented to
pharmacogenetics analysis to the overall patient distribution for
each respective clinical trial is shown in Table 3.
TABLE-US-00007 TABLE 3 RAD B251: Distribution of Pharmacogenetic
Samples Compared to the Overall Clinical Trial Samples
Pharmacogenetic samples Trial Samples Age (years) 43.34 43.49 Race
Caucasian (61) 74% (388) 68% Black (9) 11% (93) 16% Oriental (0) 0%
(11) 2% Other (12) 15% (76) 13% Gender Male (51) 62% (357) 63%
Female (31) 38% (211) 37% Treatment RAD001(1.5 mg/d) (24) 29.3%
(189) 33.3% RAD001(3.0 mg/d) (29) 35.3% (189) 33.3% MMF (2 g/d)
(29) 35% (190) 33.4% Weight (kg) 80.7 77.1 Baseline CHO (mg/dL)
168.7 162.8 HDL (mg/dL) 40.9 40.8 LDL (mg/dL) 96.6 96.8 TGC (mg/dL)
155.3 119.9 End of treatment CHO (mg/dL) 234.5 232.2 HDL (mg/dL)
52.7 53.3 LDL (mg/dL) 123.2 126.6 TGC (mg/dL) 283.0 254.6
[0072] Only one statistically significant difference was found
between the patient population used in the pharmacogenetic study
compared to the overall patient population in the RAD B251 clinical
trial: the differences in the mean TGC values among the
pharmacogenetic patient population and the overall RAD B251 patient
population were found to be statistically significant (p<0.001).
This comparison therefore suggests that the patient population used
in the pharmacogenetic study is representative of the overall
patient population tested in each trial. Of the 47 SNPs that were
tested in this study, 21 were experimentally determined to be not
polymorphic. Therefore, 26 SNPs were used in the analysis described
below.
[0073] Statistical analysis of the genotypes with the RAD B251
clinical data set identified a polymorphism within the IL-1.beta.
gene promoter at position (-511) that had a significant association
with cholesterol levels. As shown in Table 4 and FIG. 1, the
IL-1.beta. (-511) (T-T) genotype in patients from both treatment
groups together correlated with the highest increase in levels of
total cholesterol measured at their last visit (p=0.0018).
TABLE-US-00008 TABLE 4 LS Mean Total Cholesterol Levels (mg/dL) by
(-511) IL-1.beta. CC, CT or TT Genotypes and Treatment Groups
Within the RAD B251 Clinical Trial RAD MMF RAD and MMF (1.5 and 3.0
mg/day) (2 mg/day) treatment groups combined Genotype CC CT TT CC
CT TT CC CT TT No. of patients 20 25 9 12 9 7 32 34 16 LS means
217.3 242.9 290.3 200.9 229.6 254.1 211.4 239.5 272.9 P-value
0.0125 0.0125 0.0125 0.0866 0.0866 0.0866 0.0018 0.0018 0.0018
[0074] A similar association was observed for the IL-1.beta. (-31)
(C-C) genotype (p=0.0013) (Table 5 and FIG. 2).
TABLE-US-00009 TABLE 5 LS Mean Total Cholesterol Levels (mg/dL) by
(-31) IL-1.beta. CC, CT or TT Genotypes and Treatment Groups Within
the RAD B251 Clinical Trial RAD MMF RAD and MMF (1.5 and 3.0
mg/day) (2 mg/day) treatment groups combined Genotype CC CT TT CC
CT TT CC CT TT No. of patients 9 26 19 7 9 12 16 35 31 LS means
291.1 240.4 216.6 254.1 239. 6 200.9 272.9 239.5 211.4 P-value
0.009 0.009 0.009 0.0625 0.0625 0.0625 0.0013 0.0013 0.0013
[0075] To analyze this correlation further, it was tested whether
an association existed between the IL-1.beta. (-511) genotype and
levels of HDL and LDL. As shown in FIG. 3 and Table 6, the
IL-1.beta. (-511) (T-T) genotype in patients from both treatment
groups together correlated with the highest levels of HDL measured
at their last visit (p=0.0214).
TABLE-US-00010 TABLE 6 LS Mean HDL levels (mg/dL) by (-511)
IL-1.beta. CC, CT or TT Genotypes and Treatment Groups Within the
RAD B251 Clinical Trial RAD MMF RAD and MMF (1.5 and 3.0 mg/day) (2
mg/day) treatment groups combined Genotype CC CT TT CC CT TT CC CT
TT No. of patients 20 25 9 12 9 7 32 34 16 LS means 47.6 54.9 68.4
45.5 56.8 50.4 47.8 54.4 58.9 P-value 0.0164 0.0164 0.0164 0.0819
0.0819 0.0819 0.0214 0.0214 0.0214
[0076] It has been previously reported that the IL-1.beta. (-511)
polymorphism is in strong linkage disequilibrium to another
polymorphism in the IL-1.beta. promoter at position (-31). See
EI-Omar et al., Nature, Vol. 404, pp. 398-402 (2000). In this
study, 254 alleles were tested in patients consenting to
pharmacogenetic analysis in the RAD B251 clinical trial. It is
reported that the IL-1.beta. (-511) and (-31) polymorphisms are in
99.2% linkage disequilibrium. Therefore, a similar association
would occur with the IL-1.beta. (-511) polymorphism and cholesterol
levels could be detected as with the IL-1.beta. (-31) polymorphism.
Statistical analysis of the RAD B251 clinical data set to the
IL-1.beta. (-31) polymorphism identified a significant association
with cholesterol levels. As shown in Table 5 and FIG. 2, the
IL-1.beta. (-31) (C-C) genotype in patients from both treatment
groups together correlated with the highest increase in levels of
total cholesterol measured at their last visit (p=0.0013). To
analyze this correlation further, it was tested whether an
association existed between the IL-1.beta. (-31) genotype and
levels of HDL and LDL. As shown in FIG. 4 and Table 7, the
IL-1.beta. (-31) (C-C) genotype in patients from both treatment
groups together weakly correlated with the highest levels of HDL
measured at their last visit (p=0.0514).
TABLE-US-00011 TABLE 7 LS Mean HDL Levels (mg/dL) by (-31)
IL-1.beta. CC, CT or TT Genotypes and Treatment Groups Within the
RAD B251 Clinical Trial RAD MMF RAD and MMF (1.5 and 3.0 mg/day) (2
mg/day) treatment groups combined Genotype CC CT TT CC CT TT CC CT
TT No. of patients 9 26 19 7 9 12 16 35 31 LS means 65.1 55 48.4
50.4 56.8 45.5 58.9 54.4 47.8 P-value 0.0205 0.0205 0.0205 0.1893
0.1893 0.1893 0.0514 0.0514 0.0514
[0077] A similar correlation was identified with LDL levels as well
(p=0.0159, FIG. 5 and Table 8).
TABLE-US-00012 TABLE 8 LS Mean LDL Levels (mg/dL) by (-511)
IL-1.beta. CC, CT or TT Genotypes and Treatment Groups Within the
RAD B251 Clinical Trial RAD MMF RAD and MMF (1.5 and 3.0 mg/day) (2
mg/day) treatment groups combined Genotype CC CT TT CC CT TT CC CT
TT No. of patients 20 25 9 12 9 7 32 34 16 LS means 112.5 123.6
144.5 113.9 118.6 143.7 110.5 123.8 145.8 P-value 0.1646 0.1646
0.1646 0.2848 0.2848 0.2848 0.0159 0.0159 0.0159
[0078] A stronger correlation was identified with LDL levels and
the -31 IL-1.beta. polymorphism (p=0.0091, FIG. 6 and Table 9).
TABLE-US-00013 TABLE 9 LS Mean LDL Levels (mg/dL) by (-31)
IL-1.beta. CC, CT or TT Genotypes and Treatment Groups Within the
RAD B251 Clinical Trial RAD MMF RAD and MMF (1.5 and 3.0 mg/day) (2
mg/day) treatment groups combined Genotype CC CT TT CC CT TT CC CT
TT No. of patients 9 26 19 7 9 12 16 35 31 LS means 145.1 124.1
112.2 143.1 119.8 107.8 145.9 124.5 108.5 P-value 0.143 0.143 0.143
0.2061 0.2061 0.2061 0.0091 0.0091 0.0091
[0079] Importantly, the HDL to LDL ratios remained unchanged
between genotype groups. The findings presented in this study would
predict a greater likelihood of individuals with a certain allele
to experience persistent increases in cholesterol levels upon
treatment with the RAD/NEORAL.RTM. regimen than individuals that do
not possess the allele. A similar trend was identified in
individuals treated with the MMF/NEORAL.RTM. regimen, but the
results did not meet the p=0.05 statistical significance.
[0080] Since total blood cholesterol levels .gtoreq.940 mg/dL are
generally considered to be excessively elevated, it was therefore
decided to determine the odds ratio for a patient with the
IL-1.beta. (-511) or IL-1.beta. (-31) polymorphisms to encounter an
increase in total blood cholesterol levels resulting in a final
concentration .gtoreq.940 mg/dL after being treated with the
RAD/NEORAL.RTM. or MMF/NEORAL.RTM. regimens. See, Cecil textbook of
Medicine, Goldman and Bennett, editors, Saunders, 6.sup.th Edition
(2000).
[0081] As shown in Table 10 below, the odds ratio indicates that
patients are 5.67 (95% confidence limits: 1.20-9.01) times more
likely to have an increase in total blood cholesterol levels to a
final concentration .gtoreq.940 mg/dL when treated with the
RAD/NEORAL.RTM. regimen if they contain a T at position (-511) in
the IL-1.beta. gene promoter, or 7.23 (95% confidence limits:
1.20-9.01) times more likely to have an increase in total blood
cholesterol levels to a final concentration .gtoreq.240 mg/dL when
treated with the RAD/NEORAL.RTM. regimen if they contain a C at
position (-31) in the IL-1.beta. gene promoter. These findings are
statistically significant (p=0.0207 and p=0.0096, respectively) and
could be used as a precautionary measure in the treatment of
transplantation patients with the RAD/NEORAL.RTM. regimen since
hypercholesterolemia is easily treatable.
TABLE-US-00014 TABLE 10 Odds Ratio for the (-511) and (-31)
IL-1.beta. Genotypes and Cholesterol Levels (-511) IL-1.beta.
Polymorphism (-31) IL-1.beta. Polymorphism Obs. Genotype Obs.
Genotype Exp. CT-TT CC Total Exp. CC-CT TT Total >239 mg/dL 24 7
31 >239 mg/dL 25 6 31 18.90 12.10 19.28 11.72 .ltoreq.239 mg/dL
26 25 51 .ltoreq.239 mg/dL 26 25 51 31.10 19.90 31.72 19.28 50 32
82 51 31 82 Odds ratio = 5.67 (95% Cl: 1.20-9.01) Odds ratio = 7.23
(95% Cl: 1.20-9.01) p = 0.0207 (Fisher's Exact test) p = 0.0096
(Fisher's Exact test)
Bonferroni Correction for Multiple Testing
[0082] A correction factor is needed due to the number of SNPs that
were analyzed in this study. To do so, the Bonferroni correction
method was performed which dictated a p-value of 0.0019.
Bonferroni = 0.05 .eta. = 0.05 26 = 0.0019 ##EQU00001##
.eta.=RAD_number_of_tests
[0083] Therefore, the finding between the IL-1.beta. (-511) and
IL-1.beta. (-31) polymorphisms and total cholesterol levels
(p=0.0018 and p=0.0013, respectively) is still be considered as
significant.
Linkage Disequilibrium of the (-511) and (-31) IL-1.beta. SNPs
[0084] It has been reported that the IL-1.beta. (-511) C.fwdarw.T
polymorphism is in strong linkage disequilibrium (99.5%) with
another polymorphism within the IL-1.beta. promoter located at
position (-31) that results in a T.fwdarw.C base transition. See
EI-Omar et al., Nature, Vol. 404, pp. 398-402 (2000). Therefore, it
is predicted that patients with a T at position (-511) of the
IL-1.beta. promoter would have a C at position (-31). This finding
is confirmed in the patients tested in these two trials. In the
wild-type IL-1.beta. gene, T is found at position at -31. This T is
very important for the expression of IL-1.beta. because it is part
of the TATA box sequence (TATAAAA) which plays a critical role in
the transcriptional initiation of IL-1.beta.. In general, TATA box
sequences are involved in recruiting and positioning the
transcriptional machinery at the correct position within genes to
ensure that transcription begins at the correct place. The
T.fwdarw.C polymorphism at position (-31) would disrupt this
important TATA box sequence (TATAAAA to CATAAAA), thus making it
inactive and prohibiting the efficient initiation of transcription
of the IL-1.beta. gene. The lack of binding of the transcriptional
machinery to this altered IL-1.beta. TATA box sequence has been
shown. See EI-Omar, supra.
[0085] Therefore, the existence of any other polymorphism which is
in linkage disequilibrium with either the polymorphism within the
IL-1.beta. promoter (located at position (-31) that results in a
base transition) or the polymorphism located at -511 (C.fwdarw.J)
of the IL-0 promoter, would also have a predictive effect on the
degree of cholesterol elevation expected in a patient during
treatment with an IM. The means for the determination of other
polymorphisms which are in linkage disequilibrium with the (-31)
polymorphism is well known to one of skill in the art. Any such
polymorphism, now known or discovered in the future, could be used
in the methods of this invention to predict the degree of likely
cholesterol elevation in patients treated with an IM or to help
determine treatment choices for such patients.
Biological Significance of the Findings
[0086] The IL-1.beta. (-31) (C-C) genotype has clinical relevance.
IL-1.beta. has been shown to inhibit cholesterol biosynthesis by
25%. See EI-Omar et al., supra. Therefore, the result of this
polymorphism would mean that patients with the IL-1.beta. (-31)
(C-C) genotype, corresponding to the IL-1.beta. (-511) (T-T)
genotype, would have decreased levels of IL-1.beta., thereby losing
the inhibition of cholesterol biosynthesis by IL-1.beta., resulting
in elevated levels of cholesterol in the blood. This type of
finding was observed in the RAD B251 trial. As shown in Tables 4
and 5 and FIGS. 1-2, patients with the IL-1.beta. (-511) (T-T)
genotype and IL-1D (-31) (C-C) genotype had the highest least
square mean levels of total cholesterol, regardless of
treatment.
[0087] It has also been reported that IL-1.beta. increases LDL
receptor gene expression through the activation of the
extracellular signal-regulated kinases (ERKs). See Kumar et al., J.
Biol. Chem., Vol. 273, pp. 15742-15748 (1998).
[0088] Elevations in LDL receptor expression would result in an
increase in the amount of cholesterol that is internalized into
cells, thereby lowering total cholesterol levels in the blood. This
has relevance to RAD (everolimus) since this drug has been shown to
inhibit biochemical pathways that are required for cell progression
through late G1 and entry into S. Importantly, the ERKs have been
shown to be involved in this process. Therefore, it is possible
that ERK activity would be decreased by everolimus. Because
everolimus would inhibit ERK activity, LDL receptor expression
would decrease in all patients, independently of IL-1.beta.
expression, thereby causing increased levels of LDL and thus total
cholesterol in patients taking everolimus. It is unlikely that
everolimus completely inhibits ERK activation. Therefore, patients
with the IL-1.beta. (-511) (T-T) and IL-1.beta. (-31) (C-C)
genotypes would be able induce some expression of the LDL receptor.
However, those patients would have very low levels of IL-1.beta.,
and therefore less LDL receptor expression, thereby resulting in
lower amounts of cholesterol being internalized into cells and
elevating blood cholesterol levels. This explanation would thus
account for the highest levels of cholesterol observed in patients
with the IL-1.beta. (-31) (C-C) genotype. Significantly, patients
with the IL-1.beta. (-511) (T-T) genotype, corresponding to the
IL-1.beta. (-31) (C-C) genotype, had the significantly higher
levels of LDL (p=0.0159) as compared to patients with other
IL-1.beta. genotypes (FIG. 3 and Table 4).
Identification and Characterization of SNPs
[0089] Many different techniques can be used to identify and
characterize SNPs, including single-strand conformation
polymorphism analysis, heteroduplex analysis by denaturing
high-performance liquid chromatography (DHPLC), direct DNA
sequencing and computational methods. See Shi, Clin. Chem., Vol.
47, pp. 164-172 (2001). Thanks to the wealth of sequence
information in public databases, computational tools can be used to
identify SNPs in silico by aligning independently submitted
sequences for a given gene (either cDNA or genomic sequences).
Comparison of SNPs obtained experimentally and by in silico methods
showed that 55% of candidate SNPs found by SNPFinderhave also been
discovered experimentally. See, Cox et al., Hum. Mutel., Vol. 17,
pp. 141-150 (2001). However, these in silico methods could only
find 27% of true SNPs.
[0090] The most common SNP typing methods currently include
hybridization, primer extension and cleavage methods. Each of these
methods must be connected to an appropriate detection system.
Detection technologies include fluorescent polarization, (see Chan
et al., Genome Res., Vol. 9, pp. 492-499 (1999)), luminometric
detection of pyrophosphate release (pyrosequencing), (see Ahmadiian
et al., Anal. Biochem., Vol. 280, pp. 103-110 (2000)), fluorescence
resonance energy transfer (FRET)-based cleavage assays, DHPLC, and
mass spectrometry (see Shi, Clin. Chem., Vol. 47, pp. 164-172
(2001) and U.S. Pat. No. 6,300,076 B1). Other methods of detecting
and characterizing SNPs are those disclosed in U.S. Pat. Nos.
6,297,018 B1 and 6,300,063 B1. The disclosures of the above
references are incorporated herein by reference in their
entirety.
[0091] In a particularly preferred embodiment the detection of the
polymorphism can be accomplished by means of so called INVADER.TM.
technology (available from Third Wave Technologies Inc. Madison,
Wis.). In this assay, a specific upstream "invader" oligonucleotide
and a partially overlapping downstream probe together form a
specific structure when bound to complementary DNA template. This
structure is recognized and cut at a specific site by the Cleavase
enzyme, and this results in the release of the 5' flap of the probe
oligonucleotide. This fragment then serves as the "invader"
oligonucleotide with respect to synthetic secondary targets and
secondary fluorescently-labeled signal probes contained in the
reaction mixture. This results in specific cleavage of the
secondary signal probes by the Cleavase enzyme. Fluorescence signal
is generated when this secondary probe, labeled with dye molecules
capable of fluorescence resonance energy transfer, is cleaved.
Cleavases have stringent requirements relative to the structure
formed by the overlapping DNA sequences or flaps and can,
therefore, be used to specifically detect single base pair
mismatches immediately upstream of the cleavage site on the
downstream DNA strand. See Ryan et al., Molecular Diagnosis, Vol.
4, No 2, pp. 135-144 (1999); and Lyamichev et al., Nat.
Biotechnol., Vol. 17, pp. 292-296 (1999); see also U.S. Pat. Nos.
5,846,717 and 6,001,567 (the disclosures of which are incorporated
herein by reference in their entirety).
[0092] In some embodiments, a composition contains two or more
differently labeled genotyping oligonucleotides for simultaneously
probing the identity of nucleotides at two or more polymorphic
sites. It is also contemplated that primer compositions may contain
two or more sets of allele-specific primer pairs to allow
simultaneous targeting and amplification of two or more regions
containing a polymorphic site.
[0093] IL-1.beta. genotyping oligonucleotides of the invention may
also be immobilized on or synthesized on a solid surface such as a
microchip, bead or glass slide (see, e.g., WO 98/20020 and WO
98/20019). Such immobilized genotyping oligonucleotides may be used
in a variety of polymorphism detection assays, including but not
limited to probe hybridization and polymerase extension assays.
Immobilized IL-1.beta. genotyping oligonucleotides of the invention
may comprise an ordered array of oligonucleotides designed to
rapidly screen a DNA sample for polymorphisms in multiple genes at
the same time.
[0094] An allele-specific oligonucleotide primer of the invention
has a 3' terminal nucleotide, or preferably a 3' penultimate
nucleotide, that is complementary to only one nucleotide of a
particular SNP, thereby acting as a primer for polymerase-mediated
extension only if the allele containing that nucleotide is present.
Allele-specific oligonucleotide primers hybridizing to either the
coding or noncoding strand are contemplated by the invention. An
ASO primer for detecting IL-1.beta. gene polymorphisms could be
developed using techniques known to those of skill in the art.
[0095] Other genotyping oligonucleotides of the invention hybridize
to a target region located one to several nucleotides downstream of
one of the novel polymorphic sites identified herein. Such
oligonucleotides are useful in polymerase-mediated primer extension
methods for detecting one of the novel polymorphisms described
herein and therefore such genotyping oligonucleotides are referred
to herein as "primer-extension oligonucleotides". In a preferred
embodiment, the 3'-terminus of a primer-extension oligonucleotide
is a deoxynucleotide complementary to the nucleotide located
immediately adjacent to the polymorphic site.
[0096] In another embodiment, the invention provides a kit
comprising at least two genotyping oligonucleotides packaged in
separate containers. The kit may also contain other components,
such as hybridization buffer (where the oligonucleotides are to be
used as a probe) packaged in a separate container. Alternatively,
where the oligonucleotides are to be used to amplify a target
region, the kit may contain, packaged in separate containers, a
polymerase and a reaction buffer optimized for primer extension
mediated by the polymerase, such as PCR. The above described
oligonucleotide compositions and kits are useful in methods for
genotyping and/or haplotyping the IL-1.beta. gene in an
individual.
[0097] As used herein, the term "haplotype with regard to the
IL-1.beta. gene" shall refer to the haplotype consisting of the
combination of the polymorphisms at the -511 and the -31 position
of the IL-1.beta. gene and these haplotypes shall be named in the
following manner; the haplotype shall be called "high cholesterol"
if both the C to T polymorphism at the polymorphic site -511 of the
IL-1.beta. gene (position 1423 of sequence X04500) and the T to C
polymorphism at the polymorphic site -31 of the IL-1.beta. gene
(position 1903 of sequence X04500) are present in one copy of the
IL-1.beta. gene. Conversely, the haplotype shall be called "low
cholesterol" if both these polymorphisms are not present in a given
copy of the IL-1.beta. gene and therefore the nucleotide at site
-31 of this IL-1.beta. gene is a T and the nucleotide at site -511
is a C in this IL-1.beta. gene in the chromosome referred to.
[0098] One embodiment of the genotyping method involves isolating
from the individual a nucleic acid mixture comprising the two
copies of the IL-1.beta. gene, or a fragment thereof, that are
present in the individual, and determining the identity of the
nucleotide pair at one or more of the polymorphic sites in the two
copies to assign a IL-1.beta. genotype to the individual. As will
be readily understood by the skilled artisan, the two "copies" of a
gene in an individual may be the same allele or may be different
alleles. In a particularly preferred embodiment, the genotyping
method comprises determining the identity of the nucleotide pair at
each polymorphic site.
[0099] Typically, the nucleic acid mixture or protein is isolated
from a biological sample taken from the individual, such as a blood
sample or tissue sample. Suitable tissue samples include whole
blood, semen, saliva, tears, urine, fecal material, sweat, buccal
smears, skin, and biopsies of specific organ tissues, such as
muscle or nerve tissue and hair. The nucleic acid mixture may be
comprised of genomic DNA, messenger polyribonucleotide (mRNA), or
cDNA and, in the latter two cases, the biological sample must be
obtained from an organ in which the IL-1.beta. gene is expressed.
Furthermore it will be understood by the skilled artisan that mRNA
or cDNA preparations would not be used to detect polymorphisms
located in introns, in 5' and 3' non-transcribed regions or in
promoter regions. If an IL-1.beta. gene fragment is isolated, it
must contain the polymorphic site(s) to be genotyped.
[0100] One embodiment of the haplotyping method comprises isolating
from the individual a nucleic acid molecule containing only one of
the two copies of the IL-1.beta. gene, or a fragment thereof, that
is present in the individual and determining in that copy the
identity of the nucleotide at one or more of the polymorphic sites
in that copy to assign a IL-1.beta. haplotype to the individual.
The nucleic acid may be isolated using any method capable of
separating the two copies of the IL-1.beta. gene or fragment,
including but not limited to, one of the methods described above
for preparing IL-1.beta. isogenes, with targeted in vivo cloning
being the preferred approach.
[0101] As will be readily appreciated by those skilled in the art,
any individual clone will only provide haplotype information on one
of the two IL-1.beta. gene copies present in an individual. If
haplotype information is desired for the individuals other copy,
additional IL-1.beta. clones will need to be examined. Typically,
at least five clones should be examined to have more than a 90%
probability of haplotyping both copies of the IL-1.beta. gene in an
individual. In a particularly preferred embodiment, the nucleotide
at each of polymorphic site is identified.
[0102] In a preferred embodiment, a IL-1.beta. haplotype pair is
determined for an individual by identifying the phased sequence of
nucleotides at one or more of the polymorphic sites in each copy of
the IL-1.beta. gene that is present in the individual. In a
particularly preferred embodiment, the haplotyping method comprises
identifying the phased sequence of nucleotides at each polymorphic
site in each copy of the IL-1.beta. gene. When haplotyping both
copies of the gene, the identifying step is preferably performed
with each copy of the gene being placed in separate containers.
However, it is also envisioned that if the two copies are labeled
with different tags, or are otherwise separately distinguishable or
identifiable, it could be possible in some cases to perform the
method in the same container. For example, if first and second
copies of the gene are labeled with different first and second
fluorescent dyes, respectively, and an allele-specific
oligonucleotide labeled with yet a third different fluorescent dye
is used to assay the polymorphic site(s), then detecting a
combination of the first and third dyes would identify the
polymorphism in the first gene copy while detecting a combination
of the second and third dyes would identify the polymorphism in the
second gene copy.
[0103] In both the genotyping and haplotyping methods, the identity
of a nucleotide (or nucleotide pair) at a polymorphic site(s) may
be determined by amplifying a target region(s) containing the
polymorphic site(s) directly from one or both copies of the
IL-1.beta. gene, or fragment thereof, and the sequence of the
amplified region(s) determined by conventional methods. It will be
readily appreciated by the skilled artisan that the same nucleotide
will be detected twice at a polymorphic site in individuals who are
homozygous at that site, while two different nucleotides will be
detected if the individual is heterozygous for that site. The
polymorphism may be identified directly, known as positive-type
identification, or by inference, referred to as negative-type
identification. For example, where a SNP is known to be guanine and
cytosine in a reference population, a site may be positively
determined to be either guanine or cytosine for all individual
homozygous at that site, or both guanine and cytosine, if the
individual is heterozygous at that site. Alternatively, the site
may be negatively determined to be not guanine (and thus
cytosine/cytosine) or not cytosine (and thus guanine/guanine).
[0104] In addition, the identity of the allele(s) present at any of
the novel polymorphic sites described herein may be indirectly
determined by genotyping a polymorphic site not disclosed herein
that is in linkage disequilibrium with the polymorphic site that is
of interest. Two sites are said to be in linkage disequilibrium if
the presence of a particular variant at one site enhances the
predictability of another variant at the second site. See Stevens,
Mol. Diag., Vol. 4, pp. 309-317 (1999). Polymorphic sites in
linkage disequilibrium with the presently disclosed polymorphic
sites may be located in regions of the gene or in other genomic
regions not examined herein. Genotyping of a polymorphic site in
linkage disequilibrium with the novel polymorphic sites described
herein may be performed by, but is not limited to, any of the
above-mentioned methods for detecting the identity of the allele at
a polymorphic site.
[0105] The target region(s) may be amplified using any
oligonucleotide-directed amplification method, including but not
limited to polymerase chain reaction (PCR) (U.S. Pat. No.
4,965,188), ligase chain reaction (LCR) (see Barany et al., Proc.
Natl. Acad. Sci. USA, Vol. 88, pp. 189-193 (1991); and WO
90/01069), and oligonucleotide ligation assay (OLA) (see Landegren
et al., Science, Vol. 241, pp. 1077-1080 (1988)). Oligonucleotides
useful as primers or probes in such methods should specifically
hybridize to a region of the nucleic acid that contains or is
adjacent to the polymorphic site. Typically, the oligonucleotides
are between 10 and 35 nucleotides in length and preferably, between
15 and 30 nucleotides in length. Most preferably, the
oligonucleotides are 20-25 nucleotides long. The exact length of
the oligonucleotide will depend on many factors that are routinely
considered and practiced by the skilled artisan.
[0106] Other known nucleic acid amplification procedures may be
used to amplify the target region including transcription-based
amplification systems (see U.S. Pat. Nos. 5,130,238 and 5,169,766;
EP 329,822; and WO 89/06700) and isothermal methods. See Walker et
al., Proc. Natl. Acad. Sci. USA, Vol. 89, pp. 392-396 (1992).
[0107] A polymorphism in the target region may also be assayed
before or after amplification using one of several
hybridization-based methods known in the art. Typically,
allele-specific oligonucleotides are utilized in performing such
methods. The allele-specific oligonucleotides may be used as
differently labeled probe pairs, with one member of the pair
showing a perfect match to one variant of a target sequence and the
other member showing a perfect match to a different variant. In
some embodiments, more than one polymorphic site may be detected at
once using a set of allele-specific oligonucleotides or
oligonucleotide pairs. Preferably, the members of the set have
melting temperatures within 5.degree. C. and more preferably within
2.degree. C., of each other when hybridizing to each of the
polymorphic sites being detected.
[0108] Hybridization of an allele-specific oligonucleotide to a
target polynucleotide may be performed with both entities in
solution or such hybridization may be performed when either the
oligonucleotide or the target polynucleotide is covalently or
noncovalently affixed to a solid support. Attachment may be
mediated, for example, by antibody-antigen interactions,
poly-L-Lys, streptavidin or avidin-biotin, salt bridges,
hydrophobic interactions, chemical linkages, UV cross-linking
baking, etc. Allele-specific oligonucleotides may be synthesized
directly on the solid support or attached to the solid support
subsequent to synthesis. Solid-supports suitable for use in
detection methods of the invention include substrates made of
silicon, glass, plastic, paper and the like, which may be formed,
for example, into wells (as in 96-well plates), slides, sheets,
membranes, fibers, chips, dishes and beads. The solid support may
be treated, coated or derivatized to facilitate the immobilization
of the allele-specific oligonucleotide or target nucleic acid.
[0109] The genotype or haplotype for the IL-1.beta. gene of an
individual may also be determined by hybridization of a nucleic
sample containing one or both copies of the gene to nucleic acid
arrays and subarrays such as described in WO 95/11995. The arrays
would contain a battery of allele-specific oligonucleotides
representing each of the polymorphic sites to be included in the
genotype or haplotype.
[0110] The identity of polymorphisms may also be determined using a
mismatch detection technique, including but not limited to the
RNase protection method using riboprobes (see Winter et al., Proc.
Natl. Acad. Sci. USA, Vol. 82, p. 7575 (1985); and Meyers et al.,
Science, Vol. 230, p. 1242 (1985)) and proteins which recognize
nucleotide mismatches, such as the E. coli mutS protein. See
Modrich, Ann. Rev. Genet., Vol. 25, pp. 229-253 (1991).
Alternatively, variant alleles can be identified by single strand
conformation polymorphism (SSCP) analysis (see Orita et al.,
Genomics, Vol. 5, pp. 874-879 (1989); and Humphries et al.,
Molecular Diagnosis of Genetic Diseases, Elles, Ed., pp. 321-340
(1996)) or denaturing gradient gel electrophoresis (DGGE). See
Wartell et at., Nucl. Acids Res., Vol. 18, pp. 2699-2706 (1990);
and Sheffield et al., Proc. Natl. Acad. Sci. USA, Vol. 86, pp.
232-236 (1989).
[0111] A polymerase-mediated primer extension method may also be
used to identify the polymorphism(s). Several such methods have
been described in the patent and scientific literature and include
the "Genetic Bit Analysis" method (WO 92/15712) and the
ligase/polymerase mediated genetic bit analysis (U.S. Pat. No.
5,679,524). Related methods are disclosed in WO 91/02087, WO
90/09455, WO 95/17676, U.S. Pat. Nos. 5,302,509 and 5,945,283.
Extended primers containing a polymorphism may be detected by mass
spectrometry as described in U.S. Pat. No. 5,605,798. Another
primer extension method is allele-specific PCR. See Ruafio et al.,
Nucl. Acids Res., Vol. 17, p. 8392 (1989); Ruafio et al., Nucl.
Acids Res., Vol. 19, pp. 6877-6882 (1991); WO 93/22456; and Turki
et al., J. Clin. Invest., Vol. 95, pp. 1635-1641 (1995). In
addition, multiple polymorphic sites may be investigated by
simultaneously amplifying multiple regions of the nucleic acid
using sets of allele-specific primers as described in Wallace et
al. (WO 89/10414).
[0112] In a preferred embodiment, the haplotype frequency data for
each ethnogeographic group is examined to determine whether it is
consistent with Hardy-Weinberg equilibrium. Hardy-Weinberg
equilibrium (see Hartl et al., Principles of Population Genomics,
Sinauer Associates, 3.sup.rd Edition, Sunderland, Mass. (1997),
postulates that the frequency of finding the haplotype pair
H.sub.1/H.sub.2 is equal to P.sub.H-W (H.sub.1/H.sub.2)=2P(H.sub.1)
p (H.sub.2) if H.sub.1.noteq.H.sub.2 and P.sub.HW
(H.sub.1/H.sub.2)=p (H.sub.1) p (H.sub.2) if H.sub.1=H.sub.2. A
statistically significant difference between the observed and
expected haplotype frequencies could be due to one or more factors
including significant inbreeding in the population group, strong
selective pressure on the gene, sampling bias, and/or errors in the
genotyping process. If large deviations from Hardy-Weinberg
equilibrium are observed in an ethnogeographic group, the number of
individuals in that group can be increased to see if the deviation
is due to a sampling bias. If a larger sample size does not reduce
the difference between observed and expected haplotype pair
frequencies, then one may wish to consider haplotyping the
individual using a direct haplotyping method such as, for example,
CLASPER System.TM. technology (U.S. Pat. No. 5,866,404), SMD or
allele-specific long-range PCR. See Michalotos-Beloin et al., Nucl.
Acids Res., Vol. 24, pp. 4841-4843 (1996).
[0113] In one embodiment of this method for predicting an
IL-1.beta. haplotype pair, the assigning step involves performing
the following analysis. First, each of the possible haplotype pairs
is compared to the haplotype pairs in the reference population.
Generally, only one of the haplotype pairs in the reference
population matches a possible haplotype pair and that pair is
assigned to the individual. Occasionally, only one haplotype
represented in the reference haplotype pairs is consistent with a
possible haplotype pair for an individual, and in such cases the
individual is assigned a haplotype pair containing this known
haplotype and a new haplotype derived by subtracting the known
haplotype from the possible haplotype pair. In rare cases, either
no haplotype in the reference population are consistent with the
possible haplotype pairs, or alternatively, multiple reference
haplotype pairs are consistent with the possible haplotype pairs.
In such cases, the individual is preferably haplotyped using a
direct molecular haplotyping method such as, for example, CLASPER
System.TM. technology (U.S. Pat. No. 5,866,404), SMD or
allele-specific long-range PCR. See Michalotos-Beloin et al.,
supra.
GLOSSARY
[0114] Allele A particular form of a gene or DNA sequence at a
specific chromosomal location (locus). [0115] Antibodies Includes
polyclonal and monoclonal antibodies, chimeric, single-chain, and
humanized antibodies, as well as Fab fragments, including the
products of an Fab or other immunoglobulin expression library.
[0116] Candidate gene A gene which is hypothesized to be
responsible for a disease, condition, or the response to a
treatment, or to be correlated with one of these. [0117]
Full-genotype The unphased 5' to 3' sequence of nucleotide pairs
found at all known polymorphic sites in a locus on a pair of
homologous chromosomes in a single individual. [0118]
Full-haplotype The 5' to 3' sequence of nucleotides found at all
known polymorphic sites in a locus on a single chromosome from a
single individual. [0119] Gene A segment of DNA that contains all
the information for the regulated biosynthesis of an RNA product,
including promoters, exons, introns, and other untranslated regions
that control expression. [0120] Genotype An unphased 5' to 3'
sequence of nucleotide pair(s) found at one or more polymorphic
sites in a locus on a pair of homologous chromosomes in an
individual. As used herein, genotype includes a full-genotype
and/or a sub-genotype as described below. [0121] Genotyping A
process for determining a genotype of an individual. [0122]
Haplotype A 5' to 3' sequence of nucleotides found at one or more
linked polymorphic sites in a locus on a single chromosome from a
single individual. [0123] Haplotype data Information concerning one
or more of the following for a specific gene: a listing of the
haplotype pairs in each individual in a population; a listing of
the different haplotypes in a population; frequency of each
haplotype in that or other populations, and any known associations
between one or more haplotypes and a trait. [0124] Haplotype pair
Two haplotypes found for a locus in a single individual. [0125]
Haplotyping A process for determining one or more haplotypes in an
individual and includes use of family pedigrees, molecular
techniques and/or statistical inference. [0126] Homolog A generic
term used in the art to indicate a polynucleotide or polypeptide
sequence possessing a high degree of sequence relatedness to a
reference sequence. Such relatedness may be quantified by
determining the degree of identity and/or similarity between the
two sequences as hereinbefore defined. Falling within this generic
term are the terms "ortholog" and "paralog". [0127] Identity A
relationship between two or more polypeptide sequences or two or
more polynucleotide sequences, determined by comparing the
sequences. In general, identity refers to an exact nucleotide to
nucleotide or amino acid to amino acid correspondence of the two
polynucleotide or two polypeptide sequences, respectively, over the
length of the sequences being compared. [0128] Isoform A particular
form of a gene, mRNA, cDNA or the protein encoded thereby,
distinguished from other forms by its particular sequence and/or
structure. [0129] Isogene One of the isoforms of a gene found in a
population. An isogene contains all of the polymorphisms present in
the particular isoform of the gene. [0130] Isolated As applied to a
biological molecule, such as RNA, DNA, oligonucleotide or protein;
isolated means the molecule is substantially free of other
biological molecules, such as nucleic acids, proteins, lipids,
carbohydrates, or other material, such as cellular debris and
growth media. Generally, the term "isolated" is not intended to
refer to a complete absence of such material or to absence of
water, buffers, or salts, unless they are present in amounts that
substantially interfere with the methods of the present invention.
[0131] Linkage Describes the tendency of genes to be inherited
together as a result of their location on the same chromosome;
measured by percent recombination between loci. [0132] Linkage
Describes a situation in which some combinations of genetic markers
disequilibrium occur more or less frequently in the population than
would be expected from their distance apart. It implies that a
group of markers has been inherited coordinately. It can result
from reduced recombination in the region or from a founder effect,
in which there has been insufficient time to reach equilibrium
since one of the markers was introduced into the population. [0133]
Locus A location on a chromosome or DNA molecule corresponding to a
gene or a physical or phenotypic feature. [0134] Modified bases
include, e.g., tritylated bases and unusual bases, such as inosine.
A variety of modifications may be made to DNA and RNA; thus,
polynucleotide embraces chemically, enzymatically or metabolically
modified forms of polynucleotides as typically found in nature, as
well as the chemical forms of DNA and RNA characteristic of viruses
and cells. Polynucleotide also embraces relatively short
polynucleotides, often referred to as oligonucleotides. [0135]
Naturally--A term used to designate that the object it is applied
to, e.g., naturally-occurring occurring polynucleotide or
polypeptide, can be isolated from a source in nature and which has
not been intentionally modified by man. [0136] Nucleotide pair The
nucleotides found at a polymorphic site on the two copies of a
chromosome from an individual. [0137] Ortholog A polynucleotide or
polypeptide that is the functional equivalent of the polynucleotide
or polypeptide in another species. [0138] Paralog A polynucleotide
or polypeptide that within the same species which is functionally
similar. [0139] Phased As applied to a sequence of nucleotide pairs
for two or more polymorphic sites in a locus, phased means the
combination of nucleotides present at those polymorphic sites on a
single copy of the locus is known. [0140] Polymorphic site A
position within a locus at which at least two alternative sequences
are (PS) found in a population, the most frequent of which has a
frequency of no more than 99%, [0141] Polymorphic A gene, mRNA,
cDNA, polypeptide or peptide whose nucleotide or variant amino acid
sequence varies from a reference sequence due to the presence of a
polymorphism in the gene. [0142] Polymorphism Any sequence variant
present at a frequency of >1% in a population. The sequence
variation observed in an individual at a polymorphic site.
Polymorphisms include nucleotide substitutions, insertions,
deletions and microsatellites and may, but need not, result in
detectable differences in gene expression or protein function.
[0143] Polymorphism Information concerning one or more of the
following for a specific gene: data location of polymorphic sites;
sequence variation at those sites; frequency of polymorphisms in
one or more populations; the different genotypes and/or haplotypes
determined for the gene; frequency of one or more of these
genotypes and/or haplotypes in one or more populations; any known
association(s) between a trait and a genotype or a haplotype for
the gene. [0144] Polymorphism A collection of polymorphism data
arranged in a systematic or database methodical way and capable of
being individually accessed by electronic or other means. [0145]
Polynucleotide Any RNA or DNA, which may be unmodified or modified
RNA or DNA. Polynucleotides include, without limitation, single-
and double-stranded DNA, DNA that is a mixture of single- and
double-stranded regions, single- and double-stranded RNA, and RNA
that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions. In addition, polynucleotide refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The term polynucleotide also includes DNAs or RNAs containing one
or more modified bases and DNAs or RNAs with backbones modified for
stability or for other reasons. [0146] Polypeptide Any polypeptide
comprising two or more amino acids joined to each other by peptide
bonds or modified peptide bonds, i.e., peptide isosteres.
Polypeptide refers to both short chains, commonly referred to as
peptides, oligopeptides or oligomers, and to longer chains,
generally referred to as proteins. Polypeptides may contain amino
acids other than the 20 gene-encoded amino acids. Polypeptides
include amino acid sequences modified either by natural processes,
such as post-translational processing, or by chemical modification
techniques that are well known in the art. Such modifications are
well described in basic texts and in more detailed monographs, as
well as in a voluminous research literature. [0147] Population
group A group of individuals sharing a common characteristic, such
as ethnogeographic origin, medical condition, response to treatment
etc. [0148] Reference A group of subjects or individuals who are
predicted to be representative population of one or more
characteristics of the population group. Typically, the reference
population represents the genetic variation in the population at a
certainty level of at least 85%, preferably at least 90%, more
preferably at least 95% and even more preferably at least 99%.
[0149] Single The occurrence of nucleotide variability at a single
nucleotide position in Nucleotide the genome, within a population.
An SNP may occur within a gene or Polymorphism within intergenic
regions of the genome. SNPs can be assayed using (SNP) Allele
Specific Amplification (ASA). For the process at least 3 primers
are required. A common primer is used in reverse complement to the
polymorphism being assayed. This common primer can be between 50
and 1500 by from the polymorphic base. The other two (or more)
primers are identical to each other except that the final 3' base
wobbles to match one of the two (or more) alleles that make up the
polymorphism. Two (or more) PCR reactions are then conducted on
sample DNA, each using the common primer and one of the Allele
Specific Primers. [0150] Splice variant cDNA molecules produced
from RNA molecules initially transcribed from the same genomic DNA
sequence but which have undergone alternative RNA splicing.
Alternative RNA splicing occurs when a primary RNA transcript
undergoes splicing, generally for the removal of introns, which
results in the production of more than one mRNA molecule each of
which may encode different amino acid sequences. The term "splice
variant" also refers to the proteins encoded by the above cDNA
molecules. [0151] Sub-genotype The unphased 5' to 3' sequence of
nucleotides seen at a subset of the known polymorphic sites in a
locus on a pair of homologous chromosomes in a single individual.
[0152] Sub-haplotype The 5' to 3' sequence of nucleotides seen at a
subset of the known polymorphic sites in a locus on a single
chromosome from a single individual. [0153] Subject A human
individual whose genotypes or haplotypes or response to treatment
or disease state are to be determined. [0154] Treatment A stimulus
administered internally or externally to a subject. [0155] Unphased
As applied to a sequence of nucleotide pairs for two or more
polymorphic sites in a locus, unphased means the combination of
nucleotides present at those polymorphic sites on a single copy of
the locus is not known. [0156] See also, Human Molecular Genetics,
2n.sup.d edition. Tom Strachan and Andrew P. Read. John Wiley and
Sons, Inc. Publication, New York, 1999
REFERENCES CITED
[0157] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication or patent or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes. The discussion of
references herein is intended merely to summarise the assertions
made by their authors and no admission is made that any reference
constitutes prior art. Applicants reserve the right to challenge
the accuracy and pertinence of the cited references.
[0158] In addition, all GenBank accession numbers, Unigene Cluster
numbers and protein accession numbers cited herein are incorporated
herein by reference in their entirety and for all purposes to the
same extent as if each such number was specifically and
individually indicated to be incorporated by reference in its
entirety for all purposes.
[0159] The present invention is not to be limited in terms of the
particular embodiments described in this application, which are
intended as single illustrations of individual aspects of the
invention. Many modifications and variations of this invention can
be made without departing from its spirit and scope, as will be
apparent to those skilled in the art. Functionally equivalent
methods and apparatus within the scope of the invention, in
addition to those enumerated herein, will be apparent to those
skilled in the art from the foregoing description and accompanying
drawings. Such modifications and variations are intended to fall
within the scope of the appended claims. The present invention is
to be limited only by the terms of the appended claims, along with
the full scope of equivalents to which such claims are entitled.
Sequence CWU 1
1
11120DNAArtificialIL-1 (-511) - forward primer 1gcagagctca
tctggcattg 20220DNAArtificialIL-1 (-511) -reverse primer
2tatgtgggac aaagtggaag 20322DNAArtificialIL-1 (-31) -forward primer
3gcacaacgat tgtcaggaaa ac 22422DNAArtificialIL-1 (-31) -reverse
primer 4atgcatacac acaaagaggc ag 22555DNAHomo sapiens 5ctgcaattga
cagagagctc ccgaggcaga gaacagcacc caaggtagag accca 55655DNAHomo
sapiens 6ctgcaattga cagagagctc ctgaggcaga gaacagcacc caaggtagag
accca 55763DNAHomo sapiens 7tcctacttct gcttttgaaa gccataaaaa
cagcgaggga gaaactggca gataccaaac 60ctc 63863DNAHomo sapiens
8tcctacttct gcttttgaaa gctataaaaa cagcgaggga gaaactggca gataccaaac
60ctc 63955DNAHomo sapiensmisc_feature(22)..(22)n at position 22
may be c or t 9ctgcaattga cagagagctc cngaggcaga gaacagcacc
caaggtagag accca 551063DNAHomo sapiensmisc_feature(23)..(23)n at
position 23 may be c or t 10tcctacttct gcttttgaaa gcnataaaaa
cagcgaggga gaaactggca gataccaaac 60ctc 63119721DNAHomo
sapiensmisc_feature(135)..(135)n at position 135 may be c or t
11agaaagaaag agagagagaa agaaaagaaa gaggaaggaa ggaaggaagg aagaaagaca
60ggctctgagg aaggtggcag ttcctacaac gggagaacca gtggttaatt tgcaaagtgg
120atcctgtgga ggcanncaga ggagtcccct aggccaccca gacagggctt
ttagctatct 180gcaggccaga caccaaattt caggagggct cagtgttagg
aatggattat ggcttatcaa 240attcacagga aactaacatg ttgaacagct
tttagatttc ctgtggaaaa tataacttac 300taaagatgga gttcttgtga
ctgactcctg atatcaagat actgggagcc aaattaaaaa 360tcagaaggct
gcttggagag caagtccatg aaatgctctt tttcccacag tagaacctat
420ttccctcgtg tctcaaatac ttgcacagag gctcactccc ttggataatg
cagagcgagc 480acgatacctg gcacatacta atttgaataa aatgctgtca
aattcccatt cacccattca 540agcagcaaac tctatctcac ctgaatgtac
atgccaggca ctgtgctaga cttggctcaa 600aaagatttca gtttcctgga
ggaaccagga gggcaaggtt tcaactcagt gctataagaa 660gtgttacagg
ctggacacgg tggctcacgc ctgtaatccc aacatttggg aggccgaggc
720gggcagatca caaggtcagg agatcgagac catcctggct aacatggtga
aaccctgtct 780ctactaaaaa tacaaaaaat tagccgggcg ttggcggcag
gtgcctgtag tcccagctgc 840tggggaggct gaggcaggag aatggtgtga
acccgggagg cggaacttgc agggggccga 900gatcgtgcca ctgcactcca
gcctgggcga cagagtgaga ctctgtctca aaaaaaaaaa 960aaaagtgtta
tgatgcagac ctgtcaaaga ggcaaaggag ggtgttccta cactccaggc
1020actgttcata acctggactc tcattcattc tacaaatgga gggctcccct
gggcagatcc 1080ctggagcagg cactttgctg gtgtctcggt taaagagaaa
ctgataactc ttggtattac 1140caagagatag agtctcagat ggatattctt
acagaaacaa tattcccact tttcagagtt 1200caccaaaaaa tcattttagg
cagagctcat ctggcattga tctggttcat ccatgagatt 1260ggctagggta
acagcacctg gtcttgcagg gttgtgtgag cttatctcca gggttgcccc
1320aactccgtca ggagcctgaa ccctgcatac cgtatgttct ctgccccagc
caagaaaggt 1380caattttctc ctcagaggct cctgcaattg acagagagct
cccgaggcag agaacagcac 1440ccaaggtaga gacccacacc ctcaatacag
acagggaggg ctattggccc ttcattgtac 1500ccatttatcc atctgtaagt
gggaagattc ctaaacttaa gtacaaagaa gtgaatgaag 1560aaaagtatgt
gcatgtataa atctgtgtgt cttccacttt gtcccacata tactaaattt
1620aaacattctt ctaacgtggg aaaatccagt attttaatgt ggacatcaac
tgcacaacga 1680ttgtcaggaa aacaatgcat atttgcatgg tgatacattt
gcaaaatgtg tcatagtttg 1740ctactccttg cccttccatg aaccagagaa
ttatctcagt ttattagtcc cctcccctaa 1800gaagcttcca ccaatactct
tttccccttt cctttaactt gattgtgaaa tcaggtattc 1860aacagagaaa
tttctcagcc tcctacttct gcttttgaaa gctataaaaa cagcgaggga
1920gaaactggca gataccaaac ctcttcgagg cacaaggcac aacaggctgc
tctgggattc 1980tcttcagcca atcttcattg ctcaagtatg actttaatct
tccttacaac taggtgctaa 2040gggagtctct ctgtctctct gcctctttgt
gtgtatgcat attctctctc tctctctctt 2100tctttctctg tctctcctct
ccttcctctc tgcctcctct ctcagctttt tgcaaaaatg 2160ccaggtgtaa
tataatgctt atgactcggg aaatattctg ggaatggata ctgcttatct
2220aacagctgac accctaaagg ttagtgtcaa agcctctgct ccagctctcc
tagccaatac 2280attgctagtt ggggtttggt ttagcaaatg cttttctcta
gacccaaagg acttctcttt 2340cacacattca ttcatttact cagagatcat
ttctttgcat gactgccatg cactggatgc 2400tgagagaaat cacacatgaa
cgtagccgtc atggggaagt cactcatttt ctccttttta 2460cacaggtgtc
tgaagcagcc atggcagaag tacctgagct cgccagtgaa atgatggctt
2520attacaggtc agtggagacg ctgagaccag taacatgagc aggtctcctc
tttcaagagt 2580agagtgttat ctgtgcttgg agaccagatt tttcccctaa
attgcctctt tcagtggcaa 2640acagggtgcc aagtaaatct gatttaaaga
ctactttccc attacaagtc cctccagcct 2700tgggacctgg aggctatcca
gatgtgttgt tgcaagggct tcctgcagag gcaaatgggg 2760agaaaagatt
ccaagcccac aatacaagga atccctttgc aaagtgtggc ttggagggag
2820agggagagct cagattttag ctgactctgc tgggctagag gttaggcctc
aagatccaac 2880agggagcacc agggtgccca cctgccaggc ctagaatctg
ccttctggac tgttctgcgc 2940atatcactgt gaaacttgcc aggtgtttca
ggcagctttg agaggcaggc tgtttgcagt 3000ttcttatgaa cagtcaagtc
ttgtacacag ggaaggaaaa ataaacctgt ttagaagaca 3060taattgagac
atgtccctgt ttttattaca gtggcaatga ggatgacttg ttctttgaag
3120ctgatggccc taaacagatg aaggtaagac tatgggttta actcccaacc
caaggaaggg 3180ctctaacaca gggaaagctc aaagaaggga gttctgggcc
actttgatgc catggtattt 3240tgttttagaa agactttaac ctcttccagt
gagacacagg ctgcaccact tgctgacctg 3300gccacttggt catcatatca
ccacagtcac tcactaacgt tggtggtggt ggccacactt 3360ggtggtgaca
ggggaggagt agtgataatg ttcccatttc atagtaggaa gacaaccaag
3420tcttcaacat aaatttgatt atccttttaa gagatggatt cagcctatgc
caatcacttg 3480agttaaactc tgaaaccaag agatgatctt gagaactaac
atatgtctac cccttttgag 3540tagaatagtt ttttgctacc tggggtgaag
cttataacaa caagacatag atgatataaa 3600caaaaagatg aattgagact
tgaaagaaaa ccattcactt gctgtttgac cttgacaagt 3660cattttaccc
gctttggacc tcatctgaaa aataaagggc tgagctggat gatctctgag
3720attccagcat cctgcaacct ccagttctga aatattttca gttgtagcta
agggcatttg 3780ggcagcaaat ggtcattttt cagactcatc cttacaaaga
gccatgttat attcctgctg 3840tcccttctgt tttatatgat gctcagtagc
cttcctaggt gcccagccat cagcctagct 3900aggtcagttg tgcaggttgg
aggcagccac ttttctctgg ctttatttta ttccagtttg 3960tgatagcctc
ccctagcctc ataatccagt cctcaatctt gttaaaaaca tatttcttta
4020gaagttttaa gactggcata acttcttggc tgcagctgtg ggaggagccc
attggcttgt 4080ctgcctggcc tttgcccccc attgcctctt ccagcagctt
ggctctgctc caggcaggaa 4140attctctcct gctcaacttt cttttgtgca
cttacaggtc tctttaactg tctttcaagc 4200ctttgaacca ttatcagcct
taaggcaacc tcagtgaagc cttaatacgg agcttctctg 4260aataagagga
aagtggtaac atttcacaaa aagtactctc acaggatttg cagaatgcct
4320atgagacagt gttatgaaaa aggaaaaaaa agaacagtgt agaaaaattg
aatacttgct 4380gagtgagcat aggtgaatgg aaaatgttat ggtcatctgc
atgaaaaagc aaatcatagt 4440gtgacagcat tagggataca aaaagatata
gagaaggtat acatgtatgg tgtaggtggg 4500gcatgtacaa aaagatgaca
agtagaatcg ggatttattc taaagaatag cctgtaaggt 4560gtccagaagc
cacattctag tcttgagtct gcctctacct gctgtgtgcc cttgagtaca
4620cccttaacct ccttgagctt cagagaggga taatcttttt attttatttt
attttatttt 4680gttttgtttt gttttgtttt gttttatgag acagagtctc
actctgttgc ccaggctgga 4740gtgcagtggt acaatcttgg cttactgcat
cctccacctc ctgagttcaa gcgattctcc 4800ttcctcagtc tcctgaatag
ctaggattac aggtgcaccc caccacaccc agctaatttt 4860tgtattttta
gtagagaagg ggtttcgcca tgttggccag gctggttttg aagtcctgac
4920ctaaatgatt catccacctc ggcttcccaa agtgctggga ttacaggcat
gagccaccac 4980gcctggccca gagagggatg atctttagaa gctcgggatt
ctttcaagcc ctttcctcct 5040ctctgagctt tctactctct gatgtcaaag
catggttcct ggcaggacca cctcaccagg 5100ctccctccct cgctctctcc
gcagtgctcc ttccaggacc tggacctctg ccctctggat 5160ggcggcatcc
agctacgaat ctccgaccac cactacagca agggcttcag gcaggccgcg
5220tcagttgttg tggccatgga caagctgagg aagatgctgg ttccctgccc
acagaccttc 5280caggagaatg acctgagcac cttctttccc ttcatctttg
aagaaggtag ttagccaaga 5340gcaggcagta gatctccact tgtgtcctct
tggaagtcat caagccccag ccaactcaat 5400tcccccagag ccaaagccct
ttaaaggtag aaggcccagc ggggagacaa aacaaagaag 5460gctggaaacc
aaagcaatca tctctttagt ggaaactatt cttaaagaag atcttgatgg
5520ctactgacat ttgcaactcc ctcactcttt ctcaggggcc tttcacttac
attgtcacca 5580gaggttcgta acctccctgt gggctagtgt tatgaccatc
accattttac ctaagtagct 5640ctgttgctcg gccacagtga gcagtaatag
acctgaagct ggaacccatg tctaatagtg 5700tcaggtccag tgttcttagc
caccccactc ccagcttcat ccctactggt gttgtcatca 5760gactttgacc
gtatatgctc aggtgtcctc caagaaatca aattttgcca cctcgcctca
5820cgaggcctgc ccttctgatt ttatacctaa acaacatgtg ctccacattt
cagaacctat 5880cttcttcgac acatgggata acgaggctta tgtgcacgat
gcacctgtac gatcactgaa 5940ctgcacgctc cgggactcac agcaaaaaag
cttggtgatg tctggtccat atgaactgaa 6000agctctccac ctccagggac
aggatatgga gcaacaaggt aaatggaaac atcctggttt 6060ccctgcctgg
cctcctggca gcttgctaat tctccatgtt ttaaacaaag tagaaagtta
6120atttaaggca aatgatcaac acaagtgaaa aaaaatatta aaaaggaata
tacaaacttt 6180ggtcctagaa atggcacatt tgattgcact ggccagtgca
tttgttaaca ggagtgtgac 6240cctgagaaat tagacggctc aagcactccc
aggaccatgt ccacccaagt ctcttgggca 6300tagtgcagtg tcaattcttc
cacaatatgg ggtcatttga tggacatggc ctaactgcct 6360gtgggttctc
tcttcctgtt gttgaggctg aaacaagagt gctggagcga taatgtgtcc
6420atccccctcc ccagtcttcc ccccttgccc caacatccgt cccacccaat
gccaggtggt 6480tccttgtagg gaaattttac cgcccagcag gaacttatat
ctctccgctg taacgggcaa 6540aagtttcaag tgcggtgaac ccatcattag
ctgtggtgat ctgcctggca tcgtgccaca 6600gtagccaaag cctctgcaca
ggagtgtggg caactaaggc tgctgacttt gaaggacagc 6660ctcactcagg
gggaagctat ttgctctcag ccaggccaag aaaatcctgt ttctttggaa
6720tcgggtagta agagtgatcc cagggcctcc aattgacact gctgtgactg
aggaagatca 6780aaatgagtgt ctctctttgg agccactttc ccagctcagc
ctctcctctc ccagtttctt 6840cccatgggct actctctgtt cctgaaacag
ttctggtgcc tgatttctgg cagaagtaca 6900gcttcacctc tttcctttcc
ttccacattg atcaagttgt tccgctcctg tggatgggca 6960cattgccagc
cagtgacaca atggcttcct tccttccttc cttcagcatt taaaatgtag
7020accctctttc attctccgtt cctactgcta tgaggctctg agaaaccctc
aggcctttga 7080ggggaaaccc taaatcaaca aaatgaccct gctattgtct
gtgagaagtc aagttatcct 7140gtgtcttagg ccaaggaacc tcactgtggg
ttcccacaga ggctaccaat tacatgtatc 7200ctactctcgg ggctaggggt
tggggtgacc ctgcatgctg tgtccctaac cacaagaccc 7260ccttctttct
tcagtggtgt tctccatgtc ctttgtacaa ggagaagaaa gtaatgacaa
7320aatacctgtg gccttgggcc tcaaggaaaa gaatctgtac ctgtcctgcg
tgttgaaaga 7380tgataagccc actctacagc tggaggtaag tgaatgctat
ggaatgaagc ccttctcagc 7440ctcctgctac cacttattcc cagacaattc
accttctccc cgcccccatc cctaggaaaa 7500gctgggaaca ggtctatttg
acaagttttg cattaatgta aataaattta acataatttt 7560taactgcgtg
caaccttcaa tcctgctgca gaaaattaaa tcattttgcc gatgttatta
7620tgtcctacca tagttacaac cccaacagat tatatattgt tagggctgct
ctcatttgat 7680agacaccttg ggaaatagat gacttaaagg gtcccattat
cacgtccact ccactcccaa 7740aatcaccacc actatcacct ccagctttct
cagcaaaagc ttcatttcca agttgatgtc 7800attctaggac cataaggaaa
aatacaataa aaagcccctg gaaactaggt acttcaagaa 7860gctctagctt
aattttcacc cccccaaaaa aaaaaaattc tcacctacat tatgctcctc
7920agcatttggc actaagtttt agaaaagaag aagggctctt ttaataatca
cacagaaagt 7980tgggggccca gttacaactc aggagtctgg ctcctgatca
tgtgacctgc tcgtcagttt 8040cctttctggc caacccaaag aacatctttc
ccataggcat ctttgtccct tgccccacaa 8100aaattcttct ttctctttcg
ctgcagagtg tagatcccaa aaattaccca aagaagaaga 8160tggaaaagcg
atttgtcttc aacaagatag aaatcaataa caagctggaa tttgagtctg
8220cccagttccc caactggtac atcagcacct ctcaagcaga aaacatgccc
gtcttcctgg 8280gagggaccaa aggcggccag gatataactg acttcaccat
gcaatttgtg tcttcctaaa 8340gagagctgta cccagagagt cctgtgctga
atgtggactc aatccctagg gctggcagaa 8400agggaacaga aaggtttttg
agtacggcta tagcctggac tttcctgttg tctacaccaa 8460tgcccaactg
cctgccttag ggtagtgcta agaggatctc ctgtccatca gccaggacag
8520tcagctctct cctttcaggg ccaatcccca gcccttttgt tgagccaggc
ctctctcacc 8580tctcctactc acttaaagcc cgcctgacag aaaccacggc
cacatttggt tctaagaaac 8640cctctgtcat tcgctcccac attctgatga
gcaaccgctt ccctatttat ttatttattt 8700gtttgtttgt tttgattcat
tggtctaatt tattcaaagg gggcaagaag tagcagtgtc 8760tgtaaaagag
cctagttttt aatagctatg gaatcaattc aatttggact ggtgtgctct
8820ctttaaatca agtcctttaa ttaagactga aaatatataa gctcagatta
tttaaatggg 8880aatatttata aatgagcaaa tatcatactg ttcaatggtt
ctgaaataaa cttcactgaa 8940gaaaaaaaaa aaagggtctc tcctgatcat
tgactgtctg gattgacact gacagtaagc 9000aaacaggctg tgagagttct
tgggactaag cccactcctc attgctgagt gctgcaagta 9060cctagaaata
tccttggcca ccgaagacta tcctcctcac ccatcccctt tatttcgttg
9120ttcaacagaa ggatattcag tgcacatctg gaacaggatc agctgaagca
ctgcagggag 9180tcaggactgg tagtaacagc taccatgatt tatctatcaa
tgcaccaaac atctgttgag 9240caagcgctat gtactaggag ctgggagtac
agagatgaga acagtcacaa gtccctcctc 9300agataggaga ggcagctagt
tataagcaga acaaggtaac atgacaagta gagtaagata 9360gaagaacgaa
gaggagtagc caggaaggag ggaggagaac gacataagaa tcaagcctaa
9420agggataaac agaagatttc cacacatggg ctgggccaat tgggtgtcgg
ttacgcctgt 9480aatcccagca ctttgggtgg caggggcaga aagatcgctt
gagcccagga gttcaagacc 9540agcctgggca acatagtgag actcccatct
ctacaaaaaa taaataaata aataaaacaa 9600tcagccaggc atgctggcat
gcacctgtag tcctagctac ttgggaagct gacactggag 9660gattgcttga
gcccagaagt tcaagactgc agtgagctta tccgttgacc tgcaggtcga 9720c
9721
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