U.S. patent application number 16/744822 was filed with the patent office on 2020-05-21 for method for treating idiopathic pulmonary fibrosis.
The applicant listed for this patent is The University of Chicago Cornell University. Invention is credited to Fernando Martinez, Imre Noth, Ustin Oldham.
Application Number | 20200155494 16/744822 |
Document ID | / |
Family ID | 57143426 |
Filed Date | 2020-05-21 |
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United States Patent
Application |
20200155494 |
Kind Code |
A1 |
Noth; Imre ; et al. |
May 21, 2020 |
METHOD FOR TREATING IDIOPATHIC PULMONARY FIBROSIS
Abstract
Aspects of the disclosure relate to a method for treating
idiopathic pulmonary fibrosis (IPF) in a patient with
N-acetylcysteine (NAC) comprising administering NAC to a patient
after a sample from the patient has been genotyped and determined
to be any one of: a) homozygous or heterozygous for a thymine at
the single nucleotide polymorphism rs3750920; b) homozygous or
heterozygous for guanine at the single nucleotide polymorphism
rs5743894; or c) homozygous or heterozygous for thymine at the
single nucleotide polymorphism rs35705950.
Inventors: |
Noth; Imre; (Chicago,
IL) ; Oldham; Ustin; (Chicago, IL) ; Martinez;
Fernando; (Ithaca, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Chicago
Cornell University |
Chicago
Ithaca |
IL
NY |
US
US |
|
|
Family ID: |
57143426 |
Appl. No.: |
16/744822 |
Filed: |
January 16, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15567988 |
Oct 20, 2017 |
10543185 |
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PCT/US16/28361 |
Apr 20, 2016 |
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16744822 |
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62150926 |
Apr 22, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/573 20130101;
A61K 31/52 20130101; A61P 11/00 20180101; A61K 31/198 20130101;
C12N 15/11 20130101; C12Q 2600/106 20130101; C12Q 1/6883 20130101;
C12Q 2600/156 20130101; A61K 31/198 20130101; A61K 2300/00
20130101; A61K 31/573 20130101; A61K 2300/00 20130101; A61K 31/52
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/198 20060101
A61K031/198; C12Q 1/6883 20060101 C12Q001/6883; A61P 11/00 20060101
A61P011/00; A61K 31/573 20060101 A61K031/573; A61K 31/52 20060101
A61K031/52; C12N 15/11 20060101 C12N015/11 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
Nos: U10 HL080513 and T32 HL007605 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A method for treating idiopathic pulmonary fibrosis (IPF) in a
patient with N-acetylcysteine (NAC) comprising administering NAC to
a patient after a sample from the patient has been genotyped and
determined to be any one of: a) homozygous or heterozygous for a
thymine at the single nucleotide polymorphism rs3750920; b)
homozygous or heterozygous for guanine at the single nucleotide
polymorphism rs5743894; or c) homozygous or heterozygous for
thymine at the single nucleotide polymorphism rs35705950.
2. The method of claim 1, wherein the patient is determined to be
homozygous for a thymine at the single nucleotide polymorphism
rs3750920.
3. A method for treating idiopathic pulmonary fibrosis (IPF) in a
patient with a combination therapy comprising prednisone,
azathioprine, and N-acetylcysteine (PAN) comprising administering
PAN to a patient after a biological sample from the patient has
been genotyped and determined to be homozygous or heterozygous for
thymine at the single nucleotide polymorphism rs3750920.
4. The method of claim 1, wherein position 3074 upstream of the
MUC5B gene has been sequenced.
5. The method of claim 1, wherein position 581 in the nucleotide
coding sequence of TOLLIP has been sequenced.
6. The method of claim 1, wherein position 6120 in genomic DNA of
the TOLLIP gene has been sequenced.
7. The method of claim 1, wherein genotyping comprises amplifying a
nucleic acid sequence complementary or identical to a region of the
MUC5B genomic DNA 3074 nucleotides upstream of the MUC5B gene.
8. The method of claim 1, wherein the patient was receiving one or
more of NAC or PAN therapy prior to genotyping.
9. The method of claim 1, wherein sequencing comprises amplifying a
nucleic acid sequence complementary or identical to a region of the
TOLLIP coding sequence that comprises position 581 or a region of
the genomic DNA of the TOLLIP gene that comprises position
6120.
10. The method of claim 9, wherein amplifying comprises using
polymerase chain reaction (PCR).
11-12. (canceled)
13. The method of claim 1, wherein the biological sample is a blood
sample, a fecal sample, or a mouth swab.
14. The method of claim 1, wherein the biological sample comprises
genomic DNA.
15-18. (canceled)
19. A method for treating idiopathic pulmonary fibrosis in a
patient in need thereof comprising: a) obtaining information
indicating one or more of: i) the sequence of the rs3750920 SNP in
the TOLLIP gene; ii) the sequence of the rs5743894 SNP in the
non-coding region of the TOLLIP gene; or iii) the sequence of the
rs35705950 SNP in the non-coding region of the MUC5B gene; b)
treating the patient with NAC or PAN when the sequence of SNP
rs3750920 is determined to be homozygous or heterozygous thymine;
or treating the patient with NAC when the sequence of SNP rs5743894
or SNP rs35705950 is determined to be homozygous or heterozygous
guanine or thymine, respectively; or c) not treating the patient
with NAC or PAN and/or determining that NAC and/or PAN is
contraindicated when the sequence of SNP rs3750920 is determined to
be homozygous cytosine; or not treating the patient with NAC and/or
determining that NAC is contraindicated when the sequence of SNP
rs5743894 or SNP rs35705950 is determined to be homozygous alanine
or guanine, respectively.
20. The method of claim 19, wherein the patient is treated with NAC
when the patient is determined to be homozygous or heterozygous for
thymine at the single nucleotide polymorphism rs3750920 or wherein
NAC therapy is determined to be contraindicated when the patient is
determined to be homozygous for cytosine at the single nucleotide
polymorphism rs3750920.
21. The method of claim 19, wherein position 3074 upstream of
genomic DNA of MUC5B gene has been sequenced, position 581 in the
nucleotide coding sequence of TOLLIP has been sequenced, and/or
position 6120 in genomic DNA of the TOLLIP gene has been
sequenced.
22. The method of claim 19, wherein sequences were obtained by
amplifying a nucleic acid sequence complementary or identical to a
region of the MUC5B genomic DNA 3074 nucleotides upstream of the
MUC5B gene, a region of the TOLLIP coding sequence that comprises
position 581, and/or a region of the genomic DNA of the TOLLIP gene
that comprises position 6120.
23. The method of claim 22, wherein amplifying comprises using
polymerase chain reaction (PCR).
24-28. (canceled)
29. The method of claim 19, wherein the patient has symptoms of or
has been diagnosed with idiopathic pulmonary fibrosis.
30-36. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 15/567,988 filed Oct. 20, 2017, which is a national phase
application under 35 U.S.C. .sctn. 371 of International Application
No. PCT/US2016/028361 filed Apr. 20, 2016, which claims the benefit
of priority of U.S. Provisional Patent Application No. 62/150,926,
filed Apr. 22, 2015. The entire contents of each of the
above-referenced disclosures are specifically incorporated herein
by reference without disclaimer.
BACKGROUND
1. Field of the Invention
[0003] Embodiments of this disclosure are directed generally to
biology and medicine. In certain aspects methods involve treating
idiopathic pulmonary fibrosis.
2. Description of Related Art
[0004] Idiopathic pulmonary fibrosis (IPF) is a deadly fibrosing
interstitial lung disease of unknown cause. Recent genome-wide
association studies (GWAS) have shown single nucleotide
polymorphisms (SNPs) within two protein-encoding genes, toll
interacting protein (TOLLIP) and mucin 5B (MUC5B), to be associated
with IPF among individuals of European ancestry (Noth I, et al.,
The Lancet. 2013; 1(4):309-317 and Fingerlin T E, et al., Nat
Genet. 2013; 45(6):613-620). Single nucleotide polymorphisms (SNPs)
within TOLLIP and MUC5B are associated with idiopathic pulmonary
fibrosis (IPF) susceptibility and survival. Both genes play
critical roles in lung host defense, a process that can be
influenced by oxidative stress and modulated by N-acetylcysteine
(NAC). However, previous reports have shown that NAC therapy was
ineffective. A better understanding of the underlying molecular
mechanisms of the disease as well as how different patient
populations respond to therapeutic regimens will lead to more
efficacious treatments for IPF.
SUMMARY
[0005] The current disclosure fulfills the aforementioned need in
the art by providing novel therapeutic methods for treating IPF.
Previous reports have shown that N-acetylcysteine (NAC) therapy
provided no benefit in patients with IPF. However, it was found
that NAC and PAN (another therapy for IPF comprising the
combination of prednisone, azathioprine, and N-acetylcysteine)
therapy provided a benefit to patients harboring certain single
nucleotide polymorphisms (SNPs) within the TOLLIP or MUC5B genes.
Furthermore, it was also found that, for patients harboring certain
SNPs, NAC therapy is contraindicated, and could actually increase
the risk of death in these patients, compared to not treating the
patients. Therefore, the current methods not only provide for more
effective therapeutic regimens, but may also prevent serious
harmful side effects and even death by identifying individuals in
which the therapy would increase the risk of such negative
outcomes.
[0006] Aspects of the disclosure relate to a method for treating
idiopathic pulmonary fibrosis (IPF) in a patient with
N-acetylcysteine (NAC) comprising administering NAC to a patient
after a sample from the patient has been genotyped and determined
to be any one of: a) homozygous or heterozygous for a thymine at
the single nucleotide polymorphism rs3750920; b) homozygous or
heterozygous for guanine at the single nucleotide polymorphism
rs5743894; or c) homozygous or heterozygous for thymine at the
single nucleotide polymorphism rs35705950.
[0007] The term "genotype," as used herein refers to a chemical
transformation of the patient's DNA to determine the sequence at
the indicated location.
[0008] Methods of the disclosure provide for therapeutic regimens
based on the determination of a patient's genotype with respect to
certain polymorphisms. It was found that a patient may benefit or
at least not be adversely affected from NAC or PAN therapy when the
patient's genotype is CT or TT at the rs3750920 SNP. Further, it
was found that a patient may have adverse effects to NAC or PAN
therapy when the patient's genotype is CC at the rs3750920 SNP. A
patient may benefit or at least not be adversely affected from NAC
therapy with the patient's genotype is AG or GG at the rs5743894
SNP or GT or TT at the rs35705950 SNP. A patient may have adverse
effects to NAC when the patient's genotype is AA at the rs5743894
SNP or GG at the rs35705950 SNP.
[0009] In some embodiments, the patient is determined to be
homozygous for a thymine at the single nucleotide polymorphism
rs3750920.
[0010] In some embodiments, the patient is administered NAC
therapy. In some embodiments, the patient is administered a therapy
comprising NAC and excluding prednisone and azathioprine.
[0011] A further aspect relates to a method for treating idiopathic
pulmonary fibrosis (IPF) in a patient with a combination therapy
comprising prednisone, azathioprine, and N-acetylcysteine (PAN)
comprising administering PAN to a patient after a biological sample
from the patient has been genotyped and determined to be homozygous
or heterozygous for thymine at the single nucleotide polymorphism
rs3750920.
[0012] A further aspect of the disclosure relates to a method for
treating idiopathic pulmonary fibrosis in a patient in need thereof
comprising: a) obtaining information indicating one or more of: i)
the sequence of the rs3750920 SNP in the TOLLIP gene; ii) the
sequence of the rs5743894 SNP in the non-coding region of the
TOLLIP gene; or iii) the sequence of the rs35705950 SNP in the
non-coding region of the MUC5B gene; b) treating the patient with
NAC or PAN when the sequence of SNP rs3750920 is determined to be
homozygous or heterozygous thymine; or treating the patient with
NAC when the sequence of SNP rs5743894 or SNP rs35705950 is
determined to be homozygous or heterozygous guanine or thymine,
respectively; or c) not treating the patient with NAC or PAN and/or
determining that NAC and/or PAN is contraindicated when the
sequence of SNP rs3750920 is determined to be homozygous cytosine;
or not treating the patient with NAC and/or determining that NAC is
contraindicated when the sequence of SNP rs5743894 or SNP
rs35705950 is determined to be homozygous alanine or guanine,
respectively.
[0013] In some embodiments, portions or all of the MUC5B or TOLLIP
gene in the patient's genomic DNA have been sequenced. In some
embodiments, position 3074 upstream of the MUC5B gene has been
sequenced. In some embodiments, position 581 in the nucleotide
coding sequence of TOLLIP has been sequenced. In some embodiments,
position 6120 in genomic DNA of the TOLLIP gene has been sequenced.
In some embodiments, the method comprises amplifying at least a
portion of chromosome 11. In some embodiments, the portion of
chromosome 11 amplified comprises one or more SNP described herein
and/or a SNP known to be associated with IPF. In some embodiments,
sequencing comprises amplifying a nucleic acid sequence
complementary or identical to a region of the MUC5B genomic DNA
3074 nucleotides upstream of the MUC5B gene.
[0014] In some embodiments, the patient was receiving one or more
of NAC or PAN therapy prior to genotyping. In some embodiments,
sequencing comprises amplifying a nucleic acid sequence
complementary or identical to a region of the TOLLIP coding
sequence that comprises position 581 or a region of the genomic DNA
of the TOLLIP gene that comprises position 6120. In some
embodiments, amplifying comprises using polymerase chain reaction
(PCR).
[0015] Also encompassed within the methods described herein are
further steps, such as obtaining a biological sample from the
patient, evaluating a biological sample from a patient, obtaining
sequence information on a patient, preparing or obtaining a report
on the sequence of the patient. In some embodiments, the sequence
of the patient refers to the sequence at an SNP described herein.
In some embodiments, the biological sample is a blood sample, a
fecal sample, or a mouth swab. Alternatively, the biological sample
may be one described herein. In some embodiments, the biological
sample comprises genomic DNA.
[0016] Aspects of the disclosure also relate to nucleic acids that
can be used as probes or primers to detect and/or amplify a genomic
sequence. Examples include SEQ ID NO: 1-20. Also included are
nucleic acids that are complementary to these sequences and
fragments of these sequences. The sequences may be modified with a
modification known in the art or with a modification such as a
dectable label and/or modified base. Also included are nucleic
acids that hybridize adjacent to regions corresponding to SEQ ID
NO:1-20 in the genomic DNA of a mammal. The nucleic acids of the
disclosure may have a certain degree of identity such as at least,
at most, or exactly about 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,
98, 99, or 100% identity, or any derivable range therein.
[0017] In some embodiments, the patient has symptoms of or has been
diagnosed with idiopathic pulmonary fibrosis. In some embodiments,
the patient is a human patient.
[0018] In some embodiments, the patient is treated with NAC when
the patient is determined to be homozygous or heterozygous for
thymine at the single nucleotide polymorphism rs3750920 or wherein
NAC therapy is determined to be contraindicated when the patient is
determined to be homozygous for cytosine at the single nucleotide
polymorphism rs3750920.
[0019] In some embodiments, position 3074 upstream of genomic DNA
of MUC5B gene has been sequenced, position 581 in the nucleotide
coding sequence of TOLLIP has been sequenced, and/or position 6120
in genomic DNA of the TOLLIP gene has been sequenced. In some
embodiments, sequencing comprises amplifying a nucleic acid
sequence complementary or identical to a region of the MUC5B
genomic DNA 3074 nucleotides upstream of the MUC5B gene, a region
of the TOLLIP coding sequence that comprises position 581, and/or a
region of the genomic DNA of the TOLLIP gene that comprises
position 6120.
[0020] In some embodiments, the patient has IPF, has been diagnosed
with IPF, or is exhibiting symptomos or signs of IPF.
[0021] It is also contemplated that the term "knowing" is used
according to its ordinary and plain meaning to refer to having the
specified information. It is contemplated that typically a medical
practitioner will be evaluating whether to prescribe or administer
a particular therapeutic and in making that evaluation the
practitioner will order one or more tests regarding sequence
information of one or both of the patient's alleles or their
encoded proteins. In the context of the polymorphisms discussed
herein, the terms "allele" and "gene" are used interchangeably.
[0022] To achieve these methods, a doctor, medical practitioner, or
their staff may obtain a biological sample for evaluation. The
sample may be analyzed by the practitioner or their staff, or it
may be sent to an outside or independent laboratory. The medical
practitioner may be cognizant of whether the test is providing
information regarding the patient's genotype at the SNPs described
herein, or the medical practitioner may be aware only that the test
indicates directly or indirectly that the genotype of the patient
indicates that the patient should be administered a certain
therapeutic regimen.
[0023] In any instance, the medical practitioner "knows" the
relevant information that will allow him or her to determine the
appropriate (or contraindicated) therapeutic regimen. It is
contemplated that, for example, a laboratory conducts the test to
determine that patient's genotype such that its personnel also know
the appropriate information. They may report back to the
practitioner with the specific result of the test performed or the
laboratory may simply report that NAC or PAN is appropriate drug
based on the laboratory results.
[0024] It is contemplated that embodiments may involve obtaining a
biological sample from a patient. A biological sample is a sample
that contains biological material such as all or part of an organ,
tissue, cells, nucleic acids, proteins, or other such
macromolecules and substances. The sample may include sputum,
serum, blood, plasma, spinal fluid, semen, lymphatic fluid, urine,
stool, pleural effusion, ascites, a tissue sample, tissue biopsy,
cell swab, or a combination thereof. In other embodiments of the
invention, a sample may include cells that are from lung, skin,
muscle, liver, renal, colon, prostate, breast, brain, bladder,
small intestine, large intestine, cervix, stomach, pancreas,
testes, ovaries, bone, marrow, or spine. In some embodiments, the
sample is a whole blood, plasma or serum sample, while in other
embodiments, the sample is obtained by lavage, smear, or swab of an
area on or in the patient. In certain embodiments, the biological
sample is a blood sample.
[0025] In some embodiments, a patient's genotype with respect to a
SNP described herein may already have been evaluated. It is
contemplated that this analysis may have been done prior to the
patient being considered for treatment with NAC or PAN or as part
of a general examination. For example, a patient's genotype at
various SNPs may be determined and entered into a database or
entered into the patient's medical history. In this case, a medical
practitioner may come to know what the sequence is by obtaining a
patient history regarding the sequence at a particular location on
chromosome 11.
[0026] The present disclosure also involves reporting the results
of a determination of the nucleic acid sequence at the relevant
position corresponding to a SNP described herein. Such a report
would identify the patient by name, social security number, and/or
other identification number or qualifier. It may also contain the
actual data as a result of the determination or a summary of that
data.
[0027] In some embodiments, methods include identifying a patient
possibly in need of treatment with NAC or PAN. A patient for which
NAC or PAN therapy is being considered as a treatment option may
have symptoms of or may have been diagnosed with a medical
condition, such as IPF. In certain embodiments, the patient has
symptoms of or has been diagnosed with IPF.
[0028] Further aspects relate to kits for the detection of the SNPs
described herein. The kits may comprise one or more nucleic acid
probes or primers for the detection of an SNP described herein. The
nucleic acids may be labeled with a detectable label. The kits may
include one or more reagents for performing an assay described in
the disclosure.
[0029] Other information may also be considered in determining an
appropriate therapeutic regimen for the patient. This may include
race, gender, age, previous surgeries, heart failure stage, patient
history regarding cardiovascular disease, diagnosis of other
diseases or conditions, risks for other diseases or condition, drug
allergies, drug toxicity, and/or other medications being taken.
[0030] Use of the one or more compositions may be employed based on
methods described herein. Other embodiments are discussed
throughout this application. Any embodiment discussed with respect
to one aspect applies to other aspects as well and vice versa. The
embodiments in the Example section are understood to be embodiments
that are applicable to all aspects of the technology described
herein.
[0031] By "gene" is meant any polynucleotide sequence or portion
thereof with a functional role in encoding or transcribing a
protein or regulating other gene expression. The gene may consist
of all the nucleic acids responsible for encoding a functional
protein or only a portion of the nucleic acids responsible for
encoding or expressing a protein. The polynucleotide sequence may
contain a genetic abnormality within exons, introns, initiation or
termination regions, promoter sequences, other regulatory sequences
or unique adjacent regions to the gene.
[0032] As used herein, "treatment" or "therapy" is an approach for
obtaining beneficial or desired clinical results. This includes:
increasing the chance of a certain endpoint, such as
hospitalization-free survival and/or progression-free survival,
reduces the symptoms of the disease or inhibits or stops the
progression of the disease.
[0033] The term "therapeutically effective amount" refers to an
amount of the drug that achieves a therapeutically desirable
endpoint, such as treatment of symptoms, reducing the risk of
hospitalization or death, or inhibits the progression of the
disease.
[0034] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one."
[0035] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device or method being employed to determine the value.
[0036] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." It is also contemplated that anything listed using the
term "or" may also be specifically excluded.
[0037] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0038] Other objects, features and advantages will become apparent
from the following detailed description. It should be understood,
however, that the detailed description and the specific examples,
while indicating specific embodiments, are given by way of
illustration only, since various changes and modifications within
the spirit and scope of the invention will become apparent to those
skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0040] FIG. 1 shows the SNP selection. This figure is further
described in Example 1.
[0041] FIG. 2 shows the PANTHER consort diagram, and is further
described in Example 1.
[0042] FIGS. 3A-B shows replication cohort rs3750920
genotype-stratified Kaplan-Meier survival curves between those who
did and did not receive NAC therapy. Whereas rs3750920 genotype was
not associated with survival in those who did not receive NAC
therapy (a), a TT genotype in those receiving NAC therapy (b) was
associated with significantly better survival than those with a CC
or CT genotype.
[0043] FIG. 4 shows endpoint-free survival between NAC and Placebo
groups in the PANTHER clinical trial after stratification by
rs3750920 (TOLLIP) genotype.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0044] The Applicants found that a patient's harboring certain SNPs
in the TOLLIP and MUC5B genes exhibited a more favorable outcome
after NAC or PAN therapy. Applicants also found that certain SNPs
were associated with an increased risk of hospitalization or death
after NAC or PAN therapy. This allows for the development of novel
treatment methods that identify and treat patients based on their
genetic makeup. These treatment methods not only identify patients
that benefit from a certain treatment but also identify patients
that may be harmed by a certain treatment.
I. IPF THERAPY
[0045] A. N-acetylcysteine (NAC) Therapy
[0046] N-acetylcysteine is a pharmaceutical drug and nutritional
supplement used primarily as a mucolytic agent and in the
management of paracetamol (acetaminophen) overdose. NAC or
(2R)-2-acetamido-3-sulfanylpropanoic acid, is a derivative of
cysteine where an acetyl group is attached to the nitrogen atom.
NAC serves as a prodrug to L-cysteine which is a precursor to the
biologic antioxidant glutathione; hence administration of NAC
replenishes glutathione stores. L-cysteine also serves as a
precursor to cystine which in turn serves as a substrate for the
cystine-glutamate antiporter on astrocytes hence increasing
glutamate release into the extracellular space. This glutamate in
turn acts on mGluR2/3 receptors, and at higher doses of
acetylcysteine, mGluR5. Glutathione also modulates the NMDA
receptor by acting at the redox site. NAC also possesses some
anti-inflammatory effects possibly via inhibiting NF-.kappa.B and
modulating cytokine synthesis.
[0047] Whereas other investigators previously reported that NAC
therapy did not provide benefit for patients with IPF (N, Martinez
et al., N Engl J Med. 2014; 370(22):2093-2101), the examples of the
Application provide evidence that NAC therapy may reduce clinically
meaningful endpoint risk for genetically predisposed individuals,
specifically those harboring certain SNPs in the TOLLIP and/or
MUC5B genes. Without being limited to any scientific theory, it is
contemplated that NAC therapy de-represses the innate immune
response in the individuals that have SNPs associated with a
favorable outcome with NAC therapy.
[0048] B. PAN Therapy
[0049] PAN therapy refers to a combination therapy of prednisone,
azathioprine, and N-acetylcysteine. Prednisone
(17,21-dihydroxypregna-1,4-diene-3,11,20-trione) is a synthetic
corticosteroid drug that is particularly effective as an
immunosuppressant drug. It is used to treat certain inflammatory
diseases (such as moderate allergic reactions) and (at higher
doses) some types of cancer, but may have significant adverse
effects.
[0050] Azathioprine is an immunosuppressive drug used in organ
transplantation and autoimmune diseases and belongs to the chemical
class of purine analogues. Synthesized originally as a cancer drug
and a prodrug for mercaptopurine in 1957, it has been widely used
as an immunosuppressant for more than 50 years. Azathioprine acts
as a prodrug for mercaptopurine, inhibiting an enzyme required for
the synthesis of DNA. Thus, it most strongly affects proliferating
cells, such as the T cells and B cells of the immune system. One
adverse effect of azathioprine may be bone marrow suppression,
which can be life-threatening, especially in people with a genetic
deficiency of the enzyme thiopurine S-methyltransferase.
Azathioprine is produced by a number of manufacturers under
different brand names (Azasan by Salix in the U.S., Imuran by
GlaxoSmithKline in Canada, the U.S., Australia, Ireland and the
United Kingdom, Azamun in Finland, and Imurel in Scandinavia and
France, among others).
[0051] The enzyme thiopurine S-methyltransferase (TPMT) is
responsible for various activation and deactivation steps in
azathioprine's mechanism of action. The first metabolic step that
azathioprine undergoes in the body is the conversion to
6-mercaptopurine (6-MP), which is itself an immunosuppressant
prodrug. The TPMT enzyme is responsible, in part, for the
methylation of 6-MP into the inactive metabolite
6-methylmercaptopurine--this methylation prevents 6-MP from further
conversion into active, cytotoxic thioguanine nucleotide (TGN)
metabolites.
II. SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS)
[0052] A. TOLLIP
[0053] TOLLIP encodes toll-interacting protein (TOLLIP), an
inhibitory adaptor protein, acting downstream from the toll-like
receptors (TLRs), which are key mediators of the innate and
adaptive immune response. The TOLLIP gene is located on human Chr
11 at position 1.3-1.33 MB, and the mRNA and protein sequence is
represented in GenBank Accession Nos: NM_019009 and NP_061882,
respectively. The sequences associated with these GenBank Accession
Nos: are herein incorporated by reference.
[0054] 1. rs3750920 SNP
[0055] rs3750920 is a SNP known in the art and is futher described
in the NCBI SNP database that can be found on the world wide web at
ncbi.nlm.nih.gov/projects/SNP. The SNP is located at position
1288726 on chromosome 11 and at position 581 of the TOLLIP mRNA and
position 139 in the TOLLIP protein. As shown in table 2 of the
examples, the CC genotype of this SNP (homozygous cysteine) results
in a combined hazard ratio of 3.22 based on the endpoints of
hospitalization-free survival and progression-free survival.
Therefore, NAC would be contraindicated in people with the CC
genotype, and those harboring the CC genotype have an increased
risk of experiencing very harmful effects from NAC therapy. In
contrast, individuals with a TT genotype had a hazard ratio of
0.14, which was statistically significant. Therefore, contrary to
previous reports, NAC therapy does provide therapeutic benefits in
individuals with IPF. Previous work with NAC failed to identify the
population of individuals that would benefit from this therapy. The
CT genotype also showed a reduced hazard ratio of 0.76, indicating
that individuals with this genotype may have some benefits with NAC
therapy.
[0056] 2. rs5743894 SNP
[0057] rs5743894 is a SNP known in the art and is futher described
in the NCBI SNP database that can be found on the world wide web at
ncbi.nlm.nih.gov/projects/SNP. The SNP is located at position
1303542 on chromosome 11 and at position 6120 in a non-coding
region in the genomic DNA of the TOLLIP gene. As shown in table 2
of the examples, the AA genotype of this SNP (homozygous alanine)
results in a combined hazard ratio of 1.67, indicating that
patient's harboring this genotype may have increased risks with NAC
therapy. The hazard ration for patients with the AG or GG genotype
is 0.33, indicating that these patients may benefit from NAC
therapy.
[0058] B. MUC5B
[0059] MUC5B encodes a highly glycosylated mucin-5B precursor
protein (Mucin-5B) involved in airway mucus production and
maintaining immune homeostasis. The MUC5B gene is located on
chromosome 11 at positions 1.24-1.28 Mb, and the mRNA and protein
sequence is represented in GenBank Accession Nos: NM_002458 and
NP_002449, respectively. The sequences associated with these
GenBank Accession Nos: are herein incorporated by reference.
[0060] 2. rs35705950 SNP
[0061] rs35705950 is a SNP known in the art and is futher described
in the NCBI SNP database that can be found on the world wide web at
ncbi.nlm.nih.gov/projects/SNP. The SNP is located at position
1219991 on chromosome 11 and at a position of 3074 upstream of the
MUC5B gene. As shown in table 2 of the examples, the GG genotype of
this SNP (homozygous guanine) results in a combined hazard ratio of
2.02, indicating that patient's harboring this genotype may have
increased risks with NAC therapy. The hazard ration for patients
with the GT or TT genotype is 0.60, indicating that these patients
may benefit from NAC therapy.
III. ANALYSIS OF POLYMORPHISM
[0062] A. Nucleic Acids
[0063] Certain embodiments concern various nucleic acids, including
amplification primers, oligonucleotide probes, and other nucleic
acid elements involved in the analysis of genomic DNA. In certain
aspects, a nucleic acid comprises a wild-type, a mutant, or a
polymorphic nucleic acid. Embodiments of the disclosure also relate
to kits comprising reagents necessary to perform the assays
described throughout the disclosure.
[0064] The term "nucleic acid" is well known in the art. A "nucleic
acid" as used herein will generally refer to a molecule (i.e., a
strand) of DNA or RNA comprising a nucleobase. A nucleobase
includes, for example, a naturally occurring purine or pyrimidine
base found in DNA (e.g., an adenine "A," a guanine "G," a thymine
"T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a
C). The term "nucleic acid" encompass the terms "oligonucleotide"
and "polynucleotide," each as a subgenus of the term "nucleic
acid." The term "oligonucleotide" refers to a molecule of between
about 3 and about 100 nucleobases in length. The term
"polynucleotide" refers to at least one molecule of greater than
about 100 nucleobases in length. A "gene" refers to coding sequence
of a gene product, as well as introns and the promoter of the gene
product.
[0065] In some embodiments, nucleic acids comprise or are
complementary to all or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,
240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,
370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,
500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,
630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750,
760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,
890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100,
1165, 1200, 1300, 1400, 1500 or more contiguous nucleotides, or any
range derivable therein, of the human TOLLIP or MUC5B gene
sequences or genomic sequences upstream or downstream of the
gene.
[0066] These definitions generally refer to a single-stranded
molecule, but in specific embodiments will also encompass an
additional strand that is partially, substantially or fully
complementary to the single-stranded molecule. Thus, a nucleic acid
may encompass a double-stranded molecule or a triple-stranded
molecule that comprises one or more complementary strand(s) or
"complement(s)" of a particular sequence comprising a molecule. As
used herein, a single stranded nucleic acid may be denoted by the
prefix "ss", a double stranded nucleic acid by the prefix "ds", and
a triple stranded nucleic acid by the prefix "ts."
[0067] In particular aspects, a nucleic acid encodes a protein,
polypeptide, or peptide. In certain embodiments, the present
disclosure concerns novel compositions comprising at least one
proteinaceous molecule. As used herein, a "proteinaceous molecule,"
"proteinaceous composition," "proteinaceous compound,"
"proteinaceous chain," or "proteinaceous material" generally
refers, but is not limited to, a protein of greater than about 200
amino acids or the full length endogenous sequence translated from
a gene; a polypeptide of greater than about 100 amino acids; and/or
a peptide of from about 3 to about 100 amino acids. All the
"proteinaceous" terms described above may be used interchangeably
herein.
[0068] 1. Preparation of Nucleic Acids
[0069] A nucleic acid may be made by any technique known to one of
ordinary skill in the art, such as for example, chemical synthesis,
enzymatic production or biological production. Non-limiting
examples of a synthetic nucleic acid (e.g., a synthetic
oligonucleotide), include a nucleic acid made by in vitro chemical
synthesis using phosphotriester, phosphite or phosphoramidite
chemistry and solid phase techniques such as described in European
Patent 266,032, incorporated herein by reference, or via
deoxynucleoside H-phosphonate intermediates as described by
Froehler et al., 1986 and U.S. Pat. No. 5,705,629, each
incorporated herein by reference. In the methods of the disclosure,
one or more oligonucleotide may be used. Various different
mechanisms of oligonucleotide synthesis have been disclosed in for
example, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566,
4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of
which is incorporated herein by reference.
[0070] A non-limiting example of an enzymatically produced nucleic
acid include one produced by enzymes in amplification reactions
such as PCR.TM. (see for example, U.S. Pat. Nos. 4,683,202 and
4,682,195, each incorporated herein by reference), or the synthesis
of an oligonucleotide described in U.S. Pat. No. 5,645,897,
incorporated herein by reference. A non-limiting example of a
biologically produced nucleic acid includes a recombinant nucleic
acid produced (i.e., replicated) in a living cell, such as a
recombinant DNA vector replicated in bacteria (see for example,
Sambrook et al. 2001, incorporated herein by reference).
[0071] 2. Purification of Nucleic Acids
[0072] A nucleic acid may be purified on polyacrylamide gels,
cesium chloride centrifugation gradients, chromatography columns or
by any other means known to one of ordinary skill in the art (see
for example, Sambrook et al., 2001, incorporated herein by
reference). In some aspects, a nucleic acid is a pharmacologically
acceptable nucleic acid. Pharmacologically acceptable compositions
are known to those of skill in the art, and are described
herein.
[0073] In certain aspects, the present disclosure concerns a
nucleic acid that is an isolated nucleic acid. As used herein, the
term "isolated nucleic acid" refers to a nucleic acid molecule
(e.g., an RNA or DNA molecule) that has been isolated free of, or
is otherwise free of, the bulk of the total genomic and transcribed
nucleic acids of one or more cells. In certain embodiments,
"isolated nucleic acid" refers to a nucleic acid that has been
isolated free of, or is otherwise free of, bulk of cellular
components or in vitro reaction components such as for example,
macromolecules such as lipids or proteins, small biological
molecules, and the like.
[0074] a. Nucleic Acid Segments
[0075] In certain embodiments, the nucleic acid is a nucleic acid
segment. As used herein, the term "nucleic acid segment," are
fragments of a nucleic acid. Thus, a "nucleic acid segment" may
comprise any part of a gene sequence, including from about 2
nucleotides to the full length gene including promoter regions to
the polyadenylation signal and any length that includes all the
coding region.
[0076] Various nucleic acid segments may be designed based on a
particular nucleic acid sequence, and may be of any length. By
assigning numeric values to a sequence, for example, the first
residue is 1, the second residue is 2, etc., an algorithm defining
all nucleic acid segments can be created:
n to n+y
where n is an integer from 1 to the last number of the sequence and
y is the length of the nucleic acid segment minus one, where n+y
does not exceed the last number of the sequence. Thus, for a
10-mer, the nucleic acid segments correspond to bases 1 to 10, 2 to
11, 3 to 12 . . . and so on. For a 15-mer, the nucleic acid
segments correspond to bases 1 to 15, 2 to 16, 3 to 17 . . . and so
on. For a 20-mer, the nucleic segments correspond to bases 1 to 20,
2 to 21, 3 to 22 . . . and so on. In certain embodiments, the
nucleic acid segment may be a probe or primer. As used herein, a
"probe" generally refers to a nucleic acid used in a detection
method or composition. As used herein, a "primer" generally refers
to a nucleic acid used in an extension or amplification method or
composition.
[0077] b. Nucleic Acid Complements
[0078] The present disclosure also encompasses a nucleic acid that
is complementary to a nucleic acid. A nucleic acid is
"complement(s)" or is "complementary" to another nucleic acid when
it is capable of base-pairing with another nucleic acid according
to the standard Watson-Crick, Hoogsteen or reverse Hoogsteen
binding complementarity rules. As used herein "another nucleic
acid" may refer to a separate molecule or a spatial separated
sequence of the same molecule. In preferred embodiments, a
complement is a hybridization probe or amplification primer for the
detection of a nucleic acid polymorphism.
[0079] As used herein, the term "complementary" or "complement"
also refers to a nucleic acid comprising a sequence of consecutive
nucleobases or semiconsecutive nucleobases (e.g., one or more
nucleobase moieties are not present in the molecule) capable of
hybridizing to another nucleic acid strand or duplex even if less
than all the nucleobases do not base pair with a counterpart
nucleobase. However, in some diagnostic or detection embodiments,
completely complementary nucleic acids are preferred.
[0080] 3. Nucleic Acid Detection and Evaluation
[0081] Genotyping can be performed using methods known in the art.
General methods of nucleic acid detection methods are provided
below, followed by specific examples employed for the
identification of polymorphisms, including single nucleotide
polymorphisms (SNPs).
[0082] Those in the art will readily recognize that nucleic acid
molecules may be double-stranded molecules and that reference to a
particular site on one strand refers, as well, to the corresponding
site on a complementary strand. Thus, in defining a polymorphic
site, reference to an adenine, a thymine (uridine), a cytosine, or
a guanine at a particular site on the plus (sense or coding) strand
of a nucleic acid molecule is also intended to include the thymine
(uridine), adenine, guanine, or cytosine (respectively) at the
corresponding site on a minus (antisense or noncoding) strand of a
complementary strand of a nucleic acid molecule. Thus, reference
may be made to either strand and still comprise the same
polymorphic site and an oligonucleotide may be designed to
hybridize to either strand. Throughout the text, in identifying a
polymorphic site, reference is made to the sense strand, only for
the purpose of convenience.
[0083] Typically, the nucleic acid mixture is isolated from a
biological sample taken from the individual, such as a blood sample
or tissue sample using standard techniques. Suitable tissue samples
include whole blood, semen saliva, tears, urine, fecal material,
sweat, buccal, skin and hair. The nucleic acid mixture may be
comprised of genomic DNA, mRNA, or cDNA and, in the latter two
cases, the biological sample must be obtained from an organ in
which the gene of interest (i.e. TOLLIP) 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 or in 5' and 3' nontranscribed regions. If a TOLLIP or
MUC5B gene fragment is isolated, it must contain the polymorphic
site(s) to be genotyped.
[0084] In the genotyping methods used, the identity of a nucleotide
(or nucleotide pair) at a polymorphic site may be determined by
amplifying a target region(s) containing the polymorphic site(s)
directly from one or both copies of the gene in the individual and
the sequence of the amplified region(s) determined by conventional
methods. It will be readily appreciated by the skilled artisan that
only one nucleotide will be detected 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 an
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).
[0085] 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) (Barany et al., 1991;
WO90/01069), and oligonucleotide ligation assay (OLA) (Landegren et
al., 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 to 25 nucleotides
long. The exact length of the oligonucleotide will depend on many
factors that are routinely considered and practiced by the skilled
artisan.
[0086] Other known nucleic acid amplification procedures may be
used to amplify the target region including transcription-based
amplification systems (U.S. Pat. No. 5,130,238; EP 329,822; U.S.
Pat. No. 5,169,766, WO89/06700) and isothermal methods (Walker et
al., 1992).
[0087] 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.
[0088] 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 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.
[0089] The genotype for one or more polymorphic sites in a gene of
an individual may also be determined by hybridization of one or
both copies of the gene, or a fragment thereof, 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.
[0090] The identity of polymorphisms may also be determined using a
mismatch detection technique, including but not limited to the
RNase protection method using riboprobes (Winter et al., 1985;
Meyers et al., 1985) and proteins which recognize nucleotide
mismatches, such as the E. coli mutS protein (Modrich, 1991).
Alternatively, variant alleles can be identified by single strand
conformation polymorphism (SSCP) analysis (Orita et al., 1989;
Humphries, et al., 1996) or denaturing gradient gel electrophoresis
(DGGE) (Wartell et al., 1990; Sheffield et al., 1989).
[0091] 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. Extended
primers containing a polymorphism may be detected by mass
spectrometry as described in U.S. Pat. No. 5,605,798. An other
primer extension method is allele-specific PCR (Ruano et al.,
1989); Ruano et al., 1991; WO 93/22456; Turki et al., 1995).
[0092] a. Hybridization
[0093] The use of a probe or primer of between 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 60, 70, 80,
90, or 100 nucleotides, preferably between 17 and 100 nucleotides
in length, or in some aspects up to 1-2 kilobases or more in
length, allows the formation of a duplex molecule that is both
stable and selective. Molecules having complementary sequences over
contiguous stretches greater than 20 bases in length are generally
preferred, to increase stability and/or selectivity of the hybrid
molecules obtained. One will generally prefer to design nucleic
acid molecules for hybridization having one or more complementary
sequences of 20 to 30 nucleotides, or even longer where desired.
Such fragments may be readily prepared, for example, by directly
synthesizing the fragment by chemical means or by introducing
selected sequences into recombinant vectors for recombinant
production.
[0094] Accordingly, the nucleotide sequences may be used for their
ability to selectively form duplex molecules with complementary
stretches of DNAs and/or RNAs or to provide primers for
amplification of DNA or RNA from samples. Depending on the
application envisioned, one would desire to employ varying
conditions of hybridization to achieve varying degrees of
selectivity of the probe or primers for the target sequence.
[0095] For applications requiring high selectivity, one will
typically desire to employ relatively high stringency conditions to
form the hybrids. For example, relatively low salt and/or high
temperature conditions, such as provided by about 0.02 M to about
0.10 M NaCl at temperatures of about 50.degree. C. to about
70.degree. C. Such high stringency conditions tolerate little, if
any, mismatch between the probe or primers and the template or
target strand and would be particularly suitable for isolating
specific genes or for detecting a specific polymorphism. It is
generally appreciated that conditions can be rendered more
stringent by the addition of increasing amounts of formamide. For
example, under highly stringent conditions, hybridization to
filter-bound DNA may be carried out in 0.5 M NaHPO.sub.4, 7% sodium
dodecyl sulfate (SDS), 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel et al., 1989).
[0096] Conditions may be rendered less stringent by increasing salt
concentration and/or decreasing temperature. For example, a medium
stringency condition could be provided by about 0.1 to 0.25 M NaCl
at temperatures of about 37.degree. C. to about 55.degree. C.,
while a low stringency condition could be provided by about 0.15 M
to about 0.9 M salt, at temperatures ranging from about 20.degree.
C. to about 55.degree. C. Under low stringent conditions, such as
moderately stringent conditions the washing may be carried out for
example in 0.2.times.SSC/0.1% SDS at 42.degree. C. (Ausubel et al.,
1989). Hybridization conditions can be readily manipulated
depending on the desired results.
[0097] In other embodiments, hybridization may be achieved under
conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3
mM MgCl.sub.2, 1.0 mM dithiothreitol, at temperatures between
approximately 20.degree. C. to about 37.degree. C. Other
hybridization conditions utilized could include approximately 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, at temperatures
ranging from approximately 40.degree. C. to about 72.degree. C.
[0098] In certain embodiments, it will be advantageous to employ
nucleic acids of defined sequences in combination with an
appropriate means, such as a label, for determining hybridization.
A wide variety of appropriate indicator means are known in the art,
including fluorescent, radioactive, enzymatic or other ligands,
such as avidin/biotin, which are capable of being detected. In
preferred embodiments, one may desire to employ a fluorescent label
or an enzyme tag such as urease, alkaline phosphatase or
peroxidase, instead of radioactive or other environmentally
undesirable reagents. In the case of enzyme tags, colorimetric
indicator substrates are known that can be employed to provide a
detection means that is visibly or spectrophotometrically
detectable, to identify specific hybridization with complementary
nucleic acid containing samples. In other aspects, a particular
nuclease cleavage site may be present and detection of a particular
nucleotide sequence can be determined by the presence or absence of
nucleic acid cleavage.
[0099] In general, it is envisioned that the probes or primers
described herein will be useful as reagents in solution
hybridization, as in PCR, for detection of expression or genotype
of corresponding genes, as well as in embodiments employing a solid
phase. In embodiments involving a solid phase, the test DNA (or
RNA) is adsorbed or otherwise affixed to a selected matrix or
surface. This fixed, single-stranded nucleic acid is then subjected
to hybridization with selected probes under desired conditions. The
conditions selected will depend on the particular circumstances
(depending, for example, on the G+C content, type of target nucleic
acid, source of nucleic acid, size of hybridization probe, etc.).
Optimization of hybridization conditions for the particular
application of interest is well known to those of skill in the art.
After washing of the hybridized molecules to remove
non-specifically bound probe molecules, hybridization is detected,
and/or quantified, by determining the amount of bound label.
Representative solid phase hybridization methods are disclosed in
U.S. Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods of
hybridization that may be used are disclosed in U.S. Pat. Nos.
5,849,481, 5,849,486 and 5,851,772. The relevant portions of these
and other references identified in this section of the
Specification are incorporated herein by reference.
[0100] b. Amplification of Nucleic Acids
[0101] Nucleic acids used as a template for amplification may be
isolated from cells, tissues or other samples according to standard
methodologies (Sambrook et al., 2001). In certain embodiments,
analysis is performed on whole cell or tissue homogenates or
biological fluid samples with or without substantial purification
of the template nucleic acid. The nucleic acid may be genomic DNA
or fractionated or whole cell RNA. Where RNA is used, it may be
desired to first convert the RNA to a complementary DNA.
[0102] The term "primer," as used herein, is meant to encompass any
nucleic acid that is capable of priming the synthesis of a nascent
nucleic acid in a template-dependent process. Typically, primers
are oligonucleotides from ten to twenty and/or thirty base pairs in
length, but longer sequences can be employed. Primers may be
provided in double-stranded and/or single-stranded form, although
the single-stranded form is preferred.
[0103] Pairs of primers designed to selectively hybridize to
nucleic acids corresponding to the TOLLIP or MUC5B gene locus or
surrounding regions, or variants thereof, and fragments thereof are
contacted with the template nucleic acid under conditions that
permit selective hybridization. Depending upon the desired
application, high stringency hybridization conditions may be
selected that will only allow hybridization to sequences that are
completely complementary to the primers. In other embodiments,
hybridization may occur under reduced stringency to allow for
amplification of nucleic acids that contain one or more mismatches
with the primer sequences. Once hybridized, the template-primer
complex is contacted with one or more enzymes that facilitate
template-dependent nucleic acid synthesis. Multiple rounds of
amplification, also referred to as "cycles," are conducted until a
sufficient amount of amplification product is produced.
[0104] The amplification product may be detected, analyzed or
quantified. In certain applications, the detection may be performed
by visual means. In certain applications, the detection may involve
indirect identification of the product via chemiluminescence,
radioactive scintigraphy of incorporated radiolabel or fluorescent
label or even via a system using electrical and/or thermal impulse
signals (Affymax technology; Bellus, 1994).
[0105] A number of template dependent processes are available to
amplify the oligonucleotide sequences present in a given template
sample. One of the best known amplification methods is the
polymerase chain reaction (referred to as PCR.TM.) which is
described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and
4,800,159, and in Innis et al., 1988, each of which is incorporated
herein by reference in their entirety.
[0106] Another method for amplification is ligase chain reaction
("LCR"), disclosed in European Application No. 320 308,
incorporated herein by reference in its entirety. U.S. Pat. No.
4,883,750 describes a method similar to LCR for binding probe pairs
to a target sequence. A method based on PCR.TM. and oligonucleotide
ligase assay (OLA) (described in further detail below), disclosed
in U.S. Pat. No. 5,912,148, may also be used.
[0107] Alternative methods for amplification of target nucleic acid
sequences that may be used are disclosed in U.S. Pat. Nos.
5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547,
5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905, 5,928,906,
5,932,451, 5,935,825, 5,939,291 and 5,942,391, Great Britain
Application 2 202 328, and in PCT Application PCT/US89/01025, each
of which is incorporated herein by reference in its entirety. Qbeta
Replicase, described in PCT Application PCT/US87/00880, may also be
used as an amplification method.
[0108] An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
target molecules that contain nucleotide
5'-[alpha-thio]-triphosphates in one strand of a restriction site
may also be useful in the amplification of nucleic acids (Walker et
al., 1992). Strand Displacement Amplification (SDA), disclosed in
U.S. Pat. No. 5,916,779, is another method of carrying out
isothermal amplification of nucleic acids which involves multiple
rounds of strand displacement and synthesis, i.e., nick
translation
[0109] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA) and 3SR (Kwoh et al.,
1989; PCT Application WO 88/10315, incorporated herein by reference
in their entirety). European Application 329 822 disclose a nucleic
acid amplification process involving cyclically synthesizing
single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA
(dsDNA), which may be used in accordance with the methods described
herein.
[0110] PCT Application WO 89/06700 (incorporated herein by
reference in its entirety) disclose a nucleic acid sequence
amplification scheme based on the hybridization of a promoter
region/primer sequence to a target single-stranded DNA ("ssDNA")
followed by transcription of many RNA copies of the sequence. This
scheme is not cyclic, i.e., new templates are not produced from the
resultant RNA transcripts. Other amplification methods include
"RACE" and "one-sided PCR" (Frohman, 1990; Ohara et al., 1989).
[0111] c. Detection of Nucleic Acids
[0112] Following any amplification, it may be desirable to separate
the amplification product from the template and/or the excess
primer. In one embodiment, amplification products are separated by
agarose, agarose-acrylamide or polyacrylamide gel electrophoresis
using standard methods (Sambrook et al., 2001). Separated
amplification products may be cut out and eluted from the gel for
further manipulation. Using low melting point agarose gels, the
separated band may be removed by heating the gel, followed by
extraction of the nucleic acid.
[0113] Separation of nucleic acids may also be effected by spin
columns and/or chromatographic techniques known in art. There are
many kinds of chromatography which may be used, including
adsorption, partition, ion-exchange, hydroxylapatite, molecular
sieve, reverse-phase, column, paper, thin-layer, and gas
chromatography as well as HPLC.
[0114] In certain embodiments, the amplification products are
visualized, with or without separation. A typical visualization
method involves staining of a gel with ethidium bromide and
visualization of bands under UV light. Alternatively, if the
amplification products are integrally labeled with radio- or
fluorometrically-labeled nucleotides, the separated amplification
products can be exposed to x-ray film or visualized under the
appropriate excitatory spectra.
[0115] In one embodiment, following separation of amplification
products, a labeled nucleic acid probe is brought into contact with
the amplified marker sequence. The probe preferably is conjugated
to a chromophore but may be radiolabeled. In another embodiment,
the probe is conjugated to a binding partner, such as an antibody
or biotin, or another binding partner carrying a detectable
moiety.
[0116] In particular embodiments, detection is by Southern blotting
and hybridization with a labeled probe. The techniques involved in
Southern blotting are well known to those of skill in the art (see
Sambrook et al., 2001). One example of the foregoing is described
in U.S. Pat. No. 5,279,721, incorporated by reference herein, which
discloses an apparatus and method for the automated electrophoresis
and transfer of nucleic acids. The apparatus permits
electrophoresis and blotting without external manipulation of the
gel and is ideally suited to carrying out methods of the
disclosure.
[0117] Other methods of nucleic acid detection that may be used are
disclosed in U.S. Pat. Nos. 5,840,873, 5,843,640, 5,843,651,
5,846,708, 5,846,717, 5,846,726, 5,846,729, 5,849,487, 5,853,990,
5,853,992, 5,853,993, 5,856,092, 5,861,244, 5,863,732, 5,863,753,
5,866,331, 5,905,024, 5,910,407, 5,912,124, 5,912,145, 5,919,630,
5,925,517, 5,928,862, 5,928,869, 5,929,227, 5,932,413 and
5,935,791, each of which is incorporated herein by reference.
[0118] d. Other Assays
[0119] Other methods for genetic screening may be used. For
example, to detect mutations in genomic DNA, cDNA and/or RNA
samples. Methods used to detect point mutations include denaturing
gradient gel electrophoresis ("DGGE"), restriction fragment length
polymorphism analysis ("RFLP"), chemical or enzymatic cleavage
methods, direct sequencing of target regions amplified by PCR.TM.
(see above), single-strand conformation polymorphism analysis
("SSCP") and other methods well known in the art.
[0120] One method of screening for point mutations is based on
RNase cleavage of base pair mismatches in RNA/DNA or RNA/RNA
heteroduplexes. As used herein, the term "mismatch" is defined as a
region of one or more unpaired or mispaired nucleotides in a
double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This
definition thus includes mismatches due to insertion/deletion
mutations, as well as single or multiple base point mutations.
[0121] U.S. Pat. No. 4,946,773 describes an RNase A mismatch
cleavage assay that involves annealing single-stranded DNA or RNA
test samples to an RNA probe, and subsequent treatment of the
nucleic acid duplexes with RNase A. For the detection of
mismatches, the single-stranded products of the RNase A treatment,
electrophoretically separated according to size, are compared to
similarly treated control duplexes. Samples containing smaller
fragments (cleavage products) not seen in the control duplex are
scored as positive.
[0122] Other investigators have described the use of RNase I in
mismatch assays. The use of RNase I for mismatch detection is
described in literature from Promega Biotech. Promega markets a kit
containing RNase I that is reported to cleave three out of four
known mismatches. Others have described using the MutS protein or
other DNA-repair enzymes for detection of single-base
mismatches.
[0123] Alternative methods for detection of deletion, insertion or
substitution mutations that may be used are disclosed in U.S. Pat.
Nos. 5,849,483, 5,851,770, 5,866,337, 5,925,525 and 5,928,870, each
of which is incorporated herein by reference in its entirety.
[0124] e. DNA Sequencing
[0125] The most commonly used method of characterizing a
polymorphism is direct DNA sequencing of the genetic locus that
flanks and includes the polymorphism. Such analysis can be
accomplished using either the "dideoxy-mediated chain termination
method," also known as the "Sanger Method" (Sanger et al., 1975) or
the "chemical degradation method," also known as the "Maxam-Gilbert
method" (Maxam et al., 1977). Sequencing in combination with
genomic sequence-specific amplification technologies, such as the
polymerase chain reaction may be utilized to facilitate the
recovery of the desired genes (Mullis et al., 1986; European Patent
Application 50,424; European Patent Application. 84,796, European
Patent Application 258,017, European Patent Application. 237,362;
European Patent Application. 201,184; U.S. Pat. Nos. 4,683,202;
4,582,788; and 4,683,194), all of the above incorporated herein by
reference.
[0126] f. Exonuclease Resistance
[0127] Other methods that can be employed to determine the identity
of a nucleotide present at a polymorphic site utilize a specialized
exonuclease-resistant nucleotide derivative (U.S. Pat. No.
4,656,127). A primer complementary to an allelic sequence
immediately 3'- to the polymorphic site is hybridized to the DNA
under investigation. If the polymorphic site on the DNA contains a
nucleotide that is complementary to the particular
exonucleotide-resistant nucleotide derivative present, then that
derivative will be incorporated by a polymerase onto the end of the
hybridized primer. Such incorporation makes the primer resistant to
exonuclease cleavage and thereby permits its detection. As the
identity of the exonucleotide-resistant derivative is known one can
determine the specific nucleotide present in the polymorphic site
of the DNA.
[0128] g. Microsequencing Methods
[0129] Several other primer-guided nucleotide incorporation
procedures for assaying polymorphic sites in DNA have been
described (Komher et al., 1989; Sokolov, 1990; Syvanen 1990;
Kuppuswamy et al., 1991; Prezant et al., 1992; Ugozzoll et al.,
1992; Nyren et al., 1993). These methods rely on the incorporation
of labeled deoxynucleotides to discriminate between bases at a
polymorphic site. As the signal is proportional to the number of
deoxynucleotides incorporated, polymorphisms that occur in runs of
the same nucleotide result in a signal that is proportional to the
length of the run (Syvanen et al., 1990).
[0130] h. Extension in Solution
[0131] French Patent 2,650,840 and PCT Application WO91/02087
discuss a solution-based method for determining the identity of the
nucleotide of a polymorphic site. According to these methods, a
primer complementary to allelic sequences immediately 3'- to a
polymorphic site is used. The identity of the nucleotide of that
site is determined using labeled dideoxynucleotide derivatives
which are incorporated at the end of the primer if complementary to
the nucleotide of the polymorphic site.
[0132] i. Genetic Bit Analysis or Solid-Phase Extension
[0133] PCT Application WO92/15712 describes a method that uses
mixtures of labeled terminators and a primer that is complementary
to the sequence 3' to a polymorphic site. The labeled terminator
that is incorporated is complementary to the nucleotide present in
the polymorphic site of the target molecule being evaluated and is
thus identified. Here the primer or the target molecule is
immobilized to a solid phase.
[0134] j. Oligonucleotide Ligation Assay (OLA)
[0135] This is another solid phase method that uses different
methodology (Landegren et al., 1988). Two oligonucleotides, capable
of hybridizing to abutting sequences of a single strand of a target
DNA are used. One of these oligonucleotides is biotinylated while
the other is detectably labeled. If the precise complementary
sequence is found in a target molecule, the oligonucleotides will
hybridize such that their termini abut, and create a ligation
substrate. Ligation permits the recovery of the labeled
oligonucleotide by using avidin. Other nucleic acid detection
assays, based on this method, combined with PCR have also been
described (Nickerson et al., 1990). Here PCR is used to achieve the
exponential amplification of target DNA, which is then detected
using the OLA.
[0136] k. Ligase/Polymerase-Mediated Genetic Bit Analysis
[0137] U.S. Pat. No. 5,952,174 describes a method that also
involves two primers capable of hybridizing to abutting sequences
of a target molecule. The hybridized product is formed on a solid
support to which the target is immobilized. Here the hybridization
occurs such that the primers are separated from one another by a
space of a single nucleotide. Incubating this hybridized product in
the presence of a polymerase, a ligase, and a nucleoside
triphosphate mixture containing at least one deoxynucleoside
triphosphate allows the ligation of any pair of abutting hybridized
oligonucleotides. Addition of a ligase results in two events
required to generate a signal, extension and ligation. This
provides a higher specificity and lower "noise" than methods using
either extension or ligation alone and unlike the polymerase-based
assays, this method enhances the specificity of the polymerase step
by combining it with a second hybridization and a ligation step for
a signal to be attached to the solid phase.
[0138] l. Invasive Cleavage Reactions
[0139] Invasive cleavage reactions can be used to evaluate cellular
DNA for a particular polymorphism. A technology called INVADER.RTM.
employs such reactions (e.g., de Arruda et al., 2002; Stevens et
al., 2003, which are incorporated by reference). Generally, there
are three nucleic acid molecules: 1) an oligonucleotide upstream of
the target site ("upstream oligo"), 2) a probe oligonucleotide
covering the target site ("probe"), and 3) a single-stranded DNA
with the the target site ("target"). The upstream oligo and probe
do not overlap but they contain contiguous sequences. The probe
contains a donor fluorophore, such as fluoroscein, and an acceptor
dye, such as Dabcyl. The nucleotide at the 3' terminal end of the
upstream oligo overlaps ("invades") the first base pair of a
probe-target duplex. Then the probe is cleaved by a
structure-specific 5' nuclease causing separation of the
fluorophore/quencher pair, which increases the amount of
fluorescence that can be detected. See Lu et al., 2004.
[0140] In some cases, the assay is conducted on a solid-surface or
in an array format.
[0141] m. Other Methods to Detect SNPs
[0142] Several other specific methods for polymorphism detection
and identification are presented below and may be used as such or
with suitable modifications in conjunction with identifying
polymorphisms described herein. Several other methods are also
described on the SNP web site of the NCBI on the World Wide Web at
ncbi.nlm.nih.gov/SNP, incorporated herein by reference.
[0143] In a particular embodiment, extended haplotypes may be
determined at any given locus in a population, which allows one to
identify exactly which SNPs will be redundant and which will be
essential in association studies. The latter is referred to as
`haplotype tag SNPs (htSNPs)`, markers that capture the haplotypes
of a gene or a region of linkage disequilibrium. See Johnson et al.
(2001) and Ke and Cardon (2003), each of which is incorporated
herein by reference, for exemplary methods.
[0144] The VDA-assay utilizes PCR amplification of genomic segments
by long PCR methods using TaKaRa LA Taq reagents and other standard
reaction conditions. The long amplification can amplify DNA sizes
of about 2,000-12,000 bp. Hybridization of products to variant
detector array (VDA) can be performed by a Affymetrix High
Throughput Screening Center and analyzed with computerized
software.
[0145] A method called Chip Assay uses PCR amplification of genomic
segments by standard or long PCR protocols. Hybridization products
are analyzed by VDA, Halushka et al. (1999), incorporated herein by
reference. SNPs are generally classified as "Certain" or "Likely"
based on computer analysis of hybridization patterns. By comparison
to alternative detection methods such as nucleotide sequencing,
"Certain" SNPs have been confirmed 100% of the time; and "Likely"
SNPs have been confirmed 73% of the time by this method.
[0146] Other methods simply involve PCR amplification following
digestion with the relevant restriction enzyme. Yet others involve
sequencing of purified PCR products from known genomic regions.
[0147] In yet another method, individual exons or overlapping
fragments of large exons are PCR-amplified. Primers are designed
from published or database sequences and PCR-amplification of
genomic DNA is performed using the following conditions: 200 ng DNA
template, 0.5 .mu.M each primer, 8004 each of dCTP, dATP, dTTP and
dGTP, 5% formamide, 1.5 mM MgCl.sub.2, 0.5 U of Taq polymerase and
0.1 volume of the Taq buffer. Thermal cycling is performed and
resulting PCR-products are analyzed by PCR-single strand
conformation polymorphism (PCR-SSCP) analysis, under a variety of
conditions, e.g, 5 or 10% polyacrylamide gel with 15% urea, with or
without 5% glycerol. Electrophoresis is performed overnight.
PCR-products that show mobility shifts are reamplified and
sequenced to identify nucleotide variation.
[0148] In a method called CGAP-GAI (DEMIGLACE), sequence and
alignment data (from a PHRAP.ace file), quality scores for the
sequence base calls (from PHRED quality files), distance
information (from PHYLIP dnadist and neighbour programs) and
base-calling data (from PHRED `-d` switch) are loaded into memory.
Sequences are aligned and examined for each vertical chunk
(`slice`) of the resulting assembly for disagreement. Any such
slice is considered a candidate SNP (DEMIGLACE). A number of
filters are used by DEMIGLACE to eliminate slices that are not
likely to represent true polymorphisms. These include filters that:
(i) exclude sequences in any given slice from SNP consideration
where neighboring sequence quality scores drop 40% or more; (ii)
exclude calls in which peak amplitude is below the fifteenth
percentile of all base calls for that nucleotide type; (iii)
disqualify regions of a sequence having a high number of
disagreements with the consensus from participating in SNP
calculations; (iv) removed from consideration any base call with an
alternative call in which the peak takes up 25% or more of the area
of the called peak; (v) exclude variations that occur in only one
read direction. PHRED quality scores were converted into
probability-of-error values for each nucleotide in the slice.
Standard Baysian methods are used to calculate the posterior
probability that there is evidence of nucleotide heterogeneity at a
given location.
[0149] In a method called CU-RDF (RESEQ), PCR amplification is
performed from DNA isolated from blood using specific primers for
each SNP, and after typical cleanup protocols to remove unused
primers and free nucleotides, direct sequencing using the same or
nested primers.
[0150] In a method called DEBNICK (METHOD-B), a comparative
analysis of clustered EST sequences is performed and confirmed by
fluorescent-based DNA sequencing. In a related method, called
DEBNICK (METHOD-C), comparative analysis of clustered EST sequences
with phred quality >20 at the site of the mismatch, average
phred quality >=20 over 5 bases 5'-FLANK and 3' to the SNP, no
mismatches in 5 bases 5' and 3' to the SNP, at least two
occurrences of each allele is performed and confirmed by examining
traces.
[0151] In a method identified by ERO (RESEQ), new primers sets are
designed for electronically published STSs and used to amplify DNA
from 10 different mouse strains. The amplification product from
each strain is then gel purified and sequenced using a standard
dideoxy, cycle sequencing technique with .sup.33P-labeled
terminators. All the ddATP terminated reactions are then loaded in
adjacent lanes of a sequencing gel followed by all of the ddGTP
reactions and so on. SNPs are identified by visually scanning the
radiographs.
[0152] In another method identified as ERO (RESEQ-HT), new primers
sets are designed for electronically published murine DNA sequences
and used to amplify DNA from 10 different mouse strains. The
amplification product from each strain is prepared for sequencing
by treating with Exonuclease I and Shrimp Alkaline Phosphatase.
Sequencing is performed using ABI Prism Big Dye Terminator Ready
Reaction Kit (Perkin-Elmer) and sequence samples are run on the
3700 DNA Analyzer (96 Capillary Sequencer).
[0153] FGU-CBT (SCA2-SNP) identifies a method where the region
containing the SNP were PCR amplified using the primers SCA2-FP3
and SCA2-RP3. Approximately 100 ng of genomic DNA is amplified in a
50 ml reaction volume containing a final concentration of 5 mM
Tris, 25 mM KCl, 0.75 mM MgCl.sub.2, 0.05% gelatin, 20 pmol of each
primer and 0.5 U of Taq DNA polymerase. Samples are denatured,
annealed and extended and the PCR product is purified from a band
cut out of the agarose gel using, for example, the QIAquick gel
extraction kit (Qiagen) and is sequenced using dye terminator
chemistry on an ABI Prism 377 automated DNA sequencer with the PCR
primers.
[0154] In a method identified as JBLACK (SEQ/RESTRICT), two
independent PCR reactions are performed with genomic DNA. Products
from the first reaction are analyzed by sequencing, indicating a
unique Fspl restriction site. The mutation is confirmed in the
product of the second PCR reaction by digesting with Fsp I.
[0155] In a method described as KWOK(1), SNPs are identified by
comparing high quality genomic sequence data from four randomly
chosen individuals by direct DNA sequencing of PCR products with
dye-terminator chemistry (see Kwok et al., 1996). In a related
method identified as KWOK(2) SNPs are identified by comparing high
quality genomic sequence data from overlapping large-insert clones
such as bacterial artificial chromosomes (BACs) or P1-based
artificial chromosomes (PACs). An STS containing this SNP is then
developed and the existence of the SNP in various populations is
confirmed by pooled DNA sequencing (see Taillon-Miller et al.,
1998). In another similar method called KWOK(3), SNPs are
identified by comparing high quality genomic sequence data from
overlapping large-insert clones BACs or PACs. The SNPs found by
this approach represent DNA sequence variations between the two
donor chromosomes but the allele frequencies in the general
population have not yet been determined. In method KWOK(5), SNPs
are identified by comparing high quality genomic sequence data from
a homozygous DNA sample and one or more pooled DNA samples by
direct DNA sequencing of PCR products with dye-terminator
chemistry. The STSs used are developed from sequence data found in
publicly available databases. Specifically, these STSs are
amplified by PCR against a complete hydatidiform mole (CHM) that
has been shown to be homozygous at all loci and a pool of DNA
samples from 80 CEPH parents (see Kwok et al., 1994).
[0156] In another such method, KWOK
(OverlapSnpDetectionWithPolyBayes), SNPs are discovered by
automated computer analysis of overlapping regions of large-insert
human genomic clone sequences. For data acquisition, clone
sequences are obtained directly from large-scale sequencing
centers. This is necessary because base quality sequences are not
present/available through GenBank. Raw data processing involves
analyzed of clone sequences and accompanying base quality
information for consistency. Finished (`base perfect`, error rate
lower than 1 in 10,000 bp) sequences with no associated base
quality sequences are assigned a uniform base quality value of 40
(1 in 10,000 bp error rate). Draft sequences without base quality
values are rejected. Processed sequences are entered into a local
database. A version of each sequence with known human repeats
masked is also stored. Repeat masking is performed with the program
"MASKERAID." Overlap detection: Putative overlaps are detected with
the program "WUBLAST." Several filtering steps followed in order to
eliminate false overlap detection results, i.e. similarities
between a pair of clone sequences that arise due to sequence
duplication as opposed to true overlap. Total length of overlap,
overall percent similarity, number of sequence differences between
nucleotides with high base quality value "high-quality mismatches."
Results are also compared to results of restriction fragment
mapping of genomic clones at Washington University Genome
Sequencing Center, finisher's reports on overlaps, and results of
the sequence contig building effort at the NCBI. SNP detection:
Overlapping pairs of clone sequence are analyzed for candidate SNP
sites with the `POLYBAYES` SNP detection software. Sequence
differences between the pair of sequences are scored for the
probability of representing true sequence variation as opposed to
sequencing error. This process requires the presence of base
quality values for both sequences. High-scoring candidates are
extracted. The search is restricted to substitution-type single
base pair variations. Confidence score of candidate SNP is computed
by the POLYBAYES software.
[0157] In method identified by KWOK (TaqMan assay), the TaqMan
assay is used to determine genotypes for 90 random individuals. In
method identified by KYUGEN(Q1), DNA samples of indicated
populations are pooled and analyzed by PLACE-SSCP. Peak heights of
each allele in the pooled analysis are corrected by those in a
heterozygote, and are subsequently used for calculation of allele
frequencies. Allele frequencies higher than 10% are reliably
quantified by this method. Allele frequency=0 (zero) means that the
allele was found among individuals, but the corresponding peak is
not seen in the examination of pool. Allele frequency=0-0.1
indicates that minor alleles are detected in the pool but the peaks
are too low to reliably quantify.
[0158] In yet another method identified as KYUGEN (Method1), PCR
products are post-labeled with fluorescent dyes and analyzed by an
automated capillary electrophoresis system under SSCP conditions
(PLACE-SSCP). Four or more individual DNAs are analyzed with or
without two pooled DNA (Japanese pool and CEPH parents pool) in a
series of experiments. Alleles are identified by visual inspection.
Individual DNAs with different genotypes are sequenced and SNPs
identified. Allele frequencies are estimated from peak heights in
the pooled samples after correction of signal bias using peak
heights in heterozygotes. For the PCR primers are tagged to have
5'-ATT or 5'-GTT at their ends for post-labeling of both strands.
Samples of DNA (10 ng/ul) are amplified in reaction mixtures
containing the buffer (10 mM Tris-HCl, pH 8.3 or 9.3, 50 mM KCl,
2.0 mM MgCl.sub.2), 0.25 .mu.M of each primer, 20004 of each dNTP,
and 0.025 units/.mu.1 of Taq DNA polymerase premixed with anti-Taq
antibody. The two strands of PCR products are differentially
labeled with nucleotides modified with R110 and R6G by an exchange
reaction of Klenow fragment of DNA polymerase I. The reaction is
stopped by adding EDTA, and unincorporated nucleotides are
dephosphorylated by adding calf intestinal alkaline phosphatase.
For the SSCP: an aliquot of fluorescently labeled PCR products and
TAMRA-labeled internal markers are added to deionized formamide,
and denatured. Electrophoresis is performed in a capillary using an
ABI Prism 310 Genetic Analyzer. Genescan softwares (P-E Biosystems)
are used for data collection and data processing. DNA of
individuals (two to eleven) including those who showed different
genotypes on SSCP are subjected for direct sequencing using big-dye
terminator chemistry, on ABI Prism 310 sequencers. Multiple
sequence trace files obtained from ABI Prism 310 are processed and
aligned by Phred/Phrap and viewed using Consed viewer. SNPs are
identified by PolyPhred software and visual inspection.
[0159] In yet another method identified as KYUGEN (Method2),
individuals with different genotypes are searched by denaturing
HPLC (DHPLC) or PLACE-SSCP (Inazuka et al., 1997) and their
sequences are determined to identify SNPs. PCR is performed with
primers tagged with 5'-ATT or 5'-GTT at their ends for
post-labeling of both strands. DHPLC analysis is carried out using
the WAVE DNA fragment analysis system (Transgenomic). PCR products
are injected into DNASep column, and separated under the conditions
determined using WAVEMaker program (Transgenomic). The two strands
of PCR products that are differentially labeled with nucleotides
modified with R110 and R6G by an exchange reaction of Klenow
fragment of DNA polymerase I. The reaction is stopped by adding
EDTA, and unincorporated nucleotides are dephosphorylated by adding
calf intestinal alkaline phosphatase. SSCP followed by
electrophoresis is performed in a capillary using an ABI Prism 310
Genetic Analyzer. Genescan softwares (P-E Biosystems). DNA of
individuals including those who showed different genotypes on DHPLC
or SSCP are subjected for direct sequencing using big-dye
terminator chemistry, on ABI Prism 310 sequencer. Multiple sequence
trace files obtained from ABI Prism 310 are processed and aligned
by Phred/Phrap and viewed using Consed viewer. SNPs are identified
by PolyPhred software and visual inspection. Trace chromatogram
data of EST sequences in Unigene are processed with PHRED. To
identify likely SNPs, single base mismatches are reported from
multiple sequence alignments produced by the programs PHRAP, BRO
and POA for each Unigene cluster. BRO corrected possible
misreported EST orientations, while POA identified and analyzed
non-linear alignment structures indicative of gene mixing/chimeras
that might produce spurious SNPs. Bayesian inference is used to
weigh evidence for true polymorphism versus sequencing error,
misalignment or ambiguity, misclustering or chimeric EST sequences,
assessing data such as raw chromatogram height, sharpness, overlap
and spacing; sequencing error rates; context-sensitivity; cDNA
library origin, etc.
[0160] In method identified as MARSHFIELD(Method-B), overlapping
human DNA sequences which contained putative insertion/deletion
polymorphisms are identified through searches of public databases.
PCR primers which flanked each polymorphic site are selected from
the consensus sequences. Primers are used to amplify individual or
pooled human genomic DNA. Resulting PCR products are resolved on a
denaturing polyacrylamide gel and a PhosphorImager is used to
estimate allele frequencies from DNA pools.
[0161] 4. Linkage Disequilibrium
[0162] Polymorphisms in linkage disequilibrium with another
polymorphism in which identification of one polymorphism is
predictive of the identity of the linked polymorphism. "Linkage
disequilibrium" ("LD" as used herein, though also referred to as
"LED" in the art) refers to a situation where a particular
combination of alleles (i.e., a variant form of a given gene) or
polymorphisms at two loci appears more frequently than would be
expected by chance. "Significant" as used in respect to linkage
disequilibrium, as determined by one of skill in the art, is
contemplated to be a statistical p or a value that may be 0.25 or
0.1 and may be 0.1, 0.05. 0.001, 0.00001 or less. A polymorphism
described herein may be determined by evaluating the nucleic acid
sequence of a polymorphism in linkage disequilibrium with the
polymorphism described herein. The methods of the disclosure may be
implemented in this manner with respect to one or more
polymorphisms so as to allow haplotype analysis. "Haplotype" is
used according to its plain and ordinary meaning to one skilled in
the art. It refers to a collective genotype of two or more alleles
or polymorphisms along one of the homologous chromosomes.
IV. THERAPY
[0163] Once the genotype of the individual is determined with
respect to a particular polymorphism or SNP, a therapeutic course
of treatment may be individualized. In a preferred embodiment of
the method, the trait of interest is a clinical response exhibited
by a patient to some therapeutic treatment, for example, response
to a therapeutic regimen such as NAC or PAN.
[0164] A. Routes of Administration
[0165] Administration of the therapeutic may be by any number of
routes including, but not limited to oral, intravenous,
intramuscular, intra-arterial, intramedullary, intrathecal,
intraventricular, intradermal, intratracheal, intravesicle,
intraocular, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, or rectal. Further
details on techniques for formulation and administration may be
found in the latest edition of Remington's Pharmaceutical Sciences
(Maack Publishing Co., Easton, Pa.). In certain embodiments the
therapeutics are formulated for oral administration.
[0166] B. Formulations
[0167] Where clinical applications are contemplated, pharmaceutical
compositions will be prepared in a form appropriate for the
intended application. Generally, this will entail preparing
compositions that are essentially free of pyrogens, as well as
other impurities that could be harmful to humans or animals.
[0168] One will generally desire to employ appropriate salts and
buffers to render delivery vectors stable and allow for uptake by
target cells. Buffers also will be employed when recombinant cells
are introduced into a patient. Aqueous compositions comprise an
effective amount of the vector or cells, dissolved or dispersed in
a pharmaceutically acceptable carrier or aqueous medium. The phrase
"pharmaceutically or pharmacologically acceptable" refer to
molecular entities and compositions that do not produce adverse,
allergic, or other untoward reactions when administered to an
animal or a human. As used herein, "pharmaceutically acceptable
carrier" includes solvents, buffers, solutions, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like acceptable for use in
formulating pharmaceuticals, such as pharmaceuticals suitable for
administration to humans. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredients, its use in therapeutic compositions is
contemplated. Supplementary active ingredients also can be
incorporated into the compositions, provided they do not inactivate
the vectors or cells of the compositions.
[0169] The active compositions may include classic pharmaceutical
preparations. Administration of these compositions may be via any
common route so long as the target tissue is available via that
route. This includes oral, nasal, or buccal. Alternatively,
administration may be by intradermal, subcutaneous, intramuscular,
intraperitoneal or intravenous injection, or by direct injection
into cardiac tissue. Such compositions would normally be
administered as pharmaceutically acceptable compositions, as
described supra.
[0170] The active compounds may also be administered parenterally
or intraperitoneally. By way of illustration, solutions of the
active compounds as free base or pharmacologically acceptable salts
can be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations generally contain a preservative to prevent the growth
of microorganisms.
[0171] The pharmaceutical forms suitable for injectable use
include, for example, sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. Generally, these preparations
are sterile and fluid to the extent that easy injectability exists.
Preparations should be stable under the conditions of manufacture
and storage and should be preserved against the contaminating
action of microorganisms, such as bacteria and fungi. Appropriate
solvents or dispersion media may contain, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetable oils. The proper fluidity can be maintained,
for example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about by various antibacterial an
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0172] For oral administration the therapeutics generally may be
incorporated with excipients and used in the form of non-ingestible
mouthwashes and dentifrices. A mouthwash may be prepared
incorporating the active ingredient in the required amount in an
appropriate solvent, such as a sodium borate solution (Dobell's
Solution). Alternatively, the active ingredient may be incorporated
into an antiseptic wash containing sodium borate, glycerin and
potassium bicarbonate. The active ingredient may also be dispersed
in dentifrices, including: gels, pastes, powders and slurries. The
active ingredient may be added in a therapeutically effective
amount to a paste dentifrice that may include water, binders,
abrasives, flavoring agents, foaming agents, and humectants.
[0173] The compositions generally may be formulated in a neutral or
salt form. Pharmaceutically-acceptable salts include, for example,
acid addition salts (formed with the free amino groups of the
protein) derived from inorganic acids (e.g., hydrochloric or
phosphoric acids, or from organic acids (e.g., acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with the free
carboxyl groups of the protein can also be derived from inorganic
bases (e.g., sodium, potassium, ammonium, calcium, or ferric
hydroxides) or from organic bases (e.g., isopropylamine,
trimethylamine, histidine, procaine and the like.
[0174] Upon formulation, solutions are preferably administered in a
manner compatible with the dosage formulation and in such amount as
is therapeutically effective. The formulations may easily be
administered in a variety of dosage forms such as injectable
solutions, drug release capsules and the like. For parenteral
administration in an aqueous solution, for example, the solution
generally is suitably buffered and the liquid diluent first
rendered isotonic for example with sufficient saline or glucose.
Such aqueous solutions may be used, for example, for intravenous,
intramuscular, subcutaneous and intraperitoneal administration.
Preferably, sterile aqueous media are employed as is known to those
of skill in the art, particularly in light of the present
disclosure. By way of illustration, a single dose may be dissolved
in 1 ml of isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject. Moreover, for human administration, preparations should
meet sterility, pyrogenicity, general safety and purity standards
as required by FDA Office of Biologics standards.
[0175] C. Dosages
[0176] The amount of therapeutic that is administered or prescribed
to the patient can be about, at least about, or at most about 0.1,
0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,
260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,
390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500 mg,
or any range derivable therein. Alternatively, the amount
administered or prescribed may be about, at least about, or at most
about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008,
0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,
1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,
2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0,
4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg/kg, or any
range derivable therein, with respect to the weight of the
patient.
[0177] When provided in a discrete amount, each intake of a
therapeutic can be considered a "dose." A medical practitioner may
prescribe or administer multiple doses of a particular therapeutic
over a particular time course (treatment regimen) or
indefinitely.
[0178] The therapeutic may be administered 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70,
80, or more times or any range derivable therein. It is further
contemplated that the drug may be taken for an indefinite period of
time or for as long as the patient exhibits symptoms of the medical
condition for which the therapeutic was prescribed or administered.
Also, the drug may be administered every 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24
hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or any range
derivable therein. Alternatively, it may be administered
systemically over any such period of time and be extended beyond
more than a year.
[0179] D. Other Therapeutic Options
[0180] In certain embodiments, methods may involve administering a
an additional therapeutic for treating IPF or for treating or
alleviating the symptoms associated with IPF.
[0181] As a second therapeutic regimen, the agent may be
administered or taken at the same time, or either before or after
the NAC or PAN therapy. The treatment may improve one or more
symptoms of IPF and/or decrease disease-related morbidity and
mortality.
[0182] Combinations may be achieved by administering a single
composition or pharmacological formulation that includes both
agents, or by contacting the cell with two distinct compositions or
formulations, at the same time. Alternatively, the NAC or PAN
therapy may precede or follow administration of the other agent(s)
by intervals ranging from minutes to weeks. In embodiments where
the other agent are administered separately from one another, one
would generally ensure that a significant period of time did not
expire between the time of each delivery, such that the agent would
still be able to exert an advantageously combined effect.
[0183] It also is conceivable that more than one administration of
either NAC, PAN, or the other agent will be desired. In this
regard, various combinations may be employed. By way of
illustration, where NAC or PAN "A" and the other agent is "B", the
following permutations based on 3 and 4 total administrations are
exemplary:
TABLE-US-00001 A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B
B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B
[0184] Other combinations are likewise contemplated.
V. EXAMPLES
[0185] The following examples are given for the purpose of
illustrating various embodiments and are not meant to limit the
present invention in any fashion. One skilled in the art will
appreciate readily that the present invention is well adapted to
carry out the objects and obtain the ends and advantages mentioned,
as well as those objects, ends and advantages inherent herein. The
present examples, along with the methods described herein are
presently representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the invention.
Changes therein and other uses which are encompassed within the
spirit of the invention as defined by the scope of the claims will
occur to those skilled in the art.
Example 1: TOLLIP and MUC5B Polymorphisms and the Response to
N-Acetylcysteine in Patients with Idiopathic Pulmonary Fibrosis
[0186] Single nucleotide polymorphisms (SNPs) within TOLLIP and
MUC5B are associated with idiopathic pulmonary fibrosis (IPF)
susceptibility and survival. Both genes play critical roles in lung
host defense, a process that can be influenced by oxidative stress
and modulated by N-acetylcysteine (NAC). Applicants set out to
determine whether SNPs within TOLLIP and MUC5B modify the efficacy
of NAC therapy in patients with IPF. To achieve this, secondary
analysis was completed of the PANTHER-IPF clinical trial (n=154)
and an independent replication cohort (n=405) composed of patients
from the INSPIRE clinical trial and University of Chicago. SNPs
within TOLLIP (rs5743890/rs5743894/rs5743854/rs3750920) and MUC5B
(r535705950) were genotyped. Multivariable Cox regression with
interaction analysis was performed followed by genotype-stratified
models for significant interaction SNPs to determine
genotype-specific survival associated with NAC therapy. A composite
endpoint of death, FVC decline, hospitalization or transplantation
was used.
[0187] As described in further detail below, significant
interaction was observed between NAC therapy and rs3750920 (TOLLIP)
(p.sub.interaction 0.002). Compared to placebo, NAC therapy was
associated with a significant reduction in endpoint risk among
those with an rs3750920 TT genotype (HR 0.14; 95% CI 0.02-0.83;
p=0.03). A trend towards increased risk was observed among NAC
recipients with an rs3750920 CC genotype (HR 3.23; 95% CI
0.79-13.16; p=0.10). Carrying one or more rs35705950 (MUC5B) T
alleles was also associated with a significantly improved
progression-free survival (HR 0.32; 95% CI 0.11-0.94; p=0.04) in
those receiving NAC therapy. Similar interaction between NAC
therapy and rs3750920 (TOLLIP) was observed in the replication
cohort.
[0188] Whereas PANTHER investigators previously showed that NAC
therapy provided no benefit in patients with IPF, here we
demonstrate that NAC may reduce clinically meaningful endpoint risk
in genetically susceptible individuals. This study highlights the
importance of drug-gene interaction in IPF clinical trials, draws
attention to the role of genes involved in airway host defense and
reignites the question of whether NAC may be an effective therapy
for some patients with IPF.
[0189] Idiopathic pulmonary fibrosis (IPF) is a deadly fibrosing
interstitial lung disease of unknown cause. Recent genome-wide
association studies (GWAS) have shown single nucleotide
polymorphisms (SNPs) within two protein-encoding genes, toll
interacting protein (TOLLIP) and mucin 5B (MUC5B), to be associated
with IPF among individuals of European ancestry (Noth I, et al.,
The Lancet. 2013; 1(4):309-317 and Fingerlin T E, et al., Nat
Genet. 2013; 45(6):613-620). SNPs within these genes have also been
associated with survival in this population (Noth I, et al., The
Lancet. 2013; 1(4):309-317 and Peljto A L, Zhang Y, et al., JAMA:
the journal of the American Medical Association. 2013;
309(21):2232-2239). TOLLIP and MUC5B reside in close vicinity on
chromosome 11p15.5 and have distinct biological functions that may
be related to IPF pathogenesis. TOLLIP encodes toll-interacting
protein (TOLLIP), an inhibitory adaptor protein, acting downstream
from the toll-like receptors (TLRs), which are key mediators of the
innate and adaptive immune response, (Zhang G, Ghosh S. The Journal
of biological chemistry. 2002; 277(9):7059-7065 and Pruitt K D, et
al., Nucleic acids research. 2014; 42(Database issue):D756-763)
whereas MUC5B encodes a highly glycosylated mucin-5B precursor
protein (Mucin-5B) involved in airway mucus production and
maintaining immune homeostasis (Pruitt K D, et al., Nucleic acids
research. 2014; 42(Database issue):D756-763 and Roy M G, et al.,
Nature. 2014; 505(7483):412-416).
[0190] Airway host defense, a process in which TOLLIP and MUC5B
participate through immune signaling, Roy M G, et al., Nature.
2014; 505(7483):412-416; Shah J A, et al., J Immunol. 2012;
189(4):1737-1746; Saito T, et al., Cell and tissue research. 2005;
321(1):75-88; and Janardhan K S, et al., Histology and
histopathology. 2006; 21(7):687-696) appears to play a critical
role in IPF, as viral and bacterial colonization, along with
particle inhalation, has been implicated in disease onset and
progression (Tang Y W, et al., Journal of clinical microbiology.
2003; 41(6):2633-2640; Han M K, et al., The lancet. Respiratory
medicine. 2014; 2(7):548-556; Hubbard R, et al., Lancet. 1996;
347(8997):284-289; and Molyneaux P L, et al., American journal of
respiratory and critical care medicine. 2014; 190(8):906-913).
Because the airway immune response can be influenced by presence of
reactive oxygen species and other forms of oxidative stress
(Asehnoune K, et al., J Immunol. 2004; 172(4):2522-2529 and De
Flora S, et al., The European respiratory journal. 1997;
10(7):1535-1541), it was hypothesized that polymorphisms within
TOLLIP and/or MUC5B modulate the efficacy of N-acetylcysteine
(NAC), an antioxidant used to treat IPF. Using a candidate gene
approach, Applicants tested this hypothesis in patients enrolled in
the Idiopathic Pulmonary Fibrosis Clinical Research Network
(IPFnet) clinical trial "Evaluating the Effectiveness of
Prednisone, Azathioprine, and N-Acetylcysteine in Patients with
Idiopathic Pulmonary Fibrosis" (PANTHER-IPF)(Martinez F J, et al.,
N Engl. J Med. 2014; 370(22):2093-2101 and Raghu G, et al., N Engl
J Med. 2012; 366(21):1968-1977). Applicants then analyzed an
independent cohort of patients drawn from the University of Chicago
and "The INSPIRE Trial: A Study of Interferon Gamma-1b for
Idiopathic Pulmonary Fibrosis" (King T E, et al., Lancet. 2009;
374(9685):222-228) to determine whether the findings could be
replicated.
[0191] Five SNPs were chosen to conduct this analysis. rs5743890
and rs5743894 reside within non-coding TOLLIP regulatory regions
and are associated with IPF susceptibility and survival (r55743890
only). rs3750920 is a synonymous coding SNP residing within TOLLIP
exon 3, was marginally associated with IPF in two GWAS discovery
cohorts and has been shown to mediate the pulmonary immune response
vis-a-vis TLR2 and TLR4 signaling. rs5743854 is located within the
TOLLIP promoter and has potential function consequences. rs35705950
resides within the promoter of MUC5B and is associated with IPF
susceptibility and improved survival.
Methods
[0192] Study Cohorts:
[0193] The PANTHER cohort consists of individuals enrolled in the
IPFnet randomized clinical trial comparing three treatments:
prednisone/azathioprine/N-acetylcysteine (PAN) combination therapy,
N-acetylcysteine (NAC) monotherapy and placebo. Participants were
enrolled from December 2009 to October 2011, at which time the PAN
arm was stopped after interim analysis showed an increased risk of
death and hospitalization in this group. Enrollment resumed for the
NAC and placebo arms from January to July 2012, with study
completion in 2013. Clinical and genetic data for patients
providing informed consent to genetic testing were used to conduct
the analysis. Patients were followed for up to 72 weeks after
randomization.
[0194] The INSPIRE cohort consists of individuals enrolled in the
industry-sponsored randomized clinical trial comparing interferon
gamma-1b to placebo. Participants were enrolled from December 2003
to April 2006 at which time the trial was stopped early after
interim analysis showed no benefit with interferon gamma-1b
compared to placebo. Clinical and genetic data for patients
providing informed consent to genetic testing were used to conduct
the analysis. Patients were followed for up to 170 weeks after
randomization.
[0195] The University of Chicago (UChicago) cohort consists of
individuals followed in the UChicago ILD clinic from 2007-2013 who
provided informed consent to undergo DNA testing. Patients were
followed from time of initial evaluation until death, lung
transplantation, loss-to-follow up or time of censoring (Sep. 9,
2013). Vital status was determined using telephone interviews with
family members and the social security death index. Patients
participating in PANTHER and INSPIRE were removed from the UC
cohort. Patients were excluded if they did not have at least two
sets of pulmonary function tests for analysis.
[0196] To minimize the effects of population stratification, only
participants that self-declared a non-Hispanic white ethnicity were
included in the analysis. Demographic and clinical information
extracted from the datasets included age, gender, race/ethnicity,
treatment arm, pulmonary function testing (PFT) including forced
vital capacity (FVC) and diffusion capacity for carbon monoxide
(DLCO) and outcomes, including death, .gtoreq.10% decline in FVC,
hospitalization and lung transplantation.
[0197] SNP Selection and Genotyping:
[0198] Eight SNPs at the chr11p15.5 locus were genotyped. Of those,
three were excluded due to the presence of moderate-to-strong
linkage disequilibrium (LD) with another SNP based on SNP
annotation and proxy search program (SNAP) R-squared approximation
using 1000 genomes project data (FIG. 1). Genotypes for the PANTHER
cohort were determined with TaqMan allelic discrimination assays
(Applied Biosystems, Foster City, Calif.). This was followed by
Sanger sequencing to confirm TaqMan calls and account for missing
data. Genotypes for those in the INSPIRE cohort were determined
using the iPLEX Gold.TM. Platform (Sequenom, San Diego, Calif.),
while UC cohort genotypes were determined by HiSeq2000 (Illumina,
San Diego, Calif.) sequencing. DNA was extracted from peripheral
blood using QIAamp.RTM. DNA Blood Maxi kit from Qiagen (Valencia,
Calif.) following manufacturer's protocol. DNA concentration was
quantified using Cytation.TM. 3 Cell Imaging Multi-Mode Reader
(BioTek.RTM. Instruments, Inc., Winooski, Vt.). Genotypes for the
PANTHER cohort were determined with TaqMan allelic discrimination
assays (Applied Biosystems, Foster City, Calif.) on a 7900HT Fast
Real-Time PCR System with automated calls generated by using the
SDS software based on discriminating plots (95% confidence). Given
the repetitive content of the DNA sequences surrounding these genes
and missing information for some SNPs with the TaqMan assays, all
samples were also sequenced by Sanger method.
[0199] Polymorphic DNA Technologies, Inc., (Alameda, Calif.)
services were used for Sanger sequencing calls. A two-step
"boost/nest" PCR strategy was applied, in which a boost reaction
with a larger fragment was performed first, which then served as a
template for the "nested" amplification and sequencing by using the
nest primers below.
TABLE-US-00002 Primers used for Genotyping by Sanger Boost Boost
forward SEQ reverse SEQ primer ID primer ID Amplicon (5' .fwdarw.
3') NO: (5' .fwdarw. 3') NO: Size* rs5743890 TTTCTCTCA 1 GAGACACCA
6 437 TTGCCTTTT GAAGGGA GAAT rs5743894 TCTAACACT 2 CAAAAGCCA 7 458
GTCCCTTGA GTCAAGCAG rs3750920 ATATACGAT 3 GTGAGCCCT 8 502 CGTGAAGCC
GCTGTT rs5743854 TTCAGCCTC 4 ATGACGGTT 9 469 AGTTTACC GTCGGC
rs35705950 TCCACCCTG 5 CCCCTTTGT 10 478 GAACAG CTCCACT Nest Nest
forward SEQ reverse SEQ primer ID primer ID Amplicon (5' .fwdarw.
3') NO: (5' .fwdarw. 3') NO: Size* rs5743890 GTTGTTTTA 11 GAGAAACAC
16 323 TCTCAAGTA AGAAGGAAA CATACA TC rs5743894 ACAGGCCCC 12
GAGATCCAG 17 376 TGGTTA AGAGGGA rs3750920 GTGTTGGTG 13 TATGGGCTC 18
395 CAGGTG AGTGCC rs5743854 TGGAGTCGC 14 TCTGGGCAG 19 428 TCTGGT
TGGGTT rs35705950 TGACACCAA 15 TGGCCAGAA 20 402 ACAAGTGG TGAGGG
*Amplicon products in base pairs.
[0200] A comparison of TaqMan and Sanger sequencing calls is shown
below. Calls were highly correlated with regard to all SNPs with
the exception of rs5743854, where a less strong correlation was
observed.
TABLE-US-00003 Taqman and Sanger genotype calls and correlation
Taqman Sanger Pearson SNP calls calls Correlation rs5743890 148 149
1 rs5743894 147 154 0.99 rs3750920 151 153 1 rs5743854 154 154 0.81
rs35705950 149 154 0.98
[0201] Genotypes for those in the INSPIRE cohort were conducted
using the iPLEX Gold.TM. Platform following manufacturer's protocol
(Sequenom, San Diego, Calif.), while UC cohort was subjected to
custom-designed target capture the Agilent SureSelect XT2 kit
followed by manufacturer's protocols. Paired-end reads of 100 bases
were generated on HiSeq2000. Data analysis was performed by the
Center for Research Informatics at University of Chicago using a
modified Illumina's Exome pipeline, including SeqPrep, FastQC,
NovoAlign, Picard, SAMtools, GATK2, and Annovar.
[0202] Statistical Analysis:
[0203] Continuous variables are reported as means with standard
deviation (SD) and are compared using a Student's t-test or one-way
analysis of variance, as appropriate. Categorical variables are
reported as counts and percentages and compared using a Chi-square
or Fisher's exact test, as appropriate. SNP correlation was
determined using Pearson's correlation coefficient. Survival
analysis was conducted using an unadjusted logrank test and
multivariable Cox proportional hazards modeling and plotted using
the Kaplan-Meier survival estimator.
[0204] The primary analysis tested whether statistical interaction
was present between SNPs of interest and NAC therapy with regard to
composite endpoint-free survival, defined as time from trial
enrollment to death, .gtoreq.10% FVC decline, hospitalization or
transplantation. Interaction was formally tested with a
multivariable Cox regression model that included the following
variables: SNP genotype, NAC therapy, and a SNP*NAC interaction
term, age, gender, FVC (% predicted) and DLCO (% predicted).
Statistical interaction was considered present when the Wald
z-statistic for SNP*NAC interaction term corresponded to a p-value
<0.01, which adjusts for multiple testing by Bonferroni
correction. Interaction detected by this method was then further
explored by constructing genotype-stratified multivariable Cox
models to determine genotype-specific endpoint risk associated with
NAC therapy. Based on SNP distributions, an additive allelic model
was assumed for rs3750920 (TOLLIP) and a dominant model for
rs5743890 (TOLLIP), rs5743894 (TOLLIP), rs5743854 (TOLLIP) and
rs35705950 (MUC5B). Each model was checked to ensure that the
proportional hazards assumption was met.
[0205] Hospitalization data were not available for patients in the
replication cohort so composite endpoint-free survival was defined
as time from trial enrollment (INSPIRE) or blood draw (UChicago) to
death, .gtoreq.10% FVC decline or transplantation in those not
receiving NAC therapy and time from NAC initiation to death,
.gtoreq.10% FVC decline or transplantation in those receiving NAC
therapy. Because follow-up time was longer for the UChicago
compared to INSPIRE cohort, UChicago cohort survival time was
censored at 170 weeks to allow for merging of the datasets. All
statistical analysis was conducted using Stata (StataCorp. 2011.
Release 12. College Station, Tex.).
Results
[0206] PANTHER Cohort:
[0207] Of the 341 patients enrolled in the PANTHER trial, 315
self-reported a non-Hispanic white ethnicity (FIG. 2). Of those 315
individuals, 154 consented to genetic testing and were included in
the primary analysis. Among genotyped patients, fifty-four patients
received placebo therapy and sixty patients received NAC therapy.
Baseline demographic and clinical characteristics between
non-Hispanic white genotyped (n=154) and non-genotyped (n=161)
patients stratified by treatment group are shown in Table 1.
TABLE-US-00004 TABLE 1 PANTHER Baseline Characteristics and
Genotypes* Genotyped (n = 154) Non-Genotyped (n = 161) Placebo Arm
NAC Arm Placebo Arm NAC Arm Characteristic (n = 54) (n = 60) (n =
66) (n = 61) p-value Age, mean (.+-.SD) 66.1 (7.9) 67.9 (8.7) 66.8
(8.2) 67.8 (8.2) 0.62 Male, n (%) 39 (72.2) 47 (78.3) 53 (80.3) 51
(83.6) 0.5 Ever Smoker, n (%) 41 (75.9) 44 (73.3) 50 (75.8) 46
(76.7) 0.98 FVC, % predicted (.+-.SD) 73.2 (14.7) 73 (15.7) 74
(13.8) 73 (15.8) 0.98 DLCO, % predicted (.+-.SD) 46.4 (11.7) 43.9
(10.9) 44.7 (12.1) 46.2 (10.8) 0.59 Death, n (%) 2 (3.7) 1 (1.7) 1
(1.5) 3 (4.9) 0.64 FVC Decline .gtoreq. 10%, n (%) 12 (22.2) 11
(18.3) 17 (25.8) 17 (27.9) 0.62 Hospitalization, n (%) 8 (14.8) 8
(13.3) 10 (15.2) 9 (14.8) 0.99 Transplant, n (%) 1 (1.9) 3 (5) 1
(1.5) 1 (1.6) 0.67 Composite Endpoint**, n (%) 17 (31.5) 19 (31.7)
24 (36.4) 26 (42.6) 0.55 SNP (Gene) Genotype, count (%) rs5743890
(TOLLIP) AA/AG/GG 43/10/0 52/8/0 -- -- 0.42 0.81/0.19/0 0.87/0.13/0
rs5743894 (TOLLIP) AA/AG/GG 29/21/4 31/25/4 -- -- 0.96
0.54/0.39/0.07 0.52/0.42/0.06 rs3750920 (TOLLIP) CC/CT/TT 14/23/17
13/30/16 -- -- 0.68 0.26/0.43/0.31 0.22/0.51/0.27 rs5743854
(TOLLIP) CC/CG/GG 44/10/0 55/5/0 0.09 0.81/0.19/0 0.92/0.08/0
rs35705950 (MUC5B) GG/GT/TT 20/29/5 14/42/4 -- -- 0.19
0.37/0.54/0.09 0.23/0.7/0.07 Abbreviations: NAC = N-acetylcysteine;
FVC = forced vital capacity; DLCO = diffusion capacity for carbon
monoxide *Non-Hispanic white individuals by self-report **Death,
10% FVC decline, hospitalization or transplantation
[0208] No differences were observed between genotyped and
non-genotyped individuals with regard to age, gender, smoking
history and pulmonary function. Trial endpoints, including death,
FVC decline .gtoreq.10%, hospitalization and lung transplantation,
as well as the composite endpoint, were similar between genotyped
and non-genotyped individuals. SNP genotype counts (Table 1) were
similar between treatment groups.
[0209] When considering patients enrolled prior to and following
the clinical alert, a non-statistically significant imbalance in
genotype frequencies was observed between the placebo and NAC arms
with regard to rs5743894 (TOLLIP) (p=0.17), rs3750920 (TOLLIP)
(p=0.12) and rs35705950 (MUC5B) (p=0.08) (Table 2).
TABLE-US-00005 TABLE 2 Treatment-stratified SNP Comparison Before
and After PANTHER Clinical Alert SNP (Gene), Before Clinical Alert
After Clinical Alert Genotype Placebo Arm NAC Arm p-value Placebo
Arm NAC Arm p-value rs5743890 25/5/0 41/7/0 0.80 18/5/0 11/1/0 0.64
AA/AG/GG (0.83/0.17/0) (0.85/0.15/0) (0.78/0.22/0) (0.92/0.08/0)
rs5743894 14/14/3 27/18/3 0.64 15/7/1 4/7/1 0.17 AA/AG/GG
(0.45/0.45/0.10) (0.56/0.38/0.06) (0.65/0.30/0.05) (0.33/0.59/0.08)
rs3750920 6/16/9 10/22/15 0.92 8/7/8 3/8/1 0.12 CC/CT/TT
(0.19/0.52/0.29) (0.21/0.47/0.32) (0.35/0.30/0.35) (0.25/0.67/0.08)
rs5743854 26/5/0 44/4/0 0.3 18/5/0 11/1/0 0.64 CC/CG/GG 0.84/0.16/0
0.92/0.08/0 0.78/0.22/0 0.92/0.08/0 rs35705950 11/16/4 13/31/4 0.54
9/13/1 1/11/0 0.08 GG/GT/TT (0.35/0.52/0.13) (0.27/0.65/0.08)
(0.39/0.57/0.04) (0.08/0.92/0) Abbreviations: SNP = single
nucleotide polymorphism; NAC = N-acetylcysteine; PAN =
prednisone/azathioprine/N-acetylcysteine
[0210] Minor allele frequencies (MAF) were compared between PANTHER
participants and those comprising the GWAS stage 2 replication
cohort in reference 1. Groups were compared by multiplying the
total number of patients in each cohort by the MAF and conducting a
Chi-square test, which compared the expected and observed major and
minor alleles in each cohort. This is shown in Table 3:
TABLE-US-00006 TABLE 3 Minor Allele Frequency Comparison Between
PANTHER and GWAS Cohorts PANTHER GWAS SNP_minor allele (Gene) n MAF
MAF* p-value rs5743890_G (TOLLIP) 149 0.08 0.09 0.61 rs5743894_G
(TOLLIP) 154 0.29 0.24 0.08 rs3750920_T (TOLLIP) 153 0.53 0.5 0.31
rs5743854_G (TOLLIP)** 154 0.06 -- -- rs35705950_T (MUC5B) 154 0.4
0.33 0.02 Abbreviations: SNP = single nucleotide polymorphism; MAF
= minor allele frequency *Based on GWAS Stage 2 replication cohort
in reference 1 **GWAS data not available
[0211] When comparing the minor allele frequency (MAF) for each SNP
between the PANTHER cohort and a recent GWAS.sup.1 stage II
replication cohort (Table 3), the MAF of rs35705950_T (MUC5B) was
significantly higher in the PANTHER cohort compared to the GWAS
cohort (0.4 vs 0.33, respectively; p=0.02). The MAF of rs5743894_G
(TOLLIP) was also higher in the PANTHER cohort compared to the GWAS
cohort (0.29 vs 0.24, respectively), but this difference was not
statistically significant (p=0.08). No SNPs were in strong linkage
disequilibrium based on methodology outlined above, nor were any
significantly correlated in the PANTHER dataset (Table 4). Pairwise
correlation was conducted using the SNP annotation and proxy search
program (SNAP) with 1000 genomes project data (references 19,20).
PANTHER SNPs were then correlated using Pearson correlation.
TABLE-US-00007 TABLE 4 Pairwise SNP correlation SNP 1 SNP2
Rsquared* Dprime* PANTHER** rs5743890 (TOLLIP) rs5743894 (TOLLIP)
0.028 1 -0.1 rs5743890 (TOLLIP) rs3750920 (TOLLIP) 0.058 0.58 0.22
rs5743890 (TOLLIP) rs5743854 (TOLLIP) 0.019 1 0.01 rs5743890
(TOLLIP) rs35705950 (MUC5B) 0.009 0.786 -0.21 rs5743894 (TOLLIP)
rs3750920 (TOLLIP) 0.078 0.532 0.49 rs5743894 (TOLLIP) rs5743854
(TOLLIP) 0.03 1 -0.23 rs5743894 (TOLLIP) rs35705950 (MUC5B) 0.158
0.549 0.52 rs5743854 (TOLLIP) rs35705950 (MUC5B) 0 0 -0.26
rs3750920 (TOLLIP) rs35705950 (MUC5B) 0.084 0.761 0.48 *Based on
SNAP analysis using 1000 Genomes Project (references 19, 20)
**Based on Pearson correlation
[0212] Survival modeling shows that no SNP independently predicted
composite endpoint-free survival in multivariable Cox regression
adjusting for treatment arm assignment, age, gender, FVC (%
predicted) and DLCO (% predicted) (Table 5). Using a multivariable
Cox regression model adjusting for treatment arm and GAP score
(based on reference 17), no SNP was an independent predictor of
endpoint risk in either progression-free survival or
hospitalization-free survival.
TABLE-US-00008 TABLE 5 Composite Endpoint Risk Associated with
TOLLIP and MUC5B SNPs Reference SNP (Gene), allele Allele HR
p-value 95% CI rs5743890 (TOLLIP), G A 1.06 0.88 0.52-2.15
rs5743894 (TOLLIP), G A 0.8 0.43 0.46-1.38 rs3750920 (TOLLIP), T C
0.93 0.74 0.62-1.40 rs5743854 (TOLLIP), G** C 0.87 0.76 0.36-2.10
rs35705950 (MUC5B), T G 1.14 0.66 0.63-2.09 *Adjusted for treatment
arm assignment, age, gender, FVC and DLCO **This SNP did not meet
the proportional hazards assumption in this model so was omitted
from subsequent analyses
[0213] The proportional hazards assumption for rs5743854 was not
met in this modeling so this SNP was omitted from subsequent
analyses. Multivariable Cox interaction modeling (Table 6) showed
significant interaction between NAC therapy and the T allele of
rs3750920 within TOLLIP (p.sub.interaction=0.002). A suggestion of
potential interaction was observed between NAC therapy and the T
allele of rs35705950 (MUC5B) (p.sub.interaction=0.06) and the G
allele of rs5743894 (TOLLIP) (p.sub.interaction=0.05). No SNP
modified endpoint risk among those receiving placebo therapy.
[0214] Interaction is statistically significant when SNP*treatment
interaction term has p-value <0.01, based on Bonferroni
correction for multiple testing.
TABLE-US-00009 TABLE 6 Multivariable Cox Interaction Model
Estimates* Variable Coefficient (95% CI) p-value rs5743890 (TOLLIP)
0.84 (-0.32 - 1.99) 0.16 NAC Therapy 0.17 (-0.58 - 0.92) 0.66 PAN
Therapy 1.36 (0.56 - 2.17) 0.001 rs5743890*NAC interaction -1.17
(-2.91 - 0.56) 0.19 rs5743890*PAN interaction -1.08 (-2.70 - 0.55)
0.19 rs5743894 (TOLLIP) 0.05 (-0.91 - 1.00) 0.93 NAC Therapy 0.45
(-0.40 - 1.30) 0.30 PAN Therapy 0.74 (-0.30 - 1.78) 0.17
rs5743894*NAC interaction -1.42 (-2.89 - 0.06) 0.06 rs5743894*PAN
interaction 0.58 (-0.82 - 1.97) 0.42 rs3750920 (TOLLIP) 0.39 (-0.32
- 1.10) 0.28 NAC Therapy 1.34 (0.17 - 2.52) 0.03 PAN Therapy 0.89
(-0.49 - 2.27) 0.20 rs3750920*NAC interaction -1.47 (-2.45 - -0.48)
0.004 rs3750920*PAN interaction 0.19 (-0.81 - 1.19) 0.71 rs35705950
(MUC5B) 0.61 (-0.53 - 1.75) 0.29 NAC Therapy 0.91 (-0.34 - 2.51)
0.15 PAN Therapy 0.98 (-0.42 - 2.37) 0.17 rs35705950*NAC
interaction -1.40 (-2.89 - 0.09) 0.07 rs35705950*PAN interaction
0.13 (-1.48 - 1.74) 0.87 *Adjusted for age, gender, FVC and
DLCO
[0215] After stratifying by rs3750920 (TOLLIP) genotype (FIG. 4),
compared to placebo, the composite endpoint risk associated with
NAC therapy was significantly reduced among those with a TT
genotype (HR 0.14; 95% CI 0.02-0.83; p=0.03). A non-significant
reduction in composite endpoint risk was observed among those with
a CT genotype (HR 0.76; 95% CI 0.27-2.19; p=0.62) and
non-significant increase in composite endpoint risk was observed
among those with a CC genotype (HR 3.23; 95% CI 0.79-13.16;
p=0.10).
[0216] Given the potential interaction observed between NAC therapy
and rs5743894 within TOLLIP and rs35705950 within MUC5B, a
sensitivity analysis (Table 7) was conducted for these SNPs, along
with the rs3750920 (TOLLIP), to determine whether individual SNP
genotypes predicted specific endpoints. Along with our primary
composite endpoint, we assessed hospitalization-free survival,
defined as time to death, hospitalization or transplant, and
progression-free survival, defined as time to death, 10% FVC
decline or transplant. Death and transplant were not considered in
isolation, as there were not enough events for robust analysis.
TABLE-US-00010 TABLE 7 Sensitivity Analysis of NAC-associated
endpoint risk after rs3750920, rs5743894 and rs35705950
stratification* Primary Composite Hospitalization- Progression- SNP
Genotype Endpoint Free Survival Free Survival (Gene) HR (95% CI)
p-value HR (95% CI) p-value HR (95% CI) p-value rs3750920 (TOLLIP)
3.22 (0.79-13.16) 0.1 3.74 (0.35-39.80) 0.27 1.27 (0.21-7.55) 0.79
CC rs3750920 (TOLLIP) 0.76 (0.27-2.19) 0.62 0.45 (0.10-2.15) 0.32
0.63 (0.17-2.30) 0.48 CT rs3750920 (TOLLIP) 0.14 (0.02-0.83) 0.03
** ** 0.09 (0.01-0.76) 0.03 TT rs5743894 (TOLLIP) 1.67 (0.71-3.95)
0.24 1.43 (0.44-4.70) 0.56 1.68 (0.58-4.90) 0.34 AA rs5743894
(TOLLIP) 0.33 (0.10-1.14) 0.08 0.21 (0.02-1.99) 0.18 0.18
(0.04-0.90) 0.04 AG/GG rs35705950 (MUC5B) 2.02 (0.57-7.20) 0.28
1.25 (0.15-10.30) 0.84 3.72 (0.69-20.16) 0.13 GG rs35705950 (MUC5B)
0.60 (0.27-1.35) 0.22 0.49 (0.15-1.58) 0.23 0.32 (0.11-0.94) 0.04
GT/TT *Models adjusted for age, gender, FVC (% predicted) and DLCO
(% predicted) ** No events observed in this group so HR could not
be estimated Primary Composite Endpoint = death, hospitalization,
10% FVC decline or transplant Progression-Free Survival = death,
10% FVC decline or transplant Hospitalization-Free Survival =
death, hospitalization or transplant
[0217] This analysis showed that when compared to placebo, a
consistent hazard reduction occurred in patients receiving NAC
therapy with each T allele of rs3750920 (TOLLIP) with regard to all
three endpoints. It also showed that NAC recipients had a
significant reduction in endpoint risk when considering
progression-free survival with an rs5743894 (TOLLIP) AG/GG genotype
(HR 0.18; 95% CI 0.04-0.90; p=0.04) and rs35705950 (MUC5B) GT/TT
genotype (HR 0.32; 95% CI 0.11-0.94; p=0.04).
[0218] Replication Cohort: Based on the findings above, interaction
modeling was performed in an independent cohort between NAC therapy
and rs3750920 (TOLLIP), rs5743894 (TOLLIP) and rs35705950 (MUC5B).
Of the 771 non-Hispanic white individuals enrolled in the INSPIRE
trial, DNA was available for 314. In the UChicago cohort, of 151
non-Hispanic white individuals with sequencing data, 91 had
longitudinal PFT data and were included in the analysis. A summary
of baseline demographic and clinical characteristics, along with
genotype counts for the INSPIRE, UChicago and combined cohorts are
shown in Table 8.
TABLE-US-00011 TABLE 8 Replication Cohort Baseline Characteristics
and Genotypes* INSPIRE (n = 314) UChicago (n = 91) Combined Cohort
(n = 405) NAC Non-NAC NAC Non-NAC NAC Non-NAC p- Characteristic (n
= 29) (n = 285) (n = 18) (n = 73) (n = 47) (n = 358) value Age,
mean (.+-.SD) 65.8 (7.5) 66.2 (7.7) 72.4 (8.0) 67.3 (8.1) 68.3
(8.3) 66.4 (7.8) 0.12 Male, n (%) 18 (62.1) 210 (73.7) 15 (83.3) 57
(78.1) 33 (70.2) 267 (74.6) 0.52 Ever Smoker, n (%) 18 (62.1) 199
(69.8) 15 (83.3) 36 (73.5) 33 (70.2) 235 (70.4) 0.98 Azathioprine
therapy, n (%) 4 (13.8) 9 (3.2) 1 (5.9) 1 (1.4) 5 (10.9) 10 (2.8)
0.02 Prednisone therapy, n (%) 19 (65.5) 121 (42.5) 4 (23.5) 11
(15.1) 23 (50.0) 132 (36.9) 0.09 FVC, % predicted (.+-.SD) 72.3
(11.9) 71.3 (12.6) 58.9 (15.0) 70.3 (19.4) 67.3 (14.5) 71.1 (14.2)
0.09 DLCO, % predicted (.+-.SD) 47.9 (8.4) 47.3 (9.2) 37 (10.8)
50.7 (19.1) 44.0 (10.6) 48.0 (11.9) 0.03 Death, n (%) 6 (20.7) 49
(17.2) 13 (72.2) 29 (39.7) 19 (40.4) 78 (21.8) 0.01 FVC Decline
.gtoreq. 10%, n (%) 13 (44.8) 115 (40.4) 4 (22.2) 17 (23.3) 17
(36.2) 132 (36.9) 0.93 Transplant, n (%) 2 (6.9) 4 (1.4) 0 (0) 7
(9.6) 2 (4.3) 11 (3.1) 0.46 Composite Endpoint**, n (%) 14 (48.3)
146 (51.2) 13 (72.2) 45 (61.6) 27 (57.5) 167 (46.7) 0.6 SNP (Gene)
Genotype, count (%) rs5743894 (TOLLIP) 18/11/0 143/129/14 11/8/2
60/31/10 29/16/2 188/151/19 0.53 AA/AG/GG 0.62/0.38/0 0.5/0.45/0.05
0.52/0.38/0.1 0.59/0.31/0.1 0.62/0.34/0.04 0.53/0.42/0.05 rs3750920
(TOLLIP) 6/15/8 61/157/66 3/12/6 21/49/31 9/25/13 77/194/86 0.84
CC/CT/TT 0.21/0.52/0.27 0.22/0.55/0.23 0.14/0.57/0.29
0.21/0.48/0.31 0.19/0.53/0.28 0.22/0.54/0.24 rs35705950 (MUC5B)
10/16/3 82/185/15 8/13/0 31/62/8 17/27/3 105/230/20 0.57 GG/GT/TT
0.35/0.55/0.1 0.29/0.66/0.05 0.38/0.62/0 0.31/0.61/0.08
0.36/0.57/0.07 0.30/0.65/0.05 Abbreviations: NAC =
N-acetylcysteine; PAN = prednisone/azathioprine/N-acetylcysteine;
FVC = forced vital capacity; DLCO = diffusion capacity for carbon
monoxide *Non-Hispanic white individuals by self-report **Death,
10% FVC decline, hospitalization or transplantation
[0219] Those in the UChicago cohort tended to have poorer lung
function and an increased number of deaths and composite endpoint
events compared to the INSPIRE cohort. After combining cohorts,
compared to those who did not receive NAC therapy, those receiving
NAC therapy had increased concurrent azathioprine use (2.8% vs
10.9%; respectively p=0.02) and were sicker overall with worse DLCO
(48% predicted vs 44% predicted, respectively; p=0.03) and more
deaths (21.8% vs 40.4%, respectively; p=0.01). There was no
difference in genotype counts between groups.
[0220] Multivariable Cox interaction models (Table 9) showed
significant interaction between NAC therapy and rs3750920 (TOLLIP)
(p.sub.interaction=0.003) and rs35705950 (MUC5B)
(p.sub.interaction=0.005). No interaction was observed between NAC
therapy and rs5743894 (TOLLIP) (p.sub.interaction=0.41).
TABLE-US-00012 TABLE 9 Replication Cohort Multivariate Cox
Interaction Model Estimates Variable Coefficient (95% CI) p-value
rsrs5743894 (TOLLIP) -0.07 (-0.35 - 0.22) 0.67 NAC Therapy 0.54
(0.01 - 1.07) 0.05 rs3750920*NAC interaction -0.40 (-1.33 - 0.54)
0.41 rs3750920 (TOLLIP) -0.07 (-0.22 - 0.2) 0.95 NAC Therapy 1.37
(0.69 - 2.05) <0.001 rs3750920*NAC interaction -0.86 (-1.43 -
-0.29) 0.003 rs35705950 (MUC5B) -0.17 (-0.49 - 0.15) 0.31 NAC
Therapy 1.24 (0.58 - 1.90) <0.001 rs35705950*NAC interaction
-1.30 (-2.20 - 0.39) 0.005 *Adjusted for age, gender, FVC and DLCO,
prednisone use, azathioprine use and study cohort
[0221] Table 10 shows rs3750920 (TOLLIP) and rs35705950 (MUC5B)
genotype-stratified composite endpoint risk associated with NAC
therapy after adjustment for age, gender, FVC (% predicted) and
DLCO (% predicted).
TABLE-US-00013 TABLE 10 Replication Cohort Genotype-stratified
Composite Endpoint Risk Associated with NAC Therapy Genotype HR 95%
CI p-value rs3750920 (TOLLIP) CC 3.51 1.44-8.56 0.01 rs3750920
(TOLLIP) CT 2.27 1.19-4.31 0.01 rs3750920 (TOLLIP) TT 0.23
0.06-0.93 0.04 rs35705950 (MUC5B) GG 3.15 1.47-6.72 0.003
rs35705950 (MUC5B) GT/TT 0.94 0.50-1.76 0.84
[0222] Compared to those who did not receive NAC therapy, the
composite endpoint risk associated with NAC therapy was
significantly reduced among those with a rs3750920 (TOLLIP) TT
genotype (HR 0.23; 95% CI 0.06-0.93; p=0.04) but significantly
increased among those with a CT genotype (HR 2.27; 95% CI
1.19-4.31; p=0.01) and CC genotype (HR 3.51; 95% CI 1.44-8.56;
p=0.01). Unadjusted endpoint-free survival between those who did
and did not receive NAC therapy stratified by rs3750920 (TOLLIP)
genotype is shown in FIG. 3. While genotype was not associated with
outcome in those who did not receive NAC (p=0.27), among NAC
recipients those with an rs3750920 (TOLLIP) TT genotype
demonstrated significantly better overall survival compared to
those with CT and CC genotypes (p=0.03). With regard to rs35705950
(MUC5B), compared to those who did not receive NAC therapy, the
composite endpoint risk associated with NAC therapy was
significantly increased in those with a GG genotype (HR 3.15; 95%
CI 1.47-6.72; p=0.003) but no different in those with a GT or TT
genotype (HR 0.94; 95% CI 0.5-1.76; p=0.84).
DISCUSSION
[0223] In this study, Applicants showed that polymorphisms within
TOLLIP and MUC5B may influence the response to NAC therapy in
patients with IPF. This represents the first significant drug-gene
interaction observed in patients with IPF. Whereas PANTHER
investigators previously reported that NAC therapy did not provide
benefit for patients with IPF, Applicants demonstrate here that NAC
therapy may reduce c