U.S. patent application number 14/704410 was filed with the patent office on 2015-09-17 for genetic lesion associated with cancer.
The applicant listed for this patent is Yale University. Invention is credited to Lena J. Chin, Elena Ratner, Frank J. Slack, Joanne B. Weidhaas.
Application Number | 20150259752 14/704410 |
Document ID | / |
Family ID | 39767038 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150259752 |
Kind Code |
A1 |
Slack; Frank J. ; et
al. |
September 17, 2015 |
Genetic Lesion Associated With Cancer
Abstract
The invention comprises methods for identifying mutations within
the 3'UTR of genes that lead to increased risk or probability of
developing cancer.
Inventors: |
Slack; Frank J.; (Branford,
CT) ; Weidhaas; Joanne B.; (Westport, CT) ;
Chin; Lena J.; (East Brunswick, NJ) ; Ratner;
Elena; (Fairfield, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yale University |
New Haven |
CT |
US |
|
|
Family ID: |
39767038 |
Appl. No.: |
14/704410 |
Filed: |
May 5, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14158047 |
Jan 17, 2014 |
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14704410 |
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13653229 |
Oct 16, 2012 |
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14158047 |
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12600013 |
Sep 24, 2010 |
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PCT/US2008/065302 |
May 30, 2008 |
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13653229 |
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61124610 |
Apr 18, 2008 |
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61065745 |
Feb 14, 2008 |
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61001965 |
Nov 5, 2007 |
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60932575 |
May 31, 2007 |
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Current U.S.
Class: |
506/2 ; 435/6.11;
506/9 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/178 20130101; C12Q 2600/172 20130101; C12Q 1/6827
20130101; C12Q 2600/118 20130101; C12Q 2600/156 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-29. (canceled)
30. A method of identifying a human subject at increased risk of
developing cancer, the method comprising (1) amplifying a
polynucleotide comprising position 4 of LCS6 of KRAS in a
biological sample from the subject; and (2) assaying for the
presence of a uracil or thymine to guanine transition at position 4
of LCS6 of KRAS in the amplification products, wherein the subject
is identified as being at increased risk of developing cancer if
there is a transition of uracil or thymine to guanine at position 4
of LCS6 of KRAS in the amplification products.
31. The method of claim 30, wherein the assaying step comprises
digesting the amplification products with one or more restriction
enzymes and separating the resulting fragments by gel
electrophoresis in the presence of a control sample comprising a
polynucleotide consisting of SEQ ID NO:15.
32. The method of claim 30, wherein the amplifying step comprises a
polymerase chain reaction.
33. The method of claim 32, wherein the assaying step comprises a
5' nuclease assay.
34. The method of claim 32, wherein the assaying step comprises an
oligonucleotide ligation assay.
35. The method of claim 32, wherein the assaying step comprises
primer extension and mass spectrophotometry.
36. The method of claim 30, wherein the cancer is non-small cell
lung cancer.
37. The method of claim 36, wherein the subject is a smoker with
less than a 41 pack-year smoking history.
38. The method of claim 36, wherein the cancer is
radon-associated.
39. The method of claim 30, wherein the cancer is selected from
ovarian cancer, breast cancer, and melanoma.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 14/158,047, filed Jan. 17, 2014, which is a continuation
application of U.S. Ser. No. 13/653,229, filed Oct. 16, 2012, which
is a continuation of U.S. Ser. No. 12/600,013, filed Sep. 24, 2010,
which is a national stage application, filed under 35 U.S.C.
.sctn.371, of International Application No. PCT/US2008/065302,
filed May 30, 2008, which claims the benefit of U.S. Ser. No.
60/932,575, filed May 31, 2007, U.S. Ser. No. 61/001,965, filed
Nov. 5, 2007, U.S. Ser. No. 61/065,745 filed Feb. 14, 2008, and
U.S. Ser. No. 61/124,610, filed Apr. 18, 2008, the contents which
are each herein incorporated by reference in their entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The contents of the text file named "34592-501C05US_ST25,"
which was created on Jan. 17, 2014 and is 47.8 KB in size, are
hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] This invention relates generally to the fields of cancer and
molecular biology. The invention provides compositions and methods
for predicting increased risk of developing cell proliferative
diseases, such as cancer.
BACKGROUND OF THE INVENTION
[0004] Even though there has been progress in the field of cancer
detection, there still remains a need in the art for the
identification of new genetic markers for a variety of cancers that
can be easily used in clinical applications. To date, there are
relatively few options available for predicting the risk of
developing cancer.
[0005] A test for predicting the risk of developing lung cancer
would be particularly useful. Lung cancer is an enormous public
health problem, with smoking as the number one risk factor, with
44.5 million people in the United States (20.9% of the population),
and over 1.3 billion people in the world currently smoking.
Unfortunately, smoking cessation does not eliminate a person's risk
for developing lung cancer. Compared to never smokers, former
smokers have a 6.6-fold increase in relative lung cancer risk for
up to 30 years after smoking cessation (95% Confidence
Interval=5.0-8.7). Screening programs have been initiated in these
populations: The Early Lung Cancer Action Project (ELCAP) found
that a chest computed tomography (C T) scan is three times more
sensitive in detecting early-stage lung cancer than a chest X-ray
in "high-risk" populations (2.4% versus 0.7%), and may improve
survival rates. There remains considerable controversy over the use
of lung CT scans as a global screening approach for lung cancer
however, due to the expense (estimated cost 2 billion dollars
yearly in the US alone) and the very low yield of yearly cancer
detection (1.2%). A genetic marker capable of determining a
smoker's risk of developing lung cancer would be a particularly
useful test that could be used in conjunction with screening
programs to diagnose lung cancer at an earlier stage, and thus
reduce mortality of this devastating disease.
[0006] Accordingly, the identification of genetic markers for
cancer is particularly relevant to improving prognosis, diagnosis,
and treatment of the disease. As such, there is need in the art to
identify alternative genetic markers that can be quickly, easily,
and safely detected. Such markers may be used to identify those
individuals who would benefit from screening or intervention.
SUMMARY OF THE INVENTION
[0007] The invention provides compositions and methods for
identifying one or more genetic markers within let-7 family miRNA
binding sites that are predictive of the onset, development and
prognosis of a variety of disorders, such as, for example, all
varieties of lung cancer, ovarian cancer, breast cancer, uterine
cancer, head and neck cancer, pancreatic cancer, and colon cancer.
In a specific embodiment, the genetic marker of the invention is a
single nucleotide polymorphism (SNP). In another specific
embodiment of the invention, the presence of a SNP within a let-7
family miRNA binding site is predictive of the onset, development
and prognosis of cancer. Subjects carrying a particular SNP,
referred to herein as the LCS6 SNP, have a significantly increased
risk of developing cancer. Smokers who carry the LCS6 SNP are far
more likely to develop non-small cell lung cancer (NSCLC) and
ovarian cancer. Moreover, the occurrence of the LCS6 SNP is
associated with earlier onset of cancer, increased occurrence of
secondary cancers (including multiple secondary cancers), and
increased occurrence of particularly aggressive or high risk forms
of cancer. Carriers of the LCS6 SNP are particularly prone to
developing all varieties of lung cancer, ovarian cancer, breast
cancer, uterine cancer, head and neck cancer, pancreatic cancer,
and colon cancer.
[0008] In one aspect, the invention provides an isolated
polynucleotide molecule comprising of between 10-50 bases of which
at least 10 contiguous bases including a polymorphic site are from
SEQ ID NO: 21 in which the nucleotide at position 4 of SEQ ID NO:
21 is not a uracil (U) or thymine (T). Furthermore, the invention
encompasses this isolated polynucleotide molecule wherein the
nucleotide at position 4 of SEQ ID NO: 21 is a guanine (G).
Compositions of the invention also include an isolated
polynucleotide molecule that is complementary to this isolated
polynucleotide molecule.
[0009] In another aspect, the invention provides an isolated
polynucleotide molecule comprising a 3' untranslated region (UTR)
of KRAS, wherein the polynucleotide contains a single nucleotide
polymorphism (SNP) within a Let-7 Complementary Site (LCS) that
modulates the binding efficacy of a let-7 family miRNA molecule.
Furthermore, the invention comprises this isolated polynucleotide
molecule of wherein the SNP occurs at position 4 of LCS6 (SEQ ID
NO: 21) and wherein the nucleotide at position 4 of SEQ ID NO: 21
is a guanine (G). Compositions of the invention also include an
isolated polynucleotide molecule that is complementary to this
isolated polynucleotide.
[0010] The invention further provides an isolated and purified
polynucleotide comprising a sequence of at least 20 nucleotides of
a KRAS allele, wherein the polynucleotide contains at least one
mutation relative to KRAS shown in SEQ ID NO: 24, the mutation
comprising a uracil (U) or thymine (T) to guanine (G) transition at
nucleotide 3377 as shown in SEQ ID NO: 24. Alternatively, or in
addition, the invention comprises, an isolated and purified
polynucleotide comprising a sequence of at least 20 nucleotides of
a KRAS allele, wherein the polynucleotide contains at least one
mutation relative to KRAS shown in SEQ ID NO: 25, the mutation
comprising a uracil (U) or thymine (T) to guanine (G) transition at
nucleotide 3253 as shown in SEQ ID NO: 25.
[0011] Compositions of the invention provide an isolated
polynucleotide including a nucleotide sequence of SEQ ID NO: 21; a
fragment of this nucleotide sequence, provided that the fragment
includes a polymorphic site in the polymorphic sequence; a
complimentary nucleotide sequence comprising a sequence
complementary to SEQ ID NO: 21; and a fragment of the complementary
nucleotide sequence, provided that the fragment includes a
polymorphic site in the polymorphic sequence.
[0012] Compositions of the invention provide an isolated
polynucleotide including a nucleotide sequence of SEQ ID NO: 26; a
fragment of this nucleotide sequence, provided that the fragment
includes a polymorphic site in the polymorphic sequence; a
complimentary nucleotide sequence comprising a sequence
complementary to SEQ ID NO: 26; and a fragment of the complementary
nucleotide sequence, provided that the fragment includes a
polymorphic site in the polymorphic sequence.
[0013] Compositions of the invention provide an isolated
polynucleotide including a nucleotide sequence of SEQ ID NO: 27; a
fragment of this nucleotide sequence, provided that the fragment
includes a polymorphic site in the polymorphic sequence; a
complimentary nucleotide sequence comprising a sequence
complementary to SEQ ID NO: 27; and a fragment of the complementary
nucleotide sequence, provided that the fragment includes a
polymorphic site in the polymorphic sequence.
[0014] The invention further encompasses a method of detecting the
LCS6 SNP. The identity of the polymorphism can be determined by
amplifying a target region containing the polymorphic site directly
from one or both copies of the KRAS gene, or a fragment thereof.
The sequence of the amplified region can be determined by
conventional methods. The polymorphism may be identified directly,
known as positive-type identification, or by inference, referred to
as negative-type identification.
[0015] The target region(s) may be amplified using any
oligonucleotide-directed amplification method, including but not
limited to polymerase chain reaction (PCR), ligase chain reaction
(LCR), and oligonucleotide ligation assay (OLA). Other known
nucleic acid amplification procedures may be used to amplify the
target region including transcription-based amplification systems
and isothermal methods.
[0016] In a specific embodiment, the invention provides a method of
detecting the LCS6 SNP in a KRAS polynucleotide by obtaining a
sample of KRAS polynucleotide; amplifying the KRAS polynucleotide
sample by polymerase chain reaction (PCR); digesting the PCR
product with one or more restriction enzyme(s); separating these
fragments by gel electrophoresis; and comparing the pattern of
fragment migration of the polynucleotide sample to a control
sample, wherein any change from the control pattern indicates the
presence of a SNP in the polynucleotide. In specific embodiments,
the control sample contains SEQ ID NO: 15.
[0017] The invention provides a method of identifying a mutation
within a let-7 Complementary Site (LCS) of a test polynucleotide by
contacting the test polynucleotide to a let-7 family miRNA
molecule; assessing the binding efficacy of the let-7 family miRNA
molecule to the test polynucleotide; and comparing the binding
efficacy of the let-7 family miRNA molecule to the test
polynucleotide to the binding efficacy of the let-7 family miRNA
molecule to a control polynucleotide. An alternation in the binding
efficacy to the test polynucleotide compared to the control
polynucleotide indicates the presence of a mutation in the test
polynucleotide.
[0018] The invention provides methods for identifying subjects at
risk for developing cell proliferative diseases by identifying
genetic mutations in miRNA binding sites that predispose an
individual to such disorders. Moreover, the invention provides
methods of predicting the onset of cell proliferative diseases in
subjects carrying these genetic mutations.
[0019] Methods of the invention are used to identify a single
nucleotide polymorphism (SNP) in a let-7 miRNA binding site in the
KRAS 3'UTR that is implicated in a variety of disorders. In a
specific embodiment of the invention, the presence of the SNP is
predictive for development of cell proliferative disorders, such as
cancer. In another embodiment of the invention, the presence of the
SNP is indicative of an increased risk of cancer.
[0020] Additionally, the invention provides methods for the
identification of additional mutations in miRNA binding sites
located in the 3' UTR of target genes, in particular oncogenes and
proto-oncogenes, that are associated with a cell proliferative
disorder, such as cancer, and methods of using identified mutations
within screening programs to assess risk of developing a cell
proliferative disorder.
[0021] Specifically, the invention provides a method of identifying
subjects at increased risk for developing a cell proliferative
disorder. The method comprises obtaining a nucleic acid sample from
a subject; detecting the presence of a mutation in a miRNA binding
site in the 3' UTR of RAS. The presence of the mutation is
indicative of an increased risk of developing a cell proliferative
disorder.
[0022] The invention also provides a method of predicting the onset
of developing cancer in a subject at risk for developing a cell
proliferative disorder. The method includes obtaining a nucleic
acid sample from a subject and detecting the presence of a mutation
in a miRNA binding site in the 3' UTR of RAS. The presence of a
mutation indicates an earlier onset of developing a cell
proliferative disorder
[0023] Mutations of the invention include single nucleotide
polymorphisms (SNPs). Furthermore, exemplary mutations include, but
are not limited to, deletions, insertions, inversions,
substitutions, frameshifts, and recombinations. In one aspect, the
mutation occurs within one or more let-7 complementary sites
(LCSs). In one embodiment, the LCS is LCS6. In another embodiment,
the mutation is a SNP at position 4 of LCS6 (SEQ ID NOs: 15 or 21).
In a preferred embodiment, the mutation is a SNP where the
guanosine triphosphate resides at position 4 of LCS6 (SEQ ID NO:
21). Furthermore, mutations occur within methylated genomic
sequences. Alternatively, or in addition, mutations of the
invention occur within an unmethylated genomic sequence.
[0024] Mutations of the invention modulate the binding efficacy of
at least one miRNA. In a preferred embodiment, the mutation occurs
in an oncogene or a proto-oncogene. In one example, the mutation
results in increased binding of at least one miRNA.
[0025] Cell proliferative disorders of the invention include
cancer, for example, such as all varieties of lung cancer (e.g.,
non-small cell lung (NSCLC) cancer and small cell lung cancer),
ovarian cancer, breast cancer, uterine cancer, head and neck
cancer, pancreatic cancer and colon cancer.
[0026] RAS genes of the invention include HRAS, KRAS, and NRAS.
[0027] In certain preferred embodiments, miRNA molecules of the
invention belong to the let-7 family of miRNA molecules.
[0028] Moreover, the invention provides a method of identifying a
subject at risk for developing a cell proliferative disease by
obtaining a DNA sample from the subject; amplifying one or more
polynucleotides from a subject comprising proto-oncogenes or
oncogenes; sequencing the polynucleotides; comparing the
polynucleotide sequences of the subject to one or more control
sequences; and identifying mutations in the polynucleotide sequence
of a subject that modulate the binding efficacy of at least one
miRNA. Optimum control sequences contain polynucleotide sequences
to which at least one miRNA binds, thereby silencing translation of
the control sequences.
[0029] Furthermore, the invention provides a method of predicting
the occurrence of a cell proliferative disease in a subject by
obtaining a DNA sample from a subject; amplifying one or more
polynucleotides from the subject comprising proto-oncogenes or
oncogenes; sequencing the polynucleotides; comparing the
polynucleotide sequences of the subject to one or more control
sequences; and identifying mutations in the polynucleotide sequence
of the subject that diminish the binding efficacy of at least one
miRNA. The control sequences contain polynucleotide sequences to
which at least one miRNA binds. The number of identified mutations
correlates with an increased probability of developing a cell
proliferative disorder.
[0030] The invention comprises subjects that are human and animal.
Subjects are healthy individuals without any family history of
cancer. Alternatively, or in addition, subjects have developed at
least one cancer. Subjects have a family history of cancer.
Subjects encompassed by the invention are screened for a wide range
of cancers by the instant methods.
[0031] The invention comprises amplification of polynucleotide
sequences. In a preferred embodiment, the amplification step is
accomplished by polymerase chain reaction (PCR). However, all known
amplification methods are contemplated and encompassed by the
invention.
[0032] The invention includes all known endogenous miRNAs, their
sequences, their targets, and the sequences of their complementary
binding sites. As used herein, the term "complementary binding
site" is meant to encompass the sequence within a target mRNA which
is complementary to the miRNA, e.g. the mRNA sequence sufficient or
required for binding the miRNA. In a preferred embodiment, the
endogenous miRNA and/or complimentary binding site belongs to the
let-7 family of miRNA molecules.
[0033] The invention comprises mutations within miRNA complimentary
binding sites. Exemplary mutations include, but are not limited to,
deletions, insertions, inversions, substitutions, frameshifts, or
recombinations. In a preferred embodiment, the mutation is a single
nucleotide polymorphism (SNP). Alternatively, or in addition, the
mutation occurs within a let-7 complementary site (LCS). In a
preferred embodiment, the LCS is LCS6. For example, the mutation is
a SNP at position 4 of LCS6 (SEQ ID NOs: 15 or 21).
[0034] Mutations occur within a sequence encoding a 3' untranslated
region (UTR). Alternatively, or in addition, mutations occur within
a sequence encoding any portion of a mRNA transcript. Mutations of
the invention also occur within areas of DNA modification. For
instance, mutations occur within a methylated genomic sequence.
Alternatively, or in addition, mutations occur within an
unmethylated genomic sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is a schematic drawing showing microRNA (miRNA)
processing and functions.
[0036] FIG. 2 is a schematic drawing showing transcription and
processing of endogenous miRNA genes and precursor molecules.
[0037] FIG. 3 is a schematic representation of role of miRNA
molecules as oncogenes and tumor suppressor genes.
[0038] FIG. 4 is a schematic representation of let-7 miRNA function
in cell cycle regulation showing a wild type and let-7
loss-of-function mutant C. elegans.
[0039] FIG. 5 is a schematic drawing showing putative targets of
the let-7 miRNA.
[0040] FIG. 6A-D are a series of schematic representations showing
the let-7 miRNA complementary binding sites in C. elegans and Homo
sapiens RAS oncogenes.
[0041] FIG. 7 shows photographs of gel electrophoresis showing DNA
expression levels (left panel) and mutant C. elegans (middle and
right panels). Photographs show that overexpression of a let-7 gene
(mir-84, left and right) silences an activating mutation in RAS
that causes bursting from the pseudovulva (middle).
[0042] FIG. 8A-C provides sequence (A), structure (C), and
expression pattern (B) comparisons among members of the let-7
family of miRNAs in C. elegans, D. melanogaster, and H.
sapiens.
[0043] FIG. 9 provides DNA and protein expression data from three
lung cancer patients showing that decreases in let-7 miRNA
expression lead to increases in RAS protein expression in tumor
cells.
[0044] FIG. 10A-G demonstrates that let-7 regulates RAS through its
3'UTR. Panel A shows schematic representation of let-7
complementary sites within the 3'UTRs of H. sapiens NRAS and KRAS.
Panels B and C show the fold repression of RAS by let-7 miRNA and
the fold induction of RAS by silencing let-7 miRNA by RNAi, both in
HeLa cells. Panels D and E show fluorescent images of RAS protein
expression following treatment of HepG2 cells with the let-7 miRNA
or a negative control and the quantification of these images,
respectively. Panels F and G show fluorescent images of RAS protein
expression following treatment of HeLa cells with an inhibitor of
the let-7 miRNA or a negative control inhibitor and the
quantification of these images, respectively.
[0045] FIG. 11 is a table showing exemplary let-7 family miRNAs
located in "fragile regions" of chromosomes often associated with
various cancers.
[0046] FIG. 12 is a schematic representation and explanation of
strategy for sequencing 3' UTRs of KRAS.
[0047] FIG. 13 provides sequence comparisons within the LCS6 region
showing how SNP mutations can alter miRNA alignment and binding
efficacy. LCS6 is drawn 5' to 3'. The variant allele (G) is in
blue. let-7s are drawn 3' to 5'. The left column depicts the
predicted binding of let-7 with the normal LCS6. The right column
depicts the predicted binding of let-7 with the variant LCS6.
[0048] FIG. 14 is a schematic representation of method for rapidly
identifying LCS6 mutation in a DNA sample using restriction enzyme
analysis.
[0049] FIG. 15 is a schematic representation of miRNA complementary
sites within 3'UTRs of NRAS and methods for sequencing these
regions.
[0050] FIG. 16 shows an analysis of LCSs in KRAS 3'UTR with respect
to ability of mutations in these regions to decrease reporter gene
expression.
[0051] FIG. 17A-B depicts a screen shot of Ensembl report
(www.ensembl.org) of SNP in the human KRAS 3'UTR.
[0052] FIG. 18A-B depicts a screen shot of Ensembl report
(www.ensembl.org) of SNP in the human NRAS 3'UTR.
[0053] FIG. 19A-B provides schematic representations of let-7 and
lin-4 miRNA sequences (A), target sequences (B), and functions
(B).
[0054] FIG. 20A-D describe the prevalence of the LCS6 SNP in
primary cancer tissue. (A) Location of the putative LCSs in the
KRAS 3_UTR. LCS1-LCS8 had been previously identified.12 LCSs where
mutations are found are shown in red. The KRAS 3_UTR is 5016 bp,
and the markers are positioned every 1000 bp. (B) The sequence of
LCS6 with either the reference allele or the variant allele (red).
(C) The variant allele in LCS6 was seen in 20.3% of the primary
lung tumors and was present in the adjacent tissue when available.
(D) Representative sequencing traces from a tumor (T) and adjacent,
non-cancerous (A) sample from a patient with the variant allele
(18T and A) and a patient without the variant allele (7C). Solid
arrows point to heterozygosity (T/G) at the fourth nucleotide of
LCS6. The double arrow points to the homozygous T allele.
[0055] FIG. 21 demonstrates the frequency of the reference allele
across the world. Frequency of the reference allele (T) at the SNP
locus was examined in 2433 people. They represented 46 populations
from around the world, which were categorized based on geography:
Africa (blue), Europe, Southwest Asia and Western Siberia (pink),
South central Asia, East Asia and the Pacific (green), and the
Americas (orange). The frequency of the reference allele (T) across
all populations is 97.1% and the frequency of the alternative
allele (G) is 2.9%. The frequency of the G allele in African
populations is 1.9%, in European populations is 7.6%, in Asian
populations is 0.03%, and in Native American populations is 0.03%.
The allele frequencies in the individual populations are in
ALFRED.
[0056] FIG. 22A-C demonstrates the effects of let-7 miRNA silencing
on KRAS wild type LCS6 and the KRAS LCS6 SNP. (A) Luciferase
reporter constructs containing the KRAS 3'UTR. Grey boxes signify
the 3'UTR used in the reporter constructs. A blue star represents
the variant allele in LCS6. (B) Representative graph of luciferase
activity of KRAS wild-type and KRAS mLCS6 (P<0.001) in A549
cells. Triplicate repeats were conducted showing similar results.
(C) Representative graph of luciferase activity of KRAS wild-type
and KRAS mLCS6 that were co-transfected with prelet-7b (Ambion)
(P=0.001) in A549 cells. Luciferase expression values were
normalized to the average luciferase expression value of samples
treated with pre-miR Negative Control #1 (Ambion). Triplicate
repeats were conducted showing similar results.
[0057] FIG. 23A-C demonstrates overexpression of KRAS in a human
cancer patient carrying the LCS6 SNP. (A) KRAS protein levels were
measured in two autopsy samples, one without the variant allele
(Aut1) and one with the variant allele (Aut2). Actin shows similar
loading (below). Levels were analyzed using Quantity One software
from BioRad. (B) let-7a, b, d and g levels in eight tumors with and
eight without the variant allele. Samples were normalized to two
non-cancerous patients, whose let-7 levels were similar. Error bars
represent variation between PCR reactions for each sample. (C) KRAS
levels in a patient without and one with the variant allele higher
KRAS in the tumor harboring the variant allele. Actin is shown
below.
[0058] FIG. 24 shows the effect of the variant allele on proposed
let-7 binding to LCS6. Table comparing the predicted binding
energies of the different human let-7s and the wild type and
alternative LCS6. These values were generated using RNAhybrid. A
mfe represents the change in binding energy if let-7 binds to the
variant LCS6 rather than the reference LCS6. mfe=minimum free
energy. The duplexes predicted by RNAhybrid, corresponding these
mfe values are shown in FIG. 16.
[0059] FIG. 25 shows the prevalence of the LCS6 SNP in ovarian
cancers of higher stage rating. The graph illustrates that a
greater proportion of ovarian cancer cases studied, in which the
LCS6 SNP was present, presented at elevated stages resulting in
worse prognoses for the individuals carrying the LCS6 SNP than for
those who did not carry the LCS6 SNP.
DETAILED DESCRIPTION
[0060] The invention is based upon the unexpected discovery of a
novel SNP in the 3' untranslated region (UTR) of KRAS. More
specifically, the invention is based upon the discovery that the
presence of this novel SNP, referred to herein as the "LCS6 SNP,"
is predictive of the onset, severity, type and/or subtype, and in
certain individuals, the occurrence of additional, or secondary,
cancers that will develop. It was determined that the presence of
the LCS6 SNP is associated with increased risk of developing
cancer, such as, but not limited to smoking-induced non-small cell
lung cancer (NSCLC) and ovarian cancer.
[0061] The invention provides a method of identifying mutations
within mRNA transcripts targeted by tumor suppressor microRNAs that
modulate endogenous miRNA binding efficacy. Specifically, methods
of the invention have been used to identify a novel SNP, the LCS6
SNP, in a let-7 complementary site within the KRAS 3'UTR that leads
to altered KRAS expression.
[0062] The LCS6 SNP was found in 20.3% of single institution
collected lung cancer cases and in 5.8% of the world populations.
The let-7 family-of-microRNAs (miRNAs) are global genetic
regulators important in controlling lung cancer oncogene expression
by binding to the 3'UTRs (untranslated regions) of their target
messenger RNAs (mRNAs).
[0063] SNPs, including the LCS6 SNP, identified using the methods
of the invention can be used to screen individuals at increased
risk of developing cancer. There are 100 million current or
ex-smokers in the United States alone and 1.3 billion smokers
worldwide that would benefit from screening for the LCS6 SNP, as
well as other SNPs identified using methods of the invention, to
help identify those that would benefit from high-level screening
for lung cancer development, to allow identification of early
tumors and increased chance for cure for these patients.
Additionally, some identified cancers could be totally prevented in
SNP carriers, especially those carrying the LCS6 SNP, by minimally
invasive surgeries, such as ovarian cancer. Patients with a cancer
diagnosis should also be tested to help identify those at high risk
for developing additional cancers, as well as to identify families
that should be tested for the LCS6 SNP.
[0064] Currently only 3% of cancers can be attributed to a genetic
cause. The invention comprises methods of identifying SNPs within
mRNA transcripts of oncogenes that inhibit or diminish binding
efficacy of tumor suppressor miRNAs that silence translation of
these transcripts. In one preferred embodiment, method of the
invention are used to identify SNPs that disrupt a miRNA binding
site and are associated with increased risk to numerous cancers.
Because miRNAs are recently discovered global gene regulators, and
their binding region (the 3'UTR) was previously discarded as junk
DNA, the paradigm of miRNA binding site disruption and disease is a
novel and unexplored direction of study.
[0065] MiRNAs are recently identified gene regulators that are at
abnormal levels and implicated in virtually all cancer subtypes
studied (Esquela-Kerscher A. and Slack, F. 2006. Nature Reviews
Cancer 6:259-69). MiRNAs bind to the 3' untranslated regions
(3'UTRs) of their target genes, regions which are evolutionarily
highly conserved, suggesting an important role for these regions in
natural selection. Because miRNAs each regulate hundreds of mRNAs
simultaneously, the potential of cellular transformation resulting
from single miRNA disturbance is high. In particular, the let-7
family of miRNAs is linked to lung cancer: let-7 miRNAs are poorly
expressed in non-small cell lung cancer (NSCLC) (Johnson S. M. et
al. Cell. 2005; 120(5):635-47; and Calin, G. A. et al. PNAS USA
2004; 101(9): 2999-3004); let-7 miRNAs regulate multiple lung
cancer oncogenes, including RAS (Johnson C. et al. Cancer Research.
2007; 67:7713-22); and let-7 miRNAs inhibit growth of lung cancer
cell lines in vitro (Takamizawa J. et al. Cancer Res 2004; 64(11):
3753-6). The role of let-7 disturbance in the initiation of cancer
has been previously undefined.
[0066] The role of miRNA single nucleotide polymorphisms (SNPs) as
they relate to predisposition to disease is just being defined.
Recent evidence has shown that a point mutation identified in
Tourette's syndrome patients in the 3'UTR of SLITRK1 disrupts the
binding of miR-189 (Abelson J. F. Science 2005; 310: 317-20). In
addition, SNPs in miRNAs that are important in cancer have been
identified; mir-125a, which is known to be at altered levels in
breast cancer (Iorio, M. V. et al. Cancer Res 2005; 65(16):7065-70;
Scott, G. K. et al. J. Biol Chem 2007; 282(2): 1479-86), has a
variant allele at a SNP in its coding sequence that decreases its
expression (Duan, R. et al. Hum Mol Genet 2007; 16:1124-31).
Furthermore, there are SNPs in miRNA target sites in human cancer
genes with allele frequencies that vary between cancerous and
normal tissues (Landi, D. et al. DNA and Cell Biology 2007; 0:1-9).
Supporting the potential importance of SNPs in miRNA binding sites
in cancer predisposition was the identification of SNPs in miRNA
binding sites of miRNAs upregulated in papillary thyroid cancer in
the KIT oncogene (He, H. et al. PNAS 2005; 102:19075-80).
Importantly, SNPs in miRNA binding sites that predispose an
individual to a specific cancer type and act as a genetic marker of
cancer risk have not previously been identified.
[0067] To identify SNPs of the invention, the 3' UTRs of known lung
cancer oncogenes were sequenced to evaluate miRNA binding site
abnormalities in lung cancer. The LCS6 SNP was subsequently
identified that is capable of disrupting a miRNA binding site in
20% of lung cancer patients in one of these genes. Experimental
data proves in a case control design that the presence of the LCS6
SNP increased the carrier's risk of developing non-small cell lung
cancer (OR=2.3, 95% Confidence Interval, 1.1-4.6, p<0.02).
Moreover, the methods of the invention have been used to show that
the LCS6 SNP is very prevalent in numerous other cancer types,
including ovarian, breast, head and neck, uterine and pancreas,
demonstrating that the LCS6 SNP is a biomarker of increased cancer
risk for its carriers. Because the LCS6 SNP alters miRNA binding,
it is a target for therapy in its carriers. Further, miRNA binding
site SNPs are used to predict disease risk.
[0068] Specifically, the LCS6 SNP, which comprises a variant allele
in a let-7 complementary site in the KRAS 3'UTR, leads to altered
KRAS expression. The discovery that the LCS6 SNP disrupts miRNA
regulation of a known oncogene, and the ability of the LCS6 SNP to
affect cancer predisposition, creates a new paradigm. The present
invention provides methods for identification of similar SNPs in
all cancer types. This variant allele adds to our knowledge of
genetic markers of increased smoking-induced lung cancer risk,
which enriches screening programs. The invention comprises methods
of screening for increased cancer risk. Furthermore, because the
LCS6 SNP, as all other mutations encompassed by the invention, is
genetically inherited, families with cancer histories should be
screened to evaluate their genetic risk of developing cancer.
Specifically, individuals with the LCS6 SNP having families with
smoking-induced cancer histories should be screened to evaluate
their genetic risk of developing lung cancer.
Single Nucleotide Polymorphisms (SNPs)
[0069] A single nucleotide polymorphism (SNP) is a DNA sequence
variation occurring when a single nucleotide in the genome (or
other shared sequence) differs between members of a species (or
between paired chromosomes in an individual). SNPs may fall within
coding sequences of genes, non-coding regions of genes, or in the
intergenic regions between genes. SNPs within a coding sequence
will not necessarily change the amino acid sequence of the protein
that is produced, due to degeneracy of the genetic code. A SNP
mutation that results in a new DNA sequence that encodes the same
polypeptide sequence is termed synonymous (also referred to as a
silent mutation). Conversely, a SNP mutation that results in a new
DNA sequence that encodes a different polypeptide sequence is
termed non-synonymous. SNPs that are not in protein-coding regions
may still have consequences for gene splicing, transcription factor
binding, or the sequence of non-coding RNA.
[0070] For the methods of the invention, SNPs occurring within
non-coding RNA regions are particularly important because those
regions contain regulatory sequences which are complementary to
miRNA molecules and required for interaction with other regulatory
factors. SNPs occurring within genomic sequences are transcribed
into mRNA transcripts which are targeted by miRNA molecules for
degradation or translational silencing. SNPs occurring within the
3' untranslated region (UTR) of the genomic sequence or mRNA of a
gene are of particular importance to the methods of the
invention.
MicroRNAs
[0071] MicroRNAs (miRNAs) are small, non-coding RNAs, recently
identified genetic regulators that control cell metabolism,
development, cell cycle, cell differentiation and cell death (FIGS.
1 and 2). In addition, miRNAs have been found to be important in
cancer, aging, and other disease states, likely due to their
ability to regulate hundreds of genes targets. One of the first
miRNAs to be identified was let-7 in Caenorhabditis elegans (C.
elegans) (FIG. 19). In let-7 mutants, stem cells fail to exit the
cell cycle and the animals burst through an organ known as the
vulva (FIGS. 4 and 7). let-7 is highly conserved, even in humans
(FIG. 8).
[0072] MiRNAs act by inhibiting translation of messenger RNA (mRNA)
into protein by binding to the 3' untranslated region (UTR) of
their target mRNAs. It has been found that these microRNA binding
sites in 3'UTRs are very highly conserved regions in humans,
suggesting an important role in these regions in natural selection.
The high degree of conservation of the 3'UTR supports the
hypothesis that a disruption of this region would lead to disease.
While not bound by theory, miRNAs inhibit mRNA translation by
either causing mRNA degradation or inhibiting translation itself
(FIG. 1).
[0073] MiRNAs are single-stranded RNA molecules of about 21-23
nucleotides in length. MiRNAs are encoded by endogenous and
exogenous genes that are transcribed from DNA by RNA polymerase II,
however, miRNA are never translated into polypeptide sequences
(FIG. 2). As such, miRNA are considered in the art as "non-coding
RNA." The term "endogenous" gene as used herein is meant to
encompass all genes that naturally occur within the genome of an
individual. The term "exogenous" gene as used herein is meant to
encompass all genes that do not naturally occur within the genome
of an individual. For example, a miRNA could be introduced
exogenously by a virus.
[0074] While not limited by theory, the present invention includes
and is based in part on the understanding that miRNA biogenesis
occurs by the following mechanism. MiRNA are processed from primary
mRNA transcripts, called "pri-miRNA" by the nuclease Drosha and the
double-stranded RNA binding protein DGCR8/Pasha. Once processed,
these transcripts form stem-loop structures referred to as
"pre-miRNA" (FIG. 2). Pre-miRNA are processed one step further by
the endonuclease Dicer, which transforms the double-stranded
pre-miRNA molecules into the single-stranded mature miRNA and
initiates formation of the RNA-induced silencing complex (RISC).
One of the two resulting single-stranded complementary miRNA
strands, the guide strand, is selected by the argonaute protein of
the RISC and incorporated into the RISC, while the other strand,
the anti-guide or passenger strand, is degraded. Following
integration into the RISC, miRNAs bind target mRNAs and
subsequently inhibit translation.
[0075] MiRNAs are complementary to a part of one or more mRNAs.
Moreover, miRNAs do not require absolute sequence complementarity
to bind an mRNA, enabling them to regulate a wide range of target
transcripts. In particular, miRNAs are frequently complementary to
the 3' UTR of the mRNA transcript. Alternatively, or in addition,
miRNAs also target methylation genomic sites which may correspond
to genes encoding targeted mRNAs. The methylation state of genomic
DNA in part determines the accessibility of that DNA to
transcription factors. As such, DNA methylation and de-methylation
regulate gene silencing and expression, respectively.
Oncogenic and Tumor Suppressor MiRNAs
[0076] MiRNAs that silence expression of tumor suppressor genes are
oncogenes. Alternatively, miRNAs are tumor suppressor genes, which
silence the translation of mRNAs transcripts of oncogenes. The term
"oncogene" as used herein is meant to encompass any gene that, when
expressed, directly or indirectly, causes a cell to inappropriately
enter the cell cycle. Exemplary oncogenes include, but are not
limited to, growth factors, transcription factors, regulatory
proteins, e.g. GTPases and receptors, and cell cycle proteins. The
term "proto-oncogene" as used herein is meant to encompass any
gene, that if modified, directly or indirectly, causes a cell to
inappropriately enter the cell cycle. Examples of proto-oncogenes
include, but are not limited to, RAS, WNT, MYC, ERK and TRK. The
term "tumor suppressor gene" as used herein encompasses any gene
that repressed or silenced, leads deregulated cell division and/or
overexpression of a proto-oncogene or oncogene. Exemplary tumor
suppressor genes include, but are not limited to, retinoblastoma
(encoding the Rb protein), TP53 (encoding the p53 protein), PTEN,
APC, and CD95. Tumor suppressor gene products repress genes that
are essential for the continuing of the cell cycle. Effectively, if
these genes are expressed, the cell cycle will not continue,
effectively inhibiting cell division. Tumor suppressor gene
products couple the cell cycle to DNA damage. Thus, these gene
products activate cell cycle checkpoints and DNA repair mechanisms
that stall or prevent cell division. If the damage cannot be
repaired, the cell initiate apoptosis, or programmed cell death.
Some tumor suppressor gene products are involved in cell adhesion,
and thus, prevent tumor cells from dispersing, block loss of
contact inhibition, and inhibit metastasis. These proteins are also
known as metastasis suppressors.
[0077] SNPs within the binding site of a tumor suppressing miRNA
that decrease binding efficacy, and therefore oncogene silencing,
lead to an increased risk, susceptibility or probability of
presenting one or more symptoms of a cell proliferative disease
(FIG. 3). Similarly, SNPs within the binding site of an oncogenic
miRNA that increase binding, and therefore increase gene
repression, lead to an increased risk, susceptibility or
probability of presenting one or more symptoms of a cell
proliferative disease (FIG. 3).
[0078] The invention comprises methods of screening for mutations
within miRNA binding sites that lead to the development of a cell
proliferative disorder. Therefore, methods of the invention
comprise all known tumor suppressor and oncogenic miRNAs. Moreover,
all endogenous human miRNAs are encompassed by the invention, the
names, sequences, and targets of which are provided at the
following database: The Wellcome Trust Sanger Institute MicroRNA
Listing for Homo sapiens, the entirety of which is herein
incorporated by reference.
RAS Gene Superfamily
[0079] The RAS gene encodes for a protein belongs to a larger
superfamily of small GTPases that include the Ras, Rho, Arf, Rab,
and Ran families. Functionally, GTPase proteins are molecular
switches for a wide variety of signal transduction pathways that
control practically every function within a cell. Exemplary
functions regulated by GTPase proteins are cytoskeletal integrity,
cell proliferation, cell adhesion, apoptosis, and cell migration.
Thus, Ras protein deregulated within a cell often leads to
increased cell invasion, metastasis, and decreased apoptosis.
Importantly, Ras protein is attached to the cell membrane by
prenylation and couples growth factor receptors to downstream
mitogenic effectors involved in cell proliferation or
differentiation.
[0080] There are three human RAS genes comprising HRAS, KRAS, and
NRAS. Each gene comprises multiple miRNA complementary sites in the
3'UTR of their mRNA transcripts. Specifically, each human RAS gene
comprises multiple let-7 complementary sites (LCSs).
[0081] Importantly, KRAS is capable of acting as either a tumor
suppressor gene, a proto-oncogene, or an oncogene. SNPs in the
3'UTR of KRAS may lead to either increased or decreased binding
efficacy of miRNAs. In one embodiment, KRAS acts as a
proto-oncogene or oncogene, the SNP decreases the binding efficacy
of at least one miRNA, causing expressing of the oncogene to be
augmented, and the SNP is a marker of cell proliferative disease.
In another embodiment, KRAS acts as a tumor suppressor gene, the
SNP increases the binding efficacy of at least one miRNA, causing
expression of the tumor suppressor gene to be repressed, and the
SNP is a marker of cell proliferative disease. In either scenario,
subjects who carry this marker are identified as having a greater
risk of developing a cell proliferative disorder. Alternatively, or
in addition, the occurrence of this SNP is predictive of the
occurrence of a cell proliferative disorder.
[0082] The present invention comprises SNPs within any region of a
human RAS family gene. In a preferred embodiment, SNPs occur within
the 3' UTR of a RAS family gene. In another preferred embodiment,
SNPs occur within the 3'UTR of KRAS. Exemplary human RAS sequences
are included below, however, all known human RAS sequences are
encompassed by the invention.
[0083] Human HRAS, transcript variant 1, is encoded by the
following mRNA sequence (NCBI Accession No. NM.sub.--005343 and SEQ
ID NO: 22) (untranslated regions are bolded):
TABLE-US-00001 1 tgccctgcgc ccgcaacccg agccgcaccc gccgcggacg
gagcccatgc gcggggcgaa 61 ccgcgcgccc ccgcccccgc cccgccccgg
cctcggcccc ggccctggcc ccgggggcag 121 tcgcgcctgt gaacggtggg
gcaggagacc ctgtaggagg accccgggcc gcaggcccct 181 gaggagcgat
gacggaatat aagctggtgg tggtgggcgc cggcggtgtg ggcaagagtg 241
cgctgaccat ccagctgatc cagaaccatt ttgtggacga atacgacccc actatagagg
301 attcctaccg gaagcaggtg gtcattgatg gggagacgtg cctgttggac
atcctggata 361 ccgccggcca ggaggagtac agcgccatgc gggaccagta
catgcgcacc ggggagggct 421 tcctgtgtgt gtttgccatc aacaacacca
agtcttttga ggacatccac cagtacaggg 481 agcagatcaa acgggtgaag
gactcggatg acgtgcccat ggtgctggtg gggaacaagt 541 gtgacctggc
tgcacgcact gtggaatctc ggcaggctca ggacctcgcc cgaagctacg 601
gcatccccta catcgagacc tcggccaaga cccggcaggg agtggaggat gccttctaca
661 cgttggtgcg tgagatccgg cagcacaagc tgcggaagct gaaccctcct
gatgagagtg 721 gccccggctg catgagctgc aagtgtgtgc tctcctgacg
cagcacaagc tcaggacatg 781 gaggtgccgg atgcaggaag gaggtgcaga
cggaaggagg aggaaggaag gacggaagca 841 aggaaggaag gaagggctgc
tggagcccag tcaccccggg accgtgggcc gaggtgactg 901 cagaccctcc
cagggaggct gtgcacagac tgtcttgaac atcccaaatg ccaccggaac 961
cccagccctt agctcccctc ccaggcctct gtgggccctt gtcgggcaca gatgggatca
1021 cagtaaatta ttggatggtc ttgaaaaaaa aaaaaaaaaa a
[0084] Human HRAS, transcript variant 2, is encoded by the
following mRNA sequence (NCBI Accession No. NM.sub.--176795 and SEQ
ID NO: 23) (untranslated regions are bolded):
TABLE-US-00002 1 tgccctgcgc ccgcaacccg agccgcaccc gccgcggacg
gagcccatgc gcggggcgaa 61 ccgcgcgccc ccgcccccgc cccgccccgg
cctcggcccc ggccctggcc ccgggggcag 121 tcgcgcctgt gaacggtggg
gcaggagacc ctgtaggagg accccgggcc gcaggcccct 181 gaggagcgat
gacggaatat aagctggtgg tggtgggcgc cggcggtgtg ggcaagagtg 241
cgctgaccat ccagctgatc cagaaccatt ttgtggacga atacgacccc actatagagg
301 attcctaccg gaagcaggtg gtcattgatg gggagacgtg cctgttggac
atcctggata 361 ccgccggcca ggaggagtac agcgccatgc gggaccagta
catgcgcacc ggggagggct 421 tcctgtgtgt gtttgccatc aacaacacca
agtcttttga ggacatccac cagtacaggg 481 agcagatcaa acgggtgaag
gactcggatg acgtgcccat ggtgctggtg gggaacaagt 541 gtgacctggc
tgcacgcact gtggaatctc ggcaggctca ggacctcgcc cgaagctacg 601
gcatccccta catcgagacc tcggccaaga cccggcaggg cagccgctct ggctctagct
661 ccagctccgg gaccctctgg gaccccccgg gacccatgtg acccagcggc
ccctcgcgct 721 ggagtggagg atgccttcta cacgttggtg cgtgagatcc
ggcagcacaa gctgcggaag 781 ctgaaccctc ctgatgagag tggccccggc
tgcatgagct gcaagtgtgt gctctcctga 841 cgcagcacaa gctcaggaca
tggaggtgcc ggatgcagga aggaggtgca gacggaagga 901 ggaggaagga
aggacggaag caaggaagga aggaagggct gctggagccc agtcaccccg 961
ggaccgtggg ccgaggtgac tgcagaccct cccagggagg ctgtgcacag actgtcttga
1021 acatcccaaa tgccaccgga accccagccc ttagctcccc tcccaggcct
ctgtgggccc 1081 ttgtcgggca cagatgggat cacagtaaat tattggatgg
tcttgaaaaa aaaaaaaaaa 1141 aaa
[0085] Human KRAS, transcript variant a, is encoded by the
following mRNA sequence (NCBI Accession No. NM.sub.--033360 and SEQ
ID NO: 24) (untranslated regions are bolded, LCS6 is
underlined):
TABLE-US-00003 1 ggccgcggcg gcggaggcag cagcggcggc ggcagtggcg
gcggcgaagg tggcggcggc 61 tcggccagta ctcccggccc ccgccatttc
ggactgggag cgagcgcggc gcaggcactg 121 aaggcggcgg cggggccaga
ggctcagcgg ctcccaggtg cgggagagag gcctgctgaa 181 aatgactgaa
tataaacttg tggtagttgg agctggtggc gtaggcaaga gtgccttgac 241
gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag aggattccta
301 caggaagcaa gtagtaattg atggagaaac ctgtctcttg gatattctcg
acacagcagg 361 tcaagaggag tacagtgcaa tgagggacca gtacatgagg
actggggagg gctttctttg 421 tgtatttgcc ataaataata ctaaatcatt
tgaagatatt caccattata gagaacaaat 481 taaaagagtt aaggactctg
aagatgtacc tatggtccta gtaggaaata aatgtgattt 541 gccttctaga
acagtagaca caaaacaggc tcaggactta gcaagaagtt atggaattcc 601
ttttattgaa acatcagcaa agacaagaca gagagtggag gatgcttttt atacattggt
661 gagggagatc cgacaataca gattgaaaaa aatcagcaaa gaagaaaaga
ctcctggctg 721 tgtgaaaatt aaaaaatgca ttataatgta atctgggtgt
tgatgatgcc ttctatacat 781 tagttcgaga aattcgaaaa cataaagaaa
agatgagcaa agatggtaaa aagaagaaaa 841 agaagtcaaa gacaaagtgt
gtaattatgt aaatacaatt tgtacttttt tcttaaggca 901 tactagtaca
agtggtaatt tttgtacatt acactaaatt attagcattt gttttagcat 961
tacctaattt ttttcctgct ccatgcagac tgttagcttt taccttaaat gcttatttta
1021 aaatgacagt ggaagttttt ttttcctcta agtgccagta ttcccagagt
tttggttttt 1081 gaactagcaa tgcctgtgaa aaagaaactg aatacctaag
atttctgtct tggggttttt 1141 ggtgcatgca gttgattact tcttattttt
cttaccaatt gtgaatgttg gtgtgaaaca 1201 aattaatgaa gcttttgaat
catccctatt ctgtgtttta tctagtcaca taaatggatt 1261 aattactaat
ttcagttgag accttctaat tggtttttac tgaaacattg agggaacaca 1321
aatttatggg cttcctgatg atgattcttc taggcatcat gtcctatagt ttgtcatccc
1381 tgatgaatgt aaagttacac tgttcacaaa ggttttgtct cctttccact
gctattagtc 1441 atggtcactc tccccaaaat attatatttt ttctataaaa
agaaaaaaat ggaaaaaaat 1501 tacaaggcaa tggaaactat tataaggcca
tttccttttc acattagata aattactata 1561 aagactccta atagcttttc
ctgttaaggc agacccagta tgaaatgggg attattatag 1621 caaccatttt
ggggctatat ttacatgcta ctaaattttt ataataattg aaaagatttt 1681
aacaagtata aaaaattctc ataggaatta aatgtagtct ccctgtgtca gactgctctt
1741 tcatagtata actttaaatc ttttcttcaa cttgagtctt tgaagatagt
tttaattctg 1801 cttgtgacat taaaagatta tttgggccag ttatagctta
ttaggtgttg aagagaccaa 1861 ggttgcaagg ccaggccctg tgtgaacctt
tgagctttca tagagagttt cacagcatgg 1921 actgtgtccc cacggtcatc
cagtgttgtc atgcattggt tagtcaaaat ggggagggac 1981 tagggcagtt
tggatagctc aacaagatac aatctcactc tgtggtggtc ctgctgacaa 2041
atcaagagca ttgcttttgt ttcttaagaa aacaaactct tttttaaaaa ttacttttaa
2101 atattaactc aaaagttgag attttggggt ggtggtgtgc caagacatta
attttttttt 2161 taaacaatga agtgaaaaag ttttacaatc tctaggtttg
gctagttctc ttaacactgg 2221 ttaaattaac attgcataaa cacttttcaa
gtctgatcca tatttaataa tgctttaaaa 2281 taaaaataaa aacaatcctt
ttgataaatt taaaatgtta cttattttaa aataaatgaa 2341 gtgagatggc
atggtgaggt gaaagtatca ctggactagg aagaaggtga cttaggttct 2401
agataggtgt cttttaggac tctgattttg aggacatcac ttactatcca tttcttcatg
2461 ttaaaagaag tcatctcaaa ctcttagttt ttttttttta caactatgta
atttatattc 2521 catttacata aggatacact tatttgtcaa gctcagcaca
atctgtaaat ttttaaccta 2581 tgttacacca tcttcagtgc cagtcttggg
caaaattgtg caagaggtga agtttatatt 2641 tgaatatcca ttctcgtttt
aggactcttc ttccatatta gtgtcatctt gcctccctac 2701 cttccacatg
ccccatgact tgatgcagtt ttaatacttg taattcccct aaccataaga 2761
tttactgctg ctgtggatat ctccatgaag ttttcccact gagtcacatc agaaatgccc
2821 tacatcttat ttcctcaggg ctcaagagaa tctgacagat accataaagg
gatttgacct 2881 aatcactaat tttcaggtgg tggctgatgc tttgaacatc
tctttgctgc ccaatccatt 2941 agcgacagta ggatttttca aacctggtat
gaatagacag aaccctatcc agtggaagga 3001 gaatttaata aagatagtgc
tgaaagaatt ccttaggtaa tctataacta ggactactcc 3061 tggtaacagt
aatacattcc attgttttag taaccagaaa tcttcatgca atgaaaaata 3121
ctttaattca tgaagcttac tttttttttt tggtgtcaga gtctcgctct tgtcacccag
3181 gctggaatgc agtggcgcca tctcagctca ctgcaacctc catctcccag
gttcaagcga 3241 ttctcgtgcc tcggcctcct gagtagctgg gattacaggc
gtgtgccact acactcaact 3301 aatttttgta tttttaggag agacggggtt
tcaccctgtt ggccaggctg gtctcgaact 3361 cctgacctca agtgattcac
ccaccttggc ctcataaacc tgttttgcag aactcattta 3421 ttcagcaaat
atttattgag tgcctaccag atgccagtca ccgcacaagg cactgggtat 3481
atggtatccc caaacaagag acataatccc ggtccttagg tagtgctagt gtggtctgta
3541 atatcttact aaggcctttg gtatacgacc cagagataac acgatgcgta
ttttagtttt 3601 gcaaagaagg ggtttggtct ctgtgccagc tctataattg
ttttgctacg attccactga 3661 aactcttcga tcaagctact ttatgtaaat
cacttcattg ttttaaagga ataaacttga 3721 ttatattgtt tttttatttg
gcataactgt gattctttta ggacaattac tgtacacatt 3781 aaggtgtatg
tcagatattc atattgaccc aaatgtgtaa tattccagtt ttctctgcat 3841
aagtaattaa aatatactta aaaattaata gttttatctg ggtacaaata aacaggtgcc
3901 tgaactagtt cacagacaag gaaacttcta tgtaaaaatc actatgattt
ctgaattgct 3961 atgtgaaact acagatcttt ggaacactgt ttaggtaggg
tgttaagact tacacagtac 4021 ctcgtttcta cacagagaaa gaaatggcca
tacttcagga actgcagtgc ttatgagggg 4081 atatttaggc ctcttgaatt
tttgatgtag atgggcattt ttttaaggta gtggttaatt 4141 acctttatgt
gaactttgaa tggtttaaca aaagatttgt ttttgtagag attttaaagg 4201
gggagaattc tagaaataaa tgttacctaa ttattacagc cttaaagaca aaaatccttg
4261 ttgaagtttt tttaaaaaaa gctaaattac atagacttag gcattaacat
gtttgtggaa 4321 gaatatagca gacgtatatt gtatcatttg agtgaatgtt
cccaagtagg cattctaggc 4381 tctatttaac tgagtcacac tgcataggaa
tttagaacct aacttttata ggttatcaaa 4441 actgttgtca ccattgcaca
attttgtcct aatatataca tagaaacttt gtggggcatg 4501 ttaagttaca
gtttgcacaa gttcatctca tttgtattcc attgattttt tttttcttct 4561
aaacattttt tcttcaaaca gtatataact ttttttaggg gatttttttt tagacagcaa
4621 aaactatctg aagatttcca tttgtcaaaa agtaatgatt tcttgataat
tgtgtagtaa 4681 tgttttttag aacccagcag ttaccttaaa gctgaattta
tatttagtaa cttctgtgtt 4741 aatactggat agcatgaatt ctgcattgag
aaactgaata gctgtcataa aatgaaactt 4801 tctttctaaa gaaagatact
cacatgagtt cttgaagaat agtcataact agattaagat 4861 ctgtgtttta
gtttaatagt ttgaagtgcc tgtttgggat aatgataggt aatttagatg 4921
aatttagggg aaaaaaaagt tatctgcaga tatgttgagg gcccatctct ccccccacac
4981 ccccacagag ctaactgggt tacagtgttt tatccgaaag tttccaattc
cactgtcttg 5041 tgttttcatg ttgaaaatac ttttgcattt ttcctttgag
tgccaatttc ttactagtac 5101 tatttcttaa tgtaacatgt ttacctggaa
tgtattttaa ctatttttgt atagtgtaaa 5161 ctgaaacatg cacattttgt
acattgtgct ttcttttgtg ggacatatgc agtgtgatcc 5221 agttgttttc
catcatttgg ttgcgctgac ctaggaatgt tggtcatatc aaacattaaa 5281
aatgaccact cttttaattg aaattaactt ttaaatgttt ataggagtat gtgctgtgaa
5341 gtgatctaaa atttgtaata tttttgtcat gaactgtact actcctaatt
attgtaatgt 5401 aataaaaata gttacagtga caaaaaaaaa aaaaaa
[0086] Human KRAS, transcript variant b, is encoded by the
following mRNA sequence (NCBI Accession No. NM.sub.--004985 and SEQ
ID NO: 25) (untranslated regions are bolded, LCS6 is
underlined):
TABLE-US-00004 1 ggccgcggcg gcggaggcag cagcggcggc ggcagtggcg
gcggcgaagg tggcggcggc 61 tcggccagta ctcccggccc ccgccatttc
ggactgggag cgagcgcggc gcaggcactg 121 aaggcggcgg cggggccaga
ggctcagcgg ctcccaggtg cgggagagag gcctgctgaa 181 aatgactgaa
tataaacttg tggtagttgg agctggtggc gtaggcaaga gtgccttgac 241
gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag aggattccta
301 caggaagcaa gtagtaattg atggagaaac ctgtctcttg gatattctcg
acacagcagg 361 tcaagaggag tacagtgcaa tgagggacca gtacatgagg
actggggagg gctttctttg 421 tgtatttgcc ataaataata ctaaatcatt
tgaagatatt caccattata gagaacaaat 481 taaaagagtt aaggactctg
aagatgtacc tatggtccta gtaggaaata aatgtgattt 541 gccttctaga
acagtagaca caaaacaggc tcaggactta gcaagaagtt atggaattcc 601
ttttattgaa acatcagcaa agacaagaca gggtgttgat gatgccttct atacattagt
661 tcgagaaatt cgaaaacata aagaaaagat gagcaaagat ggtaaaaaga
agaaaaagaa 721 gtcaaagaca aagtgtgtaa ttatgtaaat acaatttgta
cttttttctt aaggcatact 781 agtacaagtg gtaatttttg tacattacac
taaattatta gcatttgttt tagcattacc 841 taattttttt cctgctccat
gcagactgtt agcttttacc ttaaatgctt attttaaaat 901 gacagtggaa
gttttttttt cctctaagtg ccagtattcc cagagttttg gtttttgaac 961
tagcaatgcc tgtgaaaaag aaactgaata cctaagattt ctgtcttggg gtttttggtg
1021 catgcagttg attacttctt atttttctta ccaattgtga atgttggtgt
gaaacaaatt 1081 aatgaagctt ttgaatcatc cctattctgt gttttatcta
gtcacataaa tggattaatt 1141 actaatttca gttgagacct tctaattggt
ttttactgaa acattgaggg aacacaaatt 1201 tatgggcttc ctgatgatga
ttcttctagg catcatgtcc tatagtttgt catccctgat 1261 gaatgtaaag
ttacactgtt cacaaaggtt ttgtctcctt tccactgcta ttagtcatgg 1321
tcactctccc caaaatatta tattttttct ataaaaagaa aaaaatggaa aaaaattaca
1381 aggcaatgga aactattata aggccatttc cttttcacat tagataaatt
actataaaga 1441 ctcctaatag cttttcctgt taaggcagac ccagtatgaa
atggggatta ttatagcaac 1501 cattttgggg ctatatttac atgctactaa
atttttataa taattgaaaa gattttaaca 1561 agtataaaaa attctcatag
gaattaaatg tagtctccct gtgtcagact gctctttcat 1621 agtataactt
taaatctttt cttcaacttg agtctttgaa gatagtttta attctgcttg 1681
tgacattaaa agattatttg ggccagttat agcttattag gtgttgaaga gaccaaggtt
1741 gcaaggccag gccctgtgtg aacctttgag ctttcataga gagtttcaca
gcatggactg 1801 tgtccccacg gtcatccagt gttgtcatgc attggttagt
caaaatgggg agggactagg 1861 gcagtttgga tagctcaaca agatacaatc
tcactctgtg gtggtcctgc tgacaaatca 1921 agagcattgc ttttgtttct
taagaaaaca aactcttttt taaaaattac ttttaaatat 1981 taactcaaaa
gttgagattt tggggtggtg gtgtgccaag acattaattt tttttttaaa 2041
caatgaagtg aaaaagtttt acaatctcta ggtttggcta gttctcttaa cactggttaa
2101 attaacattg cataaacact tttcaagtct gatccatatt taataatgct
ttaaaataaa 2161 aataaaaaca atccttttga taaatttaaa atgttactta
ttttaaaata aatgaagtga 2221 gatggcatgg tgaggtgaaa gtatcactgg
actaggaaga aggtgactta ggttctagat 2281 aggtgtcttt taggactctg
attttgagga catcacttac tatccatttc ttcatgttaa 2341 aagaagtcat
ctcaaactct tagttttttt tttttacaac tatgtaattt atattccatt 2401
tacataagga tacacttatt tgtcaagctc agcacaatct gtaaattttt aacctatgtt
2461 acaccatctt cagtgccagt cttgggcaaa attgtgcaag aggtgaagtt
tatatttgaa 2521 tatccattct cgttttagga ctcttcttcc atattagtgt
catcttgcct ccctaccttc 2581 cacatgcccc atgacttgat gcagttttaa
tacttgtaat tcccctaacc ataagattta 2641 ctgctgctgt ggatatctcc
atgaagtttt cccactgagt cacatcagaa atgccctaca 2701 tcttatttcc
tcagggctca agagaatctg acagatacca taaagggatt tgacctaatc 2761
actaattttc aggtggtggc tgatgctttg aacatctctt tgctgcccaa tccattagcg
2821 acagtaggat ttttcaaacc tggtatgaat agacagaacc ctatccagtg
gaaggagaat 2881 ttaataaaga tagtgctgaa agaattcctt aggtaatcta
taactaggac tactcctggt 2941 aacagtaata cattccattg ttttagtaac
cagaaatctt catgcaatga aaaatacttt 3001 aattcatgaa gcttactttt
tttttttggt gtcagagtct cgctcttgtc acccaggctg 3061 gaatgcagtg
gcgccatctc agctcactgc aacctccatc tcccaggttc aagcgattct 3121
cgtgcctcgg cctcctgagt agctgggatt acaggcgtgt gccactacac tcaactaatt
3181 tttgtatttt taggagagac ggggtttcac cctgttggcc aggctggtct
cgaactcctg 3241 acctcaagtg attcacccac cttggcctca taaacctgtt
ttgcagaact catttattca 3301 gcaaatattt attgagtgcc taccagatgc
cagtcaccgc acaaggcact gggtatatgg 3361 tatccccaaa caagagacat
aatcccggtc cttaggtagt gctagtgtgg tctgtaatat 3421 cttactaagg
cctttggtat acgacccaga gataacacga tgcgtatttt agttttgcaa 3481
agaaggggtt tggtctctgt gccagctcta taattgtttt gctacgattc cactgaaact
3541 cttcgatcaa gctactttat gtaaatcact tcattgtttt aaaggaataa
acttgattat 3601 attgtttttt tatttggcat aactgtgatt cttttaggac
aattactgta cacattaagg 3661 tgtatgtcag atattcatat tgacccaaat
gtgtaatatt ccagttttct ctgcataagt 3721 aattaaaata tacttaaaaa
ttaatagttt tatctgggta caaataaaca ggtgcctgaa 3781 ctagttcaca
gacaaggaaa cttctatgta aaaatcacta tgatttctga attgctatgt 3841
gaaactacag atctttggaa cactgtttag gtagggtgtt aagacttaca cagtacctcg
3901 tttctacaca gagaaagaaa tggccatact tcaggaactg cagtgcttat
gaggggatat 3961 ttaggcctct tgaatttttg atgtagatgg gcattttttt
aaggtagtgg ttaattacct 4021 ttatgtgaac tttgaatggt ttaacaaaag
atttgttttt gtagagattt taaaggggga 4081 gaattctaga aataaatgtt
acctaattat tacagcctta aagacaaaaa tccttgttga 4141 agttttttta
aaaaaagcta aattacatag acttaggcat taacatgttt gtggaagaat 4201
atagcagacg tatattgtat catttgagtg aatgttccca agtaggcatt ctaggctcta
4261 tttaactgag tcacactgca taggaattta gaacctaact tttataggtt
atcaaaactg 4321 ttgtcaccat tgcacaattt tgtcctaata tatacataga
aactttgtgg ggcatgttaa 4381 gttacagttt gcacaagttc atctcatttg
tattccattg attttttttt tcttctaaac 4441 attttttctt caaacagtat
ataacttttt ttaggggatt tttttttaga cagcaaaaac 4501 tatctgaaga
tttccatttg tcaaaaagta atgatttctt gataattgtg tagtaatgtt 4561
ttttagaacc cagcagttac cttaaagctg aatttatatt tagtaacttc tgtgttaata
4621 ctggatagca tgaattctgc attgagaaac tgaatagctg tcataaaatg
aaactttctt 4681 tctaaagaaa gatactcaca tgagttcttg aagaatagtc
ataactagat taagatctgt 4741 gttttagttt aatagtttga agtgcctgtt
tgggataatg ataggtaatt tagatgaatt 4801 taggggaaaa aaaagttatc
tgcagatatg ttgagggccc atctctcccc ccacaccccc 4861 acagagctaa
ctgggttaca gtgttttatc cgaaagtttc caattccact gtcttgtgtt 4921
ttcatgttga aaatactttt gcatttttcc tttgagtgcc aatttcttac tagtactatt
4981 tcttaatgta acatgtttac ctggaatgta ttttaactat ttttgtatag
tgtaaactga 5041 aacatgcaca ttttgtacat tgtgctttct tttgtgggac
atatgcagtg tgatccagtt 5101 gttttccatc atttggttgc gctgacctag
gaatgttggt catatcaaac attaaaaatg 5161 accactcttt taattgaaat
taacttttaa atgtttatag gagtatgtgc tgtgaagtga 5221 tctaaaattt
gtaatatttt tgtcatgaac tgtactactc ctaattattg taatgtaata 5281
aaaatagtta cagtgacaaa aaaaaaaaaa aa
[0087] Human KRAS, transcript variant a, comprising the LCS6 SNP,
is encoded by the following mRNA sequence (SEQ ID NO: 26)
(untranslated regions are bolded, LCS6 is underlined, SNP is
capitalized):
TABLE-US-00005 1 ggccgcggcg gcggaggcag cagcggcggc ggcagtggcg
gcggcgaagg tggcggcggc 61 tcggccagta ctcccggccc ccgccatttc
ggactgggag cgagcgcggc gcaggcactg 121 aaggcggcgg cggggccaga
ggctcagcgg ctcccaggtg cgggagagag gcctgctgaa 181 aatgactgaa
tataaacttg tggtagttgg agctggtggc gtaggcaaga gtgccttgac 241
gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag aggattccta
301 caggaagcaa gtagtaattg atggagaaac ctgtctcttg gatattctcg
acacagcagg 361 tcaagaggag tacagtgcaa tgagggacca gtacatgagg
actggggagg gctttctttg 421 tgtatttgcc ataaataata ctaaatcatt
tgaagatatt caccattata gagaacaaat 481 taaaagagtt aaggactctg
aagatgtacc tatggtccta gtaggaaata aatgtgattt 541 gccttctaga
acagtagaca caaaacaggc tcaggactta gcaagaagtt atggaattcc 601
ttttattgaa acatcagcaa agacaagaca gagagtggag gatgcttttt atacattggt
661 gagggagatc cgacaataca gattgaaaaa aatcagcaaa gaagaaaaga
ctcctggctg 721 tgtgaaaatt aaaaaatgca ttataatgta atctgggtgt
tgatgatgcc ttctatacat 781 tagttcgaga aattcgaaaa cataaagaaa
agatgagcaa agatggtaaa aagaagaaaa 841 agaagtcaaa gacaaagtgt
gtaattatgt aaatacaatt tgtacttttt tcttaaggca 901 tactagtaca
agtggtaatt tttgtacatt acactaaatt attagcattt gttttagcat 961
tacctaattt ttttcctgct ccatgcagac tgttagcttt taccttaaat gcttatttta
1021 aaatgacagt ggaagttttt ttttcctcta agtgccagta ttcccagagt
tttggttttt 1081 gaactagcaa tgcctgtgaa aaagaaactg aatacctaag
atttctgtct tggggttttt 1141 ggtgcatgca gttgattact tcttattttt
cttaccaatt gtgaatgttg gtgtgaaaca 1201 aattaatgaa gcttttgaat
catccctatt ctgtgtttta tctagtcaca taaatggatt 1261 aattactaat
ttcagttgag accttctaat tggtttttac tgaaacattg agggaacaca 1321
aatttatggg cttcctgatg atgattcttc taggcatcat gtcctatagt ttgtcatccc
1381 tgatgaatgt aaagttacac tgttcacaaa ggttttgtct cctttccact
gctattagtc 1441 atggtcactc tccccaaaat attatatttt ttctataaaa
agaaaaaaat ggaaaaaaat 1501 tacaaggcaa tggaaactat tataaggcca
tttccttttc acattagata aattactata 1561 aagactccta atagcttttc
ctgttaaggc agacccagta tgaaatgggg attattatag 1621 caaccatttt
ggggctatat ttacatgcta ctaaattttt ataataattg aaaagatttt 1681
aacaagtata aaaaattctc ataggaatta aatgtagtct ccctgtgtca gactgctctt
1741 tcatagtata actttaaatc ttttcttcaa cttgagtctt tgaagatagt
tttaattctg 1801 cttgtgacat taaaagatta tttgggccag ttatagctta
ttaggtgttg aagagaccaa 1861 ggttgcaagg ccaggccctg tgtgaacctt
tgagctttca tagagagttt cacagcatgg 1921 actgtgtccc cacggtcatc
cagtgttgtc atgcattggt tagtcaaaat ggggagggac 1981 tagggcagtt
tggatagctc aacaagatac aatctcactc tgtggtggtc ctgctgacaa 2041
atcaagagca ttgcttttgt ttcttaagaa aacaaactct tttttaaaaa ttacttttaa
2101 atattaactc aaaagttgag attttggggt ggtggtgtgc caagacatta
attttttttt 2161 taaacaatga agtgaaaaag ttttacaatc tctaggtttg
gctagttctc ttaacactgg 2221 ttaaattaac attgcataaa cacttttcaa
gtctgatcca tatttaataa tgctttaaaa 2281 taaaaataaa aacaatcctt
ttgataaatt taaaatgtta cttattttaa aataaatgaa 2341 gtgagatggc
atggtgaggt gaaagtatca ctggactagg aagaaggtga cttaggttct 2401
agataggtgt cttttaggac tctgattttg aggacatcac ttactatcca tttcttcatg
2461 ttaaaagaag tcatctcaaa ctcttagttt ttttttttta caactatgta
atttatattc 2521 catttacata aggatacact tatttgtcaa gctcagcaca
atctgtaaat ttttaaccta 2581 tgttacacca tcttcagtgc cagtcttggg
caaaattgtg caagaggtga agtttatatt 2641 tgaatatcca ttctcgtttt
aggactcttc ttccatatta gtgtcatctt gcctccctac 2701 cttccacatg
ccccatgact tgatgcagtt ttaatacttg taattcccct aaccataaga 2761
tttactgctg ctgtggatat ctccatgaag ttttcccact gagtcacatc agaaatgccc
2821 tacatcttat ttcctcaggg ctcaagagaa tctgacagat accataaagg
gatttgacct 2881 aatcactaat tttcaggtgg tggctgatgc tttgaacatc
tctttgctgc ccaatccatt 2941 agcgacagta ggatttttca aacctggtat
gaatagacag aaccctatcc agtggaagga 3001 gaatttaata aagatagtgc
tgaaagaatt ccttaggtaa tctataacta ggactactcc 3061 tggtaacagt
aatacattcc attgttttag taaccagaaa tcttcatgca atgaaaaata 3121
ctttaattca tgaagcttac tttttttttt tggtgtcaga gtctcgctct tgtcacccag
3181 gctggaatgc agtggcgcca tctcagctca ctgcaacctc catctcccag
gttcaagcga 3241 ttctcgtgcc tcggcctcct gagtagctgg gattacaggc
gtgtgccact acactcaact 3301 aatttttgta tttttaggag agacggggtt
tcaccctgtt ggccaggctg gtctcgaact 3361 cctgacctca agtgatGcac
ccaccttggc ctcataaacc tgttttgcag aactcattta 3421 ttcagcaaat
atttattgag tgcctaccag atgccagtca ccgcacaagg cactgggtat 3481
atggtatccc caaacaagag acataatccc ggtccttagg tagtgctagt gtggtctgta
3541 atatcttact aaggcctttg gtatacgacc cagagataac acgatgcgta
ttttagtttt 3601 gcaaagaagg ggtttggtct ctgtgccagc tctataattg
ttttgctacg attccactga 3661 aactcttcga tcaagctact ttatgtaaat
cacttcattg ttttaaagga ataaacttga 3721 ttatattgtt tttttatttg
gcataactgt gattctttta ggacaattac tgtacacatt 3781 aaggtgtatg
tcagatattc atattgaccc aaatgtgtaa tattccagtt ttctctgcat 3841
aagtaattaa aatatactta aaaattaata gttttatctg ggtacaaata aacaggtgcc
3901 tgaactagtt cacagacaag gaaacttcta tgtaaaaatc actatgattt
ctgaattgct 3961 atgtgaaact acagatcttt ggaacactgt ttaggtaggg
tgttaagact tacacagtac 4021 ctcgtttcta cacagagaaa gaaatggcca
tacttcagga actgcagtgc ttatgagggg 4081 atatttaggc ctcttgaatt
tttgatgtag atgggcattt ttttaaggta gtggttaatt 4141 acctttatgt
gaactttgaa tggtttaaca aaagatttgt ttttgtagag attttaaagg 4201
gggagaattc tagaaataaa tgttacctaa ttattacagc cttaaagaca aaaatccttg
4261 ttgaagtttt tttaaaaaaa gctaaattac atagacttag gcattaacat
gtttgtggaa 4321 gaatatagca gacgtatatt gtatcatttg agtgaatgtt
cccaagtagg cattctaggc 4381 tctatttaac tgagtcacac tgcataggaa
tttagaacct aacttttata ggttatcaaa 4441 actgttgtca ccattgcaca
attttgtcct aatatataca tagaaacttt gtggggcatg 4501 ttaagttaca
gtttgcacaa gttcatctca tttgtattcc attgattttt tttttcttct 4561
aaacattttt tcttcaaaca gtatataact ttttttaggg gatttttttt tagacagcaa
4621 aaactatctg aagatttcca tttgtcaaaa agtaatgatt tcttgataat
tgtgtagtaa 4681 tgttttttag aacccagcag ttaccttaaa gctgaattta
tatttagtaa cttctgtgtt 4741 aatactggat agcatgaatt ctgcattgag
aaactgaata gctgtcataa aatgaaactt 4801 tctttctaaa gaaagatact
cacatgagtt cttgaagaat agtcataact agattaagat 4861 ctgtgtttta
gtttaatagt ttgaagtgcc tgtttgggat aatgataggt aatttagatg 4921
aatttagggg aaaaaaaagt tatctgcaga tatgttgagg gcccatctct ccccccacac
4981 ccccacagag ctaactgggt tacagtgttt tatccgaaag tttccaattc
cactgtcttg 5041 tgttttcatg ttgaaaatac ttttgcattt ttcctttgag
tgccaatttc ttactagtac 5101 tatttcttaa tgtaacatgt ttacctggaa
tgtattttaa ctatttttgt atagtgtaaa 5161 ctgaaacatg cacattttgt
acattgtgct ttcttttgtg ggacatatgc agtgtgatcc 5221 agttgttttc
catcatttgg ttgcgctgac ctaggaatgt tggtcatatc aaacattaaa 5281
aatgaccact cttttaattg aaattaactt ttaaatgttt ataggagtat gtgctgtgaa
5341 gtgatctaaa atttgtaata tttttgtcat gaactgtact actcctaatt
attgtaatgt 5401 aataaaaata gttacagtga caaaaaaaaa aaaaaa
[0088] Human KRAS, transcript variant b, comprising the LCS6 SNP,
is encoded by the following mRNA sequence (SEQ ID NO:
27)(untranslated regions are bolded, LCS6 is underlined, SNP is
capitalized):
TABLE-US-00006 1 ggccgcggcg gcggaggcag cagcggcggc ggcagtggcg
gcggcgaagg tggcggcggc 61 tcggccagta ctcccggccc ccgccatttc
ggactgggag cgagcgcggc gcaggcactg 121 aaggcggcgg cggggccaga
ggctcagcgg ctcccaggtg cgggagagag gcctgctgaa 181 aatgactgaa
tataaacttg tggtagttgg agctggtggc gtaggcaaga gtgccttgac 241
gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag aggattccta
301 caggaagcaa gtagtaattg atggagaaac ctgtctcttg gatattctcg
acacagcagg 361 tcaagaggag tacagtgcaa tgagggacca gtacatgagg
actggggagg gctttctttg 421 tgtatttgcc ataaataata ctaaatcatt
tgaagatatt caccattata gagaacaaat 481 taaaagagtt aaggactctg
aagatgtacc tatggtccta gtaggaaata aatgtgattt 541 gccttctaga
acagtagaca caaaacaggc tcaggactta gcaagaagtt atggaattcc 601
ttttattgaa acatcagcaa agacaagaca gggtgttgat gatgccttct atacattagt
661 tcgagaaatt cgaaaacata aagaaaagat gagcaaagat ggtaaaaaga
agaaaaagaa 721 gtcaaagaca aagtgtgtaa ttatgtaaat acaatttgt
cttttttctt aaggcatact 781 agtacaagtg gtaatttttg tacattacac
taaattatta gcatttgttt tagcattacc 841 taattttttt cctgctccat
gcagactgtt agcttttacc ttaaatgctt attttaaaat 901 gacagtggaa
gttttttttt cctctaagtg ccagtattcc cagagttttg gtttttgaac 961
tagcaatgcc tgtgaaaaag aaactgaata cctaagattt ctgtcttggg gtttttggtg
1021 catgcagttg attacttctt atttttctta ccaattgtga atgttggtgt
gaaacaaatt 1081 aatgaagctt ttgaatcatc cctattctgt gttttatcta
gtcacataaa tggattaatt 1141 actaatttca gttgagacct tctaattggt
ttttactgaa acattgaggg aacacaaatt 1201 tatgggcttc ctgatgatga
ttcttctagg catcatgtcc tatagtttgt catccctgat 1261 gaatgtaaag
ttacactgtt cacaaaggtt ttgtctcctt tccactgcta ttagtcatgg 1321
tcactctccc caaaatatta tattttttct ataaaaagaa aaaaatggaa aaaaattaca
1381 aggcaatgga aactattata aggccatttc cttttcacat tagataaatt
actataaaga 1441 ctcctaatag cttttcctgt taaggcagac ccagtatgaa
atggggatta ttatagcaac 1501 cattttgggg ctatatttac atgctactaa
atttttataa taattgaaaa gattttaaca 1561 agtataaaaa attctcatag
gaattaaatg tagtctccct gtgtcagact gctctttcat 1621 agtataactt
taaatctttt cttcaacttg agtctttgaa gatagtttta attctgcttg 1681
tgacattaaa agattatttg ggccagttat agcttattag gtgttgaaga gaccaaggtt
1741 gcaaggccag gccctgtgtg aacctttgag ctttcataga gagtttcaca
gcatggactg 1801 tgtccccacg gtcatccagt gttgtcatgc attggttagt
caaaatgggg agggactagg 1861 gcagtttgga tagctcaaca agatacaatc
tcactctgtg gtggtcctgc tgacaaatca 1921 agagcattgc ttttgtttct
taagaaaaca aactcttttt taaaaattac ttttaaatat 1981 taactcaaaa
gttgagattt tggggtggtg gtgtgccaag acattaattt tttttttaaa 2041
caatgaagtg aaaaagtttt acaatctcta ggtttggcta gttctcttaa cactggttaa
2101 attaacattg cataaacact tttcaagtct gatccatatt taataatgct
ttaaaataaa 2161 aataaaaaca atccttttga taaatttaaa atgttactta
ttttaaaata aatgaagtga 2221 gatggcatgg tgaggtgaaa gtatcactgg
actaggaaga aggtgactta ggttctagat 2281 aggtgtcttt taggactctg
attttgagga catcacttac tatccatttc ttcatgttaa 2341 aagaagtcat
ctcaaactct tagttttttt tttttacaac tatgtaattt atattccatt 2401
tacataagga tacacttatt tgtcaagctc agcacaatct gtaaattttt aacctatgtt
2461 acaccatctt cagtgccagt cttgggcaaa attgtgcaag aggtgaagtt
tatatttgaa 2521 tatccattct cgttttagga ctcttcttcc atattagtgt
catcttgcct ccctaccttc 2581 cacatgcccc atgacttgat gcagttttaa
tacttgtaat tcccctaacc ataagattta 2641 ctgctgctgt ggatatctcc
atgaagtttt cccactgagt cacatcagaa atgccctaca 2701 tcttatttcc
tcagggctca agagaatctg acagatacca taaagggatt tgacctaatc 2761
actaattttc aggtggtggc tgatgctttg aacatctctt tgctgcccaa tccattagcg
2821 acagtaggat ttttcaaacc tggtatgaat agacagaacc ctatccagtg
gaaggagaat 2881 ttaataaaga tagtgctgaa agaattcctt aggtaatcta
taactaggac tactcctggt 2941 aacagtaata cattccattg ttttagtaac
cagaaatctt catgcaatga aaaatacttt 3001 aattcatgaa gcttactttt
tttttttggt gtcagagtct cgctcttgtc acccaggctg 3061 gaatgcagtg
gcgccatctc agctcactgc aacctccatc tcccaggttc aagcgattct 3121
cgtgcctcgg cctcctgagt agctgggatt acaggcgtgt gccactacac tcaactaatt
3181 tttgtatttt taggagagac ggggtttcac cctgttggcc aggctggtct
cgaactcctg 3241 acctcaagtg atGcacccac cttggcctca taaacctgtt
ttgcagaact catttattca 3301 gcaaatattt attgagtgcc taccagatgc
cagtcaccgc acaaggcact gggtatatgg 3361 tatccccaaa caagagacat
aatcccggtc cttaggtagt gctagtgtgg tctgtaatat 3421 cttactaagg
cctttggtat acgacccaga gataacacga tgcgtatttt agttttgcaa 3481
agaaggggtt tggtctctgt gccagctcta taattgtttt gctacgattc cactgaaact
3541 cttcgatcaa gctactttat gtaaatcact tcattgtttt aaaggaataa
acttgattat 3601 attgtttttt tatttggcat aactgtgatt cttttaggac
aattactgta cacattaagg 3661 tgtatgtcag atattcatat tgacccaaat
gtgtaatatt ccagttttct ctgcataagt 3721 aattaaaata tacttaaaaa
ttaatagttt tatctgggta caaataaaca ggtgcctgaa 3781 ctagttcaca
gacaaggaaa cttctatgta aaaatcacta tgatttctga attgctatgt 3841
gaaactacag atctttggaa cactgtttag gtagggtgtt aagacttaca cagtacctcg
3901 tttctacaca gagaaagaaa tggccatact tcaggaactg cagtgcttat
gaggggatat 3961 ttaggcctct tgaatttttg atgtagatgg gcattttttt
aaggtagtgg ttaattacct 4021 ttatgtgaac tttgaatggt ttaacaaaag
atttgttttt gtagagattt taaaggggga 4081 gaattctaga aataaatgtt
acctaattat tacagcctta aagacaaaaa tccttgttga 4141 agttttttta
aaaaaagcta aattacatag acttaggcat taacatgttt gtggaagaat 4201
atagcagacg tatattgtat catttgagtg aatgttccca agtaggcatt ctaggctcta
4261 tttaactgag tcacactgca taggaattta gaacctaact tttataggtt
atcaaaactg 4321 ttgtcaccat tgcacaattt tgtcctaata tatacataga
aactttgtgg ggcatgttaa 4381 gttacagttt gcacaagttc atctcatttg
tattccattg attttttttt tcttctaaac 4441 attttttctt caaacagtat
ataacttttt ttaggggatt tttttttaga cagcaaaaac 4501 tatctgaaga
tttccatttg tcaaaaagta atgatttctt gataattgtg tagtaatgtt 4561
ttttagaacc cagcagttac cttaaagctg aatttatatt tagtaacttc tgtgttaata
4621 ctggatagca tgaattctgc attgagaaac tgaatagctg tcataaaatg
aaactttctt 4681 tctaaagaaa gatactcaca tgagttcttg aagaatagtc
ataactagat taagatctgt 4741 gttttagttt aatagtttga agtgcctgtt
tgggataatg ataggtaatt tagatgaatt 4801 taggggaaaa aaaagttatc
tgcagatatg ttgagggccc atctctcccc ccacaccccc 4861 acagagctaa
ctgggttaca gtgttttatc cgaaagtttc caattccact gtcttgtgtt 4921
ttcatgttga aaatactttt gcatttttcc tttgagtgcc aatttcttac tagtactatt
4981 tcttaatgta acatgtttac ctggaatgta ttttaactat ttttgtatag
tgtaaactga 5041 aacatgcaca ttttgtacat tgtgctttct tttgtgggac
atatgcagtg tgatccagtt 5101 gttttccatc atttggttgc gctgacctag
gaatgttggt catatcaaac attaaaaatg 5161 accactcttt taattgaaat
taacttttaa atgtttatag gagtatgtgc tgtgaagtga 5221 tctaaaattt
gtaatatttt tgtcatgaac tgtactactc ctaattattg taatgtaata 5281
aaaatagtta cagtgacaaa aaaaaaaaaa aa
[0089] Human NRAS is encoded by the following mRNA sequence (NCBI
Accession No. NM.sub.--002524 and SEQ ID NO: 28) (untranslated
regions are bolded):
TABLE-US-00007 1 gaaacgtccc gtgtgggagg ggcgggtctg ggtgcggctg
ccgcatgact cgtggttcgg 61 aggcccacgt ggccggggcg gggactcagg
cgcctggcag ccgactgatt acgtagcggg 121 cggggccgga agtgccgctc
cttggtgggg gctgttcatg gcggttccgg ggtctccaac 181 atttttcccg
gtctgtggtc ctaaatctgt ccaaagcaga ggcagtggag cttgaggttc 241
ttgctggtgt gaaatgactg agtacaaact ggtggtggtt ggagcaggtg gtgttgggaa
301 aagcgcactg acaatccagc taatccagaa ccactttgta gatgaatatg
atcccaccat 361 agaggattct tacagaaaac aagtggttat agatggtgaa
acctgtttgt tggacatact 421 ggatacagct ggacaagaag agtacagtgc
catgagagac caatacatga ggacaggcga 481 aggcttcctc tgtgtatttg
ccatcaataa tagcaagtca tttgcggata ttaacctcta 541 cagggagcag
attaagcgag taaaagactc ggatgatgta cctatggtgc tagtgggaaa 601
caagtgtgat ttgccaacaa ggacagttga tacaaaacaa gcccacgaac tggccaagag
661 ttacgggatt ccattcattg aaacctcagc caagaccaga cagggtgttg
aagatgcttt 721 ttacacactg gtaagagaaa tacgccagta ccgaatgaaa
aaactcaaca gcagtgatga 781 tgggactcag ggttgtatgg gattgccatg
tgtggtgatg taacaagata cttttaaagt 841 tttgtcagaa aagagccact
ttcaagctgc actgacaccc tggtcctgac ttcctggagg 901 agaagtattc
ctgttgctgt cttcagtctc acagagaagc tcctgctact tccccagctc 961
tcagtagttt agtacaataa tctctatttg agaagttctc agaataacta cctcctcact
1021 tggctgtctg accagagaat gcacctcttg ttactccctg ttatttttct
gccctgggtt 1081 cttccacagc acaaacacac ctcaacacac ctctgccacc
ccaggttttt catctgaaaa 1141 gcagttcatg tctgaaacag agaaccaaac
cgcaaacgtg aaattctatt gaaaacagtg 1201 tcttgagctc taaagtagca
actgctggtg attttttttt tctttttact gttgaactta 1261 gaactatgcc
taatttttgg agaaatgtca taaattactg ttttgccaag aatatagtta 1321
ttattgctgt ttggtttgtt tataatgtta tcggctctat tctctaaact ggcatctgct
1381 ctagattcat aaatacaaaa atgaatactg aattttgagt ctatcctagt
cttcacaact 1441 ttgacgtaat taaatccaac ttttcacagt gaagtgcctt
tttcctagaa gtggtttgta 1501 gactccttta taatatttca gtggaataga
tgtctcaaaa atccttatgc atgaaatgaa 1561 tgtctgagat acgtctgtga
cttatctacc attgaaggaa agctatatct atttgagagc 1621 agatgccatt
ttgtacatgt atgaaattgg ttttccagag gcctgttttg gggctttccc 1681
aggagaaaga tgaaactgaa agcatatgaa taatttcact taataatttt tacctaatct
1741 ccactttttt cataggttac tacctataca atgtatgtaa tttgtttccc
ctagcttact 1801 gataaaccta atattcaatg aacttccatt tgtattcaaa
tttgtgtcat accagaaagc 1861 tctacatttg cagatgttca aatattgtaa
aactttggtg cattgttatt taatagctgt 1921 gatcagtgat tttcaaacct
caaatatagt atattaacaa att
Let-7 Complementary Sites (LCS)
[0090] As used herein, the term "let-7 complementary site" is meant
to describe any region of a gene or gene transcript that binds a
member of the let-7 family of miRNAs. Moreover, this term
encompasses those sequences within a gene or gene transcript that
are complementary to the sequence of a let-7 family miRNA. The term
"complementary" as used herein describes a threshold of binding
between two sequences wherein a majority of nucleotides in each
sequence are capable of binding to a majority of nucleotides within
the other sequence in trans.
[0091] The Human NRAS 3' UTR comprises 9 LCSs named LCS1-LCS9,
respectively (see FIG. 6B). For the following sequences, thymine
(T) may be substituted for uracil (U). LCS1 comprises the sequence
AGUUCUCAGAAUAACUACCUCCUCA (SEQ ID NO: 1). LCS2 comprises the
sequence GGCUGUCUGACCAGAGAAUGCACCUC (SEQ ID NO: 2). LCS3 comprises
the sequence ACAGCACAAACACACCUC (SEQ ID NO: 3). LCS4 comprises the
sequence AGCUGUGAUCAGUGAUUUUCAAACCYCA (SEQ ID NO: 4). LCS5
comprises the sequence AAUUGCCUUCAAUCCCCUUCUCACCCCACCUC (SEQ ID NO:
5). LCS6 comprises the sequence AUCUAAAUACUUACUGAGGUCCUC (SEQ ID
NO: 6). LCS7 comprises the sequence AAUUUUCCUGAGGCUUAUCACCUCA (SEQ
ID NO: 7). LCS8 comprises the sequence
GAUUGCUGAAAAGAAUUCUAGUUUACCUCA (SEQ ID NO: 8). LCS9 comprises the
sequence AACAGGAACUAUUGGCCUC (SEQ ID NO: 9).
[0092] The Human KRAS 3' UTR comprises 8 LCSs named LCS1-LCS8,
respectively (see FIG. 6C). For the following sequences, thymine
(T) may be substituted for uracil (U). LCS1 comprises the sequence
GACAGUGGAAGUUUUUUUUUCCUCG (SEQ ID NO: 10).
[0093] LCS2 comprises the sequence AUUAGUGUCAUCUUGCCUC (SEQ ID NO:
11). LCS3 comprises the sequence AAUGCCCUACAUCUUAUUUUCCUCA (SEQ ID
NO: 12). LCS4 comprises the sequence GGUUCAAGCGAUUCUCGUGCCUCG (SEQ
ID NO: 13). LCS5 comprises the sequence GGCUGGUCCGAACUCCUGACCUCA
(SEQ ID NO: 14). LCS6 comprises the sequence GAUUCACCCACCUUGGCCUCA
(SEQ ID NO: 15). LCS7 comprises the sequence
GGGUGUUAAGACUUGACACAGUACCUCG (SEQ ID NO: 16). LCS8 comprises the
sequence AGUGCUUAUGAGGGGAUAUUUAGGCCUC (SEQ ID NO: 17).
[0094] The Human HRAS 3' UTR comprises 3 LCSs named LCS1-LCS3,
respectively (see FIG. 6D). For the following sequences, thymine
(T) may be substituted for uracil (U). LCS1 comprises the sequence
GACCGUGGGCCGAGGUGACUGCAGACCCUC (SEQ ID NO: 18). LCS2 comprises the
sequence GGAACCCCAGCCCUUAGCUCCCCUC (SEQ ID NO: 19). LCS3 comprises
the sequence AGCCCUUAGCUCCCCUCCCAGGCCUC (SEQ ID NO: 20).
The LCS6 SNP
[0095] The present invention encompasses a SNP within the 3'UTR of
KRAS. Specifically, this SNP is the result of a substitution of a G
for a U at position 4 of SEQ ID NO: 21 of LCS6. This LCS6 SNP
comprises the sequence GAUGCACCCACCUUGGCCUCA (SNP bolded for
emphasis)(SEQ ID NO: 21).
[0096] let-60, the C. elegans homolog of human RAS, is a direct
target of let-7. It has multiple putative let-7 complementary sites
(LCSs) in its 3' UTR. Human RAS is a well known oncogene that often
plays a role in cancer. Knock down of let-60 by RNA interference
(RNAi) in let-7(n2853) loss-of-function mutants partially
suppresses the lethal, bursting phenotype of let-7(n2853)(FIG. 7).
Furthermore, a lacZ reporter containing the let-60 3' UTR was down
regulated in the presence of let-7.
[0097] Human RAS is also a target of let-7 (FIG. 5). There are 3
human RAS genes: HRAS, KRAS, and NRAS. Each of these genes has
multiple LCSs in their 3' UTRs (FIG. 7). Based on RAS levels in the
absence or presence of let-7 (FIG. 10), luciferase reporters with
the 3' UTRs of NRAS or KRAS (FIG. 22), and the inverse relationship
between RAS and let-7 levels in lung cancer patients (FIG. 9), it
was determined that let-7 represses RAS in a 3' UTR-dependent
manner in human cells. Human let-7 is at low levels in various
human cancers, suggesting that let-7 is a tumor suppressor, and
lung cancer patients with low levels of let-7 have decreased
survival rates, supporting the hypothesis that let-7 is important
in lung cancer.
[0098] The KRAS and NRAS 3' UTRs have been sequenced from lung
samples of lung cancer patients, normal lung samples, and human
cell lines (FIGS. 6, 10, 12, and 15). In the KRAS 3' UTR, several
LCSs containing mutations were found (FIGS. 14, 16, 20, 23, and
25). None of these mutations appeared to be associated with lung
cancer. In contrast, a single nucleotide polymorphism (SNP) at the
fourth base pair of LCS6, the LCS6 SNP was found in 20% of patient
samples. The LCS6 SNP was associated with the risk of developing
squamous cancers versus adenocarcinomas of the lung, with younger
patients with lung cancer in our population, with patients that had
additional cancers, and with patients with positive family
histories of cancer.
[0099] To further validate the importance of this SNP in lung
cancer predisposition the baseline prevalence in the 25 different
human populations was first determined. The prevalence was highest
in Caucasian populations, at 7.4%. In Caucasian lung cancer
patients the prevalence was 24%, which is significantly higher. The
association of this SNP with smoking-induced lung cancer was
further validated in a case control study of smokers who did or did
not develop lung cancer. A significant association of the SNP with
non-small cell lung cancer development in patients matched for age,
sex, race and smoking status was found. These results support the
hypothesis that the presence of the LCS6 SNP is a genetic marker
for an increased risk of lung cancer development.
[0100] The prevalence of the LCS6 SNP was further examined in
several other cancers, including head and neck cancers, breast
cancer, ovarian cancer, uterine cancer and pancreatic cancer. It
was discovered that the LCS6 SNP is at a significantly higher
prevalence than expected in these cancers. Moreover, the LCS6 SNP
has been demonstrated to be associated with a specific subtype of
each of these cancers (Table 1). Specifically, the LCS6 SNP was
shown to be associated with the subtypes associated with the worst
prognosis in each of these cancer types. With respect to ovarian
cancer, for instance, the presence of the LCS6 SNP is also
coincident with the presentation of more advanced stages of cancer
(FIG. 25). Accordingly, the LCS6 SNP predicts whether the cancer is
aggressive or resistant to current therapy, which are the most
critical to prevent. This predictive ability demonstrates that the
presence of the LCS6 SNP is a biomarker of cancer outcome.
TABLE-US-00008 TABLE 1 Prevalence of KRAS SNP in Cancer Types
Cancer Type Frequency of SNP Significance Case control Studies Lung
cancer 18.1-20.3% OR = 1.4-2.3, (Non-small cell (from 2 independent
p < 0.01 subtype case controls, 400 and 4000 pts.) Pancreatic
cancer 18.8% OR = 1.2 (Exocrine pancreas) (800 patients, ongoing)
Prevalence studies Endometrial Cancer 48% (10/21) p = .0004 (High
Risk subtypes) odds ratio = 5.57 Ovarian Cancer 51% (22/43) p <
0.00000000001 (all subtypes) odds ratio = 8.45 Head and Neck Cancer
33% (8/24) p = 0.011 (Oropharynx subtype) odds ratio = 3.07 Breast
Cancer 25% (7/22) p = 0.017 (Her-2 + subtype) odds ratio = 2.1
Colon Cancer 18.3% (249/1364) p < 0.001 odds ratio = 1.4
Melanoma 28.6% (2/7) p < 0.01 odds ratio = 2.0 Table 1.
Prevalence studies based on the expected frequency of the SNP of
14%. Significance is based on a Chi-squared analysis. Chi-squared
and OR numbers are based on the prevalence expected in Caucasian
patients of 14%, and for some of these cancers up to 1/2 of cancer
patients were AA, thus, these are underestimations.
[0101] The LCS6 SNP was examined to determine how the presence of
the SNP altered the binding efficacy of let-7 family miRNAs to
KRAS. The LCS6 SNP was engineered in a luciferase reporter
construct containing all LCSs (FIG. 22). The LCS6 SNP causes an
increase in luciferase expression as compared to the non-SNP
reporter in lung cancer cells.
[0102] In two case-controlled association studies, the presence of
the variant allele predicts for an increased risk of non-small cell
lung cancer (NSCLC) (OR=1.36-2.3, 95% CI=1.07-1.73, p=0.01, 95%
CI=1.1-4.6, p=0.02) in patients with a<40 to 41 pack-year
smoking history. One difference between the subjects of the
case-control designs and the patient cohort at Yale University is
that the case-control designs were primary lung cancer studies, and
thus people with prior cancers were excluded from both. In
contrast, in the retrospective patient cohort, 64% of the
allele-carriers had additional cancers and 89% of these cancers
were diagnosed before their lung cancer. This difference may
actually lead to an under-estimation of the lung cancer risk for
smokers carrying the variant allele. As such, these studies may
underestimate the predictive power of the LCS6 SNP comprised by the
invention.
[0103] While not limited by theory, the present invention includes
and is based in part on the understanding that alteration of let-7
binding, brought about by the presence or absence of one or more
SNP(s), impacts cellular levels of let-7. Increased let-7 binding
could lead to sequestration of let-7 and a decrease in cellular
let-7 levels. As let-7 is known to regulate cell proliferation
genes, this could lead to excess cellular proliferation and
oncogenesis. Equally as plausible, however, is the possibility that
there exists a cellular feed-back system that would detect let-7 as
too low due to its increased KRAS binding, leading to elevated
cellular let-7 levels. As let-7 has been shown to regulate genes
important in the DNA damage response pathway, a state of high let-7
could also lead to oncogenesis by leaving the cell open to excess
DNA damage.
[0104] This LCS6 SNP is a marker for increased genetic
susceptibility to smoking induced lung cancer and other cancers.
Methods of the invention demonstrating means for identifying this
SNP and similar SNPs are used to enhance screening programs to
enrich for people at the highest risk of developing lung cancer,
testing families with histories of lung cancer to determine
individual risk, setting up smoking cessation programs and
screening participants, and testing patients with smoking-induced
cancers to determine the risk of developing additional, or
secondary, cancers. This SNP variant, as well as all SNPs
encompassed by the invention, are used to predict cancer outcome,
e.g. prognosis, and to identify patients for whom therapies
designed to target particular SNPs should be applied.
[0105] The LCS6 SNP comprises the first identified 3' UTR SNP
affecting miRNA binding that is genetically linked to cancer. The
methods of the invention demonstrate particular utility as an
incentive for individuals who smoke to accurately access
smoking-induced risk for developing lung cancer and/or additional
cancers. The LCS6 SNP can also be used to assess an increased risk
of developing ovarian, breast, colon, head and neck, pancreatic and
kidney cancers.
[0106] Moreover, the presence of the LCS6 SNP indicates a greater
risk for developing radon-associated non-small cell lung cancer, as
well as other radon-associated cancers. Radon is a colorless,
naturally occurring, radioactive noble gas that is formed from the
decay of radium. The radiation decay products ionize genetic
material, causing mutations that sometimes turn cancerous. It is
one of the heaviest substances that are gases under normal
conditions and is considered to be a health hazard. Radon is a
significant contaminant that affects indoor air quality worldwide.
Radon gas from natural sources can accumulate in buildings and
reportedly causes 21,000 lung cancer deaths per year in the United
States alone. Radon is the second most frequent cause of lung
cancer, after cigarette smoking, and radon-induced lung cancer is
thought to be the 6th leading cause of cancer death overall.
[0107] Methods of the invention were used to determine the
prevalence of the LCS6 SNP among cancer patients whose occupation
was mining. Among minors who developed lung cancer, data gathered
using the methods of the invention show that the prevalence of the
LCS6 SNP was higher than expected in this population (23% in minors
with lung cancer patients versus 14% in control individuals who
represent the general, non-cancerous population). The average radon
exposure among the minors studied was 1362 work level months for
the non-LCS6 SNP subset versus 1073 work level months for the LCS6
SNP carrying subset. Work level months is a measurement that
reflects the number of hours of exposure to radon over an equal
number of months.
[0108] These data show that LCS6 SNP carrying individuals are at a
greater risk of developing radon associated lung cancer (as well as
other radon-associated cancers) than individuals who do not carry
the LCS6 SNP, because the LCS6 SNP carrying population are
over-represented as cancer patients in this study despite having
overall less exposure to radon. In other words, individuals who
carry the LCS6 SNP appear to develop radon-associated cancers
following a lower level or threshold of radon exposure. In a
preferred embodiment, methods of the invention are used to
determine an individual's risk for developing radon-associated
cancer prior to, during, or following exposure to radon.
Isolated Nucleic Acid Molecules
[0109] The present invention provides isolated nucleic acid
molecules that contain one or more SNPs. Exemplary isolated nucleic
acid molecules containing one or more SNPs include, but are not
limited to, the nucleic acid molecules of SEQ ID NOs: 21, 26, and
27. Isolated nucleic acid molecules containing one or more SNPs
disclosed herein may be interchangeably referred to throughout the
present text as "SNP-containing nucleic acid molecules". Isolated
nucleic acid molecules may optionally encode a full-length variant
protein or fragment thereof. The isolated nucleic acid molecules of
the present invention also include probes and primers (which are
described in greater detail below in the section entitled "SNP
Detection Reagents"), which may be used for assaying the disclosed
SNPs, and isolated full-length genes, transcripts, cDNA molecules,
and fragments thereof, which may be used for such purposes as
expressing an encoded protein.
[0110] As used herein, an "isolated nucleic acid molecule"
generally is one that contains a SNP of the present invention or
one that hybridizes to such molecule such as a nucleic acid with a
complementary sequence, and is separated from most other nucleic
acids present in the natural source of the nucleic acid molecule.
Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule containing a SNP of the present invention, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or chemical precursors or
other chemicals when chemically synthesized. A nucleic acid
molecule can be fused to other coding or regulatory sequences and
still be considered "isolated". Nucleic acid molecules present in
non-human transgenic animals, which do not naturally occur in the
animal, are also considered "isolated". For example, recombinant
DNA molecules contained in a vector are considered "isolated".
Further examples of "isolated" DNA molecules include recombinant
DNA molecules maintained in heterologous host cells, and purified
(partially or substantially) DNA molecules in solution. Isolated
RNA molecules include in vivo or in vitro RNA transcripts of the
isolated SNP-containing DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0111] Generally, an isolated SNP-containing nucleic acid molecule
comprises one or more SNP positions disclosed by the present
invention with flanking nucleotide sequences on either side of the
SNP positions. A flanking sequence can include nucleotide residues
that are naturally associated with the SNP site and/or heterologous
nucleotide sequences. Preferably the flanking sequence is up to
about 500, 300, 100, 60, 50, 30, 25, 20, 15, 10, 8, or 4
nucleotides (or any other length in-between) on either side of a
SNP position, or as long as the full-length gene, entire coding, or
non-coding sequence (or any portion thereof such as an exon,
intron, or a 5' or 3' untranslated region (UTR)), especially if the
SNP-containing nucleic acid molecule is to be used to produce a
protein or protein fragment.
[0112] For full-length genes and entire protein-coding sequences, a
SNP flanking sequence can be, for example, up to about 5 KB, 4 KB,
3 KB, 2 KB, 1 KB on either side of the SNP. Furthermore, in such
instances, the isolated nucleic acid molecule comprises exonic
sequences (including protein-coding and/or non-coding exonic
sequences), but may also include intronic sequences and
untranslated regulatory sequences. Thus, any protein coding
sequence may be either contiguous or separated by introns. The
important point is that the nucleic acid is isolated from remote
and unimportant flanking sequences and is of appropriate length
such that it can be subjected to the specific manipulations or uses
described herein such as recombinant protein expression,
preparation of probes and primers for assaying the SNP position,
and other uses specific to the SNP-containing nucleic acid
sequences.
[0113] An isolated SNP-containing nucleic acid molecule can
comprise, for example, a full-length gene or transcript, such as a
gene isolated from genomic DNA (e.g., by cloning or PCR
amplification), a cDNA molecule, or an mRNA transcript molecule.
Furthermore, fragments of such full-length genes and transcripts
that contain one or more SNPs disclosed herein are also encompassed
by the present invention.
[0114] Thus, the present invention also encompasses fragments of
the nucleic acid sequences including, but not limited to, SEQ ID
NOs: 21, 26 and 27, and their complements. A fragment typically
comprises a contiguous nucleotide sequence at least about 8 or more
nucleotides, more preferably at least about 10 or more nucleotides,
and even more preferably at least about 16 or more nucleotides.
Further, a fragment could comprise at least about 18, 20, 21, 22,
25, 30, 40, 50, 60, 100, 250 or 500 (or any other number
in-between) nucleotides in length. The length of the fragment will
be based on its intended use. Such fragments can be isolated using
nucleotide sequences such as, but not limited to, SEQ ID NOs: 15,
21, 24, 25, 26 and 27 for the synthesis of a polynucleotide probe.
For example, a fragment may comprise nucleotides 3370-3400,
3360-3500, 3350-3600, 3340-3700, 3330-3800, 3320-3900, 3310-4000,
3300-4100, of SEQ ID NOs: 24, 25, 26, or 27, for example, or any
range in between. A labeled probe can then be used, for example, to
screen a cDNA library, genomic DNA library, or mRNA to isolate
nucleic acid corresponding to the region of interest. Further,
primers can be used in amplification reactions, such as for
purposes of assaying one or more SNPs sites or for cloning specific
regions of a gene.
[0115] An isolated nucleic acid molecule of the present invention
further encompasses a SNP-containing polynucleotide that is the
product of any one of a variety of nucleic acid amplification
methods, which are used to increase the copy numbers of a
polynucleotide of interest in a nucleic acid sample. Such
amplification methods are well known in the art, and they include
but are not limited to, polymerase chain reaction (PCR) (U.S. Pat.
No. 4,683,195; and U.S. Pat. No. 4,683,202; PCR Technology:
Principles and Applications for DNA Amplification, ed. H. A.
Erlich, Freeman Press, NY, N.Y., 1992), ligase chain reaction (LCR)
(Wu and Wallace, Genomics 4:560, 1989; Landegren et al., Science
241:1077, 1988), strand displacement amplification (SDA) (U.S. Pat.
No. 5,270,184; and U.S. Pat. No. 5,422,252), transcription-mediated
amplification (TMA) (U.S. Pat. No. 5,399,491), linked linear
amplification (LLA) (U.S. Pat. No. 6,027,923), and the like, and
isothermal amplification methods such as nucleic acid sequence
based amplification (NASBA), and self-sustained sequence
replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87: 1874,
1990). Based on such methodologies, a person skilled in the art can
readily design primers in any suitable regions 5' and 3' to a SNP
disclosed herein. Such primers may be used to amplify DNA of any
length so long that it contains the SNP of interest in its
sequence.
[0116] As used herein, an "amplified polynucleotide" of the
invention is a SNP-containing nucleic acid molecule whose amount
has been increased at least two fold by any nucleic acid
amplification method performed in vitro as compared to its starting
amount in a test sample. In other preferred embodiments, an
amplified polynucleotide is the result of at least ten fold, fifty
fold, one hundred fold, one thousand fold, or even ten thousand
fold increase as compared to its starting amount in a test sample.
In a typical PCR amplification, a polynucleotide of interest is
often amplified at least fifty thousand fold in amount over the
unamplified genomic DNA, but the precise amount of amplification
needed for an assay depends on the sensitivity of the subsequent
detection method used.
[0117] Generally, an amplified polynucleotide is at least about 10
nucleotides in length. More typically, an amplified polynucleotide
is at least about 16 nucleotides in length. In a preferred
embodiment of the invention, an amplified polynucleotide is at
least about 20 nucleotides in length. In a more preferred
embodiment of the invention, an amplified polynucleotide is at
least about 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or 60
nucleotides in length. In yet another preferred embodiment of the
invention, an amplified polynucleotide is at least about 100, 200,
or 300 nucleotides in length. While the total length of an
amplified polynucleotide of the invention can be as long as an
exon, an intron, a 5' UTR, a 3' UTR, or the entire gene where the
SNP of interest resides, an amplified product is typically no
greater than about 1,000 nucleotides in length (although certain
amplification methods may generate amplified products greater than
1000 nucleotides in length). More preferably, an amplified
polynucleotide is not greater than about 600 nucleotides in length.
It is understood that irrespective of the length of an amplified
polynucleotide, a SNP of interest may be located anywhere along its
sequence.
[0118] In a specific embodiment of the invention, the amplified
product is at least about 21 nucleotides in length, and comprises a
SNP in a let-7 complementary site (LCS) that modifies binding of a
let-7 miRNA family member. In a specific embodiment, the amplified
product is at least about 21 nucleotides in length, and comprises
SEQ ID NOs: 21, 26, or 27. Such a product may have additional
sequences on its 5' end or 3' end or both. In another embodiment,
the amplified product is about 101 nucleotides in length, and it
contains a SNP disclosed herein. Preferably, the SNP is located at
the middle of the amplified product (e.g., at position 101 in an
amplified product that is 201 nucleotides in length, or at position
51 in an amplified product that is 101 nucleotides in length), or
within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 nucleotides
from the middle of the amplified product (however, as indicated
above, the SNP of interest may be located anywhere along the length
of the amplified product).
[0119] The present invention provides isolated nucleic acid
molecules that comprise, consist of, or consist essentially of one
or more polynucleotide sequences that contain one or more SNPs
disclosed herein, complements thereof, and SNP-containing fragments
thereof.
[0120] Accordingly, the present invention provides nucleic acid
molecules that consist of any of the nucleotide sequences of SEQ ID
NO: 21, 26 and 27. A nucleic acid molecule consists of a nucleotide
sequence when the nucleotide sequence is the complete nucleotide
sequence of the nucleic acid molecule.
[0121] The present invention further provides nucleic acid
molecules that consist essentially of any of the nucleotide
sequences of SEQ ID NO: 21, 26 and 27. A nucleic acid molecule
consists essentially of a nucleotide sequence when such a
nucleotide sequence is present with only a few additional
nucleotide residues in the final nucleic acid molecule.
[0122] The present invention further provides nucleic acid
molecules that comprise any of the nucleotide sequences of SEQ ID
NOs: 21, 26 or 27. A nucleic acid molecule comprises a nucleotide
sequence when the nucleotide sequence is at least part of the final
nucleotide sequence of the nucleic acid molecule. In such a
fashion, the nucleic acid molecule can be only the nucleotide
sequence or have additional nucleotide residues, such as residues
that are naturally associated with it or heterologous nucleotide
sequences. Such a nucleic acid molecule can have one to a few
additional nucleotides or can comprise many more additional
nucleotides. A brief description of how various types of these
nucleic acid molecules can be readily made and isolated is provided
below, and such techniques are well known to those of ordinary
skill in the art (Sambrook and Russell, 2000, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, NY).
[0123] The isolated nucleic acid molecules include, but are not
limited to, nucleic acid molecules having a sequence encoding a
peptide alone, a sequence encoding a mature peptide and additional
coding sequences such as a leader or secretory sequence (e.g., a
pre-pro or pro-protein sequence), a sequence encoding a mature
peptide with or without additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but untranslated sequences
that play a role in, for example, transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding,
and/or stability of mRNA. In addition, the nucleic acid molecules
may be fused to heterologous marker sequences encoding, for
example, a peptide that facilitates purification.
[0124] Isolated nucleic acid molecules can be in the form of RNA,
such as mRNA, or in the form DNA, including cDNA and genomic DNA,
which may be obtained, for example, by molecular cloning or
produced by chemical synthetic techniques or by a combination
thereof (Sambrook and Russell, 2000, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, NY). Furthermore,
isolated nucleic acid molecules, particularly SNP detection
reagents such as probes and primers, can also be partially or
completely in the form of one or more types of nucleic acid
analogs, such as peptide nucleic acid (PNA) (U.S. Pat. Nos.
5,539,082; 5,527,675; 5,623,049; 5,714,331). The nucleic acid,
especially DNA, can be double-stranded or single-stranded.
Single-stranded nucleic acid can be the coding strand (sense
strand) or the complementary non-coding strand (anti-sense strand).
DNA, RNA, or PNA segments can be assembled, for example, from
fragments of the human genome (in the case of DNA or RNA) or single
nucleotides, short oligonucleotide linkers, or from a series of
oligonucleotides, to provide a synthetic nucleic acid molecule.
Nucleic acid molecules can be readily synthesized using the
sequences provided herein as a reference; oligonucleotide and PNA
oligomer synthesis techniques are well known in the art (see, e.g.,
Corey, "Peptide nucleic acids: expanding the scope of nucleic acid
recognition", Trends Biotechnol. 1997 June; 15(6):224-9, and Hyrup
et al., "Peptide nucleic acids (PNA): synthesis, properties and
potential applications", Bioorg Med Chem. 1996 January; 4(1):5-23).
Furthermore, large-scale automated oligonucleotide/PNA synthesis
(including synthesis on an array or bead surface or other solid
support) can readily be accomplished using commercially available
nucleic acid synthesizers, such as the Applied Biosystems (Foster
City, Calif.) 3900 High-Throughput DNA Synthesizer or Expedite 8909
Nucleic Acid Synthesis System, and the sequence information
provided herein.
[0125] The present invention encompasses nucleic acid analogs that
contain modified, synthetic, or non-naturally occurring nucleotides
or structural elements or other alternative/modified nucleic acid
chemistries known in the art. Such nucleic acid analogs are useful,
for example, as detection reagents (e.g., primers/probes) for
detecting one or more SNPs identified in SEQ ID NOs: 21, 26 and 27.
Furthermore, kits/systems (such as beads, arrays, etc.) that
include these analogs are also encompassed by the present
invention. For example, PNA oligomers that are based on the
polymorphic sequences of the present invention are specifically
contemplated. PNA oligomers are analogs of DNA in which the
phosphate backbone is replaced with a peptide-like backbone
(Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters,
4: 1081-1082 (1994), Petersen et al., Bioorganic & Medicinal
Chemistry Letters, 6: 793-796 (1996), Kumar et al., Organic Letters
3(9): 1269-1272 (2001), WO96/04000). PNA hybridizes to
complementary RNA or DNA with higher affinity and specificity than
conventional oligonucleotides and oligonucleotide analogs. The
properties of PNA enable novel molecular biology and biochemistry
applications unachievable with traditional oligonucleotides and
peptides.
[0126] Additional examples of nucleic acid modifications that
improve the binding properties and/or stability of a nucleic acid
include the use of base analogs such as inosine, intercalators
(U.S. Pat. No. 4,835,263) and the minor groove binders (U.S. Pat.
No. 5,801,115). Thus, references herein to nucleic acid molecules,
SNP-containing nucleic acid molecules, SNP detection reagents
(e.g., probes and primers), oligonucleotides/polynucleotides
include PNA oligomers and other nucleic acid analogs. Other
examples of nucleic acid analogs and alternative/modified nucleic
acid chemistries known in the art are described in Current
Protocols in Nucleic Acid Chemistry, John Wiley & Sons, N.Y.
(2002).
[0127] Further variants of the nucleic acid molecules including,
but not limited to those identified as SEQ ID NOs: 21, 26 and 27,
such as naturally occurring allelic variants (as well as orthologs
and paralogs) and synthetic variants produced by mutagenesis
techniques, can be identified and/or produced using methods well
known in the art. Such further variants can comprise a nucleotide
sequence that shares at least 70-80%, 80-85%, 85-90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a
nucleic acid sequence disclosed as SEQ ID NOs: 21, 26 or 27 (or a
fragment thereof) and that includes a novel SNP allele disclosed as
SEQ ID NOs: 21, 26 or 27. Thus, the present invention specifically
contemplates isolated nucleic acid molecule that have a certain
degree of sequence variation compared with the sequences of SEQ ID
NOs: 21, 26 and 27, but that contain a novel SNP allele disclosed
herein. In other words, as long as an isolated nucleic acid
molecule contains a novel SNP allele disclosed herein, other
portions of the nucleic acid molecule that flank the novel SNP
allele can vary to some degree from the specific sequences
identified herein as SEQ ID NOs: 21, 26, and 27.
[0128] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. (Computational Molecular Biology, Lesk, A.
M., ed., Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, D. W., ed., Academic Press,
New York, 1993; Computer Analysis of Sequence Data, Part 1,
Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,
1994; Sequence Analysis in Molecular Biology, von Heinje, G.,
Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M Stockton Press, New York, 1991). In a
preferred embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch algorithm
(J. Mol. Biol. (48):444-453 (1970)) which has been incorporated
into the GAP program in the GCG software package, using either a
Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
[0129] In yet another preferred embodiment, the percent identity
between two nucleotide sequences is determined using the GAP
program in the GCG software package (Devereux, J., et al., Nucleic
Acids Res. 12(1):387 (1984)), using a NWSgapdna. CMP matrix and a
gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3,
4, 5, or 6. In another embodiment, the percent identity between two
amino acid or nucleotide sequences is determined using the
algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which
has been incorporated into the ALIGN program (version 2.0), using a
PAM120 weight residue table, a gap length penalty of 12, and a gap
penalty of 4.
[0130] The nucleotide and amino acid sequences of the present
invention can further be used as a "query sequence" to perform a
search against sequence databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches
can be performed with the NBLAST program, score=100, wordlength=12
to obtain nucleotide sequences homologous to the nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to the proteins of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. In addition to BLAST, examples of
other search and sequence comparison programs used in the art
include, but are not limited to, FASTA (Pearson, Methods Mol. Biol.
25, 365-389 (1994)) and KERR (Dufresne et al., Nat Biotechnol 2002
December; 20(12): 1269-71). For further information regarding
bioinformatics techniques, see Current Protocols in Bioinformatics,
John Wiley & Sons, Inc., N.Y.
SNP Detection Reagents
[0131] In a specific aspect of the present invention, the sequences
disclosed herein can be used for the design of SNP detection
reagents. In a preferred embodiment, sequences of SEQ ID NOs: 21,
24, 25, 26, and 27 are used for the design of SNP detection
reagents. Methods of the invention encompass all sequences
comprising let-7 complementary sites (LCSs). As such, any sequence
comprising at least one LCS can be used to design a SNP detection
reagent. As used herein, a "SNP detection reagent" is a reagent
that specifically detects a specific target SNP position disclosed
herein, and that is preferably specific for a particular nucleotide
(allele) of the target SNP position (i.e., the detection reagent
preferably can differentiate between different alternative
nucleotides at a target SNP position, thereby allowing the identity
of the nucleotide present at the target SNP position to be
determined). Typically, such detection reagent hybridizes to a
target SNP-containing nucleic acid molecule by complementary
base-pairing in a sequence specific manner, and discriminates the
target variant sequence from other nucleic acid sequences such as
an art-known form in a test sample. An example of a detection
reagent is a probe that hybridizes to a target nucleic acid
containing SEQ ID NO: 21. In a preferred embodiment, such a probe
can differentiate between nucleic acids having a particular
nucleotide (allele) at a target SNP position from other nucleic
acids that have a different nucleotide at the same target SNP
position. In addition, a detection reagent may hybridize to a
specific region 5' and/or 3' to a SNP position, particularly a
region corresponding the 3'UTR. Another example of a detection
reagent is a primer which acts as an initiation point of nucleotide
extension along a complementary strand of a target polynucleotide.
The SNP sequence information provided herein is also useful for
designing primers, e.g. allele-specific primers, to amplify (e.g.,
using PCR) any SNP of the present invention.
[0132] In one preferred embodiment of the invention, a SNP
detection reagent is an isolated or synthetic DNA or RNA
polynucleotide probe or primer or PNA oligomer, or a combination of
DNA, RNA and/or PNA, that hybridizes to a segment of a target
nucleic acid molecule containing a SNP located within a LCS. In a
specific embodiment, a SNP detection reagent is an isolated or
synthetic DNA or RNA polynucleotide probe or primer or PNA
oligomer, or a combination of DNA, RNA and/or PNA, that hybridizes
to a segment of a target nucleic acid molecule containing SEQ ID
NO: 21. A detection reagent in the form of a polynucleotide may
optionally contain modified base analogs, intercalators or minor
groove binders. Multiple detection reagents such as probes may be,
for example, affixed to a solid support (e.g., arrays or beads) or
supplied in solution (e.g., probe/primer sets for enzymatic
reactions such as PCR, RT-PCR, TaqMan assays, or primer-extension
reactions) to form a SNP detection kit.
[0133] A probe or primer typically is a substantially purified
oligonucleotide or PNA oligomer. Such oligonucleotide typically
comprises a region of complementary nucleotide sequence that
hybridizes under stringent conditions to at least about 8, 10, 12,
16, 18, 20, 21, 22, 25, 30, 40, 50, 60, 100 (or any other number
in-between) or more consecutive nucleotides in a target nucleic
acid molecule. Depending on the particular assay, the consecutive
nucleotides can either include the target SNP position, or be a
specific region in close enough proximity 5' and/or 3' to the SNP
position to carry out the desired assay.
[0134] It will be apparent to one of skill in the art that such
primers and probes are directly useful as reagents for genotyping
the SNPs of the present invention, and can be incorporated into any
kit/system format.
[0135] In order to produce a probe or primer specific for a target
SNP-containing sequence, the gene/transcript and/or context
sequence surrounding the SNP of interest is typically examined
using a computer algorithm which starts at the 5' or at the 3' end
of the nucleotide sequence. Typical algorithms will then identify
oligomers of defined length that are unique to the gene/SNP context
sequence, have a GC content within a range suitable for
hybridization, lack predicted secondary structure that may
interfere with hybridization, and/or possess other desired
characteristics or that lack other undesired characteristics.
[0136] A primer or probe of the present invention is typically at
least about 8 nucleotides in length. In one embodiment of the
invention, a primer or a probe is at least about 10 nucleotides in
length. In a preferred embodiment, a primer or a probe is at least
about 12 nucleotides in length. In a more preferred embodiment, a
primer or probe is at least about 16, 17, 18, 19, 20, 21, 22, 23,
24 or 25 nucleotides in length. While the maximal length of a probe
can be as long as the target sequence to be detected, depending on
the type of assay in which it is employed, it is typically less
than about 50, 60, 65, or 70 nucleotides in length. In the case of
a primer, it is typically less than about 30 nucleotides in length.
In a specific preferred embodiment of the invention, a primer or a
probe is within the length of about 18 and about 28 nucleotides.
However, in other embodiments, such as nucleic acid arrays and
other embodiments in which probes are affixed to a substrate, the
probes can be longer, such as on the order of 30-70, 75, 80, 90,
100, or more nucleotides in length (see the section below entitled
"SNP Detection Kits and Systems").
[0137] For analyzing SNPs, it may be appropriate to use
oligonucleotides specific for alternative SNP alleles. Such
oligonucleotides which detect single nucleotide variations in
target sequences may be referred to by such terms as
"allele-specific oligonucleotides", "allele-specific probes", or
"allele-specific primers". The design and use of allele-specific
probes for analyzing polymorphisms is described in, e.g., Mutation
Detection A Practical Approach, ed. Cotton et al. Oxford University
Press, 1998; Saiki et al., Nature 324, 163-166 (1986); Dattagupta,
EP235,726; and Saiki, WO 89/11548.
[0138] While the design of each allele-specific primer or probe
depends on variables such as the precise composition of the
nucleotide sequences flanking a SNP position in a target nucleic
acid molecule, and the length of the primer or probe, another
factor in the use of primers and probes is the stringency of the
condition under which the hybridization between the probe or primer
and the target sequence is performed. Higher stringency conditions
utilize buffers with lower ionic strength and/or a higher reaction
temperature, and tend to require a more perfect match between
probe/primer and a target sequence in order to form a stable
duplex. If the stringency is too high, however, hybridization may
not occur at all. In contrast, lower stringency conditions utilize
buffers with higher ionic strength and/or a lower reaction
temperature, and permit the formation of stable duplexes with more
mismatched bases between a probe/primer and a target sequence. By
way of example and not limitation, exemplary conditions for high
stringency hybridization conditions using an allele-specific probe
are as follows: Prehybridization with a solution containing
5.times. standard saline phosphate EDTA (SSPE), 0.5% NaDodSO.sub.4
(SDS) at 55.degree. C., and incubating probe with target nucleic
acid molecules in the same solution at the same temperature,
followed by washing with a solution containing 2.times.SSPE, and
0.1% SDS at 55.degree. C. or room temperature.
[0139] Moderate stringency hybridization conditions may be used for
allele-specific primer extension reactions with a solution
containing, e.g., about 50 mM KCl at about 46.degree. C.
Alternatively, the reaction may be carried out at an elevated
temperature such as 60.degree. C. In another embodiment, a
moderately stringent hybridization condition suitable for
oligonucleotide ligation assay (OLA) reactions wherein two probes
are ligated if they are completely complementary to the target
sequence may utilize a solution of about 100 mM KCl at a
temperature of 46.degree. C.
[0140] In a hybridization-based assay, allele-specific probes can
be designed that hybridize to a segment of target DNA from one
individual but do not hybridize to the corresponding segment from
another individual due to the presence of different polymorphic
forms (e.g., alternative SNP alleles/nucleotides) in the respective
DNA segments from the two individuals. Hybridization conditions
should be sufficiently stringent that there is a significant
detectable difference in hybridization intensity between alleles,
and preferably an essentially binary response, whereby a probe
hybridizes to only one of the alleles or significantly more
strongly to one allele. While a probe may be designed to hybridize
to a target sequence that contains a SNP site such that the SNP
site aligns anywhere along the sequence of the probe, the probe is
preferably designed to hybridize to a segment of the target
sequence such that the SNP site aligns with a central position of
the probe (e.g., a position within the probe that is at least three
nucleotides from either end of the probe). This design of probe
generally achieves good discrimination in hybridization between
different allelic forms.
[0141] In another embodiment, a probe or primer may be designed to
hybridize to a segment of target DNA such that the SNP aligns with
either the 5' most end or the 3' most end of the probe or primer.
In a specific preferred embodiment which is particularly suitable
for use in a oligonucleotide ligation assay (U.S. Pat. No.
4,988,617), the 3' most nucleotide of the probe aligns with the SNP
position in the target sequence.
[0142] Oligonucleotide probes and primers may be prepared by
methods well known in the art. Chemical synthetic methods include,
but are limited to, the phosphotriester method described by Narang
et al., 1979, Methods in Enzymology 68:90; the phosphodiester
method described by Brown et al., 1979, Methods in Enzymology
68:109, the diethylphosphoamidate method described by Beaucage et
al., 1981, Tetrahedron Letters 22:1859; and the solid support
method described in U.S. Pat. No. 4,458,066.
[0143] Allele-specific probes are often used in pairs (or, less
commonly, in sets of 3 or 4, such as if a SNP position is known to
have 3 or 4 alleles, respectively, or to assay both strands of a
nucleic acid molecule for a target SNP allele), and such pairs may
be identical except for a one nucleotide mismatch that represents
the allelic variants at the SNP position.
[0144] Commonly, one member of a pair perfectly matches a reference
form of a target sequence that has a more common SNP allele (i.e.,
the allele that is more frequent in the target population) and the
other member of the pair perfectly matches a form of the target
sequence that has a less common SNP allele (i.e., the allele that
is rarer in the target population). In the case of an array,
multiple pairs of probes can be immobilized on the same support for
simultaneous analysis of multiple different polymorphisms.
[0145] In one type of PCR-based assay, an allele-specific primer
hybridizes to a region on a target nucleic acid molecule that
overlaps a SNP position and only primes amplification of an allelic
form to which the primer exhibits perfect complementarity (Gibbs,
1989, Nucleic Acid Res. 17 2427-2448). Typically, the primer's
3'-most nucleotide is aligned with and complementary to the SNP
position of the target nucleic acid molecule. This primer is used
in conjunction with a second primer that hybridizes at a distal
site. Amplification proceeds from the two primers, producing a
detectable product that indicates which allelic form is present in
the test sample. A control is usually performed with a second pair
of primers, one of which shows a single base mismatch at the
polymorphic site and the other of which exhibits perfect
complementarity to a distal site. The single-base mismatch prevents
amplification or substantially reduces amplification efficiency, so
that either no detectable product is formed or it is formed in
lower amounts or at a slower pace. The method generally works most
effectively when the mismatch is at the 3'-most position of the
oligonucleotide (i.e., the 3'-most position of the oligonucleotide
aligns with the target SNP position) because this position is most
destabilizing to elongation from the primer (see, e.g., WO
93/22456). This PCR-based assay can be utilized as part of the
TaqMan assay, described below.
[0146] In a specific embodiment of the invention, a primer of the
invention contains a sequence substantially complementary to a
segment of a target SNP-containing nucleic acid molecule except
that the primer has a mismatched nucleotide in one of the three
nucleotide positions at the 3'-most end of the primer, such that
the mismatched nucleotide does not base pair with a particular
allele at the SNP site. In a preferred embodiment, the mismatched
nucleotide in the primer is the second from the last nucleotide at
the 3'-most position of the primer. In a more preferred embodiment,
the mismatched nucleotide in the primer is the last nucleotide at
the 3'-most position of the primer.
[0147] In another embodiment of the invention, a SNP detection
reagent of the invention is labeled with a fluorogenic reporter dye
that emits a detectable signal. While the preferred reporter dye is
a fluorescent dye, any reporter dye that can be attached to a
detection reagent such as an oligonucleotide probe or primer is
suitable for use in the invention. Such dyes include, but are not
limited to, Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5,
Cy7, Dabcyl, Edans, Eosin, Erythrosin, Fluorescein, 6-Fam, Tet,
Joe, Hex, Oregon Green, Rhodamine, Rhodol Green, Tamra, Rox, and
Texas Red.
[0148] In yet another embodiment of the invention, the detection
reagent may be further labeled with a quencher dye such as Tamra,
especially when the reagent is used as a self-quenching probe such
as a TaqMan (U.S. Pat. Nos. 5,210,015 and 5,538,848) or Molecular
Beacon probe (U.S. Pat. Nos. 5,118,801 and 5,312,728), or other
stemless or linear beacon probe (Livak et al., 1995, PCR Method
Appl. 4:357-362; Tyagi et al., 1996, Nature Biotechnology 14:
303-308; Nazarenko et al., 1997, Nucl. Acids Res. 25:2516-2521;
U.S. Pat. Nos. 5,866,336 and 6,117,635).
[0149] The detection reagents of the invention may also contain
other labels, including but not limited to, biotin for streptavidin
binding, hapten for antibody binding, and oligonucleotide for
binding to another complementary oligonucleotide such as pairs of
zipcodes.
[0150] The present invention also contemplates reagents that do not
contain (or that are complementary to) a SNP nucleotide identified
herein but that are used to assay one or more SNPs disclosed
herein. For example, primers that flank, but do not hybridize
directly to a target SNP position provided herein are useful in
primer extension reactions in which the primers hybridize to a
region adjacent to the target SNP position (i.e., within one or
more nucleotides from the target SNP site). During the primer
extension reaction, a primer is typically not able to extend past a
target SNP site if a particular nucleotide (allele) is present at
that target SNP site, and the primer extension product can readily
be detected in order to determine which SNP allele is present at
the target SNP site. For example, particular ddNTPs are typically
used in the primer extension reaction to terminate primer extension
once a ddNTP is incorporated into the extension product (a primer
extension product which includes a ddNTP at the 3'-most end of the
primer extension product, and in which the ddNTP corresponds to a
SNP disclosed herein, is a composition that is encompassed by the
present invention). Thus, reagents that bind to a nucleic acid
molecule in a region adjacent to a SNP site, even though the bound
sequences do not necessarily include the SNP site itself, are also
encompassed by the present invention.
SNP Detection Kits and Systems
[0151] A person skilled in the art will recognize that, based on
the SNP and associated sequence information disclosed herein,
detection reagents can be developed and used to assay any SNP of
the present invention individually or in combination, and such
detection reagents can be readily incorporated into one of the
established kit or system formats which are well known in the art.
The terms "kits" and "systems", as used herein in the context of
SNP detection reagents, are intended to refer to such things as
combinations of multiple SNP detection reagents, or one or more SNP
detection reagents in combination with one or more other types of
elements or components (e.g., other types of biochemical reagents,
containers, packages such as packaging intended for commercial
sale, substrates to which SNP detection reagents are attached,
electronic hardware components, etc.). Accordingly, the present
invention further provides SNP detection kits and systems,
including but not limited to, packaged probe and primer sets (e.g.,
TaqMan probe/primer sets), arrays/microarrays of nucleic acid
molecules, and beads that contain one or more probes, primers, or
other detection reagents for detecting one or more SNPs of the
present invention. The kits/systems can optionally include various
electronic hardware components; for example, arrays ("DNA chips")
and microfluidic systems ("lab-on-a-chip" systems) provided by
various manufacturers typically comprise hardware components. Other
kits/systems (e.g., probe/primer sets) may not include electronic
hardware components, but may be comprised of, for example, one or
more SNP detection reagents (along with, optionally, other
biochemical reagents) packaged in one or more containers.
[0152] In some embodiments, a SNP detection kit typically contains
one or more detection reagents and other components (e.g., a
buffer, enzymes such as DNA polymerases or ligases, chain extension
nucleotides such as deoxynucleotide triphosphates, and in the case
of Sanger-type DNA sequencing reactions, chain terminating
nucleotides, positive control sequences, negative control
sequences, and the like) necessary to carry out an assay or
reaction, such as amplification and/or detection of a
SNP-containing nucleic acid molecule. A kit may further contain
means for determining the amount of a target nucleic acid, and
means for comparing the amount with a standard, and can comprise
instructions for using the kit to detect the SNP-containing nucleic
acid molecule of interest. In one embodiment of the present
invention, kits are provided which contain the necessary reagents
to carry out one or more assays to detect one or more SNPs
disclosed herein. In a preferred embodiment of the present
invention, SNP detection kits/systems are in the form of nucleic
acid arrays, or compartmentalized kits, including
microfluidic/lab-on-a-chip systems.
[0153] SNP detection kits/systems may contain, for example, one or
more probes, or pairs of probes, that hybridize to a nucleic acid
molecule at or near each target SNP position. Multiple pairs of
allele-specific probes may be included in the kit/system to
simultaneously assay large numbers of SNPs, at least one of which
is a SNP of the present invention. In some kits/systems, the
allele-specific probes are immobilized to a substrate such as an
array or bead.
[0154] The terms "arrays", "microarrays", and "DNA chips" are used
herein interchangeably to refer to an array of distinct
polynucleotides affixed to a substrate, such as glass, plastic,
paper, nylon or other type of membrane, filter, chip, or any other
suitable solid support. The polynucleotides can be synthesized
directly on the substrate, or synthesized separate from the
substrate and then affixed to the substrate. In one embodiment, the
microarray is prepared and used according to the methods described
in U.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995
(Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14:
1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93:
10614-10619), all of which are incorporated herein in their
entirety by reference. In other embodiments, such arrays are
produced by the methods described by Brown et al., U.S. Pat. No.
5,807,522.
[0155] Nucleic acid arrays are reviewed in the following
references: Zammatteo et al., "New chips for molecular biology and
diagnostics", Biotechnol Annu Rev. 2002; 8:85-101; Sosnowski et
al., "Active microelectronic array system for DNA hybridization,
genotyping and pharmacogenomic applications", Psychiatr Genet. 2002
December; 12(4):181-92; Heller, "DNA microarray technology:
devices, systems, and applications", Annu Rev Biomed Eng. 2002;
4:129-53. Epub 2002 Mar. 22; Kolchinsky et al., "Analysis of SNPs
and other genomic variations using gel-based chips", Hum Mutat.
2002 April; 19(4):343-60; and McGall et al., "High-density genechip
oligonucleotide probe arrays", Adv Biochem Eng Biotechnol. 2002;
77:21-42.
[0156] Any number of probes, such as allele-specific probes, may be
implemented in an array, and each probe or pair of probes can
hybridize to a different SNP position. In the case of
polynucleotide probes, they can be synthesized at designated areas
(or synthesized separately and then affixed to designated areas) on
a substrate using a light-directed chemical process. Each DNA chip
can contain, for example, thousands to millions of individual
synthetic polynucleotide probes arranged in a grid-like pattern and
miniaturized (e.g., to the size of a dime). Preferably, probes are
attached to a solid support in an ordered, addressable array.
[0157] A microarray can be composed of a large number of unique,
single-stranded polynucleotides, usually either synthetic antisense
polynucleotides or fragments of cDNAs, fixed to a solid support.
Typical polynucleotides are preferably about 6-60 nucleotides in
length, more preferably about 15-30 nucleotides in length, and most
preferably about 18-25 nucleotides in length. For certain types of
microarrays or other detection kits/systems, it may be preferable
to use oligonucleotides that are only about 7-20 nucleotides in
length. In other types of arrays, such as arrays used in
conjunction with chemiluminescent detection technology, preferred
probe lengths can be, for example, about 15-80 nucleotides in
length, preferably about 50-70 nucleotides in length, more
preferably about 55-65 nucleotides in length, and most preferably
about 60 nucleotides in length. The microarray or detection kit can
contain polynucleotides that cover the known 5' or 3' sequence of a
gene/transcript or target SNP site, sequential polynucleotides that
cover the full-length sequence of a gene/transcript; or unique
polynucleotides selected from particular areas along the length of
a target gene/transcript sequence, particularly areas corresponding
to one or more SNPs, for instance the LCS6 SNP identified within
SEQ ID NOs: 21, 26, and 27. Polynucleotides used in the microarray
or detection kit can be specific to a SNP or SNPs of interest
(e.g., specific to a particular SNP allele at a target SNP site, or
specific to particular SNP alleles at multiple different SNP
sites), or specific to a polymorphic gene/transcript or
genes/transcripts of interest.
[0158] Hybridization assays based on polynucleotide arrays rely on
the differences in hybridization stability of the probes to
perfectly matched and mismatched target sequence variants. For SNP
genotyping, it is generally preferable that stringency conditions
used in hybridization assays are high enough such that nucleic acid
molecules that differ from one another at as little as a single SNP
position can be differentiated (e.g., typical SNP hybridization
assays are designed so that hybridization will occur only if one
particular nucleotide is present at a SNP position, but will not
occur if an alternative nucleotide is present at that SNP
position). Such high stringency conditions may be preferable when
using, for example, nucleic acid arrays of allele-specific probes
for SNP detection. Such high stringency conditions are described in
the preceding section, and are well known to those skilled in the
art and can be found in, for example, Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989),
6.3.1-6.3.6.
[0159] In other embodiments, the arrays are used in conjunction
with chemiluminescent detection technology. The following patents
and patent applications, which are all hereby incorporated by
reference, provide additional information pertaining to
chemiluminescent detection: U.S. patent application Ser. Nos.
10/620,332 and 10/620,333 describe chemiluminescent approaches for
microarray detection; U.S. Pat. Nos. 6,124,478, 6,107,024,
5,994,073, 5,981,768, 5,871,938, 5,843,681, 5,800,999, and
5,773,628 describe methods and compositions of dioxetane for
performing chemiluminescent detection; and U.S. published
application US2002/0110828 discloses methods and compositions for
microarray controls.
[0160] In one embodiment of the invention, a nucleic acid array can
comprise an array of probes of about 15-25 nucleotides in length.
In further embodiments, a nucleic acid array can comprise any
number of probes, in which at least one probe is capable of
detecting the LCS6 SNP of SEQ ID NOs: 21, 26 and 27, and/or at
least one probe comprises a fragment of one of the sequences
selected from the group consisting of those disclosed in the
Sequence Listing, sequences complementary thereto, and fragment
thereof comprising at least about 8 consecutive nucleotides,
preferably 10, 12, 15, 16, 18, 20, more preferably 22, 25, 30, 40,
47, 50, 55, 60, 65, 70, 80, 90, 100, or more consecutive
nucleotides (or any other number in-between) and containing (or
being complementary to) a novel SNP allele disclosed in SEQ ID NOs:
21, 26, and 27. In some embodiments, the nucleotide complementary
to the SNP site is within 5, 4, 3, 2, or 1 nucleotide from the
center of the probe, more preferably at the center of said
probe.
[0161] A polynucleotide probe can be synthesized on the surface of
the substrate by using a chemical coupling procedure and an ink jet
application apparatus, as described in PCT application WO95/251116
(Baldeschweiler et al.) which is incorporated herein in its
entirety by reference. In another aspect, a "gridded" array
analogous to a dot (or slot) blot may be used to arrange and link
cDNA fragments or oligonucleotides to the surface of a substrate
using a vacuum system, thermal, UV, mechanical or chemical bonding
procedures. An array, such as those described above, may be
produced by hand or by using available devices (slot blot or dot
blot apparatus), materials (any suitable solid support), and
machines (including robotic instruments), and may contain 8, 24,
96, 384, 1536, 6144 or more polynucleotides, or any other number
which lends itself to the efficient use of commercially available
instrumentation.
[0162] Using such arrays or other kits/systems, the present
invention provides methods of identifying the SNPs disclosed herein
in a test sample. Such methods typically involve incubating a test
sample of nucleic acids with an array comprising one or more probes
corresponding to at least one SNP position of the present
invention, and assaying for binding of a nucleic acid from the test
sample with one or more of the probes. Conditions for incubating a
SNP detection reagent (or a kit/system that employs one or more
such SNP detection reagents) with a test sample vary. Incubation
conditions depend on such factors as the format employed in the
assay, the detection methods employed, and the type and nature of
the detection reagents used in the assay. One skilled in the art
will recognize that any one of the commonly available
hybridization, amplification and array assay formats can readily be
adapted to detect the SNPs disclosed herein.
[0163] A SNP detection kit/system of the present invention may
include components that are used to prepare nucleic acids from a
test sample for the subsequent amplification and/or detection of a
SNP-containing nucleic acid molecule. Such sample preparation
components can be used to produce nucleic acid extracts (including
DNA and/or RNA), proteins or membrane extracts from any bodily
fluids (such as blood, serum, plasma, urine, saliva, phlegm,
gastric juices, semen, tears, sweat, etc.), skin, hair, cells
(especially nucleated cells), biopsies, buccal swabs or tissue
specimens. The test samples used in the above-described methods
will vary based on such factors as the assay format, nature of the
detection method, and the specific tissues, cells or extracts used
as the test sample to be assayed. Methods of preparing nucleic
acids, proteins, and cell extracts are well known in the art and
can be readily adapted to obtain a sample that is compatible with
the system utilized. Automated sample preparation systems for
extracting nucleic acids from a test sample are commercially
available, and examples are Qiagen's BioRobot 9600, Applied
Biosystems' PRISM 6700, and Roche Molecular Systems' COBAS
AmpliPrep System.
[0164] Another form of kit contemplated by the present invention is
a compartmentalized kit. A compartmentalized kit includes any kit
in which reagents are contained in separate containers. Such
containers include, for example, small glass containers, plastic
containers, strips of plastic, glass or paper, or arraying material
such as silica. Such containers allow one to efficiently transfer
reagents from one compartment to another compartment such that the
test samples and reagents are not cross-contaminated, or from one
container to another vessel not included in the kit, and the agents
or solutions of each container can be added in a quantitative
fashion from one compartment to another or to another vessel. Such
containers may include, for example, one or more containers which
will accept the test sample, one or more containers which contain
at least one probe or other SNP detection reagent for detecting one
or more SNPs of the present invention, one or more containers which
contain wash reagents (such as phosphate buffered saline,
Tris-buffers, etc.), and one or more containers which contain the
reagents used to reveal the presence of the bound probe or other
SNP detection reagents. The kit can optionally further comprise
compartments and/or reagents for, for example, nucleic acid
amplification or other enzymatic reactions such as primer extension
reactions, hybridization, ligation, electrophoresis (preferably
capillary electrophoresis), mass spectrometry, and/or laser-induced
fluorescent detection. The kit may also include instructions for
using the kit. Exemplary compartmentalized kits include
microfluidic devices known in the art (see, e.g., Weigl et al.,
"Lab-on-a-chip for drug development", Adv Drug Deliv Rev. 2003 Feb.
24; 55(3):349-77). In such microfluidic devices, the containers may
be referred to as, for example, microfluidic "compartments",
"chambers", or "channels".
[0165] Microfluidic devices, which may also be referred to as
"lab-on-a-chip" systems, biomedical micro-electro-mechanical
systems (bioMEMs), or multicomponent integrated systems, are
exemplary kits/systems of the present invention for analyzing SNPs.
Such systems miniaturize and compartmentalize processes such as
probe/target hybridization, nucleic acid amplification, and
capillary electrophoresis reactions in a single functional device.
Such microfluidic devices typically utilize detection reagents in
at least one aspect of the system, and such detection reagents may
be used to detect one or more SNPs of the present invention. One
example of a microfluidic system is disclosed in U.S. Pat. No.
5,589,136, which describes the integration of PCR amplification and
capillary electrophoresis in chips. Exemplary microfluidic systems
comprise a pattern of microchannels designed onto a glass, silicon,
quartz, or plastic wafer included on a microchip. The movements of
the samples may be controlled by electric, electroosmotic or
hydrostatic forces applied across different areas of the microchip
to create functional microscopic valves and pumps with no moving
parts. Varying the voltage can be used as a means to control the
liquid flow at intersections between the micro-machined channels
and to change the liquid flow rate for pumping across different
sections of the microchip. See, for example, U.S. Pat. No.
6,153,073, Dubrow et al., and U.S. Pat. No. 6,156,181, Parce et
al.
[0166] For genotyping SNPs, an exemplary microfluidic system may
integrate, for example, nucleic acid amplification, primer
extension, capillary electrophoresis, and a detection method such
as laser induced fluorescence detection. In a first step of an
exemplary process for using such an exemplary system, nucleic acid
samples are amplified, preferably by PCR. Then, the amplification
products are subjected to automated primer extension reactions
using ddNTPs (specific fluorescence for each ddNTP) and the
appropriate oligonucleotide primers to carry out primer extension
reactions which hybridize just upstream of the targeted SNP. Once
the extension at the 3' end is completed, the primers are separated
from the unincorporated fluorescent ddNTPs by capillary
electrophoresis. The separation medium used in capillary
electrophoresis can be, for example, polyacrylamide,
polyethyleneglycol or dextran. The incorporated ddNTPs in the
single nucleotide primer extension products are identified by
laser-induced fluorescence detection. Such an exemplary microchip
can be used to process, for example, at least 96 to 384 samples, or
more, in parallel.
Uses of Nucleic Acid Molecules
[0167] The nucleic acid molecules of the present invention have a
variety of uses, especially in the assessing the risk of developing
a disorder. Exemplary disorders include but are not limited to,
inflammatory, degenerative, metabolic, proliferative, circulatory,
cognitive, reproductive, and behavioral disorders. In a preferred
embodiment of the invention the disorder is cancer. For example,
the nucleic acid molecules are useful as hybridization probes, such
as for genotyping SNPs in messenger RNA, transcript, cDNA, genomic
DNA, amplified DNA or other nucleic acid molecules, and for
isolating full-length cDNA and genomic clones.
[0168] A probe can hybridize to any nucleotide sequence along the
entire length of a LCS-containing nucleic acid molecule.
Preferably, a probe of the present invention hybridizes to a region
of a target sequence that encompasses a SNP such as the sequences
of SEQ ID NOs: 21, 26, and 27. More preferably, a probe hybridizes
to a SNP-containing target sequence in a sequence-specific manner
such that it distinguishes the target sequence from other
nucleotide sequences which vary from the target sequence only by
which nucleotide is present at the SNP site. Such a probe is
particularly useful for detecting the presence of a SNP-containing
nucleic acid in a test sample, or for determining which nucleotide
(allele) is present at a particular SNP site (i.e., genotyping the
SNP site).
[0169] A nucleic acid hybridization probe may be used for
determining the presence, level, form, and/or distribution of
nucleic acid expression. The nucleic acid whose level is determined
can be DNA or RNA. Accordingly, probes specific for the SNPs
described herein can be used to assess the presence, expression
and/or gene copy number in a given cell, tissue, or organism. These
uses are relevant for diagnosis of disorders involving an increase
or decrease in gene expression relative to normal levels. In vitro
techniques for detection of mRNA include, for example, Northern
blot hybridizations and in situ hybridizations. In vitro techniques
for detecting DNA include Southern blot hybridizations and in situ
hybridizations (Sambrook and Russell, 2000, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y.).
[0170] Thus, the nucleic acid molecules of the invention can be
used as hybridization probes to detect the SNPs disclosed herein,
thereby determining whether an individual with the polymorphisms is
at risk for developing a disorder. Detection of a SNP associated
with a disease phenotype provides a prognostic tool for an active
disease and/or genetic predisposition to the disease.
[0171] The nucleic acid molecules of the invention are also useful
for designing ribozymes corresponding to all, or a part, of an mRNA
molecule expressed from a SNP-containing nucleic acid molecule
described herein.
[0172] The nucleic acid molecules of the invention are also useful
for constructing transgenic animals expressing all, or a part, of
the nucleic acid molecules and variant peptides. The production of
recombinant cells and transgenic animals having nucleic acid
molecules which contain the LCS6 SNP disclosed herein allow, for
example, effective clinical design of treatment compounds and
dosage regimens.
SNP Genotyping Methods
[0173] The process of determining which specific nucleotide (i.e.,
allele) is present at each of one or more SNP positions, such as a
SNP position in a nucleic acid molecule disclosed in SEQ ID NO: 21,
26 or 27, is referred to as SNP genotyping. The present invention
provides methods of SNP genotyping, such as for use in screening
for a variety of disorders, or determining predisposition thereto,
or determining responsiveness to a form of treatment, or prognosis,
or in genome mapping or SNP association analysis, etc.
[0174] Nucleic acid samples can be genotyped to determine which
allele(s) is/are present at any given genetic region (e.g., SNP
position) of interest by methods well known in the art. The
neighboring sequence can be used to design SNP detection reagents
such as oligonucleotide probes, which may optionally be implemented
in a kit format. Exemplary SNP genotyping methods are described in
Chen et al., "Single nucleotide polymorphism genotyping:
biochemistry, protocol, cost and throughput", Pharmacogenomics J.
2003; 3(2):77-96; Kwok et al., "Detection of single nucleotide
polymorphisms", Curr Issues Mol. Biol. 2003 April; 5(2):43-60; Shi,
"Technologies for individual genotyping: detection of genetic
polymorphisms in drug targets and disease genes", Am J
Pharmacogenomics. 2002; 2(3):197-205; and Kwok, "Methods for
genotyping single nucleotide polymorphisms", Annu Rev Genomics Hum
Genet 2001; 2:235-58. Exemplary techniques for high-throughput SNP
genotyping are described in Marnellos, "High-throughput SNP
analysis for genetic association studies", Curr Opin Drug Discov
Devel. 2003 May; 6(3):317-21. Common SNP genotyping methods
include, but are not limited to, TaqMan assays, molecular beacon
assays, nucleic acid arrays, allele-specific primer extension,
allele-specific PCR, arrayed primer extension, homogeneous primer
extension assays, primer extension with detection by mass
spectrometry, pyrosequencing, multiplex primer extension sorted on
genetic arrays, ligation with rolling circle amplification,
homogeneous ligation, OLA (U.S. Pat. No. 4,988,167), multiplex
ligation reaction sorted on genetic arrays, restriction-fragment
length polymorphism, single base extension-tag assays, and the
Invader assay. Such methods may be used in combination with
detection mechanisms such as, for example, luminescence or
chemiluminescence detection, fluorescence detection, time-resolved
fluorescence detection, fluorescence resonance energy transfer,
fluorescence polarization, mass spectrometry, and electrical
detection.
[0175] Various methods for detecting polymorphisms include, but are
not limited to, methods in which protection from cleavage agents is
used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes
(Myers et al., Science 230:1242 (1985); Cotton et al., PNAS 85:4397
(1988); and Saleeba et al., Meth. Enzymol. 217:286-295 (1992)),
comparison of the electrophoretic mobility of variant and wild type
nucleic acid molecules (Orita et al., PNAS 86:2766 (1989); Cotton
et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet.
Anal. Tech. Appl. 9:73-79 (1992)), and assaying the movement of
polymorphic or wild-type fragments in polyacrylamide gels
containing a gradient of denaturant using denaturing gradient gel
electrophoresis (DGGE) (Myers et al., Nature 313:495 (1985)).
Sequence variations at specific locations can also be assessed by
nuclease protection assays such as RNase and SI protection or
chemical cleavage methods.
[0176] In a preferred embodiment, SNP genotyping is performed using
the TaqMan assay, which is also known as the 5' nuclease assay
(U.S. Pat. Nos. 5,210,015 and 5,538,848). The TaqMan assay detects
the accumulation of a specific amplified product during PCR. The
TaqMan assay utilizes an oligonucleotide probe labeled with a
fluorescent reporter dye and a quencher dye. The reporter dye is
excited by irradiation at an appropriate wavelength, it transfers
energy to the quencher dye in the same probe via a process called
fluorescence resonance energy transfer (FRET). When attached to the
probe, the excited reporter dye does not emit a signal. The
proximity of the quencher dye to the reporter dye in the intact
probe maintains a reduced fluorescence for the reporter. The
reporter dye and quencher dye may be at the 5' most and the 3' most
ends, respectively, or vice versa. Alternatively, the reporter dye
may be at the 5' or 3' most end while the quencher dye is attached
to an internal nucleotide, or vice versa. In yet another
embodiment, both the reporter and the quencher may be attached to
internal nucleotides at a distance from each other such that
fluorescence of the reporter is reduced.
[0177] During PCR, the 5' nuclease activity of DNA polymerase
cleaves the probe, thereby separating the reporter dye and the
quencher dye and resulting in increased fluorescence of the
reporter. Accumulation of PCR product is detected directly by
monitoring the increase in fluorescence of the reporter dye. The
DNA polymerase cleaves the probe between the reporter dye and the
quencher dye only if the probe hybridizes to the target
SNP-containing template which is amplified during PCR, and the
probe is designed to hybridize to the target SNP site only if a
particular SNP allele is present.
[0178] Preferred TaqMan primer and probe sequences can readily be
determined using the SNP and associated nucleic acid sequence
information provided herein. A number of computer programs, such as
Primer Express (Applied Biosystems, Foster City, Calif.), can be
used to rapidly obtain optimal primer/probe sets. It will be
apparent to one of skill in the art that such primers and probes
for detecting the SNPs of the present invention are useful in
prognostic assays for a variety of disorders including cancer, and
can be readily incorporated into a kit format. The present
invention also includes modifications of the Taqman assay well
known in the art such as the use of Molecular Beacon probes (U.S.
Pat. Nos. 5,118,801 and 5,312,728) and other variant formats (U.S.
Pat. Nos. 5,866,336 and 6,117,635).
[0179] 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., Proc.
Natl. Acad Sci. USA 82:7575, 1985; Meyers et al., Science 230:1242,
1985) and proteins which recognize nucleotide mismatches, such as
the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253,
1991). Alternatively, variant alleles can be identified by single
strand conformation polymorphism (SSCP) analysis (Orita et al.,
Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis
of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or
denaturing gradient gel electrophoresis (DGGE) (Wartell et al.,
Nuci. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Nati.
Acad. Sci. USA 86:232-236, 1989).
[0180] A polymerase-mediated primer extension method may also be
used to identify the polymorphism(s). Several such methods have
been described in the patent and scientific literature and include
the "Genetic Bit Analysis" method (WO92/15712) and the
ligase/polymerase mediated genetic bit analysis (U.S. Pat. No.
5,679,524). Related methods are disclosed in WO91/02087,
WO90/09455, WO95/17676, U.S. Pat. Nos. 5,302,509, and 5,945,283.
Extended primers containing a polymorphism may be detected by mass
spectrometry as described in U.S. Pat. No. 5,605,798. Another
primer extension method is allele-specific PCR (Ruano et al., Nucl.
Acids Res. 17:8392, 1989; Ruano et al., Nucl. Acids Res. 19,
6877-6882, 1991; WO 93/22456; Turki et al., J Clin. Invest.
95:1635-1641, 1995). In addition, multiple polymorphic sites may be
investigated by simultaneously amplifying multiple regions of the
nucleic acid using sets of allele-specific primers as described in
Wallace et al. (WO89/10414).
[0181] Another preferred method for genotyping the SNPs of the
present invention is the use of two oligonucleotide probes in an
OLA (see, e.g., U.S. Pat. No. 4,988,617). In this method, one probe
hybridizes to a segment of a target nucleic acid with its 3' most
end aligned with the SNP site. A second probe hybridizes to an
adjacent segment of the target nucleic acid molecule directly 3' to
the first probe. The two juxtaposed probes hybridize to the target
nucleic acid molecule, and are ligated in the presence of a linking
agent such as a ligase if there is perfect complementarity between
the 3' most nucleotide of the first probe with the SNP site. If
there is a mismatch, ligation would not occur. After the reaction,
the ligated probes are separated from the target nucleic acid
molecule, and detected as indicators of the presence of a SNP.
[0182] The following patents, patent applications, and published
international patent applications, which are all hereby
incorporated by reference, provide additional information
pertaining to techniques for carrying out various types of OLA:
U.S. Pat. Nos. 6,027,889, 6,268,148, 5,494,810, 5,830,711, and
6,054,564 describe OLA strategies for performing SNP detection; WO
97/31256 and WO 00/56927 describe OLA strategies for performing SNP
detection using universal arrays, wherein a zipcode sequence can be
introduced into one of the hybridization probes, and the resulting
product, or amplified product, hybridized to a universal zip code
array; U.S. application Ser. No. 01/17,329 (and Ser. No.
09/584,905) describes OLA (or LDR) followed by PCR, wherein
zipcodes are incorporated into OLA probes, and amplified PCR
products are determined by electrophoretic or universal zipcode
array readout; U.S. application 60/427,818, 60/445,636, and
60/445,494 describe SNP1ex methods and software for multiplexed SNP
detection using OLA followed by PCR, wherein zipcodes are
incorporated into OLA probes, and amplified PCR products are
hybridized with a zipchute reagent, and the identity of the SNP
determined from electrophoretic readout of the zipchute. In some
embodiments, OLA is carried out prior to PCR (or another method of
nucleic acid amplification). In other embodiments, PCR (or another
method of nucleic acid amplification) is carried out prior to
OLA.
[0183] Another method for SNP genotyping is based on mass
spectrometry. Mass spectrometry takes advantage of the unique mass
of each of the four nucleotides of DNA. SNPs can be unambiguously
genotyped by mass spectrometry by measuring the differences in the
mass of nucleic acids having alternative SNP alleles. MALDI-TOF
(Matrix Assisted Laser Desorption Ionization--Time of Flight) mass
spectrometry technology is preferred for extremely precise
determinations of molecular mass, such as SNPs. Numerous approaches
to SNP analysis have been developed based on mass spectrometry.
Preferred mass spectrometry-based methods of SNP genotyping include
primer extension assays, which can also be utilized in combination
with other approaches, such as traditional gel-based formats and
microarrays.
[0184] Typically, the primer extension assay involves designing and
annealing a primer to a template PCR amplicon upstream (5') from a
target SNP position. A mix of dideoxynucleotide triphosphates
(ddNTPs) and/or deoxynucleotide triphosphates (dNTPs) are added to
a reaction mixture containing template (e.g., a SNP-containing
nucleic acid molecule which has typically been amplified, such as
by PCR), primer, and DNA polymerase. Extension of the primer
terminates at the first position in the template where a nucleotide
complementary to one of the ddNTPs in the mix occurs. The primer
can be either immediately adjacent (i.e., the nucleotide at the 3'
end of the primer hybridizes to the nucleotide next to the target
SNP site) or two or more nucleotides removed from the SNP position.
If the primer is several nucleotides removed from the target SNP
position, the only limitation is that the template sequence between
the 3' end of the primer and the SNP position cannot contain a
nucleotide of the same type as the one to be detected, or this will
cause premature termination of the extension primer. Alternatively,
if all four ddNTPs alone, with no dNTPs, are added to the reaction
mixture, the primer will always be extended by only one nucleotide,
corresponding to the target SNP position. In this instance, primers
are designed to bind one nucleotide upstream from the SNP position
(i.e., the nucleotide at the 3' end of the primer hybridizes to the
nucleotide that is immediately adjacent to the target SNP site on
the 5' side of the target SNP site). Extension by only one
nucleotide is preferable, as it minimizes the overall mass of the
extended primer, thereby increasing the resolution of mass
differences between alternative SNP nucleotides. Furthermore,
mass-tagged ddNTPs can be employed in the primer extension
reactions in place of unmodified ddNTPs. This increases the mass
difference between primers extended with these ddNTPs, thereby
providing increased sensitivity and accuracy, and is particularly
useful for typing heterozygous base positions. Mass-tagging also
alleviates the need for intensive sample-preparation procedures and
decreases the necessary resolving power of the mass
spectrometer.
[0185] The extended primers can then be purified and analyzed by
MALDI-TOF mass spectrometry to determine the identity of the
nucleotide present at the target SNP position. In one method of
analysis, the products from the primer extension reaction are
combined with light absorbing crystals that form a matrix. The
matrix is then hit with an energy source such as a laser to ionize
and desorb the nucleic acid molecules into the gas-phase. The
ionized molecules are then ejected into a flight tube and
accelerated down the tube towards a detector. The time between the
ionization event, such as a laser pulse, and collision of the
molecule with the detector is the time of flight of that molecule.
The time of flight is precisely correlated with the mass-to-charge
ratio (m/z) of the ionized molecule. Ions with smaller m/z travel
down the tube faster than ions with larger m/z and therefore the
lighter ions reach the detector before the heavier ions. The
time-of-flight is then converted into a corresponding, and highly
precise, m/z. In this manner, SNPs can be identified based on the
slight differences in mass, and the corresponding time of flight
differences, inherent in nucleic acid molecules having different
nucleotides at a single base position. For further information
regarding the use of primer extension assays in conjunction with
MALDI-TOF mass spectrometry for SNP genotyping, see, e.g., Wise et
al., "A standard protocol for single nucleotide primer extension in
the human genome using matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry", Rapid Commun Mass Spectrom.
2003; 17(11):1195-202.
[0186] The following references provide further information
describing mass spectrometry-based methods for SNP genotyping:
Bocker, "SNP and mutation discovery using base-specific cleavage
and MALDI-TOF mass spectrometry", Bioinformatics. 2003 July; 19
Suppl 1:144-153; Storm et al., "MALDI-TOF mass spectrometry-based
SNP genotyping", Methods Mol. Biol. 2003; 212:241-62; Jurinke et
al., "The use of MassARRAY technology for high throughput
genotyping", Adv Biochem Eng Biotechnol. 2002;77:57-74; and Jurinke
et al., "Automated genotyping using the DNA MassArray technology",
Methods Mol. Biol. 2002; 187:179-92.
[0187] SNPs can also be scored by direct DNA sequencing. A variety
of automated sequencing procedures can be utilized ((1995)
Biotechniques 19:448), including sequencing by mass spectrometry
(see, e.g., PCT International Publication No. WO94/16101; Cohen et
al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl.
Biochem. Biotechnol. 38:147-159 (1993)). The nucleic acid sequences
of the present invention enable one of ordinary skill in the art to
readily design sequencing primers for such automated sequencing
procedures. Commercial instrumentation, such as the Applied
Biosystems 377, 3100, 3700, 3730, and 3730.times.1 DNA Analyzers
(Foster City, Calif.), is commonly used in the art for automated
sequencing.
[0188] Other methods that can be used to genotype the SNPs of the
present invention include single-strand conformational polymorphism
(SSCP), and denaturing gradient gel electrophoresis (DGGE) (Myers
et al., Nature 313:495 (1985)). SSCP identifies base differences by
alteration in electrophoretic migration of single stranded PCR
products, as described in Orita et al., Proc. Nat. Acad.
Single-stranded PCR products can be generated by heating or
otherwise denaturing double stranded PCR products. Single-stranded
nucleic acids may refold or form secondary structures that are
partially dependent on the base sequence. The different
electrophoretic mobilities of single-stranded amplification
products are related to base-sequence differences at SNP positions.
DGGE differentiates SNP alleles based on the different
sequence-dependent stabilities and melting properties inherent in
polymorphic DNA and the corresponding differences in
electrophoretic migration patterns in a denaturing gradient gel
(Erlich, ed., PCR Technology, Principles and Applications for DNA
Amplification, W. H. Freeman and Co, New York, 1992, Chapter
7).
[0189] Sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can
also be used to score SNPs based on the development or loss of a
ribozyme cleavage site. Perfectly matched sequences can be
distinguished from mismatched sequences by nuclease cleavage
digestion assays or by differences in melting temperature. If the
SNP affects a restriction enzyme cleavage site, the SNP can be
identified by alterations in restriction enzyme digestion patterns,
and the corresponding changes in nucleic acid fragment lengths
determined by gel electrophoresis
[0190] SNP genotyping can include the steps of, for example,
collecting a biological sample from a human subject (e.g., sample
of tissues, cells, fluids, secretions, etc.), isolating nucleic
acids (e.g., genomic DNA, mRNA or both) from the cells of the
sample, contacting the nucleic acids with one or more primers which
specifically hybridize to a region of the isolated nucleic acid
containing a target SNP under conditions such that hybridization
and amplification of the target nucleic acid region occurs, and
determining the nucleotide present at the SNP position of interest,
or, in some assays, detecting the presence or absence of an
amplification product (assays can be designed so that hybridization
and/or amplification will only occur if a particular SNP allele is
present or absent). In some assays, the size of the amplification
product is detected and compared to the length of a control sample;
for example, deletions and insertions can be detected by a change
in size of the amplified product compared to a normal genotype.
[0191] SNP genotyping is useful for numerous practical
applications, as described below. Examples of such applications
include, but are not limited to, SNP-disease association analysis,
disease predisposition screening, disease diagnosis, disease
prognosis, disease progression monitoring, determining therapeutic
strategies based on an individual's genotype ("pharmacogenomics"),
developing therapeutic agents based on SNP genotypes associated
with a disease or likelihood of responding to a drug, stratifying a
patient population for clinical trial for a treatment regimen, and
predicting the likelihood that an individual will experience toxic
side effects from a therapeutic agent.
Disease Screening Assays
[0192] Information on association/correlation between genotypes and
disease-related phenotypes can be exploited in several ways. For
example, in the case of a highly statistically significant
association between one or more SNPs with predisposition to a
disease for which treatment is available, detection of such a
genotype pattern in an individual may justify immediate
administration of treatment, or at least the institution of regular
monitoring of the individual. In the case of a weaker but still
statistically significant association between a SNP and a human
disease, immediate therapeutic intervention or monitoring may not
be justified after detecting the susceptibility allele or SNP.
Nevertheless, the subject can be motivated to begin simple
life-style changes (e.g., diet, exercise, quit smoking, increased
monitoring/examination) that can be accomplished at little or no
cost to the individual but would confer potential benefits in
reducing the risk of developing conditions for which that
individual may have an increased risk by virtue of having the
susceptibility allele(s).
[0193] In one aspect, the invention provides methods of identifying
SNPs which increase the risk, susceptibility, or probability of
developing a disease such as a cell proliferative disorder (e.g.
cancer). In a further aspect, the invention provides methods for
identifying a subject at risk for developing a disease, determining
the prognosis a disease or predicting the onset of a disease. For
example, a subject's risk of developing a cell proliferative
disease, the prognosis of an individual with a disease, or the
predicted onset of a cell proliferative disease is are determined
by detecting a mutation in the 3' untranslated region (UTR) of a
member of the RAS gene superfamily. In a specific example, a
subject's risk of developing a cell proliferative disease, the
prognosis of an individual with a disease, or the predicted onset
of a cell proliferative disease is are determined by detecting a
mutation in the 3' untranslated region (UTR) of KRAS.
Identification of the mutation indicates an increases risk of
developing a cell proliferative disorder, poor prognosis or an
earlier onset of developing a cell proliferative disorder.
[0194] The mutation is for example a deletion, insertion,
inversion, substitution, frameshift or recombination. In one
aspect, the mutation occurs within a let-7 complementary site
(LCS). The mutation is for example, one or more SNPs in the 3'
untranslated region of RAS. RAS includes KRAS, HRAS, or NRAS. For
example the mutation is a SNP at position 4 of LCS6 of KRAS of
which results in a uracil (U) or thymine (T) to guanine (G)
conversion.
[0195] The mutation modulates, e.g. increases or decreases, the
binding efficacy of an miRNA, such as a let-7 family miRNA. By
"binding efficacy" it is meant the ability of a miRNA molecule to
bind to a target gene or transcript, and therefore, silence,
decrease, reduce, inhibit, or prevent the transcription or
translation of the target gene or transcript, respectively. Binding
efficacy is determined by the ability of the miRNA to inhibit
protein production or inhibit reporter protein production.
Alternatively, or in addition, binding efficacy is defined as
binding energy and measured in minimum free energy (mfe)
(kilocalories/mole) (see FIGS. 26 and 16).
[0196] "Risk" in the context of the present invention, relates to
the probability that an event will occur over a specific time
period, and can mean a subject's "absolute" risk or "relative"
risk. Absolute risk can be measured with reference to either actual
observation post-measurement for the relevant time cohort, or with
reference to index values developed from statistically valid
historical cohorts that have been followed for the relevant time
period. Relative risk refers to the ratio of absolute risks of a
subject compared either to the absolute risks of low risk cohorts
or an average population risk, which can vary by how clinical risk
factors are assessed. Odds ratios, the proportion of positive
events to negative events for a given test result, are also
commonly used (odds are according to the formula p/(1-p) where p is
the probability of event and (1-p) is the probability of no event)
to no-conversion.
[0197] "Risk evaluation," or "evaluation of risk" in the context of
the present invention encompasses making a prediction of the
probability, odds, or likelihood that an event or disease state may
occur, the rate of occurrence of the event or conversion from one
disease state to another, i.e., from a primary tumor to a
metastatic tumor or to one at risk of developing a metastatic, or
from at risk of a primary metastatic event to a secondary
metastatic event or from at risk of a developing a primary tumor of
one type to developing a one or more primary tumors of a different
type. Risk evaluation can also comprise prediction of future
clinical parameters, traditional laboratory risk factor values, or
other indices of cancer, either in absolute or relative terms in
reference to a previously measured population.
[0198] An "increased risk" is meant to describe an increased
probably that an individual who carries a SNP within a let-7 family
miRNA binding site, particularly the LCS6 SNP, will develop at
least one of a variety of disorders, such as cancer, compared to an
individual who does not carry a SNP within a let-7 family miRNA
binding site. In certain embodiments, a LCS6 SNP carrier is
1.5.times., 2.times., 2.5.times., 3.times., 3.5.times., 4.times.,
4.5.times., 5.times., 5.5.times., 6.times., 6.5.times., 7.times.,
7.5.times., 8.times., 8.5.times., 9.times., 9.5.times., 10.times.,
20.times., 30.times., 40.times., 50.times., 60.times., 70.times.,
80.times., 90.times., or 100.times. more likely to develop at least
one type of cancer than an individual who does not carry the LCS6
SNP. Moreover, an increased risk is meant to describe an increased
susceptibility to developing at least one of a variety of
disorders. In a specific embodiment, individuals who carry the LCS6
SNP are more susceptible to the deleterious effects of smoking and
develop smoking-induced non-small cell lung cancer (NSCLC) earlier
and more frequently than smokers who do not carry the LCS6 SNP. In
certain embodiments, LCS6 SNP carriers who smoke develop at least
one type of cancer 1, 2, 5, 7, 10, 12, 15, 17, 20, 22, 25, 27, or
30 years prior to the average age that a smoker who does not carry
the LCS6 SNP develops at least one type of cancer. In other
embodiments, a LCS6 SNP carrier who smokes is 1.5.times., 2.times.,
2.5.times., 3.times., 3.5.times., 4.times., 4.5.times., 5.times.,
5.5.times., 6.times., 6.5.times., 7.times., 7.5.times., 8.times.,
8.5.times., 9.times., 9.5.times., 10.times., 20.times., 30.times.,
40.times., 50.times., 60.times., 70.times., 80.times., 90.times.,
or 100.times. more likely to develop at least one type of cancer
than a smoking individual who does not carry the LCS6 SNP.
Moreover, carriers of the LCS6 SNP who have developed one cancer
are more likely to develop secondary cancers. In certain
embodiments, LCS6 SNP carriers who smoke develop at least one
secondary cancer 1, 2, 5, 7, 10, 12, 15, 17, 20, 22, 25, 27, or 30
years prior to the average age that a smoker who does not carry the
LCS6 SNP develops at least one secondary cancer.
[0199] By poor prognosis is meant that the probability of the
individual surviving the development of particularly aggressive or
high-risk subtypes of cancer is less than the probability of
surviving more benign forms. Poor prognosis is also meant to
describe a less satisfactory recovery, longer recovery period, more
invasive or high-risk therapeutic regime, or an increased
probability of reoccurrence of the cancer. It has been shown that
the LCS6 SNP is predicative of the occurrence of aggressive
subtypes of cancer. These aggressive subtypes of cancers are
associated with the worst prognosis of each of these cancer
resulting in a poor prognosis.
[0200] "Predicting the onset" is meant to describe a method of
detecting the presence of a SNP within an miRNA binding site that
not only predicts the development of a disorder, but also
correlates with an earlier presentation of that disorder. In a
preferred embodiment, the disorder that develops as a result of the
SNP is cancer. For example, it has been shown that cancer patients
who carry the LCS6 SNP are younger, on average, than other cancer
patients. As such, individuals who carry the LCS6 SNP will
experience the onset of particular types of cancer including, but
not limited to, all varieties of lung cancer (NSCLC and small cell
lung cancer), ovarian cancer, breast cancer, uterine cancer, head
and neck cancer, pancreatic cancer, and colon cancer at an earlier
age. In certain embodiments, the presence of the LCS6 SNP, predicts
that presentation of at least one type of cancer 1, 2, 5, 7, 10,
12, 15, 17, 20, 22, 25, 27, or 30 years prior to the average age
that an individual who does not carry the LCS6 SNP develops at
least one type of cancer. In other embodiments, the identification
of a SNP within an miRNA binding site of the invention, predicts
that presentation of at least one disorder 1, 2, 5, 7, 10, 12, 15,
17, 20, 22, 25, 27, or 30 years prior to the average age that an
individual who does not carry the same SNP develops this same
disorder.
[0201] Cell proliferative disorders include a variety of conditions
wherein cell division is deregulated. Exemplary cell proliferative
disorder include, but are not limited to, neoplasms, benign tumors,
malignant tumors, pre-cancerous conditions, in situ tumors,
encapsulated tumors, metastatic tumors, liquid tumors, solid
tumors, immunological tumors, hematological tumors, cancers,
carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing
cells. The term "rapidly dividing cell" as used herein is defined
as any cell that divides at a rate that exceeds or is greater than
what is expected or observed among neighboring or juxtaposed cells
within the same tissue.
[0202] Cancers include, but are not limited to, acute lymphoblastic
leukemia, acute myeloid leukemia, adrenocortical carcinoma,
adrenocortical carcinoma, AIDS-related cancers, AIDS-related
lymphoma, anal cancer, appendix cancer, childhood cerebellar
astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma,
skin cancer (non-melanoma), extrahepatic bile duct cancer, bladder
cancer, bone cancer, osteosarcoma and malignant fibrous
histiocytoma, brain tumor, brain stem glioma, cerebellar
astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma,
medulloblastoma, supratentorial primitive neuroectodermal tumors,
visual pathway and hypothalamic glioma, breast cancer, bronchial
adenomas/carcinoids, carcinoid tumor, gastrointestinal, central
nervous system lymphoma, cervical cancer, childhood cancers,
chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic
myeloproliferative disorders, colon cancer, colorectal cancer,
cutaneous T-cell lymphoma, mycosis fungoides, Sezary Syndrome,
endometrial cancer, esophageal cancer, extracranial germ cell
tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer,
eye cancer, intraocular melanoma, retinoblastoma, gallbladder
cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor,
gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian
germ cell tumor, gestational trophoblastic tumor glioma, head and
neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma,
hypopharyngeal cancer, intraocular melanoma, islet cell tumors
(endocrine pancreas), Kaposi Sarcoma, kidney (renal cell) cancer,
kidney cancer, laryngeal cancer, acute lymphoblastic leukemia,
acute myeloid leukemia, chronic lymphocytic leukemia, chronic
myelogenous leukemia, hairy cell leukemia, lip and oral cavity
cancer, liver cancer, non-small cell lung cancer, small cell lung
cancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary
central nervous system lymphoma, Waldenstrom macroglobulinemia,
medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell
carcinoma, mesothelioma malignant, mesothelioma, metastatic
squamous neck cancer, mouth cancer, multiple endocrine neoplasia
syndrome, mycosis fungoides, myelodysplastic syndromes,
myelodysplastic/myeloproliferative diseases, chronic myelogenous
leukemia, acute myeloid leukemia, multiple myeloma, chronic
myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma,
oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian
cancer, ovarian epithelial cancer, ovarian low malignant potential
tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal
sinus and nasal cavity cancer, parathyroid cancer, penile cancer,
pharyngeal cancer, pheochromocytoma, pineoblastoma and
supratentorial primitive neuroectodermal tumors, pituitary tumor,
plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma,
prostate cancer, rectal cancer, renal pelvis and ureter,
transitional cell cancer, retinoblastoma, rhabdomyosarcoma,
salivary gland cancer, ewing family of sarcoma tumors, Kaposi
Sarcoma, soft tissue sarcoma, uterine sarcoma, skin cancer
(nonmelanoma), skin cancer (melanoma), merkel cell skin carcinoma,
small intestine cancer, soft tissue sarcoma, squamous cell
carcinoma, stomach (gastric) cancer, supratentorial primitive
neuroectodermal tumors, testicular Cancer, throat Cancer, thymoma,
thymoma and thymic carcinoma, thyroid cancer, transitional cell
cancer of the renal pelvis and ureter, gestational trophoblastic
tumor, urethral cancer, endometrial uterine cancer, uterine
sarcoma, vaginal cancer, vulvar cancer, and Wilms Tumor.
[0203] A subject is preferably a mammal. The mammal can be a human,
non-human primate, mouse, rat, dog, cat, horse, or cow, but are not
limited to these examples. Mammals other than humans can be
advantageously used as subjects that represent animal models of a
particular disease. A subject can be male or female. A subject can
be one who has been previously diagnosed or identified as having a
disease and optionally has already undergone, or is undergoing, a
therapeutic intervention for the disease. Alternatively, a subject
can also be one who has not been previously diagnosed as having the
disease. For example, a subject can be one who exhibits one or more
risk factors for a disease.
[0204] The biological sample can be any tissue or fluid that
contains nucleic acids. Various embodiments include paraffin
imbedded tissue, frozen tissue, surgical fine needle aspirations,
cells of the skin, muscle, lung, head and neck, esophagus, kidney,
pancreas, mouth, throat, pharynx, larynx, esophagus, facia, brain,
prostate, breast, endometrium, small intestine, blood cells, liver,
testes, ovaries, uterus, cervix, colon, stomach, spleen, lymph
node, bone marrow or kidney. Other embodiments include fluid
samples such as bronchial brushes, bronchial washes, bronchial
ravages, peripheral blood lymphocytes, lymph fluid, ascites, serous
fluid, pleural effusion, sputum, cerebrospinal fluid, lacrimal
fluid, esophageal washes, and stool or urinary specimens such as
bladder washing and urine.
[0205] Linkage disequilibrium (LD) refers to the co-inheritance of
alleles (e.g., alternative nucleotides) at two or more different
SNP sites at frequencies greater than would be expected from the
separate frequencies of occurrence of each allele in a given
population. The expected frequency of co-occurrence of two alleles
that are inherited independently is the frequency of the first
allele multiplied by the frequency of the second allele. Alleles
that co-occur at expected frequencies are said to be in "linkage
equilibrium". In contrast, LD refers to any non-random genetic
association between allele(s) at two or more different SNP sites,
which is generally due to the physical proximity of the two loci
along a chromosome. LD can occur when two or more SNPs sites are in
close physical proximity to each other on a given chromosome and
therefore alleles at these SNP sites will tend to remain
unseparated for multiple generations with the consequence that a
particular nucleotide (allele) at one SNP site will show a
non-random association with a particular nucleotide (allele) at a
different SNP site located nearby. Hence, genotyping one of the SNP
sites will give almost the same information as genotyping the other
SNP site that is in LD.
[0206] For screening individuals for genetic disorders (e.g.
prognostic or risk) purposes, if a particular SNP site is found to
be useful for screening a disorder, then the skilled artisan would
recognize that other SNP sites which are in LD with this SNP site
would also be useful for screening the condition. Various degrees
of LD can be encountered between two or more SNPs with the result
being that some SNPs are more closely associated (i.e., in stronger
LD) than others. Furthermore, the physical distance over which LD
extends along a chromosome differs between different regions of the
genome, and therefore the degree of physical separation between two
or more SNP sites necessary for LD to occur can differ between
different regions of the genome.
[0207] For screening applications, polymorphisms (e.g., SNPs and/or
haplotypes) that are not the actual disease-causing (causative)
polymorphisms, but are in LD with such causative polymorphisms, are
also useful. In such instances, the genotype of the polymorphism(s)
that is/are in LD with the causative polymorphism is predictive of
the genotype of the causative polymorphism and, consequently,
predictive of the phenotype (e.g., disease) that is influenced by
the causative SNP(s). Thus, polymorphic markers that are in LD with
causative polymorphisms are useful as markers, and are particularly
useful when the actual causative polymorphism(s) is/are
unknown.
[0208] Linkage disequilibrium in the human genome is reviewed in:
Wall et al., "Haplotype blocks and linkage disequilibrium in the
human genome", Nat Rev Genet. 2003 August; 4(8):587-97; Gamer et
al., "On selecting markers for association studies: patterns of
linkage disequilibrium between two and three diallelic loci", Genet
Epidemiol. 2003 January; 24(1):57-67; Ardlie et al., "Patterns of
linkage disequilibrium in the human genome", Nat Rev Genet. 2002
April; 3(4):299-309 (erratum in Nat Rev Genet 2002 July; 3(7):566);
and Remm et al., "High-density genotyping and linkage
disequilibrium in the human genome using chromosome 22 as a model";
Curr Opin Chem Biol. 2002 February; 6(1):24-30.
[0209] The contribution or association of particular SNPs and/or
SNP haplotypes with disease phenotypes, such as cancer, enables the
SNPs of the present invention to be used to develop superior tests
capable of identifying individuals who express a detectable trait,
such as cancer, as the result of a specific genotype, or
individuals whose genotype places them at an increased or decreased
risk of developing a detectable trait at a subsequent time as
compared to individuals who do not have that genotype. As described
herein, screening may be based on a single SNP or a group of SNPs.
To increase the accuracy of predisposition/risk screening, analysis
of the SNPs of the present invention can be combined with that of
other polymorphisms or other risk factors of the disease, such as
disease symptoms, pathological characteristics, family history,
diet, environmental factors or lifestyle factors.
[0210] The screening techniques of the present invention may employ
a variety of methodologies to determine whether a test subject has
a SNP or a SNP pattern associated with an increased or decreased
risk of developing a detectable trait or whether the individual
suffers from a detectable trait as a result of a particular
polymorphism/mutation, including, for example, methods which enable
the analysis of individual chromosomes for haplotyping, family
studies, single sperm DNA analysis, or somatic hybrids. The trait
analyzed using the diagnostics of the invention may be any
detectable trait that is commonly observed in pathologies and
disorders.
EXAMPLES
Example 1
General Methods
Study Populations
[0211] Lung tissue samples from patients with a diagnosis of NSCLC
were collected following Yale University Human Investigation
Committee approval. Cases were chosen based on the availability of
frozen stored tissue from lung tumor resections from 1994 through
2003, and from recent cases with extra tissue available. Tissue was
collected from 87 patients. Seven patients were excluded due to
other risk factors for lung cancer (e.g., immunosuppression,
tuberculosis) and six were excluded due to their tumors being
non-lung primary metastatic disease. Seventy-four patients were
included in the analysis (Table 2).
TABLE-US-00009 TABLE 2 Table 2. Yale NSCLC patient characteristics.
Proportion male (M) (51.3%) and female (F) (48.6%), proportion
Caucasian (85.1%), African American (AA)(8.1%), Hispanic (2.7%) and
Asian (4.1%). For patients with multiple simultaneously diagnosed
lung cancers both cancer types are listed. The presence or absence
of the SNP is denoted as Yes (Y) or No (N). Pack- LCS6 Patient Sex
Population Age Year Cancer Type SNP 1 F Caucasian 64 150
Adenocarcinoma N 2 M Caucasian 73 20 Adenosquamous N 3 M Caucasian
64 50 Large cell N 4 F Caucasian 76 unknown Adenocarcinoma N 5 M
Hispanic 54 70 Squamous cell N 6 M Caucasian 64 10 Squamous cell Y
7 M Caucasian 86 60 Squamous cell N 8 F Caucasian 54 0
Adenocarcinoma N 9 F Caucasian 58 40 Adenosquamous N 10 F Caucasian
65 150 Adenocarcinoma N 11 M Caucasian 64 17 Adenocarcinoma Y 12 F
Hispanic 65 20 Adenocarcinoma N 13 F Caucasian 89 unknown
Adenocarcinoma N 14 F Caucasian 47 0 Large cell N 15 M Caucasian 48
45 Squamous cell Y 16 M Caucasian 85 70 Adenocarcinoma N 17 M
Caucasian 86 75 Adenosquamous N 18 F Caucasian 49 15 Squamous cell,
Y Adenocarcinoma 19 M Asian 55 40 Squamous cell N 20 F Caucasian 74
0 Adenocarcinoma N 21 F Caucasian 58 2 Adenocarcinoma Y 22 F AA 40
20 Adenocarcinoma N 23 F Caucasian 52 30 Adenocarcinoma, N BAC 24 M
Caucasian 50 40 Large cell N 25 M Caucasian 69 105 Adenocarcinoma N
26 F Caucasian 75 50 Adenocarcinoma N 27 M Caucasian 83 60 Large
cell N 28 M Caucasian 42 20 Adenocarcinoma Y 29 F Caucasian 52 35
Adenocarcinoma N 30 M Caucasian 71 70 Adenocarcinoma, N Squamous
cell 31 F AA 69 50 Adenocarcinoma N 32 M Caucasian 44 50
Adenocarcinoma N 33 F Caucasian 66 2.sup.nd hand Adenocarcinoma Y
34 F Caucasian 73 75 Adenocarcinoma N 35 M AA 72 30 Adenocarcinoma
N 36 F Caucasian 72 120 Squamous cell Y 37 F Caucasian 62 50
Adenocarcinoma N 38 M Caucasian 74 unknown Adenocarcinoma N 39 M
Caucasian 78 20 Adenocarcinoma N 40 M Caucasian 68 32
Adenocarcinoma N 41 M Caucasian 66 40 Adenosquamous N 42 M
Caucasian 88 40 Squamous cell Y 43 F Caucasian 63 60 Adenocarcinoma
N 44 F AA 69 0 Adenocarcinoma N 45 M Caucasian 60 17 Squamous cell
Y 46 F AA 49 50 Adenocarcinoma N 47 F Caucasian 65 60 Adenosquamous
N 48 M Caucasian 63 45 Adenocarcinoma N 49 F Asian 67 0
Adenocarcinoma N 50 M Caucasian 60 100 Adenocarcinoma Y 51 M
Caucasian 65 125 Carcinoma N 52 M Caucasian 52 80 Squamous cell N
53 M Caucasian 62 90 Squamous cell N 54 F Caucasian 69 140 Large
cell N 55 F Caucasian 61 40 Squamous cell N 56 F Caucasian 80 15
Squamous cell N 57 M Caucasian 73 60 Squamous cell Y 58 F Caucasian
57 120 Adenocarcinoma N 59 F Caucasian 56 15 Adenocarcinoma N 60 M
Caucasian 43 40 Adenocarcinoma Y 61 F Asian 47 10 Adenocarcinoma N
62 F Caucasian 39 2.sup.nd hand BAC N 63 M Caucasian 76 55
Adenocarcinoma N 64 F Caucasian 43 33 Adenocarcinoma Y 65 F
Caucasian 50 30 Squamous cell N 66 M Caucasian 70 100 Squamous
cell, N Adenocarcinoma 67 M Caucasian 70 56 Squamous cell N 68 F
Caucasian 73 94 Adenocarcinoma N 69 F AA 62 40 Adenocarcinoma N 70
M Caucasian 58 80 Adenocarcinoma N 71 M Caucasian 71 55
Adenocarcinoma N 72 M Caucasian 78 90 Squamous cell N 73 M
Caucasian 72 45 Squamous cell N 74 M Caucasian 65 65 Squamous cell,
Y Adenosquamous
[0212] To determine the frequency of the SNP alleles, 2433
individuals were typed from a global sample of 46 populations.
According to population ancestry and geographic locations, these 46
populations are categorized into 4 groups: European (including West
Asia), African, Asian (including the Pacific) and Native American.
Sample descriptions and samples sizes can be found in the ALlele
FREquency Database (ALFRED)(Cheung, K. et al. Nucleic Acids Res
2000; 28:361-3) by searching for the population names
(http://alfred.med.yale.edu/). DNA samples were extracted from
lymphoblastoid cell lines that have been established and/or grown
in the Yale University laboratory of K.K.K. The methods of
transformation, cell culture, and DNA purification have previously
been described (Anderson, M. and G, J. F. In Vitro 1984; 20:
856-8). All volunteers were apparently normal and otherwise healthy
adult males or females and samples were collected after receipt of
appropriate informed consent.
[0213] Lung cancer cases (n=325) for the New Mexico case-control
study were recruited from Albuquerque through two local hospitals,
the Veterans hospital and the University of New Mexico (UNM)
hospital. All stages and histological types of lung cancer were
included. Controls (n=325) with no history of any prior cancer were
recruited from two ongoing local smoker cohorts, the Veterans
Smokers Cohort (mainly veterans from Albuquerque) and the Lovelace
Smokers Cohort (general residents in Albuquerque). Those two
cohorts started to recruit participants in 2001 to conduct
longitudinal studies on molecular markers of respiratory
carcinogenesis in biological fluids such as sputum from people at
risk for lung cancer. Enrollment of lung cancer patients from these
populations began in 2004. A standardized questionnaire was used to
collect information on medical, family, and smoking exposure
history, and quality of life for both lung cancer cases and control
cohort members. Controls were randomly matched to lung cancer cases
after categorization into different age groups (5-year differences)
by sex and cohort (Table 3). Cases with small cell lung cancer were
excluded to more precisely assess the effect of the LCS6 SNP on
risk for NSCLC. Cases over 82 years old (the maximum age in the
control group), cases with any prior cancer history, never smokers
or cases with missing data on smoking-related covariates were also
excluded in the data analysis, resulting in 218 cases included in
the analysis.
TABLE-US-00010 TABLE 3 Variables Controls Cases P-value N 325 218
Age 64.8 .+-. 9.0 65.1 .+-. 9.0 0.72.sup.a Sex (male, %) 68.9 73.9
0.22.sup.a Ethnicity (%) 0.01.sup.b White 67.4 75.7 Hispanic 24.9
14.7 Others 7.7 9.6 Current smoking status 32.9 37.2 0.31.sup.a
(current smoker, %) Pack-years 41.4 .+-. 28.5 56.9 .+-. 32.4
<0.0001.sup.c Family history of cancer 44.6 59.4 0.0008.sup.b
(yes, %) Histology (%) Adenocarcinoma 45.9 Squamous cell carninoma
24.8 Others.sup.d 29.4 Table 3. Demographic New Mexico Lung Cancer
Case-Control Data. Cases indicate patients with lung cancer and
controls are non-cancerous patients. .sup.aTwo-Sided two-sample
t-test between cases and controls. .sup.b2 test for differences
between cases and controls. .sup.cTwo-sided Wilcoxon rank sum test
between cases and controls. .sup.dOthers included large cell lung
cancer, poorly differentiated and other non-small cell lung
cancer.
[0214] The Boston study population was derived from a large ongoing
molecular epidemiological study that began in 1992 and now has more
than 2205 NSCLC patients recruited at MGH. Details of this
case-control population have been described previously (Thai, R. et
al. Clin Cancer Res 2008; 14:612-7; Su, L. et al. Carcinogenesis
2006; 27:1024-9; and Zhou, W. et al. Cancer Research 2002; 62:
1377-81). This study was approved by the Human Subjects Committees
of Massachusetts General Hospital (MGH) and Harvard School of
Public Health, Boston, Mass. Briefly, all histologically confirmed,
newly diagnosed patients with NSCLC at MGH were recruited between
December 1992 and February 2006. Before 1997, only early stage
(stage I and II) patients were recruited. After 1997, all stages of
NSCLC cases were recruited in this study. Controls were recruited
at MGH from healthy friends and non-blood-related family members
(usually spouses) of several groups of hospital patients: (a)
patients with cancer, whether related or not related to a case; or
(b) patients with a cardiothoracic condition undergoing surgery. No
matching was performed. Importantly, none of the controls were
themselves patients. Potential controls who carried a previous
diagnosis of any cancer (other than non-melanoma skin cancer) were
excluded from participation. Over 85% eligible cases and over 90%
controls participated in this study and provided blood samples. A
research nurse administered questionnaires on demographic
information and a detailed smoking history of each participant. To
reduce potential variation in allele frequency by ethnicity, only
Caucasians were considered in the analysis. Detailed demographics
of the participants of this case control are in Table 4.
TABLE-US-00011 TABLE 4 Supplementary Table 3B. Demographic
characteristics among Boston NSCLC cases and controls Table 4.
Demographic characteristics among Boston NSCLC cases and controls.
Overall Male Cases Controls Cases Controls Characteristics (n =
2205) (n = 1497) p (n = 1118) (n = 665) p Age (mean .+-. SD) 64.9
.+-. 58.2 .+-. <0.01 65.8 .+-. 60.2 .+-. <0.01 10.7 12.1 10.5
12.8 Gender, N (%) Female 1087 (49.3%) 822 (55.6%) <0.01 Male
1118 (56.7%) 665 (44.4%) Smoking, N (%) Never 204 (9.3%) 522
(34.9%) <0.01 77 (6.8%) 206 (30.8%) <0.01 Ex-smoker 1174
(53.2%) 688 (45.9%) 649 (57.6%) 362 (53.8%) Current smoker 827
(37.5%) 287 (19.2%) 461 (35.6%) 105 (15.6%) Years since 12 (1-59)
18 (1-65) <0.01 14 (1-59) 20 (1-65) <0.01 quit (median)
.sup.b Pack-years 50 (0.1-231) 25 (0.1-218) <0.01 58 (0.2-231)
29 (0.1-210) <0.01 Tumor stage (%) I and II 48.6% 48.0% III and
IV 51.3% 51.0% Cell type (%) Adenocarcinoma 57.0% 50.6% Squamous
cell carcinoma 21.9% 28.1% Others 21.1% 21.3% Kresler7 genotypes TT
1805 (81.9%) 1248 (83.4%) 0.32 944 (84.4%) 549 (82.6%) 0.46 TG 378
(17.4%) 231 (15.4%) 161 (14.4%) 105 (15.8%) GG 22 (1.0%) 18 (1.2%)
13 (1.2%) 11 (1.7%) TG + GG 400 (18.1%) 249 (16.6) 0.25* 174
(15.6%) 116 (17.4%) 0.32* Female Cases Controls Characteristics (n
= 1087) (n = 832) p Age (mean .+-. SD) 64.8 .+-. 56.8 .+-. <0.01
11.0 11.4 Gender, N (%) Female Male Smoking, N (%) Never 128
(11.7%) 324 (38.6%) <0.01 Ex-smoker 535 (48.8%) 333 (39.7%)
Current smoker 434 (39.6%) 183 (21.8%) Years since 12 (1-55) 17
(1-59) quit (median) .sup.b Pack-years 44 (0.02-210) 21 (0.03-218)
<0.01 Tumor stage (%) I and II 48.0% III and IV 52.0% Cell type
(%) Adenocarcinoma 63.4% Squamous cell carcinoma 15.6% Others 21.0%
Kresler7 genotypes TT 861 (79.2%) 599 (84.0%) 0.02 TG 217 (20.0%)
126 (15.1%) GG 9 (0.8%) 7 (0.8%) TG + GG 226 (20.5%) 133 (15.8%)
0.003 Ex-smokers only; .sup.bMedian range, tested by non-parametric
Wilcoxon's rank sum test; Continuous variables tested with the
Student's t-test categorical variables tested using the test.
*Compared with TT genotype, Fisher's exact test. indicates data
missing or illegible when filed
Evaluation of 3'UTR Sequences and the LCS6 SNP
[0215] DNA was isolated from fresh-frozen and formalin-fixed
paraffin-embedded (FFPE) lung tumors and non-cancerous lungs, and
non-primary lung tumors using the DNeasy Blood and Tissue Kit
(Qiagen). The tissue samples were acquired through the Yale-New
Haven Hospital Pathology Department after HIC approval. Segments of
the KRAS 3'UTR were amplified using PfuTurbo DNA polymerase
(Stratagene) and DNA primers (Table 5). PCR products were purified
using the QIAquick PCR Purification Kit or 96 PCR Purification Kit
(Qiagen) and sequenced using the same primers. The NRAS 3'UTR was
sequenced in the same manner.
TABLE-US-00012 TABLE 5 Primer Sequences Used in the Study. Primer
Sequence (5'-3') For sequencing SMJ104
CTAGCTAGCATACAATTTGTACTTTTTTCTTAAGGCATAC the KRAS (SEQ ID NO: 29)
3' UTR LJC1 GGCACACCACCACCCCAAAATCTC (SEQ ID NO: 30) LJC2
CCATCTTCAGTGCCAGTCTTGGG (SEQ ID NO: 31) LJC3
GGGTCGTATACCAAAGGCCTTAG (SEQ ID NO: 32) LJC4
GCCTGAACTAGTTCACAGACAAGGG (SEQ ID NO: 33) LJC5
CTAGCTAGCTCAATGCAGAATTCATGCTATCCAG (SEQ ID NO: 34) For sequencing
LJC21 GGTGTCAGAGTCTCGCTCTT (SEQ ID NO: 35) only LCS6 LJC3
GGGTCGTATACCAAAGGCCTTAG (SEQ ID NO: 36) and RFLP LJC27*
CCTGAGTAGCTGGGATTACA (SEQ ID NO: 37) analysis LJC28*
GGATACCATATACCCAGTGCCTT (SEQ ID NO: 38) For sequencing LJC13
CCACTTTCAAGCTGCACTGACAC (SEQ ID NO: 39) the NRAS LJC8
CTAGCTGGAGTTACTGGTGCAATGAGC (SEQ ID NO: 40) 3' UTR LJC9
GATACCTATGAGGATTTGGAGGC (SEQ ID NO: 41) LJC10
GCATGGTAGCCTTCAGACAGAAC (SEQ ID NO: 42) LJC11
CTGCTTCTTGTAATTCATCTCTGC (SEQ ID NO: 43) LJC12
CAACTTAAAATATCGGCCCTTCC (SEQ ID NO: 44) For making SMJ104
CTAGCTAGCATACAATTTGTACTTTTTTCTTAAGGCATAC KRAS wild-type (SEQ ID NO:
29) LJC5 CTAGCTAGCTCAATGCAGAATTCATGCTATCCAG (SEQ ID NO: 34) For
making LJC16 CGAACTCCTGACCTCAAGTGATgCACCCACCTT (SEQ ID NO: 45) KRAS
mLCS6 LJC17 ATCACTTGAGGTCAGGAGTTCGAGACCAGCCT (SEQ ID NO: 46)
Restriction Fragment Length Polymorphism (RFLP) Analysis
[0216] DNA isolated for sequencing was amplified using PfuTurbo DNA
polymerase (Stratagene) and primers listed in Table 5. The PCR
product was then digested with Hin fI and analyzed on agarose
gels.
TaqMan Assay
[0217] For high-throughput genotyping, the DNA isolated from
lymphocytes, blood, or tumor samples was amplified using TaqMan PCR
assays designed specifically to identify the LSC6 SNP (Applied
Biosciences). Data was analyzed using standard software on the
real-time PCR machine used for each study.
Statistical Analysis
[0218] All statistical analyses were performed using the SAS
statistical software (SAS Institute, Cary, N.C.) and a chi-square
test was used to test for departures from Hardy-Weinberg
equilibrium (HWE) for the variant allele in the Yale study
population. To calculate significance a Chi-Square test was used
for categorical variables, a t-test was used for continuous
variables and in some cases a two-sided Fisher's exact test was
used. For the case-control association studies, to compare controls
and cases, two-sided two-sample t-tests, Chi-Square analyses and
two-sided Wilcoxon rank-sum tests were performed, as appropriate.
For evaluating the association between the KRAS LCS6 allele and
risk for NSCLC in light or heavy smokers, age, race, sex, smoking
status, pack-years of smoking and years since smoking cessation (if
ex-smokers) were adjusted with an unconditional logistic regression
model. To test the association with the allele and the pack-year
interaction for NSCLC, a likelihood ratio test was used. The median
pack year was used as the evaluation point for the gene-environment
interaction in both studies. The variant homozygotes were few and
pooled with the heterozygotes for these analyses and are referred
to collectively as those "with the variant."
Methods of Detecting SNPs
[0219] The invention encompasses methods of detecting the LCS6 SNP
including, but not limited to, polymerase chain reaction (PCR)
using either the primers disclosed herein (SEQ ID NOs: 22-39) or
with any primer that amplifies any portion of the 3'UTR of a RAS
family gene or mRNA transcript comprising the LCS6 SNP, nucleic
acid/probe hybridization (for example, all forms of DNA and RNA are
contemplated as probes), probe hybridization (for example, in vitro
assays, in situ hybridization, Northern and/or Southern blots),
sequencing, RFLP analysis, functional assays (for example,
introduction of a test polynucleotide into a cell in vivo or in
vitro and examination of resulting cell proliferation, cell death,
cell metastasis, change of morphology, degradation of extracellular
matrix, protein expression, reporter protein/marker expression),
miRNA-binding assays (for example, in vitro or in vivo assays to
determine ability of miRNAs to bind, silence, degrade, or inhibit
the translation of the test polynucleotide), translational assays
(for example, expression of polypeptides encoded for by test
polynucleotides, expression of reporter polypeptides or detectable
markers/labels linked to the test polynucleotide, Western blot
analysis to determine translation of the test polynucleotide), and
all other art-recognized methods.
[0220] Probes used to identify or detect the LCS6 SNP are
polynucleic acids, either DNA or RNA, and correspond to either the
entire 3'UTR of KRAS, or any fragment thereof. The term "fragment",
as used herein, is meant to describe a polynucleotide that is 100%
identical to the polynucleotide from which it is derived over a
span that is less than the entire length of the polynucleotide from
which it is derived. Encompassed probes comprise SEQ ID NO: 15,
e.g., wild type LCS6, or SEQ ID NO: 21,e.g. the LCS6 SNP. Probes
used to detect the LCS6 SNP comprise the sequences of SEQ ID NOs:
15 or 21, as well as any sequences complementary to SEQ ID NOs: 15
or 21. Contemplated probes also include wild type and/or modified
miRNA sequences, and fragments thereof.
Luciferase Reporters and Transient Transfections
[0221] The luciferase reporter with an altered LCS6 KRAS 3'UTR
corresponding to the LCS6 variant (pGL3-KRASm6) was constructed
through site-directed mutagenesis of pGL3-KRAS (Johnson, S. M. et
al. Cell 2005; 120(5): 635-47) using GeneTailor (Invitrogen) (Table
5). HeLa S3 and CRL-2741 cells were grown in DMEM with 10% FBS or
Keratinocyte-SFM, both with penicillin/streptomycin (Invitrogen).
Cells were transiently transfected with 700 ng pGL3-KRAS,
pGL3-KRASm6, or pGL3-Control (Promega) and 70 ng pRL-TK (Promega)
using Lipofectamine 2000 (Invitrogen) for 24 hours. Reporter
expression was analyzed with the Dual-Luciferase Reporter Assay
(Promega) and Wallac Victor 1420 (PerkinElmer)(Chen, K. et al.
Nature Genetics 2006; 38:). Two-tailed t-tests were done to verify
statistical significance of differences in luciferase expression
using GraphPad Prism.
Example 2
Identification of a Candidate Let-7 SNP
[0222] let-7 complementary sites (LCSs) were sequenced in the KRAS
3' untranslated region (UTR) from 74 non-small cell lung cancer
(NSCLC) cases to identify mutations and single nucleotide
polymorphisms (SNPs) that correlated with NSCLC. A candidate SNP
was identified and the allele frequency was determined by typing
the polymorphism in 2433 people (representing 46 human
populations). The association was further assessed between the SNP
and the risk of smoking-induced NSCLC in two independent
case-control studies.
[0223] The novel SNP was identified in an LCS in 24% of Caucasian
NSCLC patients, compared to 7.4% of the general Caucasian
population. The presence of the SNP predicted for squamous cell
carcinoma versus adenocarcinoma and a positive family history of
cancer. The variant allele at the SNP is associated with earlier
onset NSCLC (<versus >50 years of age) and additional cancer
diagnoses. The frequency of the variant is 20.3% in our cohort of
NSCLC patients and 5.8% in world populations. Both independent
case-control studies found that smokers with the variant and <40
or 41 pack-year smoking histories had an elevated risk of
developing NSCLC compared to smokers without the variant
(ORs=1.36-2.3, 95% CI=1.07-1.73, p=0.01 and 1.1-4.6, p=0.02).
Functionally, the variant allele leads to increased let-7 binding
and KRAS suppression in vitro.
[0224] A variant allele in a KRAS miRNA complementary site is
significantly associated with increased smoking-induced NSCLC risk.
These findings represent a new paradigm for miRNAs in cancer
susceptibility and are used to better direct lung cancer screening
programs.
Example 3
Identification of a SNP in a Let-7 Complementary Site in the KRAS
3'UTR
[0225] RAS expression is regulated in a 3'UTR and let-7-dependent
manner through ten putative let-7 complementary sites (LCSs) in the
human KRAS 3'UTR (FIG. 23A) (Johnson, S. M. et al. Cell 2005;
120(5): 635-47). Based on data from the HapMap (Consortium TIH.
Nature 2003; 426:789-96) and dbSNP (Sherry, S. et al. Genome Res
1999; 9:677-9) databases, only one SNP, rs712(-), is reported in an
LCS. Tissue from seventy-four patients with NSCLC exhibited changes
from the reference human sequence in at least one LCSs. Changes in
LCS1, LCS9, and LCS4 (Table 7) did not appear to correlate with
NSCLC. However, a SNP (T to G, with G the less frequent variant)
identified at the fourth nucleotide in LCS6 was found in 20.3% of
the NSCLC patients (FIG. 20B-20D). Supporting the hypothesis that
the variant allele at this SNP is a genetic marker of increased
lung cancer risk, an increased frequency of the allele was found in
younger patients, in patients with a high frequency of additional
cancers, and in patients with a reported family history of cancer
(Table 6). However, because of the small numbers of patients in
this retrospectively studied case series, firm conclusions were not
drawn.
[0226] As a control, the 3'UTR of NRAS was sequenced in the same
NSCLC patients to look for similar SNPs. NRAS is not associated
with lung cancer but contains 9 putative LCSs. No SNPs were
identified within the LCSs of the NRAS 3'UTR, supporting the idea
that the identified SNP in the KRAS 3'UTR is an important change
with respect to lung cancer.
TABLE-US-00013 TABLE 6 Among cancer patients Table 6:
Characteristics of Yale NSCLC Patients. Wildtype Variant Allele
P-Value Cancer Type Adenocarcinoma 40 (83.3%) 8 (16.7%) Squamous 11
(61.1%) 7 (38.9%) 0.096.sup.b 48 15 No. of Other Cancers None 39
(88.9%) 5 (11.1%) 1 12 (75.0%) 4 (25.0%) 2 and More 6 (54.6%) 5
(45.4%) 0.034.sup.b 57 14 Mean pack year for patients 2.sup.nd
cancers 66.7 .+-. 36.5 47.4 .+-. 39.3 0.220 Mean Packyear of cases
52.8 .+-. 38.4 37.1 .+-. 35.2 0.257 Cancer Onset <50 6 5
.gtoreq.50 51 9 0.034.sup.b Family History Negative 12 (100%) 0
(0%) Positive 19 (66.7%) 9 (33.3%) 0.038.sup.b 31 9 .sup.aP-value
is for t-test (continuous variables) or .chi.2 test (categorical
variables), except where noted. .sup.bP-value is for two-sided
Fisher's exact test.
TABLE-US-00014 TABLE 7 Alterations in KRAS 3'UTR LCSs. Non-primary
Primary Lung Lung Tumor with Tumor/Adjacent Primary or without
Non-tumor Lung Adjacent Non- Non-cancerous Tissue Tumor tumor
Tissue Lung LCS1 4th bp = A 35 16 5 3 4th bp = A/C 6.dagger. 1 11 0
25th bp = G 14 5 2 3 rs712(--) = T/G 27.sctn. 12 4 0 Total Patients
41 17 6 3 LCS9 28th bp = T 37 15 6 1 28th bp = T/C 3.dagger-dbl. 2
0 2 15th bp = C 39 17 6 3 15th bp = C/T 1.dagger. 0 0 0 12th bp = A
40 16 6 3 12th bp = A/C 0 1 0 0 Total Patients 40 17 6 3 LCS4 14th
bp = C 40 18 6 3 14th bp = C/T 1 0 0 0 Total Patients 41 18 6 3
Table 7. All LCSs in which mutations were identified in are listed
here, as are the number of NSCLC patients with these mutations. 68%
of the primary lung tumors and 67% of the non-primary lung tumors
examined were heterozygous at the rs712 locus in LCS1. A mutation
at the fourth nucleotide of LCS1 was found in patients of both
sexes and in a variety of non-small cell lung cancers types. The
change at the twentieth nucleotide of LCS9 and was found in both
sexes and in cancerous and non-cancerous lungs. The mutation at the
twelfth base of LCS9 was seen in the adjacent tissue sample of a
female, adenosquamous carcinoma patient. A mutation at the
fifteenth base pair of LCS9 was found in the adjacent tissue of a
female, squamous cell carcinoma patient, where as the primary lung
tumor sample was normal. Lastly, there was one case of a mutation
in LCS4. Both the tumor and adjacent tissue samples were
heterozygous at this site. .dagger.Only tumor sample (3 patients),
only adjacent sample (3 patients). .sctn.Tumor and adjacent samples
(24 patients), only tumor sample (1 patient), only adjacent sample
(2 patients). .dagger-dbl.Only tumor sample (2 patients), tumor and
adjacent samples (1 patient). .psi.Only adjacent sample. Tumor and
adjacent samples.
Example 4
Frequency of the Variant Allele Across Populations
[0227] To determine the allele frequencies of the SNP in the
general population, a collection of genomic DNA from 2433 healthy
individuals from a global set of 46 populations was used.
Considerable polymorphism data already exist on these samples and
can be found, along with the population descriptions in ALFRED, the
ALlele FREquency Database (http://alfred.med.yale.edu). The results
of a TaqMan assay revealed that <3% of the 4866 chromosomes
tested had the G allele (variant) at the LCS6 SNP site (FIG. 24).
The frequency of this allele varied across geographic populations,
with "European" populations exhibiting the variant allele most
frequently (7.6% of the chromosomes tested); African populations
less frequently (<2.0% of chromosomes tested); and "Asian" and
Native American populations infrequently (<0.4% of chromosomes
tested). Of note, over 85% of the patients in the retrospective
patient cohort were of European descent. It is apparent from this
data that the SNP arose in Africa and is now frequently found
outside of this geographic area, consistent with random genetic
drift involved in the bottleneck of expansion out of Africa. The
findings are also consistent with subsequent loss of the variant
allele with expansion into East Asia and the Americas.
Example 5
The Variant Allele is Associated with Smoking-Induced NSCLC
Risk
[0228] Two independent lung cancer case-control designs were used,
referred to as the New Mexico (Table 8A, 325 patients) and the
Boston (Table 8B, 3702 patients) studies, to determine the impact
of the SNP on smoking-induced lung cancer. The frequency of the
variant allele in the NSCLC cases in these studies was 18.8% and
18.1% respectively, not significantly different from the frequency
in the lung cancer patients studied at Yale (p=0.20). While the
presence of the LCS6 variant allele did not predict NSCLC risk for
the entire patient cohort in either study (Table 8A and 8B), the
variant allele was associated with increased NSCLC risk in smokers
with less than a 41 or 40 pack-year smoking history (Table 8A and
8B, New Mexico Study odds ratio (OR)=2.3, 95% confidence interval
(CI)=1.1-4.6, p=0.02, Boston Study odds ratio (OR)=1.36, 95%
confidence interval (CI)=1.07-1.73, p=0.01), which are the medians
in the respective populations. The ORs were adjusted for age,
gender, smoking status, pack-years of smoking, and years since
smoking cessation in both studies.
[0229] These findings indicate that the variant allele is a marker
for increased risk of smoking-induced NSCLC in patients with less
cigarette exposure, which in these studies was less than the mean
smoking exposure of .about.40 pack years, meaning a person has
smoked the equivalent of one pack of cigarettes per day for 40
years. The finding that the variant SNP only impacts cancer risk
for less cigarette exposure agrees with other studies showing a
dose-dependent gene-environment interaction for smoking-induced
lung cancer risk (Zhou, W. et al. Cancer Research 2002; 62:
1377-81; Zhou, W. et al. Cancer Epidemiology, Biomarkers &
Prevention 2005; 14:491-6; and Liu, G. et al. Int. J. Cancer 2007,
online); with higher smoking exposure any genetic predisposition is
hypothesized to be overwhelmed by the extent of smoking-related
damage.
TABLE-US-00015 TABLE 8A Association between KRAS variant allele and
non-small cell lung cancer in the New Mexico Case Control Table 8A.
Association between KRAS variant allele and non-small cell lung
cancer in the New Mexico Case Control. Controls Gen (n = 325) Cases
(n = 218) Crude OR Adjusted OR.sup.a TT 280 177 reference reference
TG 44 38 1.4 (0.9-2.2) p = 0.19 1.4 (0.8-2.3) p = 0.21 GG 1 3 4.7
(0.5-45.9) p = 0.17 5.3 (0.5-54.4) p = 0.17 TG 45 41 1.4 (0.9-2.3)
p = 0.13 1.5 (0.9-2.4) p = 0.13 Pack-years.sup.b Genotype Controls
Cases Crude OR Adjusted OR.sup.a <41 TT 171 57 reference
reference TG or GG 24 18 2.3 (1.1-4.4) 2.3 (1.1-4.6) p = 0.02 p =
0.02 .gtoreq.41 TT 109 120 reference reference TG or GG 21 23 1.0
(0.5-1.9) 0.9 (0.5-1.8) p = 0.99 p = 0.86 .sup.aAge, race, sex and
current smoking status were adjusted in unconditional logistic
regression model. P value for SNP-pack-years interaction was equal
to 0.08 by likelihood ratio test. .sup.b41 pack-years is the median
of 543 study subjects. The result is not sensitive to different
cutoffs.
TABLE-US-00016 TABLE 8B Association between KRAS variant allele and
non-small cell lung cancer in the Boston Case Control Table 8B.
Association between KRAS variant allele and non-small cell lung
cancer in the Boston Case Control. Adjusted Group Genotype Crude OR
p OR.sup.# p Overall TT 1.0 (2205 cases TG + GG 1.11 (0.93-1.32)
0.23 1.17 0.15 vs. 1497 (0.97-1.44) controls) Pack-years* TT 1.0
<40 TG + GG 1.28 (1.03-1.60) 0.03 1.36 0.01 (956 cases
(1.07-1.73) vs. 1214 controls) Pack-years TT 1.0 .gtoreq.40 TG + GG
0.85 (0.61-1.29) 0.34 0.89 0.49 (1249 cases (0.63-1.25) vs. 283
controls) *Pack-years is the median in smokers. .sup.#Adjusted for
age, gender, smoking status, pack-years of smoking, and years since
smoking cessation (if ex-smoker)
Example 6
The LCS6 SNP Impacts KRAS Expression
[0230] One criterion for the quality of putative miRNA binding
sites is the free energy at the proposed mRNA:miRNA interaction,
where the lower the free energy value, the higher the likelihood
for an interaction between the miRNA and the mRNA. Based on
RNAhybrid (Kruger, J. and Rehmsmeier, M. Nucleic Acids Res 2006;
34:W451-4) (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/)
values, changing the fourth nucleotide of LCS6 in the KRAS 3'UTR
from a T to a G resulted in reduced free energy values for the
proposed binding of each of the human let-7 sequences (FIGS. 24 and
13). These results demonstrate that the variant allele results in
improved let-7 miRNA binding at this site.
[0231] To determine the effect of the LCS6 variant allele on KRAS
expression, a luciferase reporter was used to represent KRAS
expression (Johnson, S. M. et al. Cell 2005; 120(5):635-47). When
the luciferase reporter with the alternative LCS6 (pGL3-KRASm6) was
transiently transfected into HeLa S3 cells, which make abundant
let-7, luciferase expression was reduced .about.5-fold as compared
to the unaltered reporter (p<0.0001, FIG. 22).
[0232] The KRAS gene was evaluated for common activating mutations
(in codons 12, 13, and 61) in 9 of our patients carrying the
variant allele and did not find any activating mutations.
Unexpectedly, activated KRAS alleles were not identified among the
LCS6 variant allele-carriers (as KRAS is activated in 30% of
adenocarcinomas) (Rodenhuis, S. Semin Cancer Biol 1992;
3:241-7).
Example 7
The LCS6 SNP is Associated with Increased NSCLC Risk
[0233] Methods of the invention and case controlled studies were
used to assess the impact of the LCS6 SNP on the occurrence and
severity of non-small cell lung cancer (NSCLC). There are a
documented 200,000 cases reported each year with an average 5-year
survival rate of 15%. Of the 74 NSCLC cases included in this study,
15 of those individuals carried the LCS6 SNP which represents 20.3%
of the population (p=0.2)(Table 1). When the odds ratio (OR) is
considered (1.4-2.3), the presence of the LCS6 SNP indicates a
40%-130% increased risk of developing lung cancer (Table 1). This
odds ratio was calculated from two independent case control studies
with 400 and 4000 patients respectively (95% CI=1.1-4.6, p<0.02;
1.1-1.7, p<0.01).
Example 8
The LCS6 SNP is Associated with Increased Ovarian Cancer Risk
[0234] There are 25,000 documented cases of Ovarian Cancer per
year, with an average 5-year survival rate of 10%. Of the 43
ovarian cancer patients included in this study, 22 of these
individuals carried the LCS6 SNP, representing 51% of the cancer
population (p<0.0000000001, odds ratio=6.4). Among those
patients who were diagnosed with high-risk subtypes, such as the
Pap serous subtype (makes up .about.75 percent of epithelial
ovarian cancer), 22 of the 38 individuals studied carried the LCS6
SNP, representing 58% of the high-risk ovarian cancer population
(p<0.0000000001, odds ratio=8.45) (Table 1). The data of the
instant study were validated using data from a set of Italian
subjects (200 cases, expected prevalence of the LCS6 SNP only 8%).
The LCS6 SNP was found to be present in 40% of these cases (all
subtypes, OR=3.86) (all subtypes except mucinous which together
make up 90% of epithelial cancers, OR=4.3) (Table 1).
Example 9
The LCS6 SNP is Associated with Increased Uterine/Endometrial
Cancer Risk
[0235] There are 45,000 documented cases of uterine/endometrial
cancer per year, with an average 5-year survival rate of 85% for
the endometriod subtype and a significantly less average 5-year
survival rate of 10% for "high risk" subtypes. Among the 25 cases
of endometriod subtype cancer included in the study, only one
individual was a carrier for the LCS6 SNP (p=0.04, significantly
not present). Importantly, of the 21 individuals included in the
study who were diagnosed with high-risk subtypes of endometrial
cancer, 10 subjects were carriers for the LCS6 SNP, representing
48% of the high-risk cancer population (p=0.0004, odds
ratio=10/11/280/1720=5.57) (Table 1). Of the high risk subtypes,
the most serious form is the pap serous subtype. Of the 9 subjects
included in this study with the pap serous subtype, 5 individuals
carried the LCS6 SNP, representing 56% of this group (p=0.0001,
odds ratio=5/4/280/1720=7.67) (Table 1). As such, the LCS6 SNP
appears to be a marker for the most serious subtypes of endometrial
cancer which lead to the worst prognosis for the individuals who
carry this marker.
Example 10
The LSC6 SNP is Associated with Increased Breast Cancer Risk
[0236] There are 230,000 documented cases of breast cancer per
year, with an average 5 year survival rate of 50%. The prevalence
of the LCS6 SNP across all subtypes of breast cancer is about 20%,
which is statistically non-significant compared to the prevalence
in the general population (of non-cancerous individuals).
Importantly, the prevalence of the LCS6 SNP in the high-risk
Her-2.sup.+ subtype (which represents about 25% of all breast
cancer with worst prognosis, only 25% 5-year survival) is 25% (11
individuals of the 44 high-risk subtype patients studied carried
the LCS6 SNP) (p=0.004, odds ratio=2.1) (Table 1).
Example 11
The LCS6 SNP is Associated with Increased Head and Neck Cancer
Risk
[0237] There are 15,000 documented cases of head and neck cancer
per year, with a 5-year average survival of 50%. Among the 21
patients studied who were diagnosed with the oropharynx subtype, 7
individuals carried the LCS6 SNP, representing 25% of the
population (p=0.03, odds ratio=3.07) (Table 1).
[0238] The LCS6 SNP is found at a significantly higher prevalence
than expected in head and neck cancers, and is usually associated
with particular subtypes, e.g. the oropharynx subtype.
Specifically, the SNP occurred in 33% of the 24 head and neck
cancer patients tested (Table 1). The statistical significance of
this number is indicated by a p-value of 0.011.
Example 12
The LCS6 SNP is Associated with Increased Pancreatic Cancer
Risk
[0239] There are a documented 50,000 cases per year, with less than
5% of those individuals surviving more than 5-years from diagnosis.
Of the 51 cases of cancer of the exocrine pancreas included in the
current study, 12 individuals carried the LCS6 SNP which represents
23.5% of the pancreatic cancer study population (p=0.05) (Table 1).
When the odds ratio (OR) is considered (1.2), the presence of the
LCS6 SNP indicates a 20% increased risk of developing pancreatic
cancer (Table 1). This odds ratio was calculated from an ongoing
analysis of a case control study with 800 patients.
Example 13
The LCS6 SNP is Associated with Increased Melanoma Risk
[0240] The LCS6 SNP is found at a significantly higher prevalence
than expected in melanoma. Specifically, the SNP occurred in 28.6%
of the 7 melanoma patients tested, (Table 1). The statistical
significance of this number is indicated by a p-value of 0.01.
Example 14
The LCS6 SNP is Associated with Increased Colon Cancer Risk
[0241] There are 108,070 documented cases of colon cancer per year
with an average 5 year survival rate of 60%. The instant study
included 1364 samples of adenocarcinomas. The LCS6 SNP was present
in 18.3% of these samples (p<0.001, odds ratio=1.4) (Table
1).
OTHER EMBODIMENTS
[0242] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
[0243] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. Genbank and NCBI
submissions indicated by accession number cited herein are hereby
incorporated by reference. All other published references,
documents, manuscripts and scientific literature cited herein are
hereby incorporated by reference.
[0244] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
94125RNAHomo sapiens 1aguucucaga auaacuaccu ccuca 25226RNAHomo
sapiens 2ggcugucuga ccagagaaug caccuc 26318RNAHomo sapiens
3acagcacaaa cacaccuc 18428RNAHomo sapiens 4agcugugauc agugauuuuc
aaaccyca 28532RNAHomo sapiens 5aauugccuuc aauccccuuc ucaccccacc uc
32624RNAHomo sapiens 6aucuaaauac uuacugaggu ccuc 24725RNAHomo
sapiens 7aauuuuccug aggcuuauca ccuca 25830RNAHomo sapiens
8gauugcugaa aagaauucua guuuaccuca 30919RNAHomo sapiens 9aacaggaacu
auuggccuc 191025RNAHomo sapiens 10gacaguggaa guuuuuuuuu ccucg
251119RNAHomo sapiens 11auuaguguca ucuugccuc 191225RNAHomo sapiens
12aaugcccuac aucuuauuuu ccuca 251324RNAHomo sapiens 13gguucaagcg
auucucgugc cucg 241424RNAHomo sapiens 14ggcugguccg aacuccugac cuca
241521RNAHomo sapiens 15gauucaccca ccuuggccuc a 211628RNAHomo
sapiens 16ggguguuaag acuugacaca guaccucg 281728RNAHomo sapiens
17agugcuuaug aggggauauu uaggccuc 281830RNAHomo sapiens 18gaccgugggc
cgaggugacu gcagacccuc 301925RNAHomo sapiens 19ggaaccccag cccuuagcuc
cccuc 252026RNAHomo sapiens 20agcccuuagc uccccuccca ggccuc
262121RNAHomo sapiens 21gaugcaccca ccuuggccuc a 21221061DNAHomo
sapiens 22tgccctgcgc ccgcaacccg agccgcaccc gccgcggacg gagcccatgc
gcggggcgaa 60ccgcgcgccc ccgcccccgc cccgccccgg cctcggcccc ggccctggcc
ccgggggcag 120tcgcgcctgt gaacggtggg gcaggagacc ctgtaggagg
accccgggcc gcaggcccct 180gaggagcgat gacggaatat aagctggtgg
tggtgggcgc cggcggtgtg ggcaagagtg 240cgctgaccat ccagctgatc
cagaaccatt ttgtggacga atacgacccc actatagagg 300attcctaccg
gaagcaggtg gtcattgatg gggagacgtg cctgttggac atcctggata
360ccgccggcca ggaggagtac agcgccatgc gggaccagta catgcgcacc
ggggagggct 420tcctgtgtgt gtttgccatc aacaacacca agtcttttga
ggacatccac cagtacaggg 480agcagatcaa acgggtgaag gactcggatg
acgtgcccat ggtgctggtg gggaacaagt 540gtgacctggc tgcacgcact
gtggaatctc ggcaggctca ggacctcgcc cgaagctacg 600gcatccccta
catcgagacc tcggccaaga cccggcaggg agtggaggat gccttctaca
660cgttggtgcg tgagatccgg cagcacaagc tgcggaagct gaaccctcct
gatgagagtg 720gccccggctg catgagctgc aagtgtgtgc tctcctgacg
cagcacaagc tcaggacatg 780gaggtgccgg atgcaggaag gaggtgcaga
cggaaggagg aggaaggaag gacggaagca 840aggaaggaag gaagggctgc
tggagcccag tcaccccggg accgtgggcc gaggtgactg 900cagaccctcc
cagggaggct gtgcacagac tgtcttgaac atcccaaatg ccaccggaac
960cccagccctt agctcccctc ccaggcctct gtgggccctt gtcgggcaca
gatgggatca 1020cagtaaatta ttggatggtc ttgaaaaaaa aaaaaaaaaa a
1061231143DNAHomo sapiens 23tgccctgcgc ccgcaacccg agccgcaccc
gccgcggacg gagcccatgc gcggggcgaa 60ccgcgcgccc ccgcccccgc cccgccccgg
cctcggcccc ggccctggcc ccgggggcag 120tcgcgcctgt gaacggtggg
gcaggagacc ctgtaggagg accccgggcc gcaggcccct 180gaggagcgat
gacggaatat aagctggtgg tggtgggcgc cggcggtgtg ggcaagagtg
240cgctgaccat ccagctgatc cagaaccatt ttgtggacga atacgacccc
actatagagg 300attcctaccg gaagcaggtg gtcattgatg gggagacgtg
cctgttggac atcctggata 360ccgccggcca ggaggagtac agcgccatgc
gggaccagta catgcgcacc ggggagggct 420tcctgtgtgt gtttgccatc
aacaacacca agtcttttga ggacatccac cagtacaggg 480agcagatcaa
acgggtgaag gactcggatg acgtgcccat ggtgctggtg gggaacaagt
540gtgacctggc tgcacgcact gtggaatctc ggcaggctca ggacctcgcc
cgaagctacg 600gcatccccta catcgagacc tcggccaaga cccggcaggg
cagccgctct ggctctagct 660ccagctccgg gaccctctgg gaccccccgg
gacccatgtg acccagcggc ccctcgcgct 720ggagtggagg atgccttcta
cacgttggtg cgtgagatcc ggcagcacaa gctgcggaag 780ctgaaccctc
ctgatgagag tggccccggc tgcatgagct gcaagtgtgt gctctcctga
840cgcagcacaa gctcaggaca tggaggtgcc ggatgcagga aggaggtgca
gacggaagga 900ggaggaagga aggacggaag caaggaagga aggaagggct
gctggagccc agtcaccccg 960ggaccgtggg ccgaggtgac tgcagaccct
cccagggagg ctgtgcacag actgtcttga 1020acatcccaaa tgccaccgga
accccagccc ttagctcccc tcccaggcct ctgtgggccc 1080ttgtcgggca
cagatgggat cacagtaaat tattggatgg tcttgaaaaa aaaaaaaaaa 1140aaa
1143245436DNAHomo sapiens 24ggccgcggcg gcggaggcag cagcggcggc
ggcagtggcg gcggcgaagg tggcggcggc 60tcggccagta ctcccggccc ccgccatttc
ggactgggag cgagcgcggc gcaggcactg 120aaggcggcgg cggggccaga
ggctcagcgg ctcccaggtg cgggagagag gcctgctgaa 180aatgactgaa
tataaacttg tggtagttgg agctggtggc gtaggcaaga gtgccttgac
240gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag
aggattccta 300caggaagcaa gtagtaattg atggagaaac ctgtctcttg
gatattctcg acacagcagg 360tcaagaggag tacagtgcaa tgagggacca
gtacatgagg actggggagg gctttctttg 420tgtatttgcc ataaataata
ctaaatcatt tgaagatatt caccattata gagaacaaat 480taaaagagtt
aaggactctg aagatgtacc tatggtccta gtaggaaata aatgtgattt
540gccttctaga acagtagaca caaaacaggc tcaggactta gcaagaagtt
atggaattcc 600ttttattgaa acatcagcaa agacaagaca gagagtggag
gatgcttttt atacattggt 660gagggagatc cgacaataca gattgaaaaa
aatcagcaaa gaagaaaaga ctcctggctg 720tgtgaaaatt aaaaaatgca
ttataatgta atctgggtgt tgatgatgcc ttctatacat 780tagttcgaga
aattcgaaaa cataaagaaa agatgagcaa agatggtaaa aagaagaaaa
840agaagtcaaa gacaaagtgt gtaattatgt aaatacaatt tgtacttttt
tcttaaggca 900tactagtaca agtggtaatt tttgtacatt acactaaatt
attagcattt gttttagcat 960tacctaattt ttttcctgct ccatgcagac
tgttagcttt taccttaaat gcttatttta 1020aaatgacagt ggaagttttt
ttttcctcta agtgccagta ttcccagagt tttggttttt 1080gaactagcaa
tgcctgtgaa aaagaaactg aatacctaag atttctgtct tggggttttt
1140ggtgcatgca gttgattact tcttattttt cttaccaatt gtgaatgttg
gtgtgaaaca 1200aattaatgaa gcttttgaat catccctatt ctgtgtttta
tctagtcaca taaatggatt 1260aattactaat ttcagttgag accttctaat
tggtttttac tgaaacattg agggaacaca 1320aatttatggg cttcctgatg
atgattcttc taggcatcat gtcctatagt ttgtcatccc 1380tgatgaatgt
aaagttacac tgttcacaaa ggttttgtct cctttccact gctattagtc
1440atggtcactc tccccaaaat attatatttt ttctataaaa agaaaaaaat
ggaaaaaaat 1500tacaaggcaa tggaaactat tataaggcca tttccttttc
acattagata aattactata 1560aagactccta atagcttttc ctgttaaggc
agacccagta tgaaatgggg attattatag 1620caaccatttt ggggctatat
ttacatgcta ctaaattttt ataataattg aaaagatttt 1680aacaagtata
aaaaattctc ataggaatta aatgtagtct ccctgtgtca gactgctctt
1740tcatagtata actttaaatc ttttcttcaa cttgagtctt tgaagatagt
tttaattctg 1800cttgtgacat taaaagatta tttgggccag ttatagctta
ttaggtgttg aagagaccaa 1860ggttgcaagg ccaggccctg tgtgaacctt
tgagctttca tagagagttt cacagcatgg 1920actgtgtccc cacggtcatc
cagtgttgtc atgcattggt tagtcaaaat ggggagggac 1980tagggcagtt
tggatagctc aacaagatac aatctcactc tgtggtggtc ctgctgacaa
2040atcaagagca ttgcttttgt ttcttaagaa aacaaactct tttttaaaaa
ttacttttaa 2100atattaactc aaaagttgag attttggggt ggtggtgtgc
caagacatta attttttttt 2160taaacaatga agtgaaaaag ttttacaatc
tctaggtttg gctagttctc ttaacactgg 2220ttaaattaac attgcataaa
cacttttcaa gtctgatcca tatttaataa tgctttaaaa 2280taaaaataaa
aacaatcctt ttgataaatt taaaatgtta cttattttaa aataaatgaa
2340gtgagatggc atggtgaggt gaaagtatca ctggactagg aagaaggtga
cttaggttct 2400agataggtgt cttttaggac tctgattttg aggacatcac
ttactatcca tttcttcatg 2460ttaaaagaag tcatctcaaa ctcttagttt
ttttttttta caactatgta atttatattc 2520catttacata aggatacact
tatttgtcaa gctcagcaca atctgtaaat ttttaaccta 2580tgttacacca
tcttcagtgc cagtcttggg caaaattgtg caagaggtga agtttatatt
2640tgaatatcca ttctcgtttt aggactcttc ttccatatta gtgtcatctt
gcctccctac 2700cttccacatg ccccatgact tgatgcagtt ttaatacttg
taattcccct aaccataaga 2760tttactgctg ctgtggatat ctccatgaag
ttttcccact gagtcacatc agaaatgccc 2820tacatcttat ttcctcaggg
ctcaagagaa tctgacagat accataaagg gatttgacct 2880aatcactaat
tttcaggtgg tggctgatgc tttgaacatc tctttgctgc ccaatccatt
2940agcgacagta ggatttttca aacctggtat gaatagacag aaccctatcc
agtggaagga 3000gaatttaata aagatagtgc tgaaagaatt ccttaggtaa
tctataacta ggactactcc 3060tggtaacagt aatacattcc attgttttag
taaccagaaa tcttcatgca atgaaaaata 3120ctttaattca tgaagcttac
tttttttttt tggtgtcaga gtctcgctct tgtcacccag 3180gctggaatgc
agtggcgcca tctcagctca ctgcaacctc catctcccag gttcaagcga
3240ttctcgtgcc tcggcctcct gagtagctgg gattacaggc gtgtgccact
acactcaact 3300aatttttgta tttttaggag agacggggtt tcaccctgtt
ggccaggctg gtctcgaact 3360cctgacctca agtgattcac ccaccttggc
ctcataaacc tgttttgcag aactcattta 3420ttcagcaaat atttattgag
tgcctaccag atgccagtca ccgcacaagg cactgggtat 3480atggtatccc
caaacaagag acataatccc ggtccttagg tagtgctagt gtggtctgta
3540atatcttact aaggcctttg gtatacgacc cagagataac acgatgcgta
ttttagtttt 3600gcaaagaagg ggtttggtct ctgtgccagc tctataattg
ttttgctacg attccactga 3660aactcttcga tcaagctact ttatgtaaat
cacttcattg ttttaaagga ataaacttga 3720ttatattgtt tttttatttg
gcataactgt gattctttta ggacaattac tgtacacatt 3780aaggtgtatg
tcagatattc atattgaccc aaatgtgtaa tattccagtt ttctctgcat
3840aagtaattaa aatatactta aaaattaata gttttatctg ggtacaaata
aacaggtgcc 3900tgaactagtt cacagacaag gaaacttcta tgtaaaaatc
actatgattt ctgaattgct 3960atgtgaaact acagatcttt ggaacactgt
ttaggtaggg tgttaagact tacacagtac 4020ctcgtttcta cacagagaaa
gaaatggcca tacttcagga actgcagtgc ttatgagggg 4080atatttaggc
ctcttgaatt tttgatgtag atgggcattt ttttaaggta gtggttaatt
4140acctttatgt gaactttgaa tggtttaaca aaagatttgt ttttgtagag
attttaaagg 4200gggagaattc tagaaataaa tgttacctaa ttattacagc
cttaaagaca aaaatccttg 4260ttgaagtttt tttaaaaaaa gctaaattac
atagacttag gcattaacat gtttgtggaa 4320gaatatagca gacgtatatt
gtatcatttg agtgaatgtt cccaagtagg cattctaggc 4380tctatttaac
tgagtcacac tgcataggaa tttagaacct aacttttata ggttatcaaa
4440actgttgtca ccattgcaca attttgtcct aatatataca tagaaacttt
gtggggcatg 4500ttaagttaca gtttgcacaa gttcatctca tttgtattcc
attgattttt tttttcttct 4560aaacattttt tcttcaaaca gtatataact
ttttttaggg gatttttttt tagacagcaa 4620aaactatctg aagatttcca
tttgtcaaaa agtaatgatt tcttgataat tgtgtagtaa 4680tgttttttag
aacccagcag ttaccttaaa gctgaattta tatttagtaa cttctgtgtt
4740aatactggat agcatgaatt ctgcattgag aaactgaata gctgtcataa
aatgaaactt 4800tctttctaaa gaaagatact cacatgagtt cttgaagaat
agtcataact agattaagat 4860ctgtgtttta gtttaatagt ttgaagtgcc
tgtttgggat aatgataggt aatttagatg 4920aatttagggg aaaaaaaagt
tatctgcaga tatgttgagg gcccatctct ccccccacac 4980ccccacagag
ctaactgggt tacagtgttt tatccgaaag tttccaattc cactgtcttg
5040tgttttcatg ttgaaaatac ttttgcattt ttcctttgag tgccaatttc
ttactagtac 5100tatttcttaa tgtaacatgt ttacctggaa tgtattttaa
ctatttttgt atagtgtaaa 5160ctgaaacatg cacattttgt acattgtgct
ttcttttgtg ggacatatgc agtgtgatcc 5220agttgttttc catcatttgg
ttgcgctgac ctaggaatgt tggtcatatc aaacattaaa 5280aatgaccact
cttttaattg aaattaactt ttaaatgttt ataggagtat gtgctgtgaa
5340gtgatctaaa atttgtaata tttttgtcat gaactgtact actcctaatt
attgtaatgt 5400aataaaaata gttacagtga caaaaaaaaa aaaaaa
5436255312DNAHomo sapiens 25ggccgcggcg gcggaggcag cagcggcggc
ggcagtggcg gcggcgaagg tggcggcggc 60tcggccagta ctcccggccc ccgccatttc
ggactgggag cgagcgcggc gcaggcactg 120aaggcggcgg cggggccaga
ggctcagcgg ctcccaggtg cgggagagag gcctgctgaa 180aatgactgaa
tataaacttg tggtagttgg agctggtggc gtaggcaaga gtgccttgac
240gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag
aggattccta 300caggaagcaa gtagtaattg atggagaaac ctgtctcttg
gatattctcg acacagcagg 360tcaagaggag tacagtgcaa tgagggacca
gtacatgagg actggggagg gctttctttg 420tgtatttgcc ataaataata
ctaaatcatt tgaagatatt caccattata gagaacaaat 480taaaagagtt
aaggactctg aagatgtacc tatggtccta gtaggaaata aatgtgattt
540gccttctaga acagtagaca caaaacaggc tcaggactta gcaagaagtt
atggaattcc 600ttttattgaa acatcagcaa agacaagaca gggtgttgat
gatgccttct atacattagt 660tcgagaaatt cgaaaacata aagaaaagat
gagcaaagat ggtaaaaaga agaaaaagaa 720gtcaaagaca aagtgtgtaa
ttatgtaaat acaatttgta cttttttctt aaggcatact 780agtacaagtg
gtaatttttg tacattacac taaattatta gcatttgttt tagcattacc
840taattttttt cctgctccat gcagactgtt agcttttacc ttaaatgctt
attttaaaat 900gacagtggaa gttttttttt cctctaagtg ccagtattcc
cagagttttg gtttttgaac 960tagcaatgcc tgtgaaaaag aaactgaata
cctaagattt ctgtcttggg gtttttggtg 1020catgcagttg attacttctt
atttttctta ccaattgtga atgttggtgt gaaacaaatt 1080aatgaagctt
ttgaatcatc cctattctgt gttttatcta gtcacataaa tggattaatt
1140actaatttca gttgagacct tctaattggt ttttactgaa acattgaggg
aacacaaatt 1200tatgggcttc ctgatgatga ttcttctagg catcatgtcc
tatagtttgt catccctgat 1260gaatgtaaag ttacactgtt cacaaaggtt
ttgtctcctt tccactgcta ttagtcatgg 1320tcactctccc caaaatatta
tattttttct ataaaaagaa aaaaatggaa aaaaattaca 1380aggcaatgga
aactattata aggccatttc cttttcacat tagataaatt actataaaga
1440ctcctaatag cttttcctgt taaggcagac ccagtatgaa atggggatta
ttatagcaac 1500cattttgggg ctatatttac atgctactaa atttttataa
taattgaaaa gattttaaca 1560agtataaaaa attctcatag gaattaaatg
tagtctccct gtgtcagact gctctttcat 1620agtataactt taaatctttt
cttcaacttg agtctttgaa gatagtttta attctgcttg 1680tgacattaaa
agattatttg ggccagttat agcttattag gtgttgaaga gaccaaggtt
1740gcaaggccag gccctgtgtg aacctttgag ctttcataga gagtttcaca
gcatggactg 1800tgtccccacg gtcatccagt gttgtcatgc attggttagt
caaaatgggg agggactagg 1860gcagtttgga tagctcaaca agatacaatc
tcactctgtg gtggtcctgc tgacaaatca 1920agagcattgc ttttgtttct
taagaaaaca aactcttttt taaaaattac ttttaaatat 1980taactcaaaa
gttgagattt tggggtggtg gtgtgccaag acattaattt tttttttaaa
2040caatgaagtg aaaaagtttt acaatctcta ggtttggcta gttctcttaa
cactggttaa 2100attaacattg cataaacact tttcaagtct gatccatatt
taataatgct ttaaaataaa 2160aataaaaaca atccttttga taaatttaaa
atgttactta ttttaaaata aatgaagtga 2220gatggcatgg tgaggtgaaa
gtatcactgg actaggaaga aggtgactta ggttctagat 2280aggtgtcttt
taggactctg attttgagga catcacttac tatccatttc ttcatgttaa
2340aagaagtcat ctcaaactct tagttttttt tttttacaac tatgtaattt
atattccatt 2400tacataagga tacacttatt tgtcaagctc agcacaatct
gtaaattttt aacctatgtt 2460acaccatctt cagtgccagt cttgggcaaa
attgtgcaag aggtgaagtt tatatttgaa 2520tatccattct cgttttagga
ctcttcttcc atattagtgt catcttgcct ccctaccttc 2580cacatgcccc
atgacttgat gcagttttaa tacttgtaat tcccctaacc ataagattta
2640ctgctgctgt ggatatctcc atgaagtttt cccactgagt cacatcagaa
atgccctaca 2700tcttatttcc tcagggctca agagaatctg acagatacca
taaagggatt tgacctaatc 2760actaattttc aggtggtggc tgatgctttg
aacatctctt tgctgcccaa tccattagcg 2820acagtaggat ttttcaaacc
tggtatgaat agacagaacc ctatccagtg gaaggagaat 2880ttaataaaga
tagtgctgaa agaattcctt aggtaatcta taactaggac tactcctggt
2940aacagtaata cattccattg ttttagtaac cagaaatctt catgcaatga
aaaatacttt 3000aattcatgaa gcttactttt tttttttggt gtcagagtct
cgctcttgtc acccaggctg 3060gaatgcagtg gcgccatctc agctcactgc
aacctccatc tcccaggttc aagcgattct 3120cgtgcctcgg cctcctgagt
agctgggatt acaggcgtgt gccactacac tcaactaatt 3180tttgtatttt
taggagagac ggggtttcac cctgttggcc aggctggtct cgaactcctg
3240acctcaagtg attcacccac cttggcctca taaacctgtt ttgcagaact
catttattca 3300gcaaatattt attgagtgcc taccagatgc cagtcaccgc
acaaggcact gggtatatgg 3360tatccccaaa caagagacat aatcccggtc
cttaggtagt gctagtgtgg tctgtaatat 3420cttactaagg cctttggtat
acgacccaga gataacacga tgcgtatttt agttttgcaa 3480agaaggggtt
tggtctctgt gccagctcta taattgtttt gctacgattc cactgaaact
3540cttcgatcaa gctactttat gtaaatcact tcattgtttt aaaggaataa
acttgattat 3600attgtttttt tatttggcat aactgtgatt cttttaggac
aattactgta cacattaagg 3660tgtatgtcag atattcatat tgacccaaat
gtgtaatatt ccagttttct ctgcataagt 3720aattaaaata tacttaaaaa
ttaatagttt tatctgggta caaataaaca ggtgcctgaa 3780ctagttcaca
gacaaggaaa cttctatgta aaaatcacta tgatttctga attgctatgt
3840gaaactacag atctttggaa cactgtttag gtagggtgtt aagacttaca
cagtacctcg 3900tttctacaca gagaaagaaa tggccatact tcaggaactg
cagtgcttat gaggggatat 3960ttaggcctct tgaatttttg atgtagatgg
gcattttttt aaggtagtgg ttaattacct 4020ttatgtgaac tttgaatggt
ttaacaaaag atttgttttt gtagagattt taaaggggga 4080gaattctaga
aataaatgtt acctaattat tacagcctta aagacaaaaa tccttgttga
4140agttttttta aaaaaagcta aattacatag acttaggcat taacatgttt
gtggaagaat 4200atagcagacg tatattgtat catttgagtg aatgttccca
agtaggcatt ctaggctcta 4260tttaactgag tcacactgca taggaattta
gaacctaact tttataggtt atcaaaactg 4320ttgtcaccat tgcacaattt
tgtcctaata tatacataga aactttgtgg ggcatgttaa 4380gttacagttt
gcacaagttc atctcatttg tattccattg attttttttt tcttctaaac
4440attttttctt caaacagtat ataacttttt ttaggggatt tttttttaga
cagcaaaaac 4500tatctgaaga tttccatttg tcaaaaagta atgatttctt
gataattgtg tagtaatgtt 4560ttttagaacc cagcagttac cttaaagctg
aatttatatt tagtaacttc tgtgttaata 4620ctggatagca tgaattctgc
attgagaaac tgaatagctg tcataaaatg aaactttctt 4680tctaaagaaa
gatactcaca tgagttcttg aagaatagtc ataactagat taagatctgt
4740gttttagttt aatagtttga agtgcctgtt tgggataatg ataggtaatt
tagatgaatt 4800taggggaaaa aaaagttatc tgcagatatg ttgagggccc
atctctcccc ccacaccccc 4860acagagctaa ctgggttaca gtgttttatc
cgaaagtttc caattccact gtcttgtgtt 4920ttcatgttga aaatactttt
gcatttttcc tttgagtgcc aatttcttac tagtactatt 4980tcttaatgta
acatgtttac ctggaatgta ttttaactat ttttgtatag tgtaaactga
5040aacatgcaca ttttgtacat tgtgctttct tttgtgggac atatgcagtg
tgatccagtt 5100gttttccatc atttggttgc gctgacctag gaatgttggt
catatcaaac attaaaaatg 5160accactcttt taattgaaat taacttttaa
atgtttatag gagtatgtgc tgtgaagtga 5220tctaaaattt gtaatatttt
tgtcatgaac tgtactactc ctaattattg taatgtaata 5280aaaatagtta
cagtgacaaa aaaaaaaaaa aa 5312265436DNAHomo sapiens 26ggccgcggcg
gcggaggcag cagcggcggc ggcagtggcg gcggcgaagg tggcggcggc 60tcggccagta
ctcccggccc ccgccatttc ggactgggag cgagcgcggc gcaggcactg
120aaggcggcgg cggggccaga ggctcagcgg ctcccaggtg cgggagagag
gcctgctgaa 180aatgactgaa tataaacttg tggtagttgg agctggtggc
gtaggcaaga gtgccttgac
240gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag
aggattccta 300caggaagcaa gtagtaattg atggagaaac ctgtctcttg
gatattctcg acacagcagg 360tcaagaggag tacagtgcaa tgagggacca
gtacatgagg actggggagg gctttctttg 420tgtatttgcc ataaataata
ctaaatcatt tgaagatatt caccattata gagaacaaat 480taaaagagtt
aaggactctg aagatgtacc tatggtccta gtaggaaata aatgtgattt
540gccttctaga acagtagaca caaaacaggc tcaggactta gcaagaagtt
atggaattcc 600ttttattgaa acatcagcaa agacaagaca gagagtggag
gatgcttttt atacattggt 660gagggagatc cgacaataca gattgaaaaa
aatcagcaaa gaagaaaaga ctcctggctg 720tgtgaaaatt aaaaaatgca
ttataatgta atctgggtgt tgatgatgcc ttctatacat 780tagttcgaga
aattcgaaaa cataaagaaa agatgagcaa agatggtaaa aagaagaaaa
840agaagtcaaa gacaaagtgt gtaattatgt aaatacaatt tgtacttttt
tcttaaggca 900tactagtaca agtggtaatt tttgtacatt acactaaatt
attagcattt gttttagcat 960tacctaattt ttttcctgct ccatgcagac
tgttagcttt taccttaaat gcttatttta 1020aaatgacagt ggaagttttt
ttttcctcta agtgccagta ttcccagagt tttggttttt 1080gaactagcaa
tgcctgtgaa aaagaaactg aatacctaag atttctgtct tggggttttt
1140ggtgcatgca gttgattact tcttattttt cttaccaatt gtgaatgttg
gtgtgaaaca 1200aattaatgaa gcttttgaat catccctatt ctgtgtttta
tctagtcaca taaatggatt 1260aattactaat ttcagttgag accttctaat
tggtttttac tgaaacattg agggaacaca 1320aatttatggg cttcctgatg
atgattcttc taggcatcat gtcctatagt ttgtcatccc 1380tgatgaatgt
aaagttacac tgttcacaaa ggttttgtct cctttccact gctattagtc
1440atggtcactc tccccaaaat attatatttt ttctataaaa agaaaaaaat
ggaaaaaaat 1500tacaaggcaa tggaaactat tataaggcca tttccttttc
acattagata aattactata 1560aagactccta atagcttttc ctgttaaggc
agacccagta tgaaatgggg attattatag 1620caaccatttt ggggctatat
ttacatgcta ctaaattttt ataataattg aaaagatttt 1680aacaagtata
aaaaattctc ataggaatta aatgtagtct ccctgtgtca gactgctctt
1740tcatagtata actttaaatc ttttcttcaa cttgagtctt tgaagatagt
tttaattctg 1800cttgtgacat taaaagatta tttgggccag ttatagctta
ttaggtgttg aagagaccaa 1860ggttgcaagg ccaggccctg tgtgaacctt
tgagctttca tagagagttt cacagcatgg 1920actgtgtccc cacggtcatc
cagtgttgtc atgcattggt tagtcaaaat ggggagggac 1980tagggcagtt
tggatagctc aacaagatac aatctcactc tgtggtggtc ctgctgacaa
2040atcaagagca ttgcttttgt ttcttaagaa aacaaactct tttttaaaaa
ttacttttaa 2100atattaactc aaaagttgag attttggggt ggtggtgtgc
caagacatta attttttttt 2160taaacaatga agtgaaaaag ttttacaatc
tctaggtttg gctagttctc ttaacactgg 2220ttaaattaac attgcataaa
cacttttcaa gtctgatcca tatttaataa tgctttaaaa 2280taaaaataaa
aacaatcctt ttgataaatt taaaatgtta cttattttaa aataaatgaa
2340gtgagatggc atggtgaggt gaaagtatca ctggactagg aagaaggtga
cttaggttct 2400agataggtgt cttttaggac tctgattttg aggacatcac
ttactatcca tttcttcatg 2460ttaaaagaag tcatctcaaa ctcttagttt
ttttttttta caactatgta atttatattc 2520catttacata aggatacact
tatttgtcaa gctcagcaca atctgtaaat ttttaaccta 2580tgttacacca
tcttcagtgc cagtcttggg caaaattgtg caagaggtga agtttatatt
2640tgaatatcca ttctcgtttt aggactcttc ttccatatta gtgtcatctt
gcctccctac 2700cttccacatg ccccatgact tgatgcagtt ttaatacttg
taattcccct aaccataaga 2760tttactgctg ctgtggatat ctccatgaag
ttttcccact gagtcacatc agaaatgccc 2820tacatcttat ttcctcaggg
ctcaagagaa tctgacagat accataaagg gatttgacct 2880aatcactaat
tttcaggtgg tggctgatgc tttgaacatc tctttgctgc ccaatccatt
2940agcgacagta ggatttttca aacctggtat gaatagacag aaccctatcc
agtggaagga 3000gaatttaata aagatagtgc tgaaagaatt ccttaggtaa
tctataacta ggactactcc 3060tggtaacagt aatacattcc attgttttag
taaccagaaa tcttcatgca atgaaaaata 3120ctttaattca tgaagcttac
tttttttttt tggtgtcaga gtctcgctct tgtcacccag 3180gctggaatgc
agtggcgcca tctcagctca ctgcaacctc catctcccag gttcaagcga
3240ttctcgtgcc tcggcctcct gagtagctgg gattacaggc gtgtgccact
acactcaact 3300aatttttgta tttttaggag agacggggtt tcaccctgtt
ggccaggctg gtctcgaact 3360cctgacctca agtgatgcac ccaccttggc
ctcataaacc tgttttgcag aactcattta 3420ttcagcaaat atttattgag
tgcctaccag atgccagtca ccgcacaagg cactgggtat 3480atggtatccc
caaacaagag acataatccc ggtccttagg tagtgctagt gtggtctgta
3540atatcttact aaggcctttg gtatacgacc cagagataac acgatgcgta
ttttagtttt 3600gcaaagaagg ggtttggtct ctgtgccagc tctataattg
ttttgctacg attccactga 3660aactcttcga tcaagctact ttatgtaaat
cacttcattg ttttaaagga ataaacttga 3720ttatattgtt tttttatttg
gcataactgt gattctttta ggacaattac tgtacacatt 3780aaggtgtatg
tcagatattc atattgaccc aaatgtgtaa tattccagtt ttctctgcat
3840aagtaattaa aatatactta aaaattaata gttttatctg ggtacaaata
aacaggtgcc 3900tgaactagtt cacagacaag gaaacttcta tgtaaaaatc
actatgattt ctgaattgct 3960atgtgaaact acagatcttt ggaacactgt
ttaggtaggg tgttaagact tacacagtac 4020ctcgtttcta cacagagaaa
gaaatggcca tacttcagga actgcagtgc ttatgagggg 4080atatttaggc
ctcttgaatt tttgatgtag atgggcattt ttttaaggta gtggttaatt
4140acctttatgt gaactttgaa tggtttaaca aaagatttgt ttttgtagag
attttaaagg 4200gggagaattc tagaaataaa tgttacctaa ttattacagc
cttaaagaca aaaatccttg 4260ttgaagtttt tttaaaaaaa gctaaattac
atagacttag gcattaacat gtttgtggaa 4320gaatatagca gacgtatatt
gtatcatttg agtgaatgtt cccaagtagg cattctaggc 4380tctatttaac
tgagtcacac tgcataggaa tttagaacct aacttttata ggttatcaaa
4440actgttgtca ccattgcaca attttgtcct aatatataca tagaaacttt
gtggggcatg 4500ttaagttaca gtttgcacaa gttcatctca tttgtattcc
attgattttt tttttcttct 4560aaacattttt tcttcaaaca gtatataact
ttttttaggg gatttttttt tagacagcaa 4620aaactatctg aagatttcca
tttgtcaaaa agtaatgatt tcttgataat tgtgtagtaa 4680tgttttttag
aacccagcag ttaccttaaa gctgaattta tatttagtaa cttctgtgtt
4740aatactggat agcatgaatt ctgcattgag aaactgaata gctgtcataa
aatgaaactt 4800tctttctaaa gaaagatact cacatgagtt cttgaagaat
agtcataact agattaagat 4860ctgtgtttta gtttaatagt ttgaagtgcc
tgtttgggat aatgataggt aatttagatg 4920aatttagggg aaaaaaaagt
tatctgcaga tatgttgagg gcccatctct ccccccacac 4980ccccacagag
ctaactgggt tacagtgttt tatccgaaag tttccaattc cactgtcttg
5040tgttttcatg ttgaaaatac ttttgcattt ttcctttgag tgccaatttc
ttactagtac 5100tatttcttaa tgtaacatgt ttacctggaa tgtattttaa
ctatttttgt atagtgtaaa 5160ctgaaacatg cacattttgt acattgtgct
ttcttttgtg ggacatatgc agtgtgatcc 5220agttgttttc catcatttgg
ttgcgctgac ctaggaatgt tggtcatatc aaacattaaa 5280aatgaccact
cttttaattg aaattaactt ttaaatgttt ataggagtat gtgctgtgaa
5340gtgatctaaa atttgtaata tttttgtcat gaactgtact actcctaatt
attgtaatgt 5400aataaaaata gttacagtga caaaaaaaaa aaaaaa
5436275312DNAHomo sapiens 27ggccgcggcg gcggaggcag cagcggcggc
ggcagtggcg gcggcgaagg tggcggcggc 60tcggccagta ctcccggccc ccgccatttc
ggactgggag cgagcgcggc gcaggcactg 120aaggcggcgg cggggccaga
ggctcagcgg ctcccaggtg cgggagagag gcctgctgaa 180aatgactgaa
tataaacttg tggtagttgg agctggtggc gtaggcaaga gtgccttgac
240gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag
aggattccta 300caggaagcaa gtagtaattg atggagaaac ctgtctcttg
gatattctcg acacagcagg 360tcaagaggag tacagtgcaa tgagggacca
gtacatgagg actggggagg gctttctttg 420tgtatttgcc ataaataata
ctaaatcatt tgaagatatt caccattata gagaacaaat 480taaaagagtt
aaggactctg aagatgtacc tatggtccta gtaggaaata aatgtgattt
540gccttctaga acagtagaca caaaacaggc tcaggactta gcaagaagtt
atggaattcc 600ttttattgaa acatcagcaa agacaagaca gggtgttgat
gatgccttct atacattagt 660tcgagaaatt cgaaaacata aagaaaagat
gagcaaagat ggtaaaaaga agaaaaagaa 720gtcaaagaca aagtgtgtaa
ttatgtaaat acaatttgta cttttttctt aaggcatact 780agtacaagtg
gtaatttttg tacattacac taaattatta gcatttgttt tagcattacc
840taattttttt cctgctccat gcagactgtt agcttttacc ttaaatgctt
attttaaaat 900gacagtggaa gttttttttt cctctaagtg ccagtattcc
cagagttttg gtttttgaac 960tagcaatgcc tgtgaaaaag aaactgaata
cctaagattt ctgtcttggg gtttttggtg 1020catgcagttg attacttctt
atttttctta ccaattgtga atgttggtgt gaaacaaatt 1080aatgaagctt
ttgaatcatc cctattctgt gttttatcta gtcacataaa tggattaatt
1140actaatttca gttgagacct tctaattggt ttttactgaa acattgaggg
aacacaaatt 1200tatgggcttc ctgatgatga ttcttctagg catcatgtcc
tatagtttgt catccctgat 1260gaatgtaaag ttacactgtt cacaaaggtt
ttgtctcctt tccactgcta ttagtcatgg 1320tcactctccc caaaatatta
tattttttct ataaaaagaa aaaaatggaa aaaaattaca 1380aggcaatgga
aactattata aggccatttc cttttcacat tagataaatt actataaaga
1440ctcctaatag cttttcctgt taaggcagac ccagtatgaa atggggatta
ttatagcaac 1500cattttgggg ctatatttac atgctactaa atttttataa
taattgaaaa gattttaaca 1560agtataaaaa attctcatag gaattaaatg
tagtctccct gtgtcagact gctctttcat 1620agtataactt taaatctttt
cttcaacttg agtctttgaa gatagtttta attctgcttg 1680tgacattaaa
agattatttg ggccagttat agcttattag gtgttgaaga gaccaaggtt
1740gcaaggccag gccctgtgtg aacctttgag ctttcataga gagtttcaca
gcatggactg 1800tgtccccacg gtcatccagt gttgtcatgc attggttagt
caaaatgggg agggactagg 1860gcagtttgga tagctcaaca agatacaatc
tcactctgtg gtggtcctgc tgacaaatca 1920agagcattgc ttttgtttct
taagaaaaca aactcttttt taaaaattac ttttaaatat 1980taactcaaaa
gttgagattt tggggtggtg gtgtgccaag acattaattt tttttttaaa
2040caatgaagtg aaaaagtttt acaatctcta ggtttggcta gttctcttaa
cactggttaa 2100attaacattg cataaacact tttcaagtct gatccatatt
taataatgct ttaaaataaa 2160aataaaaaca atccttttga taaatttaaa
atgttactta ttttaaaata aatgaagtga 2220gatggcatgg tgaggtgaaa
gtatcactgg actaggaaga aggtgactta ggttctagat 2280aggtgtcttt
taggactctg attttgagga catcacttac tatccatttc ttcatgttaa
2340aagaagtcat ctcaaactct tagttttttt tttttacaac tatgtaattt
atattccatt 2400tacataagga tacacttatt tgtcaagctc agcacaatct
gtaaattttt aacctatgtt 2460acaccatctt cagtgccagt cttgggcaaa
attgtgcaag aggtgaagtt tatatttgaa 2520tatccattct cgttttagga
ctcttcttcc atattagtgt catcttgcct ccctaccttc 2580cacatgcccc
atgacttgat gcagttttaa tacttgtaat tcccctaacc ataagattta
2640ctgctgctgt ggatatctcc atgaagtttt cccactgagt cacatcagaa
atgccctaca 2700tcttatttcc tcagggctca agagaatctg acagatacca
taaagggatt tgacctaatc 2760actaattttc aggtggtggc tgatgctttg
aacatctctt tgctgcccaa tccattagcg 2820acagtaggat ttttcaaacc
tggtatgaat agacagaacc ctatccagtg gaaggagaat 2880ttaataaaga
tagtgctgaa agaattcctt aggtaatcta taactaggac tactcctggt
2940aacagtaata cattccattg ttttagtaac cagaaatctt catgcaatga
aaaatacttt 3000aattcatgaa gcttactttt tttttttggt gtcagagtct
cgctcttgtc acccaggctg 3060gaatgcagtg gcgccatctc agctcactgc
aacctccatc tcccaggttc aagcgattct 3120cgtgcctcgg cctcctgagt
agctgggatt acaggcgtgt gccactacac tcaactaatt 3180tttgtatttt
taggagagac ggggtttcac cctgttggcc aggctggtct cgaactcctg
3240acctcaagtg atgcacccac cttggcctca taaacctgtt ttgcagaact
catttattca 3300gcaaatattt attgagtgcc taccagatgc cagtcaccgc
acaaggcact gggtatatgg 3360tatccccaaa caagagacat aatcccggtc
cttaggtagt gctagtgtgg tctgtaatat 3420cttactaagg cctttggtat
acgacccaga gataacacga tgcgtatttt agttttgcaa 3480agaaggggtt
tggtctctgt gccagctcta taattgtttt gctacgattc cactgaaact
3540cttcgatcaa gctactttat gtaaatcact tcattgtttt aaaggaataa
acttgattat 3600attgtttttt tatttggcat aactgtgatt cttttaggac
aattactgta cacattaagg 3660tgtatgtcag atattcatat tgacccaaat
gtgtaatatt ccagttttct ctgcataagt 3720aattaaaata tacttaaaaa
ttaatagttt tatctgggta caaataaaca ggtgcctgaa 3780ctagttcaca
gacaaggaaa cttctatgta aaaatcacta tgatttctga attgctatgt
3840gaaactacag atctttggaa cactgtttag gtagggtgtt aagacttaca
cagtacctcg 3900tttctacaca gagaaagaaa tggccatact tcaggaactg
cagtgcttat gaggggatat 3960ttaggcctct tgaatttttg atgtagatgg
gcattttttt aaggtagtgg ttaattacct 4020ttatgtgaac tttgaatggt
ttaacaaaag atttgttttt gtagagattt taaaggggga 4080gaattctaga
aataaatgtt acctaattat tacagcctta aagacaaaaa tccttgttga
4140agttttttta aaaaaagcta aattacatag acttaggcat taacatgttt
gtggaagaat 4200atagcagacg tatattgtat catttgagtg aatgttccca
agtaggcatt ctaggctcta 4260tttaactgag tcacactgca taggaattta
gaacctaact tttataggtt atcaaaactg 4320ttgtcaccat tgcacaattt
tgtcctaata tatacataga aactttgtgg ggcatgttaa 4380gttacagttt
gcacaagttc atctcatttg tattccattg attttttttt tcttctaaac
4440attttttctt caaacagtat ataacttttt ttaggggatt tttttttaga
cagcaaaaac 4500tatctgaaga tttccatttg tcaaaaagta atgatttctt
gataattgtg tagtaatgtt 4560ttttagaacc cagcagttac cttaaagctg
aatttatatt tagtaacttc tgtgttaata 4620ctggatagca tgaattctgc
attgagaaac tgaatagctg tcataaaatg aaactttctt 4680tctaaagaaa
gatactcaca tgagttcttg aagaatagtc ataactagat taagatctgt
4740gttttagttt aatagtttga agtgcctgtt tgggataatg ataggtaatt
tagatgaatt 4800taggggaaaa aaaagttatc tgcagatatg ttgagggccc
atctctcccc ccacaccccc 4860acagagctaa ctgggttaca gtgttttatc
cgaaagtttc caattccact gtcttgtgtt 4920ttcatgttga aaatactttt
gcatttttcc tttgagtgcc aatttcttac tagtactatt 4980tcttaatgta
acatgtttac ctggaatgta ttttaactat ttttgtatag tgtaaactga
5040aacatgcaca ttttgtacat tgtgctttct tttgtgggac atatgcagtg
tgatccagtt 5100gttttccatc atttggttgc gctgacctag gaatgttggt
catatcaaac attaaaaatg 5160accactcttt taattgaaat taacttttaa
atgtttatag gagtatgtgc tgtgaagtga 5220tctaaaattt gtaatatttt
tgtcatgaac tgtactactc ctaattattg taatgtaata 5280aaaatagtta
cagtgacaaa aaaaaaaaaa aa 5312281963DNAHomo sapiens 28gaaacgtccc
gtgtgggagg ggcgggtctg ggtgcggctg ccgcatgact cgtggttcgg 60aggcccacgt
ggccggggcg gggactcagg cgcctggcag ccgactgatt acgtagcggg
120cggggccgga agtgccgctc cttggtgggg gctgttcatg gcggttccgg
ggtctccaac 180atttttcccg gtctgtggtc ctaaatctgt ccaaagcaga
ggcagtggag cttgaggttc 240ttgctggtgt gaaatgactg agtacaaact
ggtggtggtt ggagcaggtg gtgttgggaa 300aagcgcactg acaatccagc
taatccagaa ccactttgta gatgaatatg atcccaccat 360agaggattct
tacagaaaac aagtggttat agatggtgaa acctgtttgt tggacatact
420ggatacagct ggacaagaag agtacagtgc catgagagac caatacatga
ggacaggcga 480aggcttcctc tgtgtatttg ccatcaataa tagcaagtca
tttgcggata ttaacctcta 540cagggagcag attaagcgag taaaagactc
ggatgatgta cctatggtgc tagtgggaaa 600caagtgtgat ttgccaacaa
ggacagttga tacaaaacaa gcccacgaac tggccaagag 660ttacgggatt
ccattcattg aaacctcagc caagaccaga cagggtgttg aagatgcttt
720ttacacactg gtaagagaaa tacgccagta ccgaatgaaa aaactcaaca
gcagtgatga 780tgggactcag ggttgtatgg gattgccatg tgtggtgatg
taacaagata cttttaaagt 840tttgtcagaa aagagccact ttcaagctgc
actgacaccc tggtcctgac ttcctggagg 900agaagtattc ctgttgctgt
cttcagtctc acagagaagc tcctgctact tccccagctc 960tcagtagttt
agtacaataa tctctatttg agaagttctc agaataacta cctcctcact
1020tggctgtctg accagagaat gcacctcttg ttactccctg ttatttttct
gccctgggtt 1080cttccacagc acaaacacac ctcaacacac ctctgccacc
ccaggttttt catctgaaaa 1140gcagttcatg tctgaaacag agaaccaaac
cgcaaacgtg aaattctatt gaaaacagtg 1200tcttgagctc taaagtagca
actgctggtg attttttttt tctttttact gttgaactta 1260gaactatgcc
taatttttgg agaaatgtca taaattactg ttttgccaag aatatagtta
1320ttattgctgt ttggtttgtt tataatgtta tcggctctat tctctaaact
ggcatctgct 1380ctagattcat aaatacaaaa atgaatactg aattttgagt
ctatcctagt cttcacaact 1440ttgacgtaat taaatccaac ttttcacagt
gaagtgcctt tttcctagaa gtggtttgta 1500gactccttta taatatttca
gtggaataga tgtctcaaaa atccttatgc atgaaatgaa 1560tgtctgagat
acgtctgtga cttatctacc attgaaggaa agctatatct atttgagagc
1620agatgccatt ttgtacatgt atgaaattgg ttttccagag gcctgttttg
gggctttccc 1680aggagaaaga tgaaactgaa agcatatgaa taatttcact
taataatttt tacctaatct 1740ccactttttt cataggttac tacctataca
atgtatgtaa tttgtttccc ctagcttact 1800gataaaccta atattcaatg
aacttccatt tgtattcaaa tttgtgtcat accagaaagc 1860tctacatttg
cagatgttca aatattgtaa aactttggtg cattgttatt taatagctgt
1920gatcagtgat tttcaaacct caaatatagt atattaacaa att
19632940DNAArtificial SequencePrimer Sequence 29ctagctagca
tacaatttgt acttttttct taaggcatac 403024DNAArtificial SequencePrimer
Sequence 30ggcacaccac caccccaaaa tctc 243123DNAArtificial
SequencePrimer Sequence 31ccatcttcag tgccagtctt ggg
233223DNAArtificial SequencePrimer Sequence 32gggtcgtata ccaaaggcct
tag 233325DNAArtificial SequencePrimer Sequence 33gcctgaacta
gttcacagac aaggg 253434DNAArtificial SequencePrimer Sequence
34ctagctagct caatgcagaa ttcatgctat ccag 343520DNAArtificial
SequencePrimer Sequence 35ggtgtcagag tctcgctctt 203623DNAArtificial
SequencePrimer Sequence 36gggtcgtata ccaaaggcct tag
233720DNAArtificial SequencePrimer Sequence 37cctgagtagc tgggattaca
203823DNAArtificial SequencePrimer Sequence 38ggataccata tacccagtgc
ctt 233923DNAArtificial SequencePrimer Sequence 39ccactttcaa
gctgcactga cac 234027DNAArtificial SequencePrimer Sequence
40ctagctggag ttactggtgc aatgagc 274123DNAArtificial SequencePrimer
Sequence 41gatacctatg aggatttgga ggc 234223DNAArtificial
SequencePrimer Sequence 42gcatggtagc cttcagacag aac
234324DNAArtificial SequencePrimer Sequence 43ctgcttcttg taattcatct
ctgc 244423DNAArtificial SequencePrimer Sequence 44caacttaaaa
tatcggccct tcc 234533DNAArtificial SequencePrimer Sequence
45cgaactcctg acctcaagtg atgcacccac ctt 334632DNAArtificial
SequencePrimer Sequence 46atcacttgag gtcaggagtt cgagaccagc ct
324726RNACaenorhabditis elegans 47acuuguaguc gaucuccuuc cgccuc
264822RNACaenorhabditis elegans 48auguuauaau guaugaugga gu
224925RNACaenorhabditis elegans 49ugcaucgauu gaacuuguuc ucucg
255020RNACaenorhabditis elegans 50uccuucauuc uaauuccuca
205124RNACaenorhabditis elegans 51acuagcaucc gaacccccuc cucg
245221RNACaenorhabditis elegans 52uguuauaaug uaugauggag u
215320RNACaenorhabditis elegans 53ugcuuuauuc cccuuccucg
205422RNACaenorhabditis elegans 54uucauacaaa uuauuggccu ca
225523RNACaenorhabditis elegans 55auucgaaagu uuuugcuccc ucg
235619RNACaenorhabditis elegans 56ucuauuuuuc cuauuccuc
195722RNAHomo sapiens 57uugauauguu ggaugaugga gu
225822DNAArtificial SequenceConsensus sequence 58tgaggtagta
ggttgtatag tt 225918DNAHomo sapiens 59tgaggtagta gtttgtgc
186022DNAHomo sapiens 60tgaggtagta gtgtgtacag tt 226122DNAHomo
sapiens 61tgaggtagta gtttgtacag ta 226221DNAHomo sapiens
62agaggtagta gtttgcatag t 216321DNAHomo sapiens 63agaggtagta
ggttgcatag t 216422DNAHomo sapiens 64tgaggtagta gattgtatag tt
226522DNAHomo sapiens 65tgaggtagta ggttgtatag tt 226621DNAHomo
sapiens 66tgaggtagga ggttgtatag t 216722DNAHomo sapiens
67tgaggtagta ggttgtatgg tt 226822DNAHomo sapiens 68tgaggtagta
ggttgtgtgg tt 226922DNAHomo sapiens 69tgaggtagta agttgtattg tt
227022DNAHomo sapiens 70tgaggtagta tgtaatattg ta
227120DNAArtificial SequenceConsensus sequence 71tgaggtaggt
gcgagaaatg 207223DNAHomo sapiens 72tgaggtaggc tcagtagatg cga
237365RNACaenorhabditis elegans 73ugagguagua gguuguauag uuuggaauau
uaccaccggu gaacuaugca auuuucuacc 60uuacc 657461RNADrosophila
melanogaster 74ugagguagua gguuguauag uaguaauuac acaucauacu
auacaaugug cuagcuuucu 60u 617570RNAHomo sapiens 75ugagguagua
gguuguauag uuuggggcuc ugcccugcua ugggauaacu auacaaucua 60cugucuuucc
707622RNAHomo sapiens 76ugagguagua gguuguauag uu 227722RNAHomo
sapiens 77ugagguagua gguugugugg uu 227822RNAHomo sapiens
78ugagguagua gguuguaugg uu 227921RNAHomo sapiens 79agagguagua
gguugcauag u 218020RNAHomo sapiens 80ugagguagga gguuguauau
208122RNAHomo sapiens 81ugagguagua gauuguauag uu 228220RNAHomo
sapiens 82ugagguagua guuuguacag 208321RNAHomo sapiens 83ugagguaguu
guuuguggug u 218461RNACaenorhabditis elegans 84uucccugaga
ccucaagugu gaguguacua uguaugcuuc acaccugggc ucuccgggua 60c
618573RNACaenorhabditis elegans 85uccggugagg uaguagguug uauaguuugg
aauauuacca ccggugaacu augcaauuuu 60cuaccuuacc gga
738618RNACaenorhabditis elegans 86cucacacaac ucaggaau
188723RNACaenorhabditis elegans 87gagugugacu ccagaguccc uug
238823RNACaenorhabditis elegans 88uuauacaacc cruucuacac uca
238921RNACaenorhabditis elegans 89ugauauguug gaugauggag u
219021DNAHomo sapiens 90gatkcaccca ccttggcctc a 219121DNAHomo
sapiens 91gattcaccca ccttggcctc a 219221DNAHomo sapiens
92gatgcaccca ccttggcctc a 219321DNAHomo sapiens 93gattcaccca
ccttggcctc a 219421DNAHomo sapiensmisc_feature(4)..(4)n is a, c, g,
t or u 94gatncaccca ccttggcctc a 21
* * * * *
References