GENEMAP OF THE HUMAN GENES ASSOCIATED WITH ENDOMETRIOSIS

CARTE GENETIQUE DES GENES HUMAINS ASSOCIES A L'ENDOMETRIOSE

English Abstract

The present invention relates to the selection of a set of polymorphism markers for use in genome wide association studies based on linkage disequilibrium mapping. In particular, the invention relates to the fields of pharmacogenomics, diagnostics, patient therapy and the use of genetic haplotype information to predict an individual's susceptibility to ENDOMETRIOSIS disease and/or their response to a particular drug or drugs.

French Abstract

L'invention concerne la sélection d'un ensemble de marqueurs de polymorphismes utilisés dans des études d'association sur l'ensemble du génome sur la base de la cartographie de déséquilibre de liaison. Cette invention relève en particulier des domaines de la pharmacogénomique, du diagnostic, du traitement des patients et de l'utilisation d'informations relatives aux haplotypes génétiques pour prédire, chez un individu, le risque de développer une endométriose et/ou sa réponse à un ou plusieurs médicaments particuliers.

Claims

WE CLAIM:
1. A method of constructing a GeneMap for ENDOMETRIOSIS disease
comprising identifying at least two chromosomal loci associated with
ENDOMETRIOSIS disease, wherein said at least two chromosomal
loci are selected from the genomic regions listed in Table 1.
2. The method of claim 1, wherein said population is a general population.
3. The method of claim 1, wherein said population is a founder
population.
4. The method of claim 3, wherein said founder population is the
population of Quebec.
5. The method of claim 1, wherein said at least two chromosomal regions
are selected from the genes in Table 2, 3 or 4.
6. The method of claim 5, wherein said genes are used to construct gene
networks based on the functional relationship of gene products
interactions.
7. The method of claim 6, wherein the interactions are direct, indirect, or a
combination thereof.
8. The method of claim 1, wherein the identifying comprises screening for
the presence or absence of at least one single nucleotide
polymorphisms (SNPs) from Tables 5-16 in at least one sample.
9. The method of claim 8, wherein the screening comprises the steps of:
(a) obtaining biological samples from at least one disease patient; (b)
screening for the presence or absence of at least one SNP or a group
of SNPs from Tables 5-16 within each biological sample; and (c)
evaluating whether said SNP or a group of SNPs shows a statistically
significant skewed genotype distribution between a group of patients
compared to a control.
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10. The method of claim 9, wherein said biological samples are fluid,
serum, tissue or buccal swabs, saliva, mucus, urine, stools, vaginal
secretions, lymph, amiotic liquid, pleural liquid or tears.
11. The method of claim 9, wherein said patients and controls are from a
human population.
12. The method of claim 11, wherein said patients and controls are
recruited independently according to specific phenotypic criteria.
13. The method of claim 11, wherein said patients and controls are
recruited in the form of trios comprising two parents and one child.
14. The method of claim 8, wherein said screening is performed by a
method selected from the group consisting of an allele-specific
hybridization assay, an oligonucleotide ligation assay, an allele-specific
elongation/ligation assay, an allele-specific amplification assay, a
single-base extension assay, a molecular inversion probe assay, an
invasive cleavage assay, a selective termination assay, RFLP, a
sequencing assay, SSCP, a mismatch-cleaving assay, and denaturing
gradient gel electrophoresis.
15. The method of claim 8, wherein said screening is carried out on each
individual of a cohort at each of at least one SNP or a group of SNPs
from Tables 5-16.
16. The method of claim 8, wherein said screening is carried out on pools
of patients and pools of controls.
17. The method of claim 8, wherein the genotype distribution is compared
one SNP at a time.
18. The method of claim 8, wherein the genotype distribution is compared
with a group of markers from Tables 5-16 forming a haplotype.
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19. The method of claim 17, wherein the genotype distribution is compared
using the allelic frequencies between the patient pools and control
pools.
20. The method of claim 1, wherein the GeneMap comprises all of the
genes of Tables 2-4.
21. A method of diagnosing ENDOMETRIOSIS disease, the predisposition
to ENDOMETRIOSIS disease, or the progression or prognostication of
ENDOMETRIOSIS disease, comprising determining the amount and/or
concentration of at least one polypeptide from Tables 2-4 and/or at
least one nucleic acid encoding the polypeptide present in said
biological sample
22. The method of claim 21, wherein the diagnosing comprises the steps
of: (a) obtaining a biological sample of mammalian body fluid or tissue
to be diagnosed; (b) comparing the amount and/or concentration of
said polypeptide and/or nucleic acid encoding the polypeptide
determine in said biological sample with the amount and/or
concentration of said polypeptide and/or nucleic acid encoding the
polypeptide as determined in a control sample, wherein the difference
in the amount of said polypeptide and/or nucleic acid encoding the
polypeptide is indicative of ENDOMETRIOSIS disease or the stage of
ENDOMETRIOSIS disease.
23. The method of claim 21, wherein a nucleic acid probe is used for
determining the amount and/or concentration of at least one nucleic
acid sequence from Tables 2-4 encoding the polypeptide.
24. The method of claim 23, wherein said nucleic acid probe is selected
from the nucleic acid sequences designated as SEQ ID NO: 1 to 6723.
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25. The method of claim 23, wherein said nucleic acid probe comprises
nucleic acids hybridizing to the nucleic acid sequences designated as
SEQ ID NO: 1 to 6723, and/or fragments thereof.
26. The method of claim 23, wherein said nucleic acid probe comprises
nucleic acids hybridizing to at least five nucleic acid sequences from
Table 2, 3 or 4.
27. The method of claim 23, wherein said nucleic acid probe specifically
hybridizes to at least 10 nucleic acid sequences from Tables 2-4.
28. The method of claim 23, wherein said nucleic acid probe specifically
hybridizes to at least 20 nucleic acid sequences from Tables 2-4.
29. The method of claim 23, wherein said nucleic acid probe specifically
hybridizes to at least 50 nucleic acid sequences from Tables 2-4.
30. The method of claim 23, wherein said nucleic acid probe specifically
hybridizes to at least 100 nucleic acid sequences from Tables 2-4.
31. The method of claim 23, wherein said nucleic acid probe specifically
hybridizes to at least 100 nucleic acid sequences from Tables 2-4.
32. The method of claim 23, wherein said nucleic acid probe is at least
about 10 nucleotides in length.
33. The method of claim 23, wherein said nucleic acid probe is at least
about 30 nucleotides in length.
34. The method of claim 23, wherein said nucleic acid probe is at least
about 50 nucleotides in length.
35. The method of claim 23, wherein a PCR technique is used for
determining the amount and/or concentration of at least one nucleic
acid from Tables 2-4.
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36. The method of claim 21, wherein a specific antibody is used for
determining the amount and/or concentration of at least one
polypeptide from Tables 2-4.
37. The method of claim 36 wherein said antibody is selected from the
group comprising polyclonal antiserum, polyclonal antibody,
monoclonal antibody, antibody fragments, single chain antibodies and
diabodies.
38. The method of claim 21, wherein the amounts and/or concentrations of
at least five polypeptides or nucleic acids are determined.
39. A method of detecting susceptibility to ENDOMETRIOSIS disease
comprising detecting at least one mutation or polymorphism in the
nucleic acid molecule selected from Tables 2-4 in a patient.
40. The method of claim 39, wherein said method comprises hybridizing a
probe to said patient's sample of DNA or RNA under stringent
conditions which allow hybridization of said probe to nucleic acid
comprising said mutation or polymorphism, wherein the presence of a
hybridization signal indicates the presence of said mutation or
polymorphism in at least one gene from Tables 2-4.
41. The method of claim 39, wherein the patient's DNA or RNA has been
amplified and said amplified DNA or RNA is hybridized.
42. The method of claim 39, wherein said method comprises using a
single-stranded conformation polymorphism technique to assay for said
mutation.
43. The method of claim 39, wherein said method comprises sequencing at
least one gene from Tables 2-4 in a sample of RNA or DNA from a
patient.
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44. The method of claim 39, wherein said method comprises determining
the sequence of at least one gene from Tables 2-4 by preparing cDNA
from RNA taken from said patient and sequencing said cDNA to
determine the presence or absence of a mutation.
45. The method of claim 39, wherein said method comprises performing an
RNAse assay.
46. The method of claim 39, wherein said probe is attached to a microarray
or a bead.
47. The method of claim 39, wherein said probes are oligonucleotides.
48. The method of claim 40, wherein said sample is selected from the
group consisting of blood, normal tissue and tumor tissue.
49. The method of claim 39, wherein the mutation is selected from the
group consisting of at least one of the SNPs from Tables 5-16, alone or
in combination.
50. The method of claim 21, further comprising comparing the level of
expression or activity of a polypeptide of Tables 2-4 in a test sample
from a patient with the level of expression or activity of the same
polypeptide in a control sample wherein a difference in the level of
expression or activity between the test sample and control sample is
indicative of ENDOMETRIOSIS disease.
51. A method of treatment of ENDOMETRIOSIS disease in a mammal in
need thereof, comprising the steps of: performing steps a) to c)
according to claim 22; and treating the mammal in need of said
treatment; wherein said medical treatment is based on the stage of the
disease.
52. A method of diagnosing susceptibility to ENDOMETRIOSIS disease in
an individual, comprising screening for an at-risk haplotype of at least
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one gene or gene region from Tables 2-4, that is more frequently
present in an individual susceptible to ENDOMETRIOSIS disease
compared to a control individual, wherein the presence of the at-risk
haplotype is indicative of a susceptibility to ENDOMETRIOSIS disease.
53. The method of claim 52 wherein the at-risk haplotype is indicative of
increased risk for ENDOMETRIOSIS disease.
54. The method of claim 53, wherein the risk is increased at least about
20%.
55. The method of claim 52, wherein the at-risk haplotype is characterized
by the presence of at least one single nucleotide polymorphism from
Tables 5-16.
56. The method of claim 52, wherein screening for the presence of an at-
risk haplotype in at least one gene from Tables 2-4, comprises
enzymatic amplification of nucleic acid from said individual or
amplification using universal oligos on elongation/ligation products.
57. The method of claim 56, wherein the nucleic acid is DNA.
58. The method of claim 57, wherein the DNA is human DNA.
59. The method of claim 52, wherein screening for the presence of an at-
risk haplotype in at least one gene from Tables 2-4 comprises: (a)
obtaining material containing nucleic acid from the individual; (b)
amplifying said nucleic acid; and (c) determining the presence or
absence of an at-risk haplotype in said amplified nucleic acid.
60. The method of claim 59, wherein determining the presence of an at-risk
haplotype is performed by electrophoretic analysis.
61. The method of claim 59, wherein determining the presence of an at-risk
haplotype is performed by restriction length polymorphism analysis.
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62. The method of claim 59, wherein determining the presence of an at-risk
haplotype is performed by sequence analysis.
63. The method of claim 59, wherein determining the presence of an at-risk
haplotype is performed by hybridization analysis.
64. A method of diagnosing a susceptibility to ENDOMETRIOSIS disease,
comprising detecting an alteration in the expression or composition of a
polypeptide encoded by at least one gene from Tables 2-4 in a test
sample, in comparison with the expression or composition of a
polypeptide encoded by said gene in a control sample, wherein the
presence of an alteration in expression or composition of the
polypeptide in the test sample is indicative of a susceptibility to
ENDOMETRIOSIS Disease.
65. The method of claim 64, wherein the alteration in the expression or
composition of a polypeptide encoded by said gene comprises
expression of a splicing variant polypeptide in a test sample that differs
from a splicing variant polypeptide expressed in a control sample.
66. A drug screening assay comprising: a)administering a test compound
to an animal having ENDOMETRIOSIS disease, or a cell population
isolated therefrom; and (b) comparing the level of gene expression of
at least one gene from Tables 2-4 in the presence of the test
compound with the level of said gene expression in normal cells;
wherein test compounds which provide the level of expression of one
or more genes from Tables 2-4 similar to that of the normal cells are
candidates for drugs to treat ENDOMETRIOSIS disease.
67. A pharmaceutical preparation for treating an animal having
ENDOMETRIOSIS disease comprising a compound identified by the
assay of claim 66 and a pharmaceutically acceptable excipient.
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68. A method for treating an animal having ENDOMETRIOSIS disease
comprising administering a compound identified by the assay of claim
66.
69. A method for predicting the efficacy of a drug for treating
ENDOMETRIOSIS disease in a human patient, comprising: (a)
obtaining a sample of cells from the patient; (b) obtaining a gene
expression profile from the sample in the absence and presence of the
drug ; the gene expression profile comprising one or more genes from
Tables 2-4; and (c) comparing the gene expression profile of the
sample with a reference gene expression profile, wherein similarity
between the sample expression profile and the reference expression
profile predicts the efficacy of the drug for treating ENDOMETRIOSIS
disease in the patient.
70. The method of claim 69, further comprising exposing the sample to the
drug for treating ENDOMETRIOSIS disease prior to obtaining the gene
expression profile of the sample.
71. The method of claim 69, wherein the sample of cells is derived from a
tissue selected from the group consisting of: the brain, reproductive
system, digestive system, skin, scalp, muscle and nervous tissue.
72. The method of claim 71, wherein the cells are selected from the group
consisting of: ovarian cell, uterine cell, vaginal cell, hair cell, brain
cell,
muscle cell, neutrophil, dentric cell, T cell, mast cell, CD4+ lymphocyte,
monocyte, macrophage, dendritic cell, and epithelial cell.
73. The method of claim 69, wherein the sample is obtained via brain
biopsy.
74. The method of claim 69, wherein the gene expression profile
comprises expression values for all of the genes listed in Tables 2-4.
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75. The method of claim 74, wherein the gene expression profile of the
sample is obtained by detecting the protein products of said genes.
76. The method of claim 69, wherein the gene expression profile of the
sample is obtained using a hybridization assay to oligonucleotides
contained in a microarray.
77. The method of claim 76, wherein the oligonucleotides comprises
nucleic acid molecules at least 95% identical to the gene sequences
from Tables 2-4.
78. The method of claim 69, wherein the reference expression profile is
that of cells derived from patients that do not have ENDOMETRIOSIS
disease.
79. The method of claim 69, wherein the drug is selected from the group
consisting of symptom relievers.
80. The method of claim 69, wherein said patient's sample of DNA has
been amplified or cloned.
81. A method for predicting the efficacy of a drug for treating
ENDOMETRIOSIS disease in a human patient, comprising: a)
obtaining a sample of cells from the patient; b) obtaining a set of
genotypes from the sample, wherein the set of genotypes comprises
genotypes of one or more polymorphic loci from Tables 2-16; and c)
comparing the set of genotypes of the sample with a set of genotypes
associated with efficacy of the drug, wherein similarity between the set
of genotypes of the sample and the set of genotypes associated with
efficacy of the drug predicts the efficacy of the drug for treating
ENDOMETRIOSIS disease in the patient.
82. The method of claim 81, wherein the sample of cells is derived from a
tissue selected from the group consisting of: ovarian cell, uterine cell,
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vaginal cell, skin, brain, nervous system, digestive system, respiratory
system, scalp and reproductive system.
83. The method of claim 82, wherein the cells are selected from the group
consisting of: ovarian cell, uterine cell, vaginal cell, hair cell, brain
cell,
muscle cell, neutrophil, dentric cell, T cell, mast cell, CD4+ lymphocyte,
monocyte, macrophage, dendritic cell, and epithelial cell.
84. The method of claim 81, wherein the sample is obtained via biopsy.
85. The method of claim 81, wherein the set of genotypes from the sample
comprises genotypes of at least two of the polymorphic loci listed in
Tables 2-16.
86. The method of claim 81 wherein the set of genotypes from the sample
is obtained by hybridization to allele-specific oligonucleotides
complementary to the polymorphic loci from Tables 2-16, wherein said
allele-specific oligonucleotides are contained on a microarray.
87. The method of claim 86, wherein the oligonucleotides comprise nucleic
acid molecules at least 95% identical to SEQ ID from Tables 2-16.
88. The method of claim 81 wherein the set of genotypes from the sample
is obtained by sequencing said polymorphic loci in said sample.
89. The method of claim 81, wherein the drug is selected from the group
consisting of symptom relievers and drugs for ENDOMETRIOSIS
disease.
90. A method of treating ENDOMETRIOSIS disease in a patient in need
thereof, comprising expressing in vivo at least one gene from Tables 2-
4 in an amount sufficient to treat the disease.
91. The method of claim 90, comprising: (a) administering to a patient a
vector comprising a gene selected from Tables 2-4 that encodes the
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protein; and (b) allowing said protein to be expressed from said gene in
said patient in an amount sufficient to treat the disease.
92. The method of claim 91, wherein said vector is selected from the group
consisting of an adenoviral vector, and a lentiviral vector.
93. The method of claim 91, wherein said vector is administered by a route
selected from the group consisting of: topical administration, intraocular
administration, parenteral administration, intranasal administration,
intratracheal administration, intrabronchial administration and
subcutaneous administration.
94. The method of claim 91, wherein said vector is a replication-defective
viral vector.
95. The method of claim 91, wherein said gene encodes a human protein.
96. A method of treating ENDOMETRIOSIS disease in a patient in need
thereof, comprising administering an agent that regulates the
expression, activity or physical state of at least one gene or its
encoding RNA from Tables 2-4 in the patient.
97. The method of claim 96, wherein the encoded protein from said gene
comprises an alteration.
98. The method of claim 96, wherein said gene comprises a mutation that
modulates the expression of the encoded protein.
99. The method of claim 96, wherein said agent is selected from the group
consisting of chemical compounds, oligonucleotides, peptides and
antibodies.
100. The method of claim 99, wherein said agent is an antisense molecule
or interfering RNA.
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101. The method of claim 99, wherein said agent is an expression
modulator.
102. The method of claim 101, wherein said modulator is an activator.
103. The method of claim 101, wherein said modulator is a repressor.
104. The method of claim 96, wherein said gene comprises a mutation that
modifies at least one property or function of the encoded protein.
105. The method of claim 96, wherein the agent modulates at least one
property or function of said gene.
106. A method of treating ENDOMETRIOSIS disease in a patient in need
thereof, comprising administering an agent that regulates the
expression, activity or physical state of at least one polypeptide
encoded by a gene from Tables 2-4 in the patient.
107. The method of claim 106, wherein the encoded protein from said gene
comprises an alteration, wherein said alteration is encoded by a
polymorphic locus in said gene.
108. The method of claim 106, wherein said gene comprises an associated
allele, a particular allele of a polymorphic locus, or the like that
modulates the expression of the encoded protein.
109. The method of claim 106, wherein said agent is selected from the
group consisting of chemical compounds, oligonucleotides, peptides
and antibodies.
110. The method of claim 106, wherein said agent is an antisense molecule
or interfering RNA.
111. The method of claim 106, wherein said agent is an expression
modulator.
112. The method of claim 111, wherein said modulator is an activator.
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113. The method of claim 111, wherein said modulator is a repressor.
114. The method of claim 106, wherein said gene comprises an associated
allele, a particular allele of a polymorphic locus, or the like that modifies
at least one property or function of the encoded protein.
115. A method for preventing the occurrence of ENDOMETRIOSIS disease
in an individual in need thereof, comprising regulating the level of at
least one gene from Tables 2-4 compared to a control.
116. The method of claim 115, wherein said level is regulated by regulating
expression of at least one gene from Tables 2-4 by a binding agent, a
receptor to said gene, a peptidomimetic, a fusion protein, a prodrug, an
antibody or a ribozyme.
117. The method of claim 115, wherein said level is controlled by genetically
altering the expression level of at least one gene from Tables 2-4,
whereby the regulated level of said gene mimics the level in a healthy
individual.
118. A method for identifying a gene that regulates drug response in
ENDOMETRIOSIS disease, comprising: (a) obtaining a gene
expression profile for at least one gene from Tables 2-4 in a resident
tissue cell induced for a pro-inflammatory like state in the presence of
the candidate drug; and (b) comparing the expression profile of said
gene to a reference expression profile for said gene in a cell induced
for the pro-inflammatory like state in the absence of the candidate drug,
wherein genes whose expression relative to the reference expression
profile is altered by the drug may identifies the gene as a gene that
regulates drug response in ENDOMETRIOSIS disease.
119. A method for identifying an agent that alters the level of activity or
expression of a polypeptide of Tables 2-4 for use in diagnostics,
prognostics, prevention, treatment, or study of ENDOMETRIOSIS
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disease, comprising: (a) contacting a cell, cell lysate, or the
polypeptide, with an agent to be tested; (b) assessing a level of activity
or expression of the polypeptide; and (c) comparing the level of activity
or expression of the polypeptide with a control sample in an absence of
the agent, wherein if the level of activity or expression of the
polypeptide in the presence of the agent differs by an amount that is
statistically significant from the level in the absence of the agent then
the agent alters the activity or expression of the polypeptide.
120. A kit for diagnosing susceptibility to ENDOMETRIOSIS disease in an
individual comprising: primers for nucleic acid amplification of a region
of at least one gene from Tables 2-4.
121. The kit of claim 120, wherein the primers comprise a segment of
nucleic acids of length suitable for nucleic acid amplification of a target
sequence, selected from the group consisting of: single nucleotide
polymorphism from Tables 5-16, and combinations thereof.
122. A kit for assessing a patient's risk of having or developing
ENDOMETRIOSIS disease, comprising: (a) detection means for
detecting the differential expression, relative to a normal cell, of at least
one gene shown in Table 4 or the gene product thereof; and (b)
instructions for correlating the differential expression of said gene or
gene product with a patient's risk of having or developing
ENDOMETRIOSIS disease.
123. The kit of claim 122, wherein the detection means includes nucleic acid
probes for detecting the level of mRNA of said genes.
124. A kit for assessing a patients risk of having or developing
ENDOMETRIOSIS disease, comprising: (a) at least one means for
amplifying or detecting a sequence of at least one gene in Tables 2-4,
wherein the detection means includes nucleic acid probes or primers
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for detecting the presence or absence of mutations or changes to at
least one sequence of Tables 2-4.
125. The kit of claim 124, wherein the detection means includes an
immunoassay for detecting the level of at least one gene product from
Tables 2-4.
126. A kit for assessing a patient's risk of having or developing
ENDOMETRIOSIS disease, comprising: a) a detection means for
detecting the genotype of at least one polymorphic locus shown in
Tables 2-16; and b) instructions for correlating the genotype of said at
least one polymorphic locus with a patient's risk of having or
developing ENDOMETRIOSIS disease.
127. The kit of claim 126, wherein the detection means includes nucleic acid
probes for detecting the genotype of said at least one polymorphic
locus.
128. A diagnostic composition for diagnosing or detecting susceptibility to
ENDOMETRIOSIS disease comprising a set of oligonucleotide probes
that specifically hybridizes to at least two geneonic regions listed in
Table 1.
129. The composition of claim 128, wherein said set of oligonucleotide
probes specifically hybridize to sequences of at least two genes
selected from the genes in Tables 2-4.
130. The composition of claim 128, wherein the oligonucleotide probes are
detectably labeled with an agent selected from the group consisting of
a fluorescent dye, a radioisotope, a bioluminescent compound, a
chemiluminescent compound, a fluorescent compound, a metal chelate
and an enzyme.
131. The composition of claim 130, wherein the oligonucleotide probes are
labeled with different fluorescent compounds.
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132. The composition of claim 128, wherein the set of oligonucleotide
probes hybridizes in situ.
133. The composition of claim 128, wherein the set of oligonucleotide
probes hybridizes at a gradually changing temperature.
134. The composition of claim 128, wherein the oligonucleotide probes are
between 2 to 100 bases.
135. The composition of claim 128, wherein the oligonucleotide probes are
between 3 to 50 bases.
136. The composition of claim 128, wherein the oligonucleotide probes are
between 8 to 25 bases.
137. A method of assessing a patient's risk of having or developing
ENDOMETRIOSIS disease, comprising: (a) determining the level of
expression of at least one gene from Tables 2-4 or gene products
thereof, and comparing the level of expression to a normal cell; and (b)
assessing a patient's risk of having or developing ENDOMETRIOSIS
disease by determining the correlation between the differential
expression of said genes or gene products with known changes in
expression of said genes measured in at least one patent suffering
from ENDOMETRIOSIS disease.
138. A method of assessing a patient's risk of having or developing
ENDOMETRIOSIS disease, comprising (a) determining a genotype for
at least one polymorphic locus from Tables 2-16 in a patient; (b)
comparing said genotype of (a) to a genotype for at least one
polymorphic locus from Tables 2-16 that is associated with
ENDOMETRIOSIS disease; and (c) assessing the patient's risk of
having or developing ENDOMETRIOSIS disease, wherein said patient
has a higher risk of having or developing ENDOMETRIOSIS disease if
the genotype for at least one polymorphic locus from Tables 2-16 in
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said patient is the same as said genotype for at least one polymorphic
locus from Tables 2-16 that is associated with ENDOMETRIOSIS
disease.
139. A method for assaying the presence of a nucleic acid associated with
resistance or susceptibility to ENDOMETRIOSIS disease in a sample,
comprising: contacting said sample with a nucleic acid recited in claim
under stringent hybridization conditions; and detecting a presence of
a hybridization complex.
140. A method for assaying the presence or amount of a polypeptide
encoded by a gene of Tables 2-4 for use in diagnostics, prognostics,
prevention, treatment, or study of ENDOMETRIOSIS disease,
comprising: contacting a sample with an antibody that specifically binds
to a gene of Tables 8, 9 or 10 under conditions appropriate for binding;
and assessing the sample for the presence or amount of binding of the
antibody to the polypeptide.
153

Description

CA 02676415 2009-07-23
WO 2008/123901 PCT/US2008/001529
GENEMAP OF THE HUMAN GENES ASSOCIATED WITH
ENDOMETRIOSIS
INVENTORS: Abdelmajid Belouchi, John Verner Raelson, Bruno Paquin, Sandie
Briand, Daniel Dubois, Paul Van Eerdewegh, Jonathan Segal, Randall David
Little and Tim Keith.
PRIORITY
[0001] This application is entitled to priority to U.S. Provisional
Application No.
60/899,615, filed February 6, 2007 and U.S. Provisional Application No.
60/948,565, filed July 9, 2007, which are hereby incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to the field of genomics and genetics, including
genome analysis and the study of DNA variations. In particular, the invention
relates to the fields of pharmacogenomics, diagnostics, patient therapy and
the
use of genetic haplotype information to predict an individual's susceptibility
to
ENDOMETRIOSIS disease and/or their response to a particular drug or drugs, so
that drugs tailored to genetic differences of population groups may be
developed
and/or administered to the appropriate population.
[0003] The invention also relates to a GeneMap for ENDOMETRIOSIS
disease, which links variations in DNA (including both genic and non-genic
regions) to an individual's susceptibility to ENDOMETRIOSIS disease and/or
response to a particular drug or drugs. The invention further relates to the
genes
disclosed in the GeneMap (see Tables 2-4 and examples of the GeneMap in the
Example section herein), which is related to methods and reagents for
detection
of an individual's increased or decreased risk for ENDOMETRIOSIS disease and
related sub-phenotypes, by identifying at least one polymorphism in one or a
combination of the genes from the GeneMap. Also related are the candidate
regions identified in Table 1, which are associated with ENDOMETRIOSIS
SUBSTITUTE SHEET (RULE 26)

CA 02676415 2009-07-23
WO 2008/123901 PCT/US2008/001529
disease. In addition, the invention further relates to nucleotide sequences of
those genes including genomic DNA sequences, DNA sequences, single
nucleotide polymorphisms (SNPs), other types of polymorphisms (insertions,
deletions, microsatellites), alleles and haplotypes (see Sequence Listing and
Tables 5-16).
[0004] The invention further relates to isolated nucleic acids comprising
these
nucleotide sequences and isolated polypeptides or peptides encoded thereby.
Also related are expression vectors and host 'cells comprising the disclosed
nucleic acids or fragments thereof, as well as antibodies that bind to the
encoded
polypeptides or peptides.
[0005] The present invention further relates to ligands that modulate the
activity
of the disclosed genes or gene products. In addition, the invention relates to
diagnostics and therapeutics for ENDOMETRIOSIS disease, utilizing the
disclosed nucleic acids, polymorphisms, chromosomal regions, GeneMaps,
polypeptides or peptides, antibodies and/or ligands and small molecules that
activate or repress relevant signaling events.
BACKGROUND OF THE INVENTION
[0006] ENDOMETRIOSIS is defined as the presence of endometrial-like
tissue growing outside the uterine cavity. It is also associated with
significant
impairment in quality of life for affected women due to severe pain during
menstruation and sexual intercourse, and infertility. The pathophysiology of
ENDOMETRIOSIS remains enigmatic. As a result, current therapeutic strategies
are mainly palliative and non-curative. Surgery is the first-line treatment to
remove ovarian endometriomas and to correct ENDOMETRIOSIS-associated
adhesions that can distort pelvic anatomy. Nevertheless, patients who undergo
surgical procedures have recurrence of ENDOMETRIOSIS in up to 47% of cases
and recurrence of adhesions in up to 89% of cases. New research treatments
include the use of aromatase inhibitors together with progestin or together
with
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oral contraceptives. However, ENDOMETRIOSIS recurs once all these
treatments are stopped.
[0007] Medical pharmacological treatments such as the androgenic therapies,
danazol and gestrinone, the constellation of GnRH agonists, buserelin,
goserelin,
leuprolide, nafarelin and triptorelin, GnRH antagonists, cetrorelix and
abarelix, as
well as the progestogens, including medroxyprogesterone acetate, induce lesion
atrophy by suppressing the production of estrogen. These approaches are not
without unwanted side effects. Danazol and gestrinone include weight gain,
hirsuitism, acne, mood changes and metabolic effects on the cardiovascular
system. The group of GnRH agonists and antagonists are found to cause a
profound suppression of estrogen leading to vasomotor effects (hot flashes)
and
depletion of bone mineral density, which restricts their use to only six
months of
therapy. The group of progestogens, including medroxyprogesterone acetate,
suppress the gonadotropins, but do not down-regulate ovarian estrogen
production to the same extent as the GnRH analogues. The side effects include
irregular bleeding, bloating, weight gain and metabolic effects on the
cardiovascular system.
[0008] Current treatments do not address the root cause of the disease.
Despite a preponderance of evidence showing inheritance of a risk for
ENDOMETRIOSIS disease through epidemiological studies and genome wide
linkage analyses, the genes affecting ENDOMETRIOSIS disease have yet to be
discovered. There is a need in the art for identifying specific genes related
to
ENDOMETRIOSIS disease to enable the development of therapeutics that
address the causes of the disease rather than relieving its symptoms. The
failure
in past studies to identify causative genes in complex diseases, such as
ENDOMETRIOSIS disease, has been due to the lack of appropriate methods to
detect a sufficient number of variations in genomic DNA samples (markers), the
insufficient quantity of necessary markers available, and the number of needed
individuals to enable such a study. The present invention addresses these
issues.
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[0009] The present invention relates specifically to a set of ENDOMETRIOSIS
disease-causing genes (GeneMap) and targets which present attractive points of
therapeutic intervention and diagnostics.
[00010] In view of the foregoing, identifying susceptibility genes associated
with
ENDOMETRIOSIS disease and their respective biochemical pathways will
facilitate the identification of diagnostic markers as well as novel targets
for
improved therapeutics. It will also improve the quality of life for those
afflicted by
this disease and will reduce the economic costs of these afflictions at the
individual and societal level. The identification of those genetic markers
would
provide the basis for novel genetic tests and eliminate or reduce the
therapeutic
methods currently used. The identification of those genetic markers will also
provide the development of effective therapeutic intervention for the battery
of
laboratory, phsychological and clinical evaluations typically required to
diagnose
ENDOMETRIOSIS. The present invention satisfies this need.
[00011] DESCRIPTION OF THE FILES CONTAINED ON THE CD-R
[00012] The contents of the submission on compact discs submitted herewith
are incorporated herein by reference in their entirety: A compact disc copy of
the
Sequence Listing (COPY 1) (filename: GENI 024 01WO SeqList.txt, date
recorded: February 06, 2008, file size: 14,920 kilobytes); a duplicate compact
disc copy of the Sequence Listing (COPY 2) (filename: GENI 024 01 WO
SeqList.txt, date recorded: February 06, 2008, file size: 14,920 kilobytes); a
duplicate compact disc copy of the Sequence Listing (COPY 3) (filename: GENI
024 01 WO SeqList.txt, date recorded: February 06, 2008, file size: 14,920
kilobytes); a computer readable format copy of the Sequence Listing (CRF
COPY) (filename: GENI 024 01WO SeqList.txt, date recorded: February 06,
2008, file size: 14,920 kilobytes).
[00013] Three compact disc copies (COPY 1, COPY 2 and COPY3) of Tables
1-18 are herewith submitted and are incorporated herein by reference in their
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entirety. Each Table may be viewed properly in the Courier font. Each compact
disc contains a copy of the following files:
[00014] filename: Table1.txt, date recorded: February 6, 2008, file size: 11
kilobytes;
[00015] filename: Table2.txt, date recorded: February 6, 2008, file size: 35
kilobytes;
[00016] filename: Table3.txt, date recorded: February 6, 2008, file size: 278
kilobytes;
[00017] filename: Table4.txt, date recorded: February 6, 2008, file size: 2
kilobytes;
[00018] filename: Table5.1.txt, date recorded: February 6, 2008, file size: 81
kilobytes;
[00019] filename: Table5.2.txt, date recorded: February 6, 2008, file size: 65
kilobytes;
[00020] filename: Table6.1.txt, date recorded: February 6, 2008, file size: 6
kilobytes;
[00021] filename: Table6.2.txt, date recorded: February 6, 2008, file size: 12
kilobytes;
[00022] filename: Table7.1.txt, date recorded: February 6, 2008, file size: 15
kilobytes;
[00023] filename: Table7.2.txt, date recorded: February 6, 2008, file size: 14
kilobytes;
[00024] filename: Table8.l.txt, date recorded: February 6, 2008, file size: 23
kilobytes;
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[00025] filename: Table8.2.txt, date recorded: February 6, 2008, file size: 22
kilobytes;
[00026] filename: Table9.1, date recorded: February 6, 2008, file size: 4
kilobytes;
[00027] filename: Table9.2, date recorded: February 6, 2008, file size: 3
kilobytes;
[00028] filename: Table10.1, date recorded: February 6, 2008, file size: 44
kilobytes;
[00029] filename: Table10.2, date recorded: February 6, 2008, file size: 17
kilobytes;
[00030] filename: Table11.1, date recorded: February 6, 2008, file size: 31
kilobytes;
[00031] filename: Table11.2, date recorded: February 6, 2008, file size: 42
kilobytes;
[00032] filename: Table12.1, date recorded: February 6, 2008, file size: 17
kilobytes;
[00033] filename: Table12.2, date recorded: February 6, 2008, file size: 11
kilobytes;
[00034] filename: Table13.1, date recorded: February 6, 2008, file size: 27
kilobytes;
[00035] filename: Table13.2, date recorded: February 6, 2008, file size: 16
kilobytes;
[00036] filename: Table14.1, date recorded: February 6, 2008, file size: 6
kilobytes;
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[00037] filename: Table14.2, date recorded: February 6, 2008, file size: 4
kilobytes;
[00038] filename: Table15.1, date recorded: February 6, 2008, file size: 13
kilobytes;
[00039] filename: Table15.2, date recorded: February 6, 2008, file size: 8
kilobytes;
[00040] filename: Table16.1, date recorded: February 6, 2008, file size: 4
kilobytes;
[00041] filename: Table16.2, date recorded: February 6, 2008, file size: 3
kilobytes;
[00042] filename: Table17, date recorded: February 6, 2008, file size: 16
kilobytes; and
[00043] filename: Table18, date recorded: February 6, 2008, file size: 11
kilobytes.
[00044] Brief Description of Drawings
[00045] Figure 1. Emerging endometriosis GeneMap.
[00046] Figure 2. Emerging endometriosis GeneMap with sub-phenotype.
[00047] Figure 3. Targeting the signaling pathway.
[00048] Figure 4. Mouse mRNA localization matrix applied to single and
multiple
mRNA localization assessment & comparative studies, cresyl violet staining.
Figs. 4A-G: All-Stage, Whole-Body Sections throughout the embryonic (1 and 2),
postnatal developmental stages (3 and 5) and adulthood (6 and 7). Fig. 4H:
Adult
Mouse Reproductive Organs: 1. Uterus, control; 2. Uterus, gestation day 5.5;
3.
Uterus, gestation day 7.5; 4. Ovary; 5. Mammary gland; 6. Prostate; 7.
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Epididymis; 8. Testis; 9. Seminal vesicle; Fig. 41: Adult Mouse Tissue Array,
General: 10. Brain, sagittal sections; 11. Thyroid; 12. Pituitary gland; 13.
Adrenal
gland; 14. Trigeminal ganglion; 15. Ovary; 16. Uterus; 17. Kidney; 18. Testis;
19.
Thymus; 20. Seminal vesicle; 21. Salivary gland; 22. Urinary Bladder; 23.
Lung;
24. Prostate; 25. Liver; 26. Gallbladder; 27. Epididymis; 28. Adipose tissue;
Fig.
4J: Adult Mouse Brain Arrays.
[00049] Figure 5. H2AFY expression in the embryonic (e10.5, e12.5 and e15.5)
and postnatal (p1 and p10) mice. Figs. A-D) X-ray film autoradiography
following
hybridization with antisense riboprobe (Seq ID: 6717) after 3-day exposure,
showing a pattern of H2AFY mRNA distribution seen as bright labeling on dark
field. Fig. E) Control (sense, Seq ID: 6716) hybridization of the section
comparable to D. Abbreviations: Br - brain; Cb - cerebellum; E - eye; K -
kidney; Li - liver; Lu - lung; OC - olfactory cavity; Sk - skin; St - stomach;
Te -
testis; Th - thymus; Ve - vertebrae; (s) - sense. Magnification x 1.6.
[00050] Figure 6. H2AFY expression in the adult mouse. Fig. 6A) Anatomical
view of the adult mouse after staining with cresyl violet. Fig. 6B) X-ray film
autoradiography after hybridization with antisense (Seq ID: 6717) riboprobe
showing the presence of H2AFY mRNA in the brain, skin, lymph node, thymus,
spleen, liver, stomach, kidney and large intestine, seen as bright labeling
under
darkfield illumination. Fig. 6C) Control (sense, Seq ID: 6716) hybridization
of an
adjacent section comparable to B. Abbreviations: BM - bone marrow; Br - brain;
Cb - cerebellum; H - heart; K - kidney; Li - liver; LI - large intestine; LN -
lymph
node; Lu - lung; Ri - ribs; SG - salivary gland; Sk - skin; Sp - spleen; St -
stomach; Th - thymus; (as) - antisense; (s) - sense. Magnification x 2.7.
[00051] Figure 7. H2AFY expression in the adult mouse tissue arrays. Fig. 7 A)
X-ray film autoradiography after hybridization with antisense (Seq ID: 6717)
riboprobe showing H2AFY mRNA distribution in the reproductive organs (RO)
seen as bright labeling on dark field. Expression sites are evident in the
ovary,
control uterus and uterine tissue at gestation stages day 5.5 and 7.5. Fig. 7
B)
H2AFY mRNA shown in the general tissue array (TA). Low to medium levels of
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expression are evident in most tissue including the brain, pituitary gland,
adrenal
gland, thyroid, testis, splee, kidney and prostate. High H2AFY mRNA
concentrations occur in the thymus and ovary. Fig. 7 C) H2AFY mRNA in the
brain tissue arrays; expression is evident in the olfactory lobe, hippocampus,
hypothalamus and cerebellum. Fig. 7 D) Control (sense, Seq ID: 6716)
hybridization of the section comparable to B. Abbreviations: Adr - adrenal
gland;
Br - brain; Hip - hippocampus; Cb - cerebellum; Hy - hypothalamus; K - kidney;
Li - liver; Lu - lung; OL - olfactory lobe; Ov - ovary; Pit - pituitary gland;
Pr -
prostate; SG - salivary gland; Sp - spleen; Td - thyroid gland; Te - testis;
Th -
thymus; UB - urinary bladder; Ut - uterus; UtO - uterus at day 0; Ut5.5
(Ut7.5) -
uterus at gestation day 5.5 (and 7.5); (s) - sense. Magnification x 1.6.
[00052] Figure 8. H2AFY expression in the adult mouse brain hippocampus and
cerebellum. Fig. 8A) Emulsion autoradiography at low magnification, after
hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA
labeling in the hippocampus seen as bright on darkfield illumination. Fig. 8B)
Fragment of the hippocampus with H2AFY mRNA labeled area CAl neurons
(arrow) Fig. 8C) Control (sense, Seq ID: 6716) hybridization of an adjacent
section comparable to B. Fig. 8D) Cerebellum at low magnification, after
hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA
labeling in Purkinje cells (arrows). Fig. 8E) Fragment of the cerebellum
showing
Purkinje cells layer at higher magnification (arrows). Fig. 8F) Control
(sense, Seq
ID: 6716) hybridization of an adjacent section comparable to E. Abbreviations:
CAl - cornu Ammonis area 1; Cb - cerebellum; DG - dentate gyrus; PC -
Purkinje cells layer; (s) - sense. Magnifications: (A and D) x 23; (B, C, E
and F) x
405.
[00053] Figure 9. H2AFY expression in the adult mouse brain bone marrow and
dorsal root ganglion. Fig. 9A) Emulsion autoradiography at low magnification,
after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY
mRNA labeling in the bone marrow region and dorsal root ganglion seen as
bright on darkfield illumination. Fig. 9B) Fragment of the bone marrow with
H2FY
mRNA labeled cells (arrow), bone unlabeled, at higher magnification. Fig. 9C)
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Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable
to
B. Fig. 9D) Dorsal root ganglion revealing the sensory neurons labeled (heavy
arrows) at higher magnification, note the satellite glial cells unlabeled
(small
arrows). Fig. 9E) Control (sense, Seq ID: 6716) hybridization of an adjacent
section comparable to D. Abbreviations: B - bone; BM - bone marrow; DRG -
dorsal root ganglion; Ve - vertebrae; (s) - sense. Magnifications: (A) x 50;
(B to
E) x 405.
[00054] Figure 10. H2AFY expression in the thymus. Fig. 10A) Emulsion
autoradiography at low magnification, after hybridization with antisense (Seq
ID:
6717) riboprobe, showing H2AFY mRNA as bright labeling under darkfield
illumination. Fig. 10B) At higher magnification, it is seen that H2AFY mRNA
labeling follow the cell density which is higher in the cortex and lower in
the
medulla. Fig. 10C) Control (sense, Seq ID: 6716) hybridization of an adjacent
section comparable to A. Abbreviations: Cx - cortex; Me - medulla; (s) -
sense.
Magnifications: (A) x 25; (B and C) x 405.
[00055] Figure 11. H2AFY expression in the ovary. Fig. 11A) Emulsion
autoradiography at low magnification, after hybridization with antisense (Seq
ID:
6717) riboprobe, showing H2AFY mRNA seen as bright labeling under darkfield
illumination mostly in the corpus luteum. Fig. 11 B) At higher magnification
H2AFY
mRNA labeling is seen in the corpus luteum cells and in the follicular cells
(arrows). Theca cells seem to not express H2AFY. Fig. 11 C) Control (sense,
Seq
ID: 6716) hybridization of an adjacent section comparable to B. Abbreviations:
CL - corpus luteum; FC - follicular cells; T - theca; (s) - sense.
Magnifications:
(A) x 25; (B and C) x 405.
[00056] Figure 12. H2AFY expression in the intact adult mouse uterus. Fig.
12A)
Emulsion autoradiography at low magnification, after hybridization with
antisense
(Seq ID: 6717) riboprobe, showing H2AFY mRNA labeling in the endometrium
epithelial cells layer (arrow) seen as bright under darkfield illumination.
Fig. 12B)
The same section seen at lightfield illumination and cresyl violet staining.
Fig.
12C) Fragment of the uterine epithelium, labeled (arrow) at high
magnification.
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Fig. 12D) Control (sense, Seq ID: 6716) hybridization of an adjacent section
comparable to A at darkfield illumination. Fig. 12E) The same section seen at
Iightfield illumination and cresyl violet staining. Fig. 12F) Fragment of the
uterine
epithelium following control (sense, Seq ID: 6716) hybridization.
Abbreviations: E
- endometrium; Ep - epithelium; M - myometrium, (as) - antisense; (s) - sense.
Magnifications: (A, B, D and E) x 25; (C and F) x 614.
[00057] Figure 13. H2AFY expression in the female uterus 7,5 days pregnant.
Fig. 13A) Emulsion autoradiography, after hybridization with antisense (Seq
ID:
6717) riboprobe, throughout the peripheral region of the uterus. H2AFY mRNA
labeling is present in mostly endometrium cells and much less in the
myometrium. Fig. 13B) Centrally located deciduas with labeled giant cells
originated from the ectoplacental cone (heavy arrows) and the presumptive
trophoblasts of trophectoderm origin (small arrows). Fig. 13C) Control (sense,
SEQIDPROBE2]) hybridization of an adjacent section comparable to B.
Abbreviations: BV - blood vessels; E - endometrium; - H - hondrion; M -
myometrium; (s) - sense. Magnification: x 380.
[00058] Figure 14. H2AFY expression in the testis. Fig. 14A) Emulsion
autoradiography, after hybridization with antisense (Seq ID: 6717) riboprobe,
throughout the testis showing H2AFY mRNA labeling as bright under lightfield
illumination. Labeling is present in a proportion of seminiferous tubules
(arrow).
Fig. 14B) Fragment of the seminiferous tubule showing H2AFY mRNA labeling
concentrated mostly in the layer of spermatogonia and much less in
spermatocyte layer. There is no detectable labeling in the spermatozoa. Fig.
14C)
Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable
to
B. Abbreviations: Sc - spermatocyte; SfT - seminiferous tubule; Sg -
spermatogonia; Sz - spermatozoa; (s) - sense. Magnifications: (A) x 25; (B and
C) x 380.
[00059] Figure 15. MAD2L2 expression in the embryonic (elO.5, e12.5 and
e15.5) and postnatal (p1 and p10) mice. Figs. 15A-D) X-ray film
autoradiography
following hybridization with antisense (Seq ID: 6719) riboprobe after 4-day
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exposure, showing a pattern of MAD2L2 mRNA distribution seen as bright
labeling on dark field. Fig. 15E: Control (sense, Seq ID: 6718) hybridization
of the
section comparable to D. Abbreviations: Br - brain; Cb - cerebellum; DRG -
dorsal root ganglia; K - kidney; Ov - ovary; Re - retina; SC - spinal cord; St
-
stomach; (s) - sense. Magnification x 1.6.
[00060] Figure 16. MAD2L2 expression in the adult mouse. Fig.16A) Anatomical
view of the adult mouse after staining with cresyl violet. Fig.16B) X-ray film
autoradiography following hybridization with antisense (Seq ID: 6719)
riboprobe
showing the presence of MAD2L2 mRNA in the salivary gland, skin, lymph node,
thymus, spleen, liver, stomach, kidney and large intestine, seen as bright
labeling
under darkfield illumination. Fig.16C: Control (sense, Seq ID: 6718)
hybridization
of an adjacent section comparable to Fig.16B. Abbreviations: Br - brain; Cb -
cerebellum; H - heart; K - kidney; Li - liver; LI - large intestine; Lu -
lung; SG -
salivary gland; Sk - skin; Sp - spleen; St - stomach; Th - thymus; (as) -
antisense; (s) - sense. Magnification x 2.7.
[00061] Figure 17. MAD2L2 expression in the adult mouse tissue arrays. Fig.
17A) X-ray film autoradiography, following hybridization with antisense (Seq
ID:
6719) riboprobe, showing MAD2L2 mRNA distribution in the reproductive organs
(RO) seen as bright labeling on dark field. High expression sites are evident
in
the ovary and testis and in the uterine tissue at gestation stages day 5.5 and
7.5.
Fig. 17B) MAD2L2 mRNA shown in the general tissue array (TA). Low to medium
level expression levels are evident in most tissue including brain, adrenal
gland,
spleen, thymus and liver. High MAD2L2 expression levels are confirmed in the
testis and ovary. Fig. 17C) MAD2L2 mRNA in the brain tissue arrays. Expression
is evident in the choroids plexus within Ilird ventricle. Fig. 17D) Control
(sense,
Seq ID: 6718) hybridization of the section comparable to B. Abbreviations: Adr
-
adrenal gland; Br - brain; ChPI - choroids plexus; K - kidney; Li - liver; OL -
olfactory lobe; Ov - ovary; Te - testis; Th - thymus; UtO - uterus at day 0;
Ut5.5
(Ut7.5) - uterus at gestation day 5.5 (and 7.5); (s) - sense. Magnification x
1.6.
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[00062] Figure 18. MAD2L2 expression in the adult mouse testis. Fig. 18A)
Emulsion autoradiography, following hybridization with antisense (Seq ID:
6719)
riboprobe, showing MAD2L2 mRNA labeling in the wall of the seminiferous
tubules seen as dark silver grains under lightfield illumination; cresyl
violet
staining of cell nuclei. By topography, the labeled cells may be identified a
spermatocytes. Spermatogonia and spermatozoa appears as unlabeled. There is
no labeling in the interstitial space Leydig cells. Fig. 18B) Control (sense,
Seq ID:
6718) hybridization of an adjacent section comparable to A. Abbreviations: IS -
interstitial space; Sc -spermatocyte; SfT - seminiferous tubule; Sg -
spermatogonia; Sz - spermatozoa; (s) - sense. Magnification x 425.
[00063] Figure 19. MAD2L2 expression in the pregnant female uterus. Fig. 19A)
Emulsion autoradiography, following hybridization with antisense (Seq ID:
6719)
riboprobe, showing MAD2L2 mRNA labeling in the uterus on day 7.5 post
coitum. Silver labeling is seen as dark under lightfield illumination; cresyl
violet
staining of cell nuclei. By topography, the labeled cells may be identified as
endometrial cells. Peripherally located myometrium seems to be free of
labeling.
Fig. 19B) MAD2L2 labeling in the endometrium region with high PCNA activity
(not shown). Fig. 19C) Fragment of the deciduas with unlabeled giant cells.
Fig.
19D) Control (sense, Seq ID: 6718) hybridization of an adjacent section
comparable to A. Abbreviations: G - giant cells; E - endometrium stroma cells;
M
- myometrium muscle cells layer; (s) - sense. Magnification x 304.
[00064] Figure 20. MCM3AP expression in the embryonic (elO.5, e12.5 and
e15.5) and postnatal (p1 and p10) mice. Figs. 20A-D) X-ray film
autoradiography
following hybridization with antisense riboprobe (Seq ID: 6721) after 5-day
exposure, showing a pattern of MCM3AP mRNA distribution seen as bright
labeling on darkfield. Fig. 20E) Control (sense, Seq ID: 6720) hybridization
of the
section comparable to D. Abbreviations: Br - brain; Cb - cerebellum; K -
kidney;
Re - retina; Sp - spleen; Th - thymus; (s) - sense. Magnification x 1.6.
[00065] Figure 21. MCM3AP expression in the adult mouse. Fig. 21A)
Anatomical view of the adult mouse after staining with cresyl violet. Fig.
2113) X-
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ray film autoradiography after hybridization with antisense riboprobe (Seq ID:
6721) showing the presence of MCM3AP mRNA in the brain, skin, lymph node,
thymus, spleen, liver, stomach, kidney and large intestine, seen as bright
labeling
under darkfield illumination. Fig. 21 C) Control (sense, Seq ID: 6720)
hybridization
of an adjacent section comparable to B. Abbreviations: BM - bone marrow; Br -
brain; Cb - cerebellum; H - heart; K - kidney; Li - liver; LI - large
intestine; LN -
lymph node; Lu - lung; SG - salivary gland; SI - small intestine; Sk - skin;
Sp -
spleen; St - stomach; Th - thymus; (as) - antisense; (s) - sense.
Magnification x
2.7.
[00066] Figure 22. MCM3AP expression in the adult mouse tissue arrays. Fig.
22A) X-ray film autoradiography, after hybridization with antisense riboprobe
(Seq
ID: 6721), showing MCM3AP mRNA distribution in the reproductive organs (RO)
seen as bright labeling on dark field. Overall low mRNA concentration is
evident.
Fig. 22B) MCM3AP mRNA shown in the general tissue array (TA). MCM3AP
expression levels are at the limit of the detection by ISH in most tissues
including
the brain, trigeminal ganglion, adrenal gland and spleen. Slightly elevated
mRNA
concentrations occur in the thymus. Fig. 22C) MCM3AP mRNA in the brain tissue
arrays; low-level mRNA concentrations are evident in the olfactory lobe,
hippocampus, hypothalamus and cerebellum. Fig. 22D) Control (sense, Seq ID:
6720) hybridization of the section comparable to B. Abbreviations: Adr -
adrenal
gland; Br - brain; Hip - hippocampus; Cb - cerebellum; OL - olfactory lobe; Ov
-
ovary; SG - salivary gland; Sp - spleen; Te - testis; Th - thymus; Ut -
uterus;
UtO - uterus at day 0; Ut5.5 (Ut7.5) - uterus at gestation day 5.5 (and 7.5);
(s) -
sense. Magnification x 1.6.
[00067] Figure 23. MCM3AP expression in the adult mouse brain hippocampus
and cerebellum. Fig. 23A) Emulsion autoradiography, after hybridization with
antisense riboprobe (Seq ID: 6721), showing MCM3AP mRNA labeling (arrow) in
the hippocampus area CAl seen as bright labeling under darkfield illumination.
Fig. 23B) The same fragment of the hippocampus seen under brightfield
illumination. Staining tissue with cresyl violet reveals a high density of
labeled cell
layer. Fig. 23C) Control (sense, Seq ID: 6720) hybridization of an adjacent
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section comparable to A under darkfield. Fig. 23D) The same section under
brightfield illumination. Abbreviations: CA1 - cornu Ammonis area 1; (as) -
antisense; (s) - sense. Magnification: x 192.
[00068] Figure 24. NRXN1 expression in the embryonic (e10.5, e12.5 and e15.5)
and postnatal (p1 and p10) mice. Figs. 24A-D) X-ray film autoradiography
following hybridization with antisense Seq ID: 6723 riboprobe after 2-day
exposure, showing a pattern of NRXN1 mRNA distribution seen as bright labeling
on dark field. Fig. 24E) Control (sense, Seq ID: 6722) hybridization of the
section
comparable to D. Abbreviations: Br - brain; Cb - cerebellum; DRG - dorsal root
ganglia; SC - spinal cord; (s) - sense. Magnification x 1.6.
[00069] Figure 25. NRXN1 expression in the adult mouse. Fig. 25A) Anatomical
view of the adult mouse after staining with cresyl violet. Fig. 25B) X-ray
film
autoradiography after hybridization with antisense riboprobe (Seq ID: 6723)
showing the presence of NRXN1 mRNA in the brain, spinal cord, dorsal root
ganglia and trigeminal ganglion. Fig. 25C) Control (sense, Seq ID: 6722)
hybridization of an adjacent section comparable to B. Abbreviations: Br -
brain;
Cb - cerebellum; Cx - cortex; DRG - dorsal root ganglia; H - heart; SC -
spinal
cord; Sk - skin; St - stomach; TG - trigeminal ganglion; Th - thymus; (as) -
antisense; (s) - sense. Magnification x 2.7.
[00070] Figure 26. NRXN1 expression in the adult mouse tissue arrays. Fig.
26A) Two-day X-ray film autoradiography, after hybridization with antisense
riboprobe (Seq ID: 6723), showing NRXN1 mRNA distribution in the reproductive
organs (RO) seen as bright labeling on dark field. Overall low mRNA
concentration is evident. Fig. 26B) NRXN1 mRNA shown in the general tissue
array (TA). NRXN1 expression is detectable in the CNS (brain), PNS (trigeminal
ganglion) and endocrine glands (pituitary and adrenals). Fig. 26C) NRXN1 mRNA
in the brain tissue arrays. Medium to high level mRNA concentration with
exception of the striatum. Fig. 26D) Control (sense, Seq ID: 6722)
hybridization of
the section comparable to B. Abbreviations: Adr - adrenal gland; Br - brain;
Cb -
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cerebellum; CxVlb - cortex, deep layer Vib; Hip - hippocampus; Hy -
hypothalamus; Str - striatum; Th - thalamus; (s) - sense. Magnification x 1.6.
[00071] Figure 27. NRXN1 expression in the adult mouse CNS hippocampus,
cortex and PNS trigeminal ganglion. Fig. 27A) Emulsion autoradiography, after
hybridization with antisense riboprobe (Seq ID: 6723), showing NRXN1 mRNA
labeling in the cortex and hippocampus area CAl seen as bright on darkfield
illumination. Note strongly labeled deep cortical sub layer Vlb. Fig. 27B)
Control
(sense, Seq ID: 6722) hybridization of an adjacent section comparable to A
under
darkfield illumination. Fig. 27C) Fragment of the trigeminal ganglion seen
under
brightfield illumination. Fig. 27D) Control (sense, Seq ID: 6722)
hybridization of
an adjacent section comparable to C. Fig. 27E) Cerebral cortex at higher
magnification. Large arrow indicates a labeled neuron. Thin arrow points an
unlabeled presumptive glial cell. Fig. 27F) Trigeminal ganglion at higher
magnification. Large arrows indicate the sensory neurons, labeled. Thin arrows
point the unlabeled satellite cells. Schwann cell seen in the nerve tissue
appear
unlabeled. Magnifications: (A to D) x 60; (E and F) x 250.
[00072] Figure 28. NRXN1 expression in the newborn (p1) mouse PNS sensory
dorsal root ganglion and ortosympathetic paravertebral ganglion. Fig. 28A)
Emulsion autoradiography, after hybridization with antisense riboprobe (Seq
ID:
6723), showing NRXN1 mRNA labeling in the dorsal root ganglion and
paravertebral ganglion on darkfield illumination. Fig. 28B) The same section
seen
under brightfield illumination. Fig. 28C) Control (sense, Seq ID: 6722)
hybridization of an adjacent section comparable to A under darkfield
illumination.
D) The same section seen under brightfield illumination. Abbreviations: DRG -
dorsal root ganglion; PVG - paravertebral ganglion; Ve - vertebrae; (as) -
antisense; (s) - sense. Magnification x 60.
[00073] Figure 29. NRXN1 expression in the postnatal and adult mouse PNS
visceral Auerbach plexus. Fig. 29A) Emulsion autoradiography, after
hybridization
with antisense riboprobe (Seq ID: 6723), showing NRXN1 mRNA labeling in the
intestine of p10 mouse. Arrows indicate group of neurons present in the smooth
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muscle cell layer. Fig. 29B) The same section seen under brightfield
illumination.
Fig. 29C) Control (sense, Seq ID: 6722) hybridization of an adjacent section
comparable to A under darkfield illumination. Fig. 29D) The same section seen
under brightfield illumination. Fig. 29E) NRXN1 mRNA-labeled neuron in the
Auerbach plexus (arrows) in the postnatal mouse intestine found in the space
between circular and longitudinal smooth muscles layer showing . Fig. 29F)
NRXN1 mRNA-labeled neuron in Auerbach plexus in the adult mouse intestine
showing an inferior labeling intensity when compared to that of plO mouse
plexus. Abbreviations: M - smooth muscle fibers; MC - circular musclular
layer;
ML - longitudinal muscular layer; V - intestinal villi; (as) - antisense; (s) -
sense.
Magnifications: (A to D) x 60; (E and F) x 250.
[00074] TABLE DESCRIPTION
[00075] Table 1. List of Endometriosis disease candidate regions identified
from the Genome Wide Scan association analyses. The first column denotes the
region identifier. The second and third columns correspond to the chromosome
and cytogenetic band, respectively. The fourth and fifth columns correspond to
the chromosomal start and end coordinates of the NCBI genome assembly
derived from build 36.
[00076] Table 2. List of candidate genes from the regions identified from the
genome wide association analysis. The first column corresponds to the region
identifier provided in Table 1. The second and third columns correspond to the
chromosome and cytogenetic band, respectively. The fourth and fifth columns
corresponds to the chromosomal start coordinates of the NCBI genome assembly
derived from build 36 (B36) and the end coordinates (the start and end
position
relate to the + orientation of the NCBI assembly and don't necessarily
correspond
to the orientation of the gene). The sixth and seventh columns correspond to
the
official gene symbol and gene name, respectively, and were obtained from the
NCBI Entrez Gene database. The eighth column corresponds to the NCBI Entrez
Gene Identifier (GenelD). The ninth and tenth columns correspond to the
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Sequence IDs from nucleotide (cDNA) and protein entries in the Sequence
Listing.
[00077] Table 3. List of candidate genes based on EST clustering from the
regions identified from the various genome wide analyses. The first column
corresponds to the region identifier provided in Table 1. The second column
corresponds to the chromosome number. The third and fourth columns
correspond to the chromosomal start and end coordinates of the NCBI genome
assemblies derived from build 36 (B36). The fifth column corresponds to the
ECGene Identifier, corresponding to the ECGene track of UCSC. These ECGene
entries were determined by their overlap with the regions from Table 1, based
on
the start and end coordinates of both Region and ECGene identifiers. The sixth
and seventh columns correspond to the Sequence IDs from nucleotide and
protein entries in the Sequence Listing.
[00078] Table 4. List of micro RNA (miRNA) from the regions identified from
the
genome wide association analyses derived from build 36 (B36). To identify the
miRNA from B36, these miRNA entries were determined by their overlap with the
regions from Table 1, based on the start and end coordinates of both Region
and
miRNA identifiers. The first column corresponds to the region identifier
provided
in Table 1. The second column corresponds to the chromosome number. The
third and fourth columns correspond to the chromosomal start and end
coordinates of the NCBI genome assembly derived from build 36 (the start and
end position relate to the + orientation of the NCBI assembly and do not
necessarily correspond to the orientation of the miRNA). The fifth and sixth
columns correspond to the miRNA accession and miRNA id, respectively, and
were obtained from the miRBase database. The seventh column corresponds to
the NCBI Entrez Gene Identifier (GenelD). The eighth column corresponds to the
Sequence ID from nucleotide (RNA) in the Sequence Listing.
[00079] Table 5.1. Genome wide association study results in the Quebec
Founder Population (QFP). SNP markers found to be associated with
Endometriosis from the analysis of genome wide scan (GWS) data: GWS win1.
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Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp);
rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical
identifier for this patent application; Sequence, 21 bp of sequence covering
10
base pair of unique sequence flanking either side of central polymorphic SNP; -
Iog10 P values for GWS, - Iog10 of the P value for statistical significance
from the
GWS for single SNP markers (both T test and Permutation test p-values are
displayed; see Example section) and for the most highly associated multi-
marker
haplotypes centered at the reference marker and defined by the sliding windows
of specified sizes.
[00080] Table 5. List of significantly associated haplotypes based on the
Endometriosis Disease results using the Quebec Founder Population (QFP).
Individual haplotypes with associated relative risks are presented in each row
of
the table; these values were extracted from the associated marker haplotype
window with the most significant p value for each SNP in Table 5.1. The first
column lists the region ID as presented in Table 1. The Haplotype column lists
the specific nucleotides for the individual SNP alleles contributing to the
haplotype reported. The Case and Control columns correspond to the numbers of
cases and controls, respectively, containing the haplotype variant noted in
the
Haplotype column. The Total Case and Total Control columns list the total
numbers of cases and controls for which genotype data was available for the
haplotype in question. The RR column gives to the relative risk for each
particular haplotype. The remainder of the columns lists the SeqlDs for the
SNPs
contributing to the haplotype and their relative location with respect to the
central
marker. The Central marker (0) column lists the SeqID for the central marker
on
which the haplotype is based. Flanking markers are identified by minus (-) or
plus (+) signs to indicate the relative location of flanking SNPs.
[00081] Table 6.1. Genome wide association study results in the Quebec
Founder Population (QFP). SNP markers found to be associated with
Endometriosis from the analysis of genome wide scan (GWS) data:
HAS_INFERTILE. Columns include: Region ID; Chromosome; Build 36 location
in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence
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ID, unique numerical identifier for this patent application; Sequence, 21 bp
of
sequence covering 10 base pair of unique sequence flanking either side of
central polymorphic SNP; - Iog10 P values for GWS, -Iog10 of the P value for
statistical significance from the GWS for single SNP markers (both T test and
Permutation test p-values are displayed; see Example section) and for the most
highly associated multi-marker haplotypes centered at the reference marker and
defined by the sliding windows of specified sizes.
[00082] Table 6.2. List of significantly associated haplotypes based on the
Endometriosis Disease results using the Quebec Founder Population (QFP).
Individual haplotypes with associated relative risks are presented in each row
of
the table; these values were extracted from the associated marker haplotype
window with the most significant p value for each SNP in Table 6.1. The first
column lists the region ID as presented in Table 1. The Haplotype column lists
the specific nucleotides for the individual SNP alleles contributing to the
haplotype reported. The Case and Control columns correspond to the numbers of
cases and controls, respectively, containing the haplotype variant noted in
the
Haplotype column. The Total Case and Total Control columns list the total
numbers of cases and controls for which genotype data was available for the
haplotype in question. The RR column gives to the relative risk for each
particular haplotype. The remainder of the columns lists the SeqlDs for the
SNPs
contributing to the haplotype and their relative location with respect to the
central
marker. The Central marker (0) column lists the SeqID for the central marker
on
which the haplotype is based. Flanking markers are identified by minus (-) or
plus
(+) signs to indicate the relative location of flanking SNPs.
[00083] Table 7.1. Genome wide association study results in the Quebec
Founder Population (QFP). SNP markers found to be associated with
Endometriosis from the analysis of genome wide scan (GWS) data: NOT
INFERTILE. Columns include: Region ID; Chromosome; Build 36 location in base
pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique
numerical identifier for this patent application; Sequence, 21 bp of sequence
covering 10 base pair of unique sequence flanking either side of central
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polymorphic SNP; - loglO P values for GWS, - loglO of the P value for
statistical
significance from the GWS for single SNP markers (both T test and Permutation
test p-values are displayed; see Example section) and for the most highly
associated multi-marker haplotypes centered at the reference marker and
defined
by the sliding windows of specified sizes.
[00084] Table 7.2. List of significantly associated haplotypes based on the
Endometriosis Disease results using the Quebec Founder Population (QFP).
Individual haplotypes with associated relative risks are presented in each row
of
the table; these values were extracted from the associated marker haplotype
window with the most significant p value for each SNP in Table 7.1. The first
column lists the region ID as presented in Table 1. The Haplotype column lists
the specific nucleotides for the individual SNP alleles contributing to the
haplotype reported. The Case and Control columns correspond to the numbers of
cases and controls, respectively, containing the haplotype variant noted in
the
Haplotype column. The Total Case and Total Control columns list the total
numbers of cases and controls for which genotype data was available for the
haplotype in question. The RR column gives to the relative risk for each
particular haplotype. The remainder of the columns lists the SeqlDs for the
SNPs
contributing to the haplotype and their relative location with respect to the
central
marker. The Central marker (0) column lists the SeqID for the central marker
on
which the haplotype is based. Flanking markers are identified by minus (-) or
plus (+) signs to indicate the relative location of flanking SNPs.
[00085] Table 8.1. Genome wide association study results in the Quebec
Founder Population (QFP). SNP markers found to be associated with
Endometriosis from the analysis of genome wide scan (GWS) data: NOT PELVIC
PAIN. Columns include: Region ID; Chromosome; Build 36 location in base pairs
(bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique
numerical identifier for this patent application; Sequence, 21 bp of sequence
covering 10 base pair of unique sequence flanking either side of central
polymorphic SNP; - loglO P values for GWS, - loglO of the P value for
statistical
significance from the GWS for single SNP markers (both T test and Permutation
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test p-values are displayed; see Example section) and for the most highly
associated multi-marker haplotypes centered at the reference marker and
defined
by the sliding windows of specified sizes.
[00086] Table 8.2. List of significantly associated haplotypes based on the
Endometriosis Disease results using the Quebec Founder Population (QFP).
Individual haplotypes with associated relative risks are presented in each row
of
the table; these values were extracted from the associated marker haplotype
window with the most significant p value for each SNP in Table 8.1. The first
column lists the region ID as presented in Table 1. The Haplotype column lists
the specific nucleotides for the individual SNP alleles contributing to the
haplotype reported. The Case and Control columns correspond to the numbers of
cases and controls, respectively, containing the haplotype variant noted in
the
Haplotype column. The Total Case and Total Control columns list the total
numbers of cases and controls for which genotype data was available for the
haplotype in question. The RR column gives to the relative risk for each
particular haplotype. The remainder of the columns lists the SeqlDs for the
SNPs
contributing to the haplotype and their relative location with respect to the
central
marker. The Central marker (0) column lists the SeqID for the central marker
on
which the haplotype is based. Flanking markers are identified by minus (-) or
plus (+) signs to indicate the relative location of flanking SNPs.
[00087] Table 9.1. Genome wide association study results in the Quebec
Founder Population (QFP). SNP markers found to be associated with
Endometriosis from the analysis of genome wide scan (GWS) data: HAS
OVARIAN CYST. Columns include: Region ID; Chromosome; Build 36 location in
base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID,
unique numerical identifier for this patent application; Sequence, 21 bp of
sequence covering 10 base pair of unique sequence flanking either side of
central polymorphic SNP; - log10 P values for GWS, - Iog10 of the P value for
statistical significance from the GWS for single SNP markers (both T test and
Permutation test p-values are displayed; see Example section) and for the most
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highly associated multi-marker haplotypes centered at the reference marker and
defined by the sliding windows of specified sizes.
[00088] Table 9.2. List of significantly associated haplotypes based on the
Endometriosis Disease results using the Quebec Founder Population (QFP).
Individual haplotypes with associated relative risks are presented in each row
of
the table; these values were extracted from the associated marker haplotype
window with the most significant p value for each SNP in Table 9.1. The first
column lists the region ID as presented in Table 1. The Haplotype column lists
the specific nucleotides for the individual SNP alieles contributing to the
haplotype reported. The Case and Control columns correspond to the numbers of
cases and controls, respectively, containing the haplotype variant noted in
the
Haplotype column. The Total Case and Total Control columns list the total
numbers of cases and controls for which genotype data was available for the
haplotype in question. The RR column gives to the relative risk for each
particular haplotype. The remainder of the columns lists the SeqlDs for the
SNPs
contributing to the haplotype and their relative location with respect to the
central
marker. The Central marker (0) column lists the SeqID for the central marker
on
which the haplotype is based. Flanking markers are identified by minus (-) or
plus (+) signs to indicate the relative location of flanking SNPs.
[00089] Table 10.1. Genome wide association study results in the Quebec
Founder Population (QFP). SNP markers found to be associated with
Endometriosis from the analysis of genome wide scan (GWS) data: NOT
OVARYAN CYSTS. Columns include: Region ID; Chromosome; Build 36 location
in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence
ID, unique numerical identifier for this patent application; Sequence, 21 bp
of
sequence covering 10 base pair of unique sequence flanking either side of
central polymorphic SNP; - log10 P values for GWS, - loglO of the P value for
statistical significance from the GWS for single SNP markers (both T test and
Permutation test p-values are displayed; see Example section) and for the most
highly associated multi-marker haplotypes centered at the reference marker and
defined by the sliding windows of specified sizes.
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[00090] Table 10.2. List of significantly associated haplotypes based on the
Endometriosis Disease results using the Quebec Founder Population (QFP).
Individual haplotypes with associated relative risks are presented in each row
of
the table; these values were extracted from the associated marker haplotype
window with the most significant p value for each SNP in Table 10.1. The first
column lists the region ID as presented in Table 1. The Haplotype column lists
the specific nucleotides for the individual SNP alleles contributing to the
haplotype reported. The Case and Control columns correspond to the numbers of
cases and controls, respectively, containing the haplotype variant noted in
the
Haplotype column. The Total Case and Total Control columns list the total
numbers of cases and controls for which genotype data was available for the
haplotype in question. The RR column gives to the relative risk for each
particular haplotype. The remainder of the columns lists the SeqlDs for the
SNPs
contributing to the haplotype and their relative location with respect to the
central
marker. The Central marker (0) column lists the SeqID for the central marker
on
which the haplotype is based. Flanking markers are identified by minus (-) or
plus
(+) signs to indicate the relative location of flanking SNPs.
[00091] Table 11.1. Genome wide association study results in the Quebec
Founder Population (QFP). SNP markers found to be associated with
Endometriosis from the analysis of genome wide scan (GWS) data: Stage 3 & 4.
Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp);
rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical
identifier for this patent application; Sequence, 21 bp of sequence covering
10
base pair of unique sequence flanking either side of central polymorphic SNP; -
Iog10 P values for GWS, - Iog10 of the P value for statistical significance
from the
GWS for single SNP markers (both T test and Permutation test p-values are
displayed; see Example section) and for the most highly associated multi-
marker
haplotypes centered at the reference marker and defined by the sliding windows
of specified sizes.
[00092] Table 11.2. List of significantly associated haplotypes based on the
Endometriosis Disease results using the Quebec Founder Population (QFP).
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Individual haplotypes with associated relative risks are presented in each row
of
the table; these values were extracted from the associated marker haplotype
window with the most significant p value for each SNP in Table 11.1. The first
column lists the region ID as presented in Table 1. The Haplotype column lists
the specific nucleotides for the individual SNP alleles contributing to the
haplotype reported. The Case and Control columns correspond to the numbers of
cases and controls, respectively, containing the haplotype variant noted in
the
Haplotype column. The Total Case and Total Control columns list the total
numbers of cases and controls for which genotype data was available for the
haplotype in question. The RR column gives to the relative risk for each
particular haplotype. The remainder of the columns lists the SeqlDs for the
SNPs
contributing to the haplotype and their relative location with respect to the
central
marker. The Central marker (0) column lists the SeqID for the central marker
on
which the haplotype is based. Flanking markers are identified by minus (-) or
plus
(+) signs to indicate the relative location of flanking SNPs.
[00093] Table 12.1. Genome wide association study results in the Quebec
Founder Population (QFP). SNP markers found to be associated with
Endometriosis from the analysis of genome wide scan (GWS) data: has_PRKCE-
1-1_cr. Columns include: Region ID; Chromosome; Build 36 location in base
pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique
numerical identifier for this patent application; Sequence, 21 bp of sequence
covering 10 base pair of unique sequence flanking either side of central
polymorphic SNP; - log10 P values for GWS, - log10 of the P value for
statistical
significance from the GWS for single SNP markers (both T test and Permutation
test p-values are displayed; see Example section) and for the most highly
associated multi-marker haplotypes centered at the reference marker and
defined
by the sliding windows of specified sizes.
[00094] Table 12.2. List of significantly associated haplotypes based on the
Endometriosis Disease results using the Quebec Founder Population (QFP).
Individual haplotypes with associated relative risks are presented in each row
of
the table; these values were extracted from the associated marker haplotype
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window with the most significant p value for each SNP in Table 12.1. The first
column lists the region ID as presented in Table 1. The Haplotype column lists
the specific nucleotides for the individual SNP alleles contributing to the
haplotype reported. The Case and Control columns correspond to the numbers of
cases and controls, respectively, containing the haplotype variant noted in
the
Haplotype column. The Total Case and Total Control columns list the total
numbers of cases and controls for which genotype data was available for the
haplotype in question. The RR column gives to the relative risk for each
particular haplotype. The remainder of the columns lists the SeqlDs for the
SNPs
contributing to the haplotype and their relative location with respect to the
central
marker. The Central marker (0) column lists the SeqID for the central marker
on
which the haplotype is based. Flanking markers are identified by minus (-) or
plus
(+) signs to indicate the relative location of flanking SNPs.
[00095] Table 13.1. Genome wide association study results in the Quebec
Founder Population (QFP). SNP markers found to be associated with
Endometriosis from the analysis of genome wide scan (GWS) data: not_RAF-
1_cr. Columns include: Region ID; Chromosome; Build 36 location in base pairs
(bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique
numerical identifier for this patent application; Sequence, 21 bp of sequence
covering 10 base pair of unique sequence flanking either side of central
polymorphic SNP; - log10 P values for GWS, - Iog10 of the P value for
statistical
significance from the GWS for single SNP markers (both T test and Permutation
test p-values are displayed; see Example section) and for the most highly
associated multi-marker haplotypes centered at the reference marker and
defined
by the sliding windows of specified sizes.
[00096] Table 13.2. List of significantly associated haplotypes based on the
Endometriosis Disease results using the Quebec Founder Population (QFP).
Individual haplotypes with associated relative risks are presented in each row
of
the table; these values were extracted from the associated marker haplotype
window with the most significant p value for each SNP in Table 13.1. The first
column lists the region ID as presented in Table 1. The Haplotype column lists
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the specific nucleotides for the individual SNP alleles contributing to the
haplotype reported. The Case and Control columns correspond to the numbers of
cases and controls, respectively, containing the haplotype variant noted in
the
Haplotype column. The Total Case and Total Control columns list the total
numbers of cases and controls for which genotype data was available for the
haplotype in question. The RR column gives to the relative risk for each
particular haplotype. The remainder of the columns lists the SeqlDs for the
SNPs
contributing to the haplotype and their relative location with respect to the
central
marker. The Central marker (0) column lists the SeqID for the central marker
on
which the haplotype is based. Flanking markers are identified by minus (-) or
plus (+) signs to indicate the relative location of flanking SNPs.
[00097] Table 14.1. Genome wide association study results in the Quebec
Founder Population (QFP). SNP markers found to be associated with
Endometriosis from the analysis of genome wide scan (GWS) data: has_DNAH5-
1_cr. Columns include: Region ID; Chromosome; Build 36 location in base pairs
(bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique
numerical identifier for this patent application; Sequence, 21 bp of sequence
covering 10 base pair of unique sequence flanking either side of central
polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for
statistical
significance from the GWS for single SNP markers (both T test and Permutation
test p-values are displayed; see Example section) and for the most highly
associated multi-marker haplotypes centered at the reference marker and
defined
by the sliding windows of specified sizes.
[00098] Table 14.2. List of significantly associated haplotypes based on the
Endometriosis Disease results using the Quebec Founder Population (QFP).
Individual haplotypes with associated relative risks are presented in each row
of
the table; these values were extracted from the associated marker haplotype
window with the most significant p value for each SNP in Table 14.1. The first
column lists the region ID as presented in Table 1. The Haplotype column lists
the specific nucleotides for the individual SNP alleles contributing to the
haplotype reported. The Case and Control columns correspond to the numbers of
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cases and controls, respectively, containing the haplotype variant noted in
the
Haplotype column. The Total Case and Total Control columns list the total
numbers of cases and controls for which genotype data was available for the
haplotype in question. The RR column gives to the relative risk for each
particular haplotype. The remainder of the columns lists the SeqlDs for the
SNPs
contributing to the haplotype and their relative location with respect to the
central
marker. The Central marker (0) column lists the SeqID for the central marker
on
which the haplotype is based. Flanking markers are identified by minus (-) or
plus (+) signs to indicate the relative location of flanking SNPs.
[00099] Table 15.1. Genome wide association study results in the Quebec
Founder Population (QFP). SNP markers found to be associated with
Endometriosis from the analysis of genome wide scan (GWS) data: has SYNE1-1
cr. Columns include: Region ID; Chromosome; Build 36 location in base pairs
(bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique
numerical identifier for this patent application; Sequence, 21 bp of sequence
covering 10 base pair of unique sequence flanking either side of central
polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for
statistical
significance from the GWS for single SNP markers (both T test and Permutation
test p-values are displayed; see Example section) and for the most highly
associated multi-marker haplotypes centered at the reference marker and
defined
by the sliding windows of specified sizes.
[000100] Table 15.2. List of significantly associated haplotypes based on the
Endometriosis Disease results using the Quebec Founder Population (QFP).
Individual haplotypes with associated relative risks are presented in each row
of
the table; these values were extracted from the associated marker haplotype
window with the most significant p value for each SNP in Table 15.1. The first
column lists the region ID as presented in Table 1. The Haplotype column lists
the specific nucleotides for the individual SNP alleles contributing to the
haplotype reported. The Case and Control columns correspond to the numbers of
cases and controls, respectively, containing the haplotype variant noted in
the
Haplotype column. The Total Case and Total Control columns list the total
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numbers of cases and controls for which genotype data was available for the
haplotype in question. The RR column gives to the relative risk for each
particular haplotype. The remainder of the columns lists the SeqlDs for the
SNPs
contributing to the haplotype and their relative location with respect to the
central
marker. The Central marker (0) column lists the SeqID for the central marker
on
which the haplotype is based. Flanking markers are identified by minus (-) or
plus (+) signs to indicate the relative location of flanking SNPs.
[000101] Table 16.1. Genome wide association study results in the Quebec
Founder Population (QFP). SNP markers found to be associated with
Endometriosis from the analysis of genome wide scan (GWS) data: not SYNE1-1
cr. Columns include: Region ID; Chromosome; Build 36 location in base pairs
(bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique
numerical identifier for this patent application; Sequence, 21 bp of sequence
covering 10 base pair of unique sequence flanking either side of central
polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for
statistical
significance from the GWS for single SNP markers (both T test and Permutation
test p-values are displayed; see Example section) and for the most highly
associated multi-marker haplotypes centered at the reference marker and
defined
by the sliding windows of specified sizes.
[000102] Table 16.2. List of significantly associated haplotypes based on the
Endometriosis Disease results using the Quebec Founder Population (QFP).
Individual haplotypes with associated relative risks are presented in each row
of
the table; these values were extracted from the associated marker haplotype
window with the most significant p value for each SNP in Table 16.1. The first
column lists the region ID as presented in Table 1. The Haplotype column lists
the specific nucleotides for the individual SNP alieles contributing to the
haplotype reported. The Case and Control columns correspond to the numbers of
cases and controls, respectively, containing the haplotype variant noted in
the
Haplotype column. The Total Case and Total Control columns list the total
numbers of cases and controls for which genotype data was available for the
haplotype in question. The RR column gives to the relative risk for each
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particular haplotype. The remainder of the columns lists the SeqlDs for the
SNPs
contributing to the haplotype and their relative location with respect to the
central
marker. The Central marker (0) column lists the SeqID for the central marker
on
which the haplotype is based. Flanking markers are identified by minus (-) or
plus (+) signs to indicate the relative location of flanking SNPs.
[000103] Table 17. Description of primer sequences used for the semi-
quantitative gene expression profiling by RT-PCR (see Example section for
details).
[000104] Table 18. Probes used for the in situ hybridization (ISH) study (see
Example section for details).
DEFINITIONS
[000105] Throughout the description of the present invention, several terms
are
used that are specific to the science of this field. For the sake of clarity
and to
avoid any misunderstanding, these definitions are provided to aid in the
understanding of the specification and claims.
[000106] Allele: One of a pair, or series, of forms of a gene or non-genic
region
that occur at a given locus in a chromosome. Alleles are symbolized with the
same basic symbol (e.g., B for dominant and b for recessive; B1, B2, Bn for n
additive alleles at a locus). In a normal diploid cell there are two alleles
of any
one gene (one from each parent), which occupy the same relative position
(locus)
on homologous chromosomes. Within a population there may be more than two
alleles of a gene. See multiple alleles. SNPs also have alleles, i.e., the two
(or
more) nucleotides that characterize the SNP.
[000107] Amplification of nucleic acids: refers to methods such as polymerase
chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR)
and
amplification methods based on the use of Q-beta replicase. These methods are
well known in the art and are described, for example, in U.S. Patent Nos.
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4,683,195 and 4,683,202. Reagents and hardware for conducting PCR are
commercially available. Primers useful for amplifying sequences from the
disorder region are preferably complementary to, and preferably hybridize
specifically to, sequences in the disorder region or in regions that flank a
target
region therein. Genes from Tables 2-4 generated by amplification may be
sequenced directly. Alternatively, the amplified sequence(s) may be cloned
prior
to sequence analysis.
[000108] Antigenic component: is a moiety that binds to its specific antibody
with sufficiently high affinity to form a detectable antigen-antibody complex.
[000109] Antibodies: refer to polyclonal and/or monoclonal antibodies and
fragments thereof, and immunologic binding equivalents thereof, that can bind
to
proteins and fragments thereof or to nucleic acid sequences from the disorder
region, particularly from the disorder gene products or a portion thereof. The
term
antibody is used both to refer to a homogeneous molecular entity, or a mixture
such as a serum product made up of a plurality of different molecular
entities.
Proteins may be prepared synthetically in a protein synthesizer and coupled to
a
carrier molecule and injected over several months into rabbits. Rabbit sera
are
tested for immunoreactivity to the protein or fragment. Monoclonal antibodies
may be made by injecting mice with the proteins, or fragments thereof.
Monoclonal antibodies can be screened by ELISA and tested for specific
immunoreactivity with protein or fragments thereof (Harlow et al. 1988,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY). These antibodies will be useful in developing assays as well as
therapeutics.
[000110] Associated allele: refers to an allele at a polymorphic locus that is
associated with a particular phenotype of interest, e.g., a predisposition to
a
disorder or a particular drug response.
[000111] cDNA: refers to complementary or copy DNA produced from an RNA
template by the action of RNA-dependent DNA polymerase (reverse
transcriptase). Thus, a cDNA clone means a duplex DNA sequence
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complementary to an RNA molecule of interest, included in a cloning vector or
PCR amplified. This term includes genes from which the intervening sequences
have been removed.
[000112] cDNA library: refers to a collection of recombinant DNA molecules
containing cDNA inserts that together comprise essentially all of the
expressed
genes of an organism or tissue. A cDNA library can be prepared by methods
known to one skilled in the art (see, e.g., Cowell and Austin, 1997, "DNA
Library
Protocols," Methods in Molecular Biology). Generally, RNA is first isolated
from
the cells of the desired organism, and the RNA is used to prepare cDNA
molecules.
[000113] Cloning: refers to the use of recombinant DNA techniques to insert a
particular gene or other DNA sequence into a vector molecule. In order to
successfully clone a desired gene, it is necessary to use methods for
generating
DNA fragments, for joining the fragments to vector molecules, for introducing
the
composite DNA molecule into a host cell in which it can replicate, and for
selecting the clone having the target gene from amongst the recipient host
cells.
[000114] Cloning vector: refers to a plasmid or phage DNA or other DNA
molecule that is able to replicate in a host cell. The cloning vector is
typically
characterized by one or more endonuclease recognition sites at which such DNA
sequences may be cleaved in a determinable fashion without loss of an
essential
biological function of the DNA, and which may contain a selectable marker
suitable for use in the identification of cells containing the vector.
[000115] Coding sequence or a protein-coding sequence: is a polynucleotide
sequence capable of being transcribed into mRNA and/or capable of being
translated into a polypeptide or peptide. The boundaries of the coding
sequence
are typically determined by a translation start codon at the 5'-terminus and a
translation stop codon at the 3'-terminus.
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[000116] Complement of a nucleic acid sequence: refers to the antisense
sequence that participates in Watson-Crick base-pairing with the original
sequence.
[000117] Disorder region: refers to the portions of the human chromosomes
displayed in Table 1 bounded by the markers from Tables 2-16.
[000118] Disorder-associated nucleic acid or polypeptide sequence: refers to a
nucleic acid sequence that maps to region of Table 1 or the polypeptides
encoded therein (Tables 2-4, nucleic acids, and polypeptides). For nucleic
acids,
this encompasses sequences that are identical or complementary to the gene
sequences from Tables 2-4, as well as sequence-conservative, function-
conservative, and non-conservative variants thereof. For polypeptides, this
encompasses sequences that are identical to the polypeptide, as well as
function-conservative and non-conservative variants thereof. Included are the
alleles of naturally-occurring polymorphisms causative of ENDOMETRIOSIS
disease such as, but not limited to, alleles that cause altered expression of
genes
of Tables 2-4 and alleles that cause altered protein levels or stability
(e.g.,
decreased levels, increased levels, expression in an inappropriate tissue
type,
increased stability, and decreased stability).
[000119] Expression vector: refers to a vehicle or plasmid that is capable of
expressing a gene that has been cloned into it, after transformation or
integration
in a host cell. The cloned gene is usually placed under the control of (i.e.,
operably linked to) a regulatory sequence.
[000120] Function-conservative variants: are those in which a change in one or
more nucleotides in a given codon position results in a polypeptide sequence
in
which a given amino acid residue in the polypeptide has been replaced by a
conservative amino acid substitution. Function-conservative variants also
include
analogs of a given polypeptide and any polypeptides that have the ability to
elicit
antibodies specific to a designated polypeptide.
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[000121] Founder population: Also a population isolate, this is a large number
of people who have mostly descended, in genetic isolation from other
populations, from a much smaller number of people who lived many generations
ago.
[000122] Gene: Refers to a DNA sequence that encodes through its template
or messenger RNA a sequence of amino acids characteristic of a specific
peptide, polypeptide, or protein. The term "gene" also refers to a DNA
sequence
that encodes an RNA product. The term gene as used herein with reference to
genomic DNA includes intervening, non-coding regions, as well as regulatory
regions, and can include 5' and 3' ends. A gene sequence is wild-type if such
sequence is usually found in individuals unaffected by the disorder or
condition of
interest. However, environmental factors and other genes can also play an
important role in the ultimate determination of the disorder. In the context
of
complex disorders involving multiple genes (oligogenic disorder), the wild
type, or
normal sequence can also be associated with a measurable risk or
susceptibility,
receiving its reference status based on its frequency in the general
population.
[000123] GeneMaps: are defined as groups of gene(s) that are directly or
indirectly involved in at least one phenotype of a disorder (some non-limiting
example of GeneMaps comprises varius combinations of genes from Tables 2-4).
As such, GeneMaps enable the development of synergistic diagnostic products,
creating "theranostics".
[000124] Genotype: Set of alieles at a specified locus or loci.
[000125] Haplotype: The allelic pattern of a group of (usually contiguous) DNA
markers or other polymorphic loci along an individual chromosome or double
helical DNA segment. Haplotypes identify individual chromosomes or
chromosome segments. The presence of shared haplotype patterns among a
group of individuals implies that the locus defined by the haplotype has been
inherited, identical by descent (IBD), from a common ancestor. Detection of
identical by descent haplotypes is the basis of linkage disequilibrium (LD)
mapping. Haplotypes are broken down through the generations by recombination
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and mutation. In some instances, a specific allele or haplotype may be
associated with susceptibility to a disorder or condition of interest, e.g.,
ENDOMETRIOSIS disease. In other instances, an allele or haplotype may be
associated with a decrease in susceptibility to a disorder or condition of
interest,
i.e., a protective sequence.
[000126] Host: includes prokaryotes and eukaryotes. The term includes an
organism or cell that is the recipient of an expression vector (e.g.,
autonomously
replicating or integrating vector).
[000127] Hybridizable: nucleic acids are hybridizable to each other when at
least one strand of the nucleic acid can anneal to another nucleic acid strand
under defined stringency conditions. In some embodiments, hybridization
requires that the two nucleic acids contain at least 10 substantially
complementary nucleotides; depending on the stringency of hybridization,
however, mismatches may be tolerated. The appropriate stringency for
hybridizing nucleic acids depends on the length of the nucleic acids and the
degree of complementarity, and can be determined in accordance with the
methods described herein.
[000128] Identity by descent (IBD): Identity among DNA sequences for
different individuals that is due to the fact that they have all been
inherited from a
common ancestor. LD mapping identifies IBD haplotypes as the likely location
of
disorder genes shared by a group of patients.
[000129] Identity: as known in the art, is a relationship between two or more
polypeptide sequences or two or more polynucleotide sequences, as determined
by comparing the sequences. In the art, identity also means the degree of
sequence relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such sequences.
Identity and similarity can be readily calculated by known methods, including
but
not limited to those described in A.M. Lesk (ed), 1988, Computational
Molecular
Biology, Oxford University Press, NY; D.W. Smith (ed), 1993, Biocomputing.
Informatics and Genome Projects, Academic Press, NY; A.M. Griffin and H.G.
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Griffin, H. G (eds), 1994, ComputerAnalysis of Sequence Data, Part 1, Humana
Press, NJ; G. von Heinje, 1987, Sequence Analysis in Molecular Biology,
Academic Press; and M. Gribskov and J. Devereux (eds), 1991, Sequence
Analysis Primer, M Stockton Press, NY; H. Carillo and D. Lipman, 1988, SIAM J.
Applied Math., 48:1073.
[000130] Immunogenic component: is a moiety that is capable of eliciting a
humoral and/or cellular immune response in a host animal.
[000131] Isolated nucleic acids: are nucleic acids separated away from other
components (e.g., DNA, RNA, and protein) with which they are associated (e.g.,
as obtained from cells, chemical synthesis systems, or phage or nucleic acid
libraries). Isolated nucleic acids are at least 60% free, preferably 75% free,
and
most preferably 90% free from other associated components. In accordance with
the present invention, isolated nucleic acids can be obtained by methods
described herein, or other established methods, including isolation from
natural
sources (e.g., cells, tissues, or organs), chemical synthesis, recombinant
methods, combinations of recombinant and chemical methods, and library
screening methods.
[000132] Isolated polypeptides or peptides: are those that are separated from
other components (e.g., DNA, RNA, and other polypeptides or peptides) with
which they are associated (e.g., as obtained from cells, translation systems,
or
chemical synthesis systems). In a preferred embodiment, isolated polypeptides
or
peptides are at least 10% pure; more preferably, 80% or 90% pure. Isolated
polypeptides and peptides include those obtained by methods described herein,
or other established methods, including isolation from natural sources (e.g.,
cells,
tissues, or organs), chemical synthesis, recombinant methods, or combinations
of
recombinant and chemical methods. Proteins or polypeptides referred to herein
as recombinant are proteins or polypeptides produced by the expression of
recombinant nucleic acids. A portion as used herein with regard to a protein
or
polypeptide, refers to fragments of that protein or polypeptide. The fragments
can
range in size from 5 amino acid residues to all but one residue of the entire
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protein sequence. Thus, a portion or fragment can be at least 5, 5-50, 50-100,
100-200, 200-400, 400-800, or more consecutive amino acid residues of a
protein
or polypeptide.
[000133] Linkage disequilibrium (LD): the situation in which the alleles for
two
or more loci do not occur together in individuals sampled from a population at
frequencies predicted by the product of their individual allele frequencies.
In
other words, markers that are in LD do not follow Mendel's second law of
independent random segregation. LD can be caused by any of several
demographic or population artifacts as well as by the presence of genetic
linkage
between markers. However, when these artifacts are controlled and eliminated
as sources of LD, then LD results directly from the fact that the loci
involved are
located close to each other on the same chromosome so that specific
combinations of alleles for different markers (haplotypes) are inherited
together.
Markers that are in high LD can be assumed to be located near each other and a
marker or haplotype that is in high LD with a genetic trait can be assumed to
be
located near the gene that affects that trait. The physical proximity of
markers can
be measured in family studies where it is called linkage or in population
studies
where it is called linkage disequilibrium.
[000134] LD mapping: population based gene mapping, which locates disorder
genes by identifying regions of the genome where haplotypes or marker
variation
patterns are shared statistically more frequently among disorder patients
compared to healthy controls. This method is based upon the assumption that
many of the patients will have inherited an allele associated with the
disorder
from a common ancestor (IBD), and that this allele will be in LD with the
disorder
gene.
[000135] Locus: a specific position along a chromosome or DNA sequence.
Depending upon context, a locus could be a gene, a marker, a chromosomal
band or a specific sequence of one or more nucleotides.
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[000136] Minor allele frequency (MAF): the population frequency of one of the
alleles for a given polymorphism, which is equal or less than 50%. The sum of
the
MAF and the Major allele frequency equals one.
[000137] Markers: an identifiable DNA sequence that is variable (polymorphic)
for different individuals within a population. These sequences facilitate the
study
of inheritance of a trait or a gene. Such markers are used in mapping the
order of
genes along chromosomes and in following the inheritance of particular genes;
genes closely linked to the marker or in LD with the marker will generally be
inherited with it. Two types of markers are commonly used in genetic analysis,
microsatellites and SNPs.
[000138] Microsatellite: DNA of eukaryotic cells comprising a repetitive,
short
sequence of DNA that is present as tandem repeats and in highly variable copy
number, flanked by sequences unique to that locus.
[000139] Mutant sequence: if it differs from one or more wild-type sequences.
For example, a nucleic acid from a gene listed in Tables 2-4 containing a
particular allele of a single nucleotide polymorphism may be a mutant
sequence.
In some cases, the individual carrying this allele has increased
susceptibility
toward the disorder or condition of interest. In other cases, the mutant
sequence
might also refer to an allele that decreases the susceptibility toward a
disorder or
condition of interest and thus acts in a protective manner. The term mutation
may
also be used to describe a specific allele of a polymorphic locus.
[000140] Non-conservative variants: are those in which a change in one or
more nucleotides in a given codon position results in a polypeptide sequence
in
which a given amino acid residue in a polypeptide has been replaced by a non-
conservative amino acid substitution. Non-conservative variants also include
polypeptides comprising non-conservative amino acid substitutions.
[000141] Nucleic acid or polynucleotide: purine- and pyrimidine-containing
polymers of any length, either polyribonucleotides or polydeoxyribonucleotide
or
mixed polyribo polydeoxyribonucleotides. This includes single-and double-
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stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as
protein nucleic acids (PNA) formed by conjugating bases to an amino acid
backbone. This also includes nucleic acids containing modified bases.
[000142] Nucleotide: a nucleotide, the unit of a DNA molecule, is composed of
a base, a 2'-deoxyribose and phosphate ester(s) attached at the 5' carbon of
the
deoxyribose. For its incorporation in DNA, the nucleotide needs to possess
three
phosphate esters but it is converted into a monoester in the process.
[000143] Operably linked: means that the promoter controls the initiation of
expression of the gene. A promoter is operably linked to a sequence of
proximal
DNA if upon introduction into a host cell the promoter determines the
transcription
of the proximal DNA sequence(s) into one or more species of RNA. A promoter is
operably linked to a DNA sequence if the promoter is capable of initiating
transcription of that DNA sequence.
[000144] Ortholog: denotes a gene or polypeptide obtained from one species
that has homology to an analogous gene or polypeptide from a different
species.
[000145] Paralog: denotes a gene or polypeptide obtained from a given
species that has homology to a distinct gene or polypeptide from that same
species.
[000146] Phenotype: any visible, detectable or otherwise measurable property
of an organism such as symptoms of, or susceptibility to, a disorder.
[000147] Polymorphism: occurrence of two or more alternative genomic
sequences or alleles between or among different genomes or individuals at a
single locus. A polymorphic site thus refers specifically to the locus at
which the
variation occurs. In some cases, an individual carrying a particular allele of
a
polymorphism has an increased or decreased susceptibility toward a disorder or
condition of interest.
[000148] Portion and fragment: are synonymous. A portion as used with regard
to a nucleic acid or polynucleotide refers to fragments of that nucleic acid
or
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polynucleotide. The fragments can range in size from 8 nucleotides to all but
one
nucleotide of the entire gene sequence. Preferably, the fragments are at least
about 8 to about 10 nucleotides in length; at least about 12 nucleotides in
length;
at least about 15 to about 20 nucleotides in length; at least about 25
nucleotides
in length; or at least about 35 to about 55 nucleotides in length.
[000149] Probe or primer: refers to a nucleic acid or oligonucleotide that
forms
a hybrid structure with a sequence in a target region of a nucleic acid due to
complementarity of the probe or primer sequence to at least one portion of the
target region sequence.
[000150] Protein and polypeptide: are synonymous. Peptides are defined as
fragments or portions of polypeptides, preferably fragments or portions having
at
least one functional activity (e.g., proteolysis, adhesion, fusion, antigenic,
or
intracellular activity) as the complete polypeptide sequence.
[000151] Recombinant nucleic acids: nucleic acids which have been produced
by recombinant DNA methodology, including those nucleic acids that are
generated by procedures which rely upon a method of artificial replication,
such
as the polymerase chain reaction (PCR) and/or cloning into a vector using
restriction enzymes. Portions of recombinant nucleic acids which code for
polypeptides can be identified and isolated by, for example, the method of M.
Jasin et al., U.S. Patent No. 4,952,501.
[000152] Regulatory sequence: refers to a nucleic acid sequence that controls
or regulates expression of structural genes when operably linked to those
genes.
These include, for example, the lac systems, the trp system, major operator
and
promoter regions of the phage lambda, the control region of fd coat protein
and
other sequences known to control the expression of genes in prokaryotic or
eukaryotic cells. Regulatory sequences will vary depending on whether the
vector
is designed to express the operably linked gene in a prokaryotic or eukaryotic
host, and may contain transcriptional elements such as enhancer elements,
termination sequences, tissue-specificity elements and/or translational
initiation
and termination sites.
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[000153] Sample: as used herein refers to a biological sample, such as, for
example, tissue or fluid isolated from an individual or animal (including,
without
limitation, plasma, serum, cerebrospinal fluid, lymph, tears, nails, hair,
saliva,
milk, pus, and tissue exudates and secretions) or from in vitro cell culture-
constituents, as well as samples obtained from, for example, a laboratory
procedure.
[000154] Single nucleotide polymorphism (SNP): variation of a single
nucleotide. This includes the replacement of one nucleotide by another and
deletion or insertion of a single nucleotide. Typically, SNPs are biallelic
markers
although tri- and tetra-alielic markers also exist. For example, SNP A\C may
comprise allele C or allele A (Tables 5-16). Thus, a nucleic acid molecule
comprising SNP A\C may include a C or A at the polymorphic position. For
clarity
purposes, an ambiguity code is used in Tables 5-16 and the sequence listing,
to
represent the variations. For a combination of SNPs, the term "haplotype" is
used, e.g. the genotype of the SNPs in a single DNA strand that are linked to
one
another. In certain embodiments, the term "haplotype" is used to describe a
combination of SNP alleles, e.g., the alleles of the SNPs found together on a
single DNA molecule. In specific embodiments, the SNPs in a haplotype are in
linkage disequilibrium with one another.
[000155] Sequence-conservative: variants are those in which a change of one
or more nucleotides in a given codon position results in no alteration in the
amino
acid encoded at that position (i.e., silent mutation).
[000156] Substantially homologous: a nucleic acid or fragment thereof is
substantially homologous to another if, when optimally aligned (with
appropriate
nucleotide insertions and/or deletions) with the other nucleic acid (or its
complementary strand), there is nucleotide sequence identity in at least 60%
of
the nucleotide bases, usually at least 70%, more usually at least 80%,
preferably
at least 90%, and more preferably at least 95-98% of the nucleotide bases.
Alternatively, substantial homology exists when a nucleic acid or fragment
thereof
will hybridize, under selective hybridization conditions, to another nucleic
acid (or
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a complementary strand thereof). Selectivity of hybridization exists when
hybridization which is substantially more selective than total lack of
specificity
occurs. Typically, selective hybridization will occur when there is at least
about
55% sequence identity over a stretch of at least about nine or more
nucleotides,
preferably at least about 65%, more preferably at least about 75%, and most
preferably at least about 90% (M. Kanehisa, 1984, NucL Acids Res. 11:203-213).
The length of homology comparison, as described, may be over longer stretches,
and in certain embodiments will often be over a stretch of at least 14
nucleotides,
usually at least 20 nucleotides, more usually at least 24 nucleotides,
typically at
least 28 nucleotides, more typically at least 32 nucleotides, and preferably
at
least 36 or more nucleotides.
[000157] Wild-type gene from Tables 2-4: refers to the reference sequence.
The wild-type gene sequences from Tables 2-4 used to identify the variants
(polymorphisms, alleles, and haplotypes) described in detail herein.
[000158] Technical and scientific terms used herein have the meanings
commonly understood by one of ordinary skill in the art to which the present
invention pertains, unless otherwise defined. Reference is made herein to
various
methodologies known to those of skill in the art. Publications and other
materials
setting forth such known methodologies to which reference is made are
incorporated herein by reference in their entireties as though set forth in
full.
Standard reference works setting forth the general principles of recombinant
DNA
technology include J. Sambrook et al., 1989, Molecular Cloning: A Laboratory
Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY;
P.B. Kaufman et al., (eds), 1995, Handbook of Molecular and Cellular Methods
in
Biology and Medicine, CRC Press, Boca Raton; M.J. McPherson (ed), 1991,
Directed Mutagenesis: A Practical Approach, IRL Press, Oxford; J. Jones, 1992,
Amino Acid and Peptide Synthesis, Oxford Science Publications, Oxford; B.M.
Austen and O.M.R. Westwood, 1991, Protein Targeting and Secretion, IRL
Press, Oxford; D.N Glover (ed), 1985, DNA Cloning, Volumes I and 11; M.J. Gait
(ed), 1984, Oligonucleotide Synthesis; B.D. Hames and S.J. Higgins (eds),
1984,
Nucleic Acid Hybridization; Quirke and Taylor (eds), 1991, PCR-A Practical
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Approach; Harries and Higgins (eds), 1984, Transcription and Translation; R.I.
Freshney (ed), 1986, Animal Cell Culture; Immobilized Cells and Enzymes, 1986,
IRL Press; Perbal, 1984, A Practical Guide to Molecular Cloning, J. H. Miller
and
M. P. Calos (eds), 1987, Gene Transfer Vectors for Mammalian Cells, Cold
Spring Harbor Laboratory Press; M.J. Bishop (ed), 1998, Guide to Human
Genome Computing, 2d Ed., Academic Press, San Diego, CA; L.F. Peruski and
A.H. Peruski, 1997, The Internet and the New Biology. Tools for Genomic and
Molecular Research, American Society for Microbiology, Washington, D.C.
Standard reference works setting forth the general principles of immunology
include S. Sell, 1996, Immunology, lmmunopathology & Immunity, 5th Ed.,
Appleton & Lange, Publ., Stamford, CT; D. Male et al., 1996, Advanced
Immunology, 3d Ed., Times Mirror Int'I Publishers Ltd., Publ., London; D.P.
Stites
and A.L Terr, 1991, Basic and Clinical Immunology, 7th Ed., Appleton & Lange,
Publ., Norwalk, CT; and A.K. Abbas et al., 1991, Cellular and Molecular
Immunology, W. B. Saunders Co., Pubi., Philadelphia, PA. Any suitable
materials
and/or methods known to those of skill can be utilized in carrying out the
present
invention; however, preferred materials and/or methods are described.
Materials,
reagents, and the like to which reference is made in the following description
and
examples are generally obtainable from commercial sources, and specific
vendors are cited herein.
DETAILED DESCRIPTION OF THE INVENTION
Genome wide association study to construct a GeneMap for
ENDOMETR/OSIS
[000159] The present invention is based on the discovery of genes associated
with ENDOMETRIOSIS disease. In the preferred embodiment, disease-
associated Ioci (candidate regions; Table 1) are identified by the
statistically
significant differences in allele or haplotype frequencies between the cases
and
the controls. For the purpose of the present invention candidate regions are
identified in Table 1.
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[000160] The invention provides a method for the discovery of genes
associated with ENDOMETRIOSIS disease and the construction of a GeneMap
for ENDOMETRIOSIS disease in a human population, comprising the following
steps (see also Example section herein). The GeneMaps of the invention,
presented in the Example section, is provided for clarity purposes and other
GeneMaps with various other combinations of genes from Tables 2-4 and/or
other genes involved in the related networks or pathways are obtained by the
methods of the invention:
[000161] Step 1: Recruit patients (cases) and controls
[000162] In the preferred embodiment, 500 patients diagnosed for
ENDOMETRIOSIS disease along with 500 independent controls samples are
recruited from the Quebec Founder Population (QFP).
[000163] In another embodiment, more or less than 500 patients and controls
are recruited.
[000164] In another embodiment, 500 patients diagnosed for
ENDOMETRIOSIS disease along with two family members are recruited from the
Quebec Founder Population (QFP). The preferred trios recruited are parent-
parent-child (PPC) trios. Trios can also be recruited as parent-child-child
(PCC)
trios. In another preferred embodiment, more or less than 500 trios are
recruited
[000165] In yet another embodiment, the present invention is performed as a
whole or partially with DNA samples from individuals of another founder
population than the Quebec population or from the general population.
[000166] Step 2: DNA extraction and quantitation
[000167] Any sample comprising cells or nucleic acids from patients or
controls
may be used. Preferred samples are those easily obtained from the patient or
control. Such samples include, but are not limited to blood, peripheral
lymphocytes, buccal swabs, epithelial cell swabs, nails, hair, bronchoalveolar
lavage fluid, sputum, or other body fluid or tissue obtained from an
individual.
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[000168] In one embodiment, DNA is extracted from such samples in the
quantity and quality necessary to perform the invention using conventional DNA
extraction and quantitation techniques. The present invention is not linked to
any
DNA extraction or quantitation platform in particular.
[000169] Step 3: Genotype the recruited individuals
[000170] In one embodiment, assay-specific and/or locus-specific and/or allele-
specific oligonucleotides for every SNP marker of the present invention
(Tables
5-16) are organized onto one or more arrays. The genotype at each SNP locus is
revealed by hybridizing short PCR fragments comprising each SNP locus onto
these arrays. The arrays permit a high-throughput genome wide association
study using DNA samples from individuals of the Quebec founder population.
Such assay-specific and/or locus-specific and/or allele-specific
oligonucleotides
necessary for scoring each SNP of the present invention are preferably
organized
onto a solid support. Such supports can be arrayed on wafers, glass slides,
beads or any other type of solid support.
[000171] In another embodiment, the assay-specific and/or locus-specific
and/or allele-specific oligonucleotides are not organized onto a solid support
but
are still used as a whole, in panels or one by one. The present invention is
therefore not linked to any genotyping platform in particular.
[000172] In another embodiment, one or more portions of the SNP maps
(publicly available maps and our own proprietary QLDM map) are used to screen
the whole genome, a subset of chromosomes, a chromosome, a subset of
genomic regions or a single genomic region.
[000173] In the preferred embodiment, the individuals composing the cases
and controls or the trios are preferably individually genotyped with at least
80,000
markers, generating at least a few million genotypes; more preferably, at
least a
hundred million. In another embodiment, individuals are pooled in cases and
control pools for genotyping and genetic analysis.
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[000174] Step 4: Exclude the markers that did not pass the quality control of
the assay.
[000175] Preferably, the quality controls comprises, but are not limited to,
the
following criteria: eliminate SNPs that had a high rate of Mendelian errors
(cut-off
at 1% Mendelian error rate), that deviate from the Hardy-Weinberg equilibrium,
that are non-polymorphic in the Quebec founder population or have too many
missing data (cut-off at 1% missing values or higher), or simply because they
are
non-polymorphic in the Quebec founder population (cut-off at 1%:5 10% minor
allele frequency (MAF)).
[000176] Step 5: Perform the genetic analysis on the results obtained using
haplotype information as well as single-marker association.
[000177] In the preferred embodiment, genetic analysis is performed on all the
genotypes from Step 3.
[000178] In another embodiment, genetic analysis is performed on a subset of
markers from Step 3 or from markers that passed the quality controls from Step
4.
[000179] In one embodiment, the genetic analysis consists of, but is not
limited
to features corresponding to Phase information and haplotype structures. Phase
information and haplotype structures are preferably deduced from trio
genotypes
using Phasefinder. Since chromosomal assignment (phase) cannot be estimated
when all trio members are heterozygous, an Expectation-Maximization (EM)
algorithm may be used to resolve chromosomal assignment ambiguities after
Phasefinder.
[000180] In yet another embodiment, the PL-EM algorithm (Partition-Ligation
EM; Niu et al.., Am. J. Hum. Genet. 70:157 (2002)) can be used to estimate
haplotypes from the "genotype" data as a measured estimate of the reference
allele frequency of a SNP in 15-marker windows that advance in increments of
one marker across the data set. The results from such algorithms are converted
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into 15-marker haplotype files. Subsequently, the individual 15-marker block
files
are assembled into one continuous block of haplotypes for the entire
chromosome. These extended haplotypes can then be used for further analysis.
Such haplotype assembly algorithms take the consensus estimate of the aliele
call at each marker over all separate estimations (most markers are estimated
15
different times as the 15 marker blocks pass over their position).
[000181] In another embodiment, the haplotype frequencies among patients
are compared to those among the controls using LDSTATS, a program that
assesses the association of haplotypes with the disease. Such program defines
haplotypes using multi-marker windows that advance across the marker map in
one-marker increments. Such windows can be 1, 3, 5, 7 or 9 markers wide, and
all these window sizes are tested concurrently. Larger multi-marker haplotype
windows can also be used. At each position the frequency of haplotypes in
cases is compared to the frequency of haplotypes in controls. Such allele
frequency differences for single marker windows can be tested using Pearson's
Chi-square with any degree of freedom. Multi-allelic haplotype association can
be
tested using Smith's normalization of the square root of Pearson's Chi-square.
Such significance of association can be reported in two ways:
[000182] The significance of association within any one haplotype window is
plotted against the marker that is central to that window.
[000183] P-values of association for each specific marker are calculated as a
pooled P-value across all haplotype windows in which they occur. The pooled P-
value is calculated using an expected value and variance calculated using a
permutation test that considers covariance between individual windows. Such
pooled P-values can yield narrower regions of gene location than the window
data (see Example 3 herein for details on various analysis methods, such as
LDSTATS v2.0 and v4.0).
[000184] In another embodiment, conditional haplotype and subtype analyses
can be performed on subsets of the original set of cases and controls using
the
program LDSTATS. For conditional analyses, the selection of a subset of cases
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and their matched controls can be based on the carrier status of cases at a
gene
or locus of interest (see conditional analysis section in Example 3 herein).
Various conditional haplotypes can be derived, such as protective haplotypes
and
risk haplotypes.
[000185] Step 6: SNP and DNA polymorphism discovery
[000186] In the preferred embodiment, all the candidate genes and regions
identified in step 5 are sequenced for polymorphism identification.
[000187] In another embodiment, the entire region, including all introns, is
sequenced to identify all polymorphisms.
[000188] In yet another embodiment, the candidate genes are prioritized for
sequencing, and only functional gene elements (promoters, conserved noncoding
sequences, exons and splice sites) are sequenced.
[000189] In yet another embodiment, previously identified polymorphisms in
the candidate regions can also be used. For example, SNPs from dbSNP, or
others can also be used rather than resequencing the candidate regions to
identify polymorphisms.
[000190] The discovery of SNPs and DNA polymorphisms generally comprises
a step consisting of determining the major haplotypes in the region to be
sequenced. The preferred samples are selected according to which haplotypes
contribute to the association signal observed in the region to be sequenced.
The
purpose is to select a set of samples that covers all the major haplotypes in
the
given region. Each major haplotype is preferably analyzed in at least a few
individuals.
[000191] Any analytical procedure may be used to detect the presence or
absence of variant nucleotides at one or more polymorphic positions of the
invention. In general, the detection of allelic variation requires a mutation
discrimination technique, optionally an amplification reaction and optionally
a
signal generation system. Any means of mutation detection or discrimination
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may be used. For instance, DNA sequencing, scanning methods, hybridization,
extension based methods, incorporation based methods, restriction enzyme-
based methods and ligation-based methods may be used in the methods of the
invention.
[000192] Sequencing methods include, but are not limited to, direct
sequencing, and sequencing by hybridization. Scanning methods include, but are
not limited to, protein truncation test (PTT), single-strand conformation
polymorphism analysis (SSCP), denaturing gradient gel electrophoresis (DGGE),
temperature gradient gel electrophoresis (TGGE), cleavage, heteroduplex
analysis, chemical mismatch cleavage (CMC), and enzymatic mismatch
cleavage. Hybridization-based methods of detection include, but are not
limited
to, solid phase hybridization such as dot blots, multiple allele specific
diagnostic
assay (MASDA), reverse dot blots, and oligonucleotide arrays (DNA Chips).
Solution phase hybridization amplification methods may also be used, such as
Taqman. Extension based methods include, but are not limited to, amplification
refraction mutation systems (ARMS), amplification refractory mutation systems
(ALEX), and competitive oligonucleotide priming systems (COPS). Incorporation
based methods include, but are not limited to, mini-sequencing and arrayed
primer extension (APEX). Restriction enzyme-based detection systems include,
but are not limited to, restriction site generating PCR. Lastly, ligation
based
detection methods include, but are not limited to, oligonucleotide ligation
assays
(OLA). Signal generation or detection systems that may be used in the methods
of the invention include, but are not limited to, fluorescence methods such as
fluorescence resonance energy transfer (FRET), fluorescence quenching,
fluorescence polarization as well as other chemiluminescence,
electrochemiluminescence, Raman, radioactivity, colometric methods,
hybridization protection assays and mass spectrometry methods. Further
amplification methods include, but are not limited to self sustained
replication
(SSR), nucleic acid sequence based amplification (NASBA), ligase chain
reaction
(LCR), strand displacement amplification (SDA) and branched DNA (B-DNA).
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[000193] Sequencing can also be performed using a proprietary sequencing
technology (Cantaloupe; PCT/EP2005/002870).
[000194] Step 7: Ultrafine Mapping
[000195] This step further maps the candidate regions and genes confirmed in
the previous step to identify and validate the responsible polymorphisms
associated with ENDOMETRIOSIS disease in the human population.
[000196] In a preferred embodiment, the discovered SNPs and polymorphisms
of step 6 are ultrafine mapped at a higher density of markers than the GWS
described herein using the same technology described in step 3.
[000197] Step 8: GeneMap construction
[000198] The confirmed variations in DNA (including both genic and non-genic
regions) are used to build a GeneMap for ENDOMETRIOSIS disease. The gene
content of this GeneMap is described in more detail below. Such GeneMap can
be used for other methods of the invention comprising the diagnostic methods
described herein, the susceptibility to ENDOMETRIOSIS disease, the response
to a particular drug, the efficacy of a particular drug, the screening methods
described herein and the treatment methods described herein.
[000199] As is evident to one of ordinary skill in the art, all of the above
steps
or the steps do not need to be performed, or performed in a given order to
practice or use the SNPs, genomic regions, genes, proteins, etc. in the
methods
of the invention.
Genes from the GeneMap
[000200] In one embodiment the GeneMap consists of genes and targets, in a
variety of combinations, identified from the candidate regions listed in Table
1. In
another embodiment, all genes from Tables 2-4 are present in the GeneMap. In
another preferred embodiment, the GeneMap consists of a selection of genes
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from Tables 2-4. The genes of the invention (Tables 2-4) are arranged by
candidate regions and by their chromosomal location. Such order is for the
purpose of clarity and does not reflect any other criteria of selection in the
association of the genes with ENDOMETRIOSIS. In yet another embodiment, the
GeneMaps of the invention consists of a selection of genes from Tables 2-4 and
a selection of genes that are interactors (direct or indirect) with the genes
from
the Tables. For clarity purposes, those interactor genes are not present in
Tables
2-4, but know in the art from various public documents (scientific articles,
patent
literature etc.). The GeneMaps represent the knowledge that is needed for
therapeutic and diagnostic intervention for a particular disease. The GeneMaps
aid in the selection of the best target to intervene in a disease state. Each
disease can be subdivided into various disease states and sub-phenotypes, thus
various GeneMaps are needed to address various disease sub-phenopypes, and
a clinical population can be stratified by sub-phenotype, which would be
covered
by a particular GeneMap.
[000201] In one embodiment, genes identified in the WGAS and subsequent
studies are evaluated using the Ingenuity Pathway Analysis application (IPA,
Ingenuity systems) in order to identify direct biological interactions between
these
genes, and also to identify molecular regulators acting on those genes
(indirect
interactions) that could be also involved in ENDOMETRIOSIS. The purpose of
this effort is to decipher the molecules involved in contributing to
ENDOMETRIOSIS. These gene interaction networks are very valuable tools in
the sense that they facilitate extension of the map of gene products that
could
represent potential drug targets for ENDOMETRIOSIS.
[000202] In another embodiment, other means (such as fuctional biochemical
assays and genetic asssays) are used to identify the biological interactions
between genes to create a GeneMap (see Example section herein for description
of the various GeneMaps).
Nucleic acid sequences
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[000203] The nucleic acid sequences of the present invention may be derived
from a variety of sources including DNA, cDNA, synthetic DNA, synthetic RNA,
derivatives, mimetics or combinations thereof. Such sequences may comprise
genomic DNA, which may or may not include naturally occurring introns, genic
regions, nongenic regions, and regulatory regions. Moreover, such genomic DNA
may be obtained in association with promoter regions or poly (A) sequences.
The
sequences, genomic DNA, or cDNA may be obtained in any of several ways.
Genomic DNA can be extracted and purified from suitable cells by means well
known in the art. Alternatively, mRNA can be isolated from a cell and used to
produce cDNA by reverse transcription or other means. The nucleic acids
described herein are used in certain embodiments of the methods of the present
invention for production of RNA, proteins or polypeptides, through
incorporation
into cells, tissues, or organisms. In one embodiment, DNA containing all or
part of
the coding sequence for the genes described in Tables 2-4, or the SNP markers
described in Tables 5-16, is incorporated into a vector for expression of the
encoded polypeptide in suitable host cells. The invention also comprises the
use
of the nucleotide sequence of the nucleic acids of this invention to identify
DNA
probes for the genes described in Tables 2-4 or the SNP markers described in
Tables 5-16, PCR primers to amplify the genes described in Tables 2-4 or the
SNP markers described in Tables 5-16, nucleotide polymorphisms in the genes
described in Tables 2-4, and regulatory elements of the genes described in
Tables 2-4. The nucleic acids of the present invention find use as primers and
templates for the recombinant production of ENDOMETRIOSIS disease-
associated peptides or polypeptides, for chromosome and gene mapping, to
provide antisense sequences, for tissue distribution studies, to locate and
obtain
full length genes, to identify and obtain homologous sequences (wild-type and
mutants), and in diagnostic applications.
Antisense oligonucleotides
[000204] In a particular embodiment of the invention, an antisense nucleic
acid
or oligonucleotide is wholly or partially complementary to, and can hybridize
with,
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a target nucleic acid (either DNA or RNA) having the sequence of SEQ ID NO:1,
NO:3 or any SEQ ID from any Tables of the invention. For example, an antisense
nucleic acid or oligonucleotide comprising 16 nucleotides can be sufficient to
inhibit expression of at least one gene from Tables 2-4. Alternatively, an
antisense nucleic acid or oligonucleotide can be complementary to 5' or 3'
untranslated regions, or can overlap the translation initiation codon (5'
untranslated and translated regions) of at least one gene from Tables 2-4, or
its
functional equivalent. In another embodiment, the antisense nucleic acid is
wholly
or partially complementary to, and can hybridize with, a target nucleic acid
that
encodes a polypeptide from a gene described in Tables 2-4.
[000205] In addition, oligonucleotides can be constructed which will bind to
duplex nucleic acid (i.e., DNA:DNA or DNA:RNA), to form a stable triple helix
containing or triplex nucleic acid. Such triplex oligonucleotides can inhibit
transcription and/or expression of a gene from Tables 2-4, or its functional
equivalent (M.D. Frank-Kamenetskii et al., 1995). Triplex oligonucleotides are
constructed using the basepairing rules of triple helix formation and the
nucleotide sequence of the genes described in Tables 2-4.
[000206] The present invention encompasses methods of using
oligonucleotides in antisense inhibition of the function of the genes from
Tables 2-
4. In the context of this invention, the term "oligonucleotide" refers to
naturally-
occurring species or synthetic species formed from naturally-occurring
subunits
or their close homologs. The term may also refer to moieties that function
similarly to oligonucleotides, but have non-naturally-occurring portions.
Thus,
oligonucleotides may have altered sugar moieties or inter-sugar linkages.
Exemplary among these are phosphorothioate and other sulfur containing
species which are known in the art. In preferred embodiments, at least one of
the
phosphodiester bonds of the oligonucleotide has been substituted with a
structure that functions to enhance the ability of the compositions to
penetrate
into the region of cells where the RNA whose activity is to be modulated is
located. It is preferred that such substitutions comprise phosphorothioate
bonds,
methyl phosphonate bonds, or short chain alkyl or cycloalkyl structures. In
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accordance with other preferred embodiments, the phosphodiester bonds are
substituted with structures which are, at once, substantially non-ionic and
non-
chiral, or with structures which are chiral and enantiomerically specific.
Persons
of ordinary skill in the art will be able to select other linkages for use in
the
practice of the invention. Oligonucleotides may also include species that
include
at least some modified base forms. Thus, purines and pyrimidines other than
those normally found in nature may be so employed. Similarly, modifications on
the furanosyl portions of the nucleotide subunits may also be effected, as
long as
the essential tenets of this invention are adhered to. Examples of such
modifications are 2'-O-alkyl- and 2'-halogen-substituted nucleotides. Some non-
limiting examples of modifications at the 2' position of sugar moieties which
are
useful in the present invention include OH, SH, SCH3, F, OCH3, OCN, O(CH2),
NH2 and O(CH2)n CH3, where n is from 1 to about 10. Such oligonucleotides are
functionally interchangeable with natural oligonucleotides or synthesized
oligonucleotides, which have one or more differences from the natural
structure.
All such analogs are comprehended by this invention so long as they function
effectively to hybridize with at least one gene from Tables 2-4 DNA or RNA to
inhibit the function thereof.
[000207] The oligonucleotides in accordance with this invention preferably
comprise from about 3 to about 50 subunits. It is more preferred that such
oligonucleotides and analogs comprise from about 8 to about 25 subunits and
still
more preferred to have from about 12 to about 20 subunits. As defined herein,
a
"subunit" is a base and sugar combination suitably bound to adjacent subunits
through phosphodiester or other bonds.
[000208] Antisense nucleic acids or oligonulcleotides can be produced by
standard techniques (see, e.g., Shewmaker et al., U.S. Patent No. 6,107,065).
The oligonucleotides used in accordance with this invention may be
conveniently
and routinely made through the well-known technique of solid phase synthesis.
Any other means for such synthesis may also be employed; however, the actual
synthesis of the oligonucleotides is well within the abilities of the
practitioner. It is
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also well known to prepare other oligonucleotides such as phosphorothioates
and
alkylated derivatives.
[000209] The oligonucleotides of this invention are designed to be
hybridizable
with RNA (e.g., mRNA) or DNA from genes described in Tables 2-4. For
example, an oligonucleotide (e.g., DNA oligonucleotide) that hybridizes to
mRNA
from a gene described in Tables 2-4 can be used to target the mRNA for RnaseH
digestion. Alternatively an oligonucleotide that can hybridize to the
translation
initiation site of the mRNA of a gene described in Tables 2-4 can be used to
prevent translation of the mRNA. In another approach, oligonucleotides that
bind
to the double-stranded DNA of a gene from Tables 2-4 can be administered.
Such oligonucleotides can form a triplex construct and inhibit the
transcription of
the DNA encoding polypeptides of the genes described in Tables 2-4. Triple
helix pairing prevents the double helix from opening sufficiently to allow the
binding of polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using triplex DNA have been described (see, e.g., J.E.
Gee
et al., 1994, Molecular and Immunologic Approaches, Futura Publishing Co., Mt.
Kisco, NY).
[000210] As non-limiting examples, antisense oligonucleotides may be
targeted to hybridize to the following regions: mRNA cap region; translation
initiation site; translational termination site; transcription initiation
site;
transcription termination site; polyadenylation signal; 3' untranslated
region; 5'
untranslated region; 5' coding region; mid coding region; 3' coding region;
DNA
replication initiation and elondation sites. Preferably, the complementary
oligonucleotide is designed to hybridize to the most unique 5' sequence of a
gene
described in Tables 2-4, including any of about 15-35 nucleotides spanning the
5'
coding sequence. In accordance with the present invention, the antisense
oligonucleotide can be synthesized, formulated as a pharmaceutical
composition,
and administered to a subject. The synthesis and utilization of antisense and
triplex oligonucleotides have been previously described (e.g., Simon et al.,
1999;
Barre et al., 2000; Elez et al., 2000; Sauter et al., 2000).
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[000211] Alternatively, expression vectors derived from retroviruses,
adenovirus, herpes or vaccinia viruses or from various bacterial plasmids may
be
used for delivery of nucleotide sequences to the targeted organ, tissue or
cell
population. Methods which are well known to those skilled in the art can be
used
to construct recombinant vectors which will express nucleic acid sequence that
is
complementary to the nucleic acid sequence encoding a polypeptide from the
genes described in Tables 2-4. These techniques are described both in
Sambrook et al., 1989 and in Ausubel et al., 1992. For example, expression of
at
least one gene from Tables 2-4 can be inhibited by transforming a cell or
tissue
with an expression vector that expresses high levels of untranslatable sense
or
antisense sequences. Even in the absence of integration into the DNA, such
vectors may continue to transcribe RNA molecules until they are disabled by
endogenous nucleases. Transient expression may last for a month or more with a
nonreplicating vector, and even longer if appropriate replication elements are
included in the vector system. Various assays may be used to test the ability
of
gene-specific antisense oligonucleotides to inhibit the expression of at least
one
gene from Tables 2-4. For example, mRNA levels of the genes described in
Tables 2-4 can be assessed by Northern blot analysis (Sambrook et al., 1989;
Ausubel et al., 1992; J.C. Alwine et al. 1977; I.M. Bird, 1998), quantitative
or
semi-quantitative RT-PCR analysis (see, e.g., W.M. Freeman et al., 1999; Ren
et
al., 1998; J.M. Cale et al., 1998), or in situ hybridization (reviewed by A.K.
Raap,
1998). Alternatively, antisense oligonucleotides may be assessed by measuring
levels of the polypeptide from the genes described in Tables 2-4, e.g., by
western
blot analysis, indirect immunofluorescence and immunoprecipitation techniques
(see, e.g., J.M. Walker, 1998, Protein Protocols on cD-ROM, Humana Press,
Totowa, NJ). Any other means for such detection may also be employed, and is
well within the abilities of the practitioner.
Mapping Technologies
[000212] The present invention includes various methods which employ
mapping technologies to map SNPs and polymorphisms. For purpose of clarity,
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this section comprises, but is not limited to, the description of mapping
technologies that can be utilized to achieve the embodiments described herein.
Mapping technologies may be based on amplification methods, restriction
enzyme cleavage methods, hybridization methods, sequencing methods, and
cleavage methods using agents.
[000213] Amplification methods include: self sustained sequence replication
(Guatelli et al., 1990), transcriptional amplification system (Kwoh et al.,
1989), Q-
Beta Replicase (Lizardi et al., 1988), isothermal amplification (e.g. Dean et
al.,
2002; and Hafner et al., 2001), or any other nucleic acid amplification
method,
followed by the detection of the amplified molecules using techniques well
known
to those of ordinary skill in the art. These detection schemes are especially
useful
for the detection of nucleic acid molecules if such molecules are present in
very
low number.
[000214] Restriction enzyme cleavage methods include: isolating sample and
control DNA, amplification (optional), digestion with one or more restriction
endonucleases, determination of fragment length sizes by gel electrophoresis
and comparing samples and controls. Differences in fragment length sizes
between sample and control DNA indicates mutations in the sample DNA.
Moreover, sequence specific ribozymes (see, e.g., U.S. Pat. No. 5,498,531 or
DNAzyme e.g. U.S. Pat. No. 5,807,718) can be used to score for the presence of
specific mutations by development or loss of a ribozyme or DNAzyme cleavage
site.
[000215] Hybridization methods include any measurement of the hybridization
or gene expression levels, of sample nucleic acids to probes corresponding to
about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 75, 100, 200, 500, 1000
or more
genes, or ranges of these numbers, such as about 5-20, about 10-20, about 20-
50, about 50-100, or about 100-200 genes of Tables 2-4.
[000216] SNPs and SNP maps of the invention can be identified or generated
by hybridizing sample nucleic acids, e.g., DNA or RNA, to high density arrays
or
bead arrays containing oligonucleotide probes corresponding to the
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polymorphisms of Tables 5-16 (see the Affymetrix arrays and Illumina bead sets
at www.affymetrix.com and www.illumina.com and see Cronin et al., 1996; or
Kozal et al., 1996).
[000217] Methods of forming high density arrays of oligonucleotides with a
minimal number of synthetic steps are known. The oligonucleotide analogue
array can be synthesized on a single or on multiple solid substrates by a
variety
of methods, including, but not limited to, light-directed chemical coupling,
and
mechanically directed coupling (see Pirrung, U.S. Patent No. 5,143,854).
[000218] In brief, the light-directed combinatorial synthesis of
oligonucleotide
arrays on a glass surface precedes using automated phosphoramidite chemistry
and chip masking techniques. In one specific implementation, a glass surface
is
derivatized with a silane reagent containing a functional group, e.g., a
hydroxyl or
amine group blocked by a photolabile protecting group. Photolysis through a
photolithogaphic mask is used selectively to expose functional groups which
are
then ready to react with incoming 5' photoprotected nucleoside
phosphoramidites. The phosphoramidites react only with those sites which are
illuminated (and thus exposed by removal of the photolabile blocking group).
Thus, the phosphoramidites only add to those areas selectively exposed from
the
preceding step. These steps are repeated until the desired array of sequences
have been synthesized on the solid surface. Combinatorial synthesis of
different
oligonucleotide analogues at different locations on the array is determined by
the
pattern of illumination during synthesis and the order of addition of coupling
reagents.
[000219] In addition to the foregoing, additional methods which can be used to
generate an array of oligonucleotides on a single substrate are described in
PCT
Publication Nos. WO 93/09668 and WO 01/23614. High density nucleic acid
arrays can also be fabricated by depositing pre-made or natural nucleic acids
in
predetermined positions. Synthesized or natural nucleic acids are deposited on
specific locations of a substrate by light directed targeting and
oligonucleotide
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directed targeting. Another embodiment uses a dispenser that moves from
region to region to deposit nucleic acids in specific spots.
[000220] Nucleic acid hybridization simply involves contacting a probe and
target nucleic acid under conditions where the probe and its complementary
target can form stable hybrid duplexes through complementary base pairing. See
WO 99/32660. The nucleic acids that do not form hybrid duplexes are then
washed away leaving the hybridized nucleic acids to be detected, typically
through detection of an attached detectable label. It is generally recognized
that
nucleic acids are denatured by increasing the temperature or decreasing the
salt
concentration of the buffer containing the nucleic acids. Under low stringency
conditions (e.g., low temperature and/or high salt) hybrid duplexes (e.g.,
DNA:DNA, RNA:RNA, or RNA:DNA) will form even where the annealed
sequences are not perfectly complementary. Thus, specificity of hybridization
is
reduced at lower stringency. Conversely, at higher stringency (e.g., higher
temperature or lower salt) successful hybridization tolerates fewer
mismatches.
One of skill in the art will appreciate that hybridization conditions may be
selected
to provide any degree of stringency.
[000221] In a preferred embodiment, hybridization is performed at low
stringency to ensure hybridization and then subsequent washes are performed at
higher stringency to eliminate mismatched hybrid duplexes. Successive washes
may be performed at increasingly higher stringency until a desired level of
hybridization specificity is obtained. Stringency can also be increased by
addition
of agents such as formamide. Hybridization specificity may be evaluated by
comparison of hybridization to the test probes with hybridization to the
various
controls that can be present (e.g., expression level control, normalization
control,
mismatch controls, etc.).
[000222] In general, there is a tradeoff between hybridization specificity
(stringency) and signal intensity. Thus, in a preferred embodiment, the wash
is
performed at the highest stringency that produces consistent results and that
provides a signal intensity greater than approximately 10% of the background
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intensity. Thus, in a preferred embodiment, the hybridized array may be washed
at successively higher stringency solutions and read between each wash.
Analysis of the data sets thus produced will reveal a wash stringency above
which the hybridization pattern is not appreciably altered and which provides
adequate signal for the particular oligonucleotide probes of interest.
[000223] Probes based on the sequences of the genes described above may
be prepared by any commonly available method. Oligonucleotide probes for
screening or assaying a tissue or cell sample are preferably of sufficient
length to
specifically hybridize only to appropriate, complementary genes or
transcripts.
Typically the oligonucleotide probes will be at least about 10, 12, 14, 16,
18, 20 or
25 nucleotides in length. In some cases, longer probes of at least 30, 40, or
50
nucleotides will be desirable.
[000224] As used herein, oligonucleotide sequences that are complementary
to one or more of the genes or gene fragments described in Tables 2-4 refer to
oligonucleotides that are capable of hybridizing under stringent conditions to
at
least part of the nucleotide sequences of said genes. Such hybridizable
oligonucleotides will typically exhibit at least about 75% sequence identity
at the
nucleotide level to said genes, preferably about 80% or 85% sequence identity
or
more preferably about 90% or 95% or more sequence identity to said genes (see
GeneChip Expression Analysis Manual, Affymetrix, Rev. 3, which is herein
incorporated by reference in its entirety).
[000225] The phrase "hybridizing specifically to" or "specifically hybridizes"
refers to the binding, duplexing, or hybridizing of a molecule substantially
to or
only to a particular nucleotide sequence or sequences under stringent
conditions
when that sequence is present in a complex mixture (e.g., total cellular) DNA
or
RNA.
[000226] As used herein a "probe" is defined as a nucleic acid, capable of
binding to a target nucleic acid of complementary sequence through one or more
types of chemical bonds, usually through complementary base pairing, usually
through hydrogen bond formation. As used herein, a probe may include natural
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(i.e., A, G, U, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
In
addition, the bases in probes may be joined by a linkage other than a
phosphodiester bond, so long as it does not interfere with hybridization.
Thus,
probes may be peptide nucleic acids in which the constituent bases are joined
by
peptide bonds rather than phosphodiester linkages.
[000227] A variety of sequencing reactions known in the art can be used to
directly sequence nucleic acids for the presence or the absence of one or more
polymorphisms of Tables 5-16. Examples of sequencing reactions include those
based on techniques developed by Maxam and Gilbert (1977) or Sanger (1977).
It is also contemplated that any of a variety of automated sequencing
procedures
can be utilized, including sequencing by mass spectrometry (see, e.g. PCT
International Publication No. WO 94/16101; Cohen et al., 1996; and Griffin et
a1.,1993), real-time pyrophosphate sequencing method (Ronaghi et a1.,1998; and
Permutt et al., 2001) and sequencing by hybridization (see e.g. Drmanac et
al.,
2002).
[000228] Other methods of detecting polymorphisms include methods in which
protection from cleavage agents is used to detect mismatched bases in
RNA/RNA, DNA/DNA or RNA/DNA heteroduplexes (Myers et al., 1985). In
general, the technique of "mismatch cleavage" starts by providing
heteroduplexes
formed by hybridizing (labeled) RNA or DNA containing a wild-type sequence
with potentially mutant RNA or DNA obtained from a sample. The double-
stranded duplexes are treated with an agent who cleaves single-stranded
regions
of the duplex such as which will exist due to basepair mismatches between the
control and sample strands. For instance, RNA/DNA duplexes can be treated
with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically
digest the mismatched regions. In other embodiments, either DNA/DNA or
RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and
with piperidine in order to digest mismatched regions. After digestion of the
mismatched regions, the resulting material is then separated by size on
denaturing polyacrylamide gels to determine the site of a mutation or SNP
(see,
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for example, Cotton et al., 1988; and Saleeba et al., 1992). In a preferred
embodiment, the control DNA or RNA can be labeled for detection.
[000229] In still another embodiment, the mismatch cleavage reaction employs
one or more proteins that recognize mismatched base pairs in double-stranded
DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting
and mapping polymorphisms. For example, the mutY enzyme of E. coli cleaves A
at G/A mismatches (Hsu et al., 1994). Other examples include, but are not
limited
to, the MutHLS enzyme complex of E. coli (Smith and Modrich Proc. 1996) and
Cel 1 from the celery (Kulinski et al., 2000) both cleave the DNA at various
mismatches. According to an exemplary embodiment, a probe based on a
polymorphic site corresponding to a polymorphism of Tables 5-16 is hybridized
to
a cDNA or other DNA product from a test cell or cells. The duplex is treated
with
a DNA mismatch repair enzyme, and the cleavage products, if any, can be
detected from electrophoresis protocols or the like. See, for example, U.S.
Pat.
No. 5,459,039. Alternatively, the screen can be performed in vivo following
the
insertion of the heteroduplexes in an appropriate vector. The whole procedure
is
known to those ordinary skilled in the art and is referred to as mismatch
repair
detection (see e.g. Fakhrai-Rad et al., 2004).
[000230] In other embodiments, alterations in electrophoretic mobility can be
used to identify polymorphisms in a sample. For example, single strand
conformation polymorphism (SSCP) analysis can be used to detect differences in
electrophoretic mobility between mutant and wild type nucleic acids (Orita et
al.,
1989; Cotton et al., 1993; and Hayashi 1992). Single-stranded DNA fragments of
case and control nucleic acids will be denatured and allowed to renature. The
secondary structure of single-stranded nucleic acids varies according to
sequence. The resulting alteration in electrophoretic mobility enables the
detection of even a single base change. The DNA fragments may be labeled or
detected with labeled probes. The sensitivity of the assay may be enhanced by
using RNA (rather than DNA), in which the secondary structure is more
sensitive
to a change in sequence. In a preferred embodiment, the method utilizes
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heteroduplex analysis to separate double stranded heteroduplex molecules on
the basis of changes in electrophoretic mobility (Kee et al., 1991).
[000231] In yet another embodiment, the movement of mutant or wild-type
fragments in a polyacrylamide gel containing a gradient of denaturant is
assayed
using denaturing gradient gel electrophoresis (DGGE) (Myers et al., 1985).
When
DGGE is used as the method of analysis, DNA will be modified to insure that it
does not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a further
embodiment, a temperature gradient is used in place of a denaturing gradient
to
identify differences in the mobility of control and sample DNA (Rosenbaum et
al.,
1987). In another embodiment, the mutant fragment is detected using denaturing
HPLC (see e.g. Hoogendoorn et al., 2000).
[000232] Examples of other techniques for detecting polymorphisms include,
but are not limited to, selective oligonucleotide hybridization, selective
amplification, selective primer extension, selective ligation, single-base
extension,
selective termination of extension or invasive cleavage assay. For example,
oligonucleotide primers may be prepared in which the polymorphism is placed
centrally and then hybridized to target DNA under conditions which permit
hybridization only if a perfect match is found (Saiki et al., 1986; Saiki et
al., 1989).
Such oligonucleotides are hybridized to PCR amplified target DNA or a number
of
different mutations when the oligonucleotides are attached to the hybridizing
membrane and hybridized with labeled target DNA. Alternatively, the
amplification, the allele-specific hybridization and the detection can be done
in a
single assay following the principle of the 5' nuclease assay (e.g. see Livak
et al.,
1995). For example, the associated allele, a particular allele of a
polymorphic
locus, or the like is amplified by PCR in the presence of both allele-specific
oligonucleotides, each specific for one or the other allele. Each probe has a
different fluorescent dye at the 5' end and a quencher at the 3' end. During
PCR,
if one or the other or both allele-specific oligonucleotides are hybridized to
the
template, the Taq polymerase via its 5' exonuclease activity will release the
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corresponding dyes. The latter will thus reveal the genotype of the amplified
product.
[000233] Hybridization assays may also be carried out with a temperature
gradient following the principle of dynamic allele-specific hybridization or
like e.g.
Jobs et al., (2003); and Bourgeois and Labuda, (2004). For example, the
hybridization is done using one of the two allele-specific oligonucleotides
labeled
with a fluorescent dye, and an intercalating quencher under a gradually
increasing temperature. At low temperature, the probe is hybridized to both
the
mismatched and full-matched template. The probe melts at a lower temperature
when hybridized to the template with a mismatch. The release of the probe is
captured by an emission of the fluorescent dye, away from the quencher. The
probe melts at a higher temperature when hybridized to the template with no
mismatch. The temperature-dependent fluorescence signals therefore indicate
the absence or presence of an associated allele, a particular allele of a
polymorphic locus, or the like (e.g. Jobs et al., 2003). Alternatively, the
hybridization is done under a gradually decreasing temperature. In this case,
both
allele-specific oligonucleotides are hybridized to the template competitively.
At
high temperature none of the two probes are hybridized. Once the optimal
temperature of the full-matched probe is reached, it hybridizes and leaves no
target for the mismatched probe (e.g. Bourgeois and Labuda, 2004). In the
latter
case, if the allele-specific probes are differently labeled, then they are
hybridized
to a single PCR-amplified target. If the probes are labeled with the same dye,
then the probe cocktail is hybridized twice to identical templates with only
one
labeled probe, different in the two cocktails, in the presence of the
unlabeled
competitive probe.
[000234] Alternatively, allele specific amplification technology that depends
on
selective PCR amplification may be used in conjunction with the present
invention. Oligonucleotides used as primers for specific amplification may
carry
the associated allele, a particular allele of a polymorphic locus, or the
like, also
referred to as "mutation" of interest in the center of the molecule, so that
amplification depends on differential hybridization (Gibbs et al., 1989) or at
the
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extreme 3' end of one primer where, under appropriate conditions, mismatch can
prevent, or reduce polymerase extension (Prossner, 1993). In addition it may
be
desirable to introduce a novel restriction site in the region of the mutation
to
create cleavage-based detection (Gasparini et al., 1992). It is anticipated
that in
certain embodiments, amplification may also be performed using Taq ligase for
amplification (Barany, 1991). In such cases, ligation will occur only if there
is a
perfect match at the 3' end of the 5' sequence making it possible to detect
the
presence of a known associated allele, a particular allele of a polymorphic
locus,
or the like at a specific site by looking for the presence or absence of
amplification. The products of such an oligonucleotide ligation assay can also
be
detected by means of gel electrophoresis. Furthermore, the oligonucleotides
may
contain universal tags used in PCR amplification and zip code tags that are
different for each allele. The zip code tags are used to isolate a specific,
labeled
oligonucleotide that may contain a mobility modifier (e.g. Grossman et al.,
1994).
[000235] In yet another alternative, allele-specific elongation followed by
ligation will form a template for PCR amplification. In such cases, elongation
will
occur only if there is a perfect match at the 3' end of the allele-specific
oligonucleotide using a DNA polymerase. This reaction is performed directly on
the genomic DNA and the extension/ligation products are amplified by PCR. To
this end, the oligonucleotides contain universal tags allowing amplification
at a
high multiplex level and a zip code for SNP identification. The PCR tags are
designed in such a way that the two alleles of a SNP are amplified by
different
forward primers, each having a different dye. The zip code tags are the same
for
both alieles of a given SNPs and they are used for hybridization of the PCR-
amplified products to oligonucleotides bound to a solid support, chip, bead
array
or like. For an example of the procedure, see Fan et al. (Cold Spring Harbor
Symposia on Quantitative Biology, Vol. LXVIII, pp. 69-78 2003).
[000236] Another alternative includes the single-base extension/ligation assay
using a molecular inversion probe, consisting of a single, long
oligonucleotide
(see e.g. Hardenbol et al., 2003). In such an embodiment, the oligonucleotide
hybridizes on both side of the SNP locus directly on the genomic DNA, leaving
a
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one-base gap at the SNP locus. The gap-filling, one-base extension/ligation is
performed in four tubes, each having a different dNTP. Following this
reaction,
the oligonucleotide is circularized whereas unreactive, linear
oligonucleotides are
degraded using an exonuclease such as exonuclease I of E. coli. The circular
oligonucleotides are then linearized and the products are amplified and
labeled
using universal tags on the oligonucleotides. The original oligonucleotide
also
contains a SNP-specific zip code allowing hybridization to oligonucleotides
bound
to a solid support, chip, and bead array or like. This reaction can be
performed at
a high multiplexed level.
[000237] In another alternative, the associated allele, a particular allele of
a
polymorphic locus, or the like is scored by single-base extension (see e.g.
U.S.
Pat. No. 5,888,819). The template is first amplified by PCR. The extension
oligonucleotide is then hybridized next to the SNP locus and the extension
reaction is performed using a thermostable polymerase such as
ThermoSequenase (GE Healthcare) in the presence of labeled ddNTPs. This
reaction can therefore be cycled several times. The identity of the labeled
ddNTP
incorporated will reveal the genotype at the SNP locus. The labeled products
can
be detected by means of gel electrophoresis, fluorescence polarization (e.g.
Chen et al., 1999) or by hybridization to oligonucleotides bound to a solid
support, chip, and bead array or like. In the latter case, the extension
oligonucleotide will contain a SNP-specific zip code tag.
[000238] In yet another alternative, a SNP is scored by selective termination
of
extension. The template is first amplified by PCR and the extension
oligonucleotide hybridizes in the vicinity of the SNP locus, close to but not
necessarily adjacent to it. The extension reaction is carried out using a
thermostable polymerase such as ThermoSequenase (GE Healthcare) in the
presence of a mix of dNTPs and at least one ddNTP. The latter has to terminate
the extension at one of the allele of the interrogated SNP, but not both such
that
the two alleles will generate extension products of different sizes. The
extension
product can then be detected by means of gel electrophoresis, in which case
the
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extension products need to be labeled, or by mass spectrometry (see e.g. Storm
et al., 2003).
[000239] In another alternative, SNPs are detected using an invasive cleavage
assay (see U.S. Pat. No. 6,090,543). There are five oligonucleotides per SNP
to
interrogate but these are used in a two step-reaction. During the primary
reaction,
three of the designed oligonucleotides are first hybridized directly to the
genomic
DNA. One of them is locus-specific and hybridizes up to the SNP locus (the
pairing of the 3' base at the SNP locus is not necessary). There are two
allele-
specific oligonucleotides that hybridize in tandem to the locus-specific probe
but
also contain a 5' flap that is specific for each allele of the SNP. Depending
upon
hybridization of the allele-specific oligonucleotides at the base of the SNP
locus,
this creates a structure that is recognized by a cleavase enzyme (U.S. Pat.
No.
6,090,606) and the allele-specific flap is released. During the secondary
reaction,
the flap fragments hybridize to a specific cassette to recreate the same
structure
as above except that the cleavage will release a small DNA fragment labeled
with
a fluorescent dye that can be detected using regular fluorescence detector. In
the
cassette, the emission of the dye is inhibited by a quencher.
Methods to identify agents that modulate the expression of a nucleic acid
encoding a gene involved in ENDOMETRIOSIS
[000240] The present invention provides methods for identifying agents that
modulate the expression of a nucleic acid encoding a gene from Tables 2-4.
Such methods may utilize any available means of monitoring for changes in the
expression level of the nucleic acids of the invention. As used herein, an
agent is
said to modulate the expression of a nucleic acid of the invention if it is
capable of
up- or down- regulating expression of the nucleic acid in a cell. Such cells
can be
obtained from any parts of the body such as the hair, mouth, rectum, scalp,
blood, dermis, epidermis, skin cells, cutaneous surfaces, intertrigious areas,
genitalia and fluids, vessels and endothelium. Some non-limiting examples of
cells that can be used are: ovarian cells, uterus cells and other cells of the
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reproductive system, muscle cells, nervous cells, blood and vessels cells, T
cell,
mast cell, lymphocyte, monocyte, macrophage, and epithelial cells.
[000241] In one assay format, the expression of a nucleic acid encoding a
gene of the invention (see Tables 2-4) in a cell or tissue sample is monitored
directly by hybridization to the nucleic acids of the invention. Cell lines or
tissues
are exposed to the agent to be tested under appropriate conditions and time
and
total RNA or mRNA is isolated by standard procedures such as those disclosed
in Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press).
[000242] Probes to detect differences in RNA expression levels between cells
exposed to the agent and control cells may be prepared as described above.
Hybridization conditions are modified using known methods, such as those
described by Sambrook et al., and Ausubel et al., as required for each probe.
Hybridization of total cellular RNA or RNA enriched for polyA RNA can be
accomplished in any available format. For instance, total cellular RNA or RNA
enriched for polyA RNA can be affixed to a solid support and the solid support
exposed to at least one probe comprising at least one, or part of one of the
sequences of the invention under conditions in which the probe will
specifically
hybridize. Alternatively, nucleic acid fragments comprising at least one, or
part of
one of the sequences of the invention can be affixed to a solid support, such
as a
silicon chip or a porous glass wafer. The chip or wafer can then be exposed to
total cellular RNA or polyA RNA from a sample under conditions in which the
affixed sequences will specifically hybridize to the RNA. By examining for the
ability of a given probe to specifically hybridize to an RNA sample from an
untreated cell population and from a cell population exposed to the agent,
agents
which up or down regulate expression are identified.
Methods to identify agents that modulate the activity of a protein encoded
by a gene involved in ENDOMETRIOSIS and antibodies of the invention
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[000243] The present invention provides methods for identifying agents that
modulate at least one activity of the proteins described in Tables 2-4. Such
methods may utilize any means of monitoring or detecting the desired activity.
As
used herein, an agent is said to modulate the expression of a protein of the
invention if it is capable of up- or down- regulating expression of the
protein in a
cell. Such cells can be obtained from any parts of the body such as the hair,
mouth, rectum, scalp, blood, dermis, epidermis, skin cells, cutaneous
surfaces,
intertrigious areas, genitalia and fluids, vessels and endothelium. Some non-
limiting examples of cells that can be used are: ovarian cells, uterus cells
and
other cells of the reproductive system, muscle cells, nervous cells, blood and
vessels cells, T cell, mast cell, lymphocyte, monocyte, macrophage, and
epithelial cells.
[000244] In one format, the specific activity of a protein of the invention,
normalized to a standard unit, may be assayed in a cell population that has
been
exposed to the agent to be tested and compared to an unexposed control cell
population. Cell lines or populations are exposed to the agent to be tested
under
appropriate conditions and times. Cellular lysates may be prepared from the
exposed cell line or population and a control, unexposed cell line or
population.
The cellular lysates are then analyzed with a probe, such as an antibody
probe.
[000245] Antibodies and Antibody probes can be prepared by immunizing
suitable mammalian (e.g. mice or transgenic mice) hosts utilizing appropriate
immunization protocols using the proteins of the invention or antigen-
containing
fragments thereof. To enhance immunogenicity, these proteins or fragments can
be conjugated to suitable carriers. Methods for preparing immunogenic
conjugates with carriers such as BSA, KLH or other carrier proteins are well
known in the art. In some circumstances, direct conjugation using, for
example,
carbodiimide reagents may be effective; in other instances linking reagents
such
as those supplied by Pierce Chemical Co. (Rockford, IL) may be desirable to
provide accessibility to the hapten. The hapten peptides can be extended at
either the amino or carboxy terminus with a cysteine residue or interspersed
with
cysteine residues, for example, to facilitate linking to a carrier.
Administration of
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the immunogens is conducted generally by injection over a suitable time period
and with use of suitable adjuvants, as is generally understood in the art.
During
the immunization schedule, titers of antibodies are taken to determine
adequacy
of antibody formation. While the polyclonal antisera produced in this way may
be
satisfactory for some applications, for pharmaceutical compositions, use of
monoclonal preparations is preferred. Immortalized cell lines which secrete
the
desired monoclonal antibodies may be prepared using standard methods, see
e.g., Kohler & Milstein (1992) or modifications which affect immortalization
of
lymphocytes or spleen cells, as is generally known. The immortalized cell
lines
secreting the desired antibodies can be screened by immunoassay in which the
antigen is the peptide hapten, polypeptide or protein. When the appropriate
immortalized cell culture secreting the desired antibody is identified, the
cells can
be cultured either in vitro or by production in ascites fluid. The desired
monoclonal antibodies may be recovered from the culture supernatant or from
the ascites supernatant. Fragments of the monoclonal antibodies or the
polyclonal antisera which contain the immunologically significant portion(s)
can
be used as antagonists, as well as the intact antibodies. Use of
immunologically
reactive fragments, such as Fab or Fab' fragments, is often preferable,
especially
in a therapeutic context, as these fragments are generally less immunogenic
than
the whole immunoglobulin. The antibody chains (light and heavy) can be cloned
into the vector by methods known in the art. The antibodies or fragments may
also be produced, using current technology, by recombinant means. Antibody
regions that bind specifically to the desired regions of the protein can also
be
produced in the context of chimeras derived from multiple species. Antibody
regions that bind specifically to the desired regions of the protein can also
be
produced in the context of chimeras from multiple species, for instance,
humanized antibodies. The antibody can therefore be a humanized antibody or a
human antibody, as described in U.S. Patent 5,585,089 or Riechmann et al.
(1988).
[000246] Phage display techniques can be used to provide libraries containing
a repertoire of antibodies with varying affinities for proteins, or fragments
thereof,
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described in Tables 2-4. Techniques for the identification of high affinity
human
antibodies from such libraries are described by Griffiths et al., EMBO J.,
13:3245-
3260 (1994); Nissim et al., ibid, pp. 692-698 and by Griffiths et al., ibid,
12:725-
734. The antibody of the invention also comprise humanized and human
antibodies. Such antibodies are made by methods known in the art.
[000247] Agents that are assayed in the above method can be randomly
selected or rationally selected or designed. As used herein, an agent is said
to be
randomly selected when the agent is chosen randomly without considering the
specific sequences involved in the association of the protein of the invention
alone or with its associated substrates, binding partners, etc. An example of
randomly selected agents is the use of a chemical library or a peptide
combinatorial library, or a growth broth of an organism. As used herein, an
agent
is said to be rationally selected or designed when the agent is chosen on a
non-
random basis which takes into account the sequence of the target site or its
conformation in connection with the agent's action. Agents can be rationally
selected or rationally designed by utilizing the peptide sequences that make
up
these sites. For example, a rationally selected peptide agent can be a peptide
whose amino acid sequence is identical to or a derivative of any functional
consensus site. The agents of the present invention can be, as examples,
oligonucleotides, antisense polynucleotides, interfering RNA, peptides,
peptide
mimetics, antibodies, antibody fragments, small molecules, vitamin
derivatives,
as well as carbohydrates. Peptide agents of the invention can be prepared
using
standard solid phase (or solution phase) peptide synthesis methods, as is
known
in the art. In addition, the DNA encoding these peptides may be synthesized
using commercially available oligonucleotide synthesis instrumentation and
produced recombinantly using standard recombinant production systems. The
production using solid phase peptide synthesis is necessitated if non-gene-
encoded amino acids are to be included.
[000248] Another class of agents of the present invention includes antibodies
or fragments thereof that bind to a protein encoded by a gene in Tables 2-4.
Antibody agents can be obtained by immunization of suitable mammalian
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subjects with peptides, containing as antigenic regions, those portions of the
protein intended to be targeted by the antibodies (see section above of
antibodies
as probes for standard antibody preparation methodologies).
[000249] In yet another class of agents, the present invention includes
peptide
mimetics that mimic the three-dimensional structure of the protein encoded by
a
gene from Tables 2-4. Such peptide mimetics may have significant advantages
over naturally occurring peptides, including, for example: more economical
production, greater chemical stability, enhanced pharmacological properties
(half-
life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-
spectrum
of biological activities), reduced antigenicity and others. In one form,
mimetics are
peptide-containing molecules that mimic elements of protein secondary
structure.
The underlying rationale behind the use of peptide mimetics is that the
peptide
backbone of proteins exists chiefly to orient amino acid side chains in such a
way
as to facilitate molecular interactions, such as those of antibody and
antigen. A
peptide mimetic is expected to permit molecular interactions similar to the
natural
molecule. In another form, peptide analogs are commonly used in the
pharmaceutical industry as non-peptide drugs with properties analogous to
those
of the template peptide. These types of non-peptide compounds are also
referred
to as peptide mimetics or peptidomimetics (Fauchere, 1986; Veber & Freidinger,
1985; Evans et al., 1987) which are usually developed with the aid of
computerized molecular modeling. Peptide mimetics that are structurally
similar
to therapeutically useful peptides may be used to produce an equivalent
therapeutic or prophylactic effect. Generally, peptide mimetics are
structurally
similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical
property or pharmacological activity), but have one or more peptide linkages
optionally replaced by a linkage using methods known in the art. Labeling of
peptide mimetics usually involves covalent attachment of one or more labels,
directly or through a spacer (e.g., an amide group), to non-interfering
position(s)
on the peptide mimetic that are predicted by quantitative structure-activity
data
and molecular modeling. Such non-interfering positions generally are positions
that do not form direct contacts with the macromolecule(s) to which the
peptide
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mimetic binds to produce the therapeutic effect. Derivitization (e.g.,
labeling) of
peptide mimetics should not substantially interfere with the desired
biological or
pharmacological activity of the peptide mimetic. The use of peptide mimetics
can
be enhanced through the use of combinatorial chemistry to create drug
libraries.
The design of peptide mimetics can be aided by identifying amino acid
mutations
that increase or decrease binding of the protein to its binding partners.
Approaches that can be used include the yeast two hybrid method (see Chien et
a/., 1991) and the phage display method. The two hybrid method detects protein-
protein interactions in yeast (Fields et al., 1989). The phage display method
detects the interaction between an immobilized protein and a protein that is
expressed on the. surface of phages such as lambda and M13 (Amberg et al.,
1993; Hogrefe et al., 1993). These methods allow positive and negative
selection
for protein-protein interactions and the identification of the sequences that
determine these interactions.
Method to diagnose ENDOMETRIOSIS
[000250] The present invention also relates to methods for diagnosing
ENDOMETRIOSIS or a related disease, preferably a subtype of
ENDOMETRIOSIS, a predisposition to such a disease and/or disease
progression. In some methods, the steps comprise contacting a target sample
with (a) nucleic acid molecule(s) or fragments thereof and comparing the
concentration of individual mRNA(s) with the concentration of the
corresponding
mRNA(s) from at least one healthy donor. An aberrant (increased or decreased)
mRNA level of at least one gene from Tables 2-4, at least 5 or 10 genes from
Tables 2-4, at least 50 genes from Tables 2-4, at least 100 genes from Tables
2-
4 or at least 200 genes from Tables 2-4 determined in the sample in comparison
to the control sample is an indication of ENDOMETRIOSIS disease or a related
subtype or a disposition to such kinds of diseases. For diagnosis, samples
are,
preferably, obtained from any parts of the body such as the hair, mouth,
rectum,
scalp, blood, dermis, epidermis, skin cells, cutaneous surfaces, intertrigious
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areas, genitalia and fluids, vessels and endothelium. Some non-limiting
examples
of cells that can be used are: ovarian cells, uterus cells and other cells of
the
reproductive system, muscle cells, nervous cells, blood and vessels cells, T
cell,
mast cell, lymphocyte, monocyte, macrophage, and epithelial cells.
[000251] For analysis of gene expression, total RNA is obtained from cells
according to standard procedures and, preferably, reverse-transcribed.
Preferably, a DNAse treatment (in order to get rid of contaminating genomic
DNA) is performed.
[000252] The nucleic acid molecule or fragment is typically a nucleic acid
probe for hybridization or a primer for PCR. The person skilled in the art is
in a
position to design suitable nucleic acids probes based on the information
provided in the Tables of the present invention. The target cellular
component,
i.e. mRNA, e.g., in brain tissue, may be detected directly in situ, e.g. by in
situ
hybridization or it may be isolated from other cell components by common
methods known to those skilled in the art before contacting with a probe.
Detection methods include Northern blot analysis, RNase protection, in situ
methods, e.g. in situ hybridization, in vitro amplification methods (PCR, LCR,
QRNA replicase or RNA-transcription/amplification (TAS, 3SR), reverse dot blot
disclosed in EP-B10237362) and other detection assays that are known to those
skilled in the art. Products obtained by in vitro amplification can be
detected
according to established methods, e.g. by separating the products on agarose
or
polyacrylamide gels and by subsequent staining with ethidium bromide or any
other dye or reagent. Alternatively, the amplified products can be detected by
using labeled primers for amplification or labeled dNTPs. Preferably,
detection is
based on a microarray.
[000253] The probes (or primers) (or, alternatively, the reverse-transcribed
sample mRNAs) can be detectably labeled, for example, with a radioisotope, a
bioluminescent compound, a chemiluminescent compound, a fluorescent
compound, a metal chelate, or an enzyme.
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[000254] The present invention also relates to the use of the nucleic acid
molecules or fragments described above for the preparation of a diagnostic
composition for the diagnosis of ENDOMETRIOSIS or a subtype or
predisposition to such a disease.
[000255] The present invention also relates to the use of the nucleic acid
molecules of the present invention for the isolation or development of a
compound which is useful for therapy of ENDOMETRIOSIS. For example, the
nucleic acid molecules of the invention and the data obtained using said
nucleic
acid molecules for diagnosis of ENDOMETRIOSIS might allow for the
identification of further genes which are specifically dysregulated, and thus
may
be considered as potential targets for therapeutic interventions. Furthermore,
such diagnostic might also be used for selection of patients that might
respond
positively or negatively to a potential target for therapeutic interventions
(as for
the pharmacogenomics and personalized medicine concept well know in the art;
see prognostic assays text below).
[000256] The invention further provides prognostic assays that can be used to
identify subjects having or at risk of developing ENDOMETRIOSIS. In such
method, a test sample is obtained from a subject and the amount and/or
concentration of the nucleic acid described in Tables 2-4 is determined;
wherein
the presence of an associated aliele, a particular aliele of a polymorphic
locus, or
the likes in the nucleic acids sequences of this invention (see SEQ ID from
Tables 5-16) can be diagnostic for a subject having or at risk of developing
ENDOMETRIOSIS. As used herein, a "test sample" refers to a biological sample
obtained from a subject of interest. For example, a test sample can be a
biological fluid, a cell sample, or tissue. A biological fluid can be, but is
not limited
to saliva, serum, mucus, urine, stools, spermatozoids, vaginal secretions,
lymph,
amiotic liquid, pleural liquid and tears. Cells can be, but are not limited
to: ovarian
cells, uterus cells and other cells of the reproductive system, hair cells,
muscle
cells, nervous cells, blood and vessels cells, dermis, epidermis and other
skin
cells, and various brain cells.
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[000257] Furthermore, the prognostic assays described herein can be used to
determine whether a subject can be administered an agent (e.g., an agonist,
antagonist, peptidomimetic, polypeptide, nucleic acid such as antisense DNA or
interfering RNA (RNAi), small molecule or other drug candidate) to treat
ENDOMETRIOSIS. Specifically, these assays can be used to predict whether an
individual will have an efficacious response or will experience adverse events
in
response to such an agent. For example, such methods can be used to
determine whether a subject can be effectively treated with an agent that
modulates the expression and/or activity of a gene from Tables 2-4 or the
nucleic
acids described herein. In another example, an association study may be
performed to identify polymorphisms from Tables 5-16 that are associated with
a
given response to the agent, e.g., an efficacious response or the likelihood
of one
or more adverse events. Thus, one embodiment of the present invention provides
methods for determining whether a subject can be effectively treated with an
agent for a disease associated with aberrant expression or activity of a gene
from
Tables 2-4 in which a test sample is obtained and nucleic acids or
polypeptides
from Tables 2-4 are detected (e.g., wherein the presence of a particular level
of
expression of a gene from Tables 2-4 or a particular allelic variant of such
gene,
such as polymorphisms from Tables 5-16 is diagnostic for a subject that can be
administered an agent to treat a disorder such as ENDOMETRIOSIS). In one
embodiment, the method includes obtaining a sample from a subject suspected
of having ENDOMETRIOSIS or an affected individual and exposing such sample
to an agent. The expression and/or activity of the nucleic acids and/or genes
of
the invention are monitored before and after treatment with such agent to
assess
the effect of such agent. After analysis of the expression values, one skilled
in the
art can determine whether such agent can effectively treat such subject. In
another embodiment, the method includes obtaining a sample from a subject
having or susceptible to developing ENDOMETRIOSIS and determining the
allelic constitution of polymorphisms from Tables 5-16 that are associated
with a
particular response to an agent. After analysis of the allelic constitution of
the
individual at the associated polymorphisms, one skilled in the art can
determine
whether such agent can effectively treat such subject.
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[000258] The methods of the invention can also be used to detect genetic
alterations in a gene from Tables 2-4, thereby determining if a subject with
the
lesioned gene is at risk for a disease associated with ENDOMETRIOSIS. In
preferred embodiments, the methods include detecting, in a sample of cells
from
the subject, the presence or absence of a genetic alteration characterized by
at
least one alteration linked to or affecting the integrity of a gene from
Tables 2-4
encoding a polypeptide or the misexpression of such gene. For example, such
genetic alterations can be detected by ascertaining the existence of at least
one
of: (1) a deletion of one or more nucleotides from a gene from Tables 2-4; (2)
an
addition of one or more nucleotides to a gene from Tables 2-4; (3) a
substitution
of one or more nucleotides of a gene from Tables 2-4; (4) a chromosomal
rearrangement of a gene from Tables 2-4; (5) an alteration in the level of a
messenger RNA transcript of a gene from Tables 2-4; (6) aberrant modification
of
a gene from Tables 2-4, such as of the methylation pattern of the genomic DNA,
(7) the presence of a non-wild type splicing pattern of a messenger RNA
transcript of a gene from Tables 2-4; (8) inappropriate post-translational
modification of a polypeptide encoded by a gene from Tables 2-4; and (9)
alternative promoter use. As described herein, there are a large number of
assay
techniques known in the art which can be used for detecting alterations in a
gene
from Tables 2-4. A preferred biological sample is a peripheral blood sample
obtained by conventional means from a subject. Another preferred biological
sample is a buccal swab. Other biological samples can be, but are not limited
to,
urine, stools, vaginal secretions, lymph, amiotic liquid, pleural liquid and
tears.
[000259] In certain embodiments, detection of the alteration involves the use
of
a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos.
4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or alternatively,
in a ligation chain reaction (LCR) (see, e.g., Landegran et a1.,1988; and
Nakazawa et al., 1994), the latter of which can be particularly useful for
detecting
point mutations in a gene from Tables 2-4 (see Abavaya et al., 1995). This
method can include the steps of collecting a sample of cells from a patient,
isolating nucleic acid (e.g., genomic DNA, mRNA, or both) from the cells of
the
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sample, contacting the nucleic acid sample with one or more primers which
specifically hybridize to a gene from Tables 2-4 under conditions such that
hybridization and amplification of the nucleic acid from Tables 2-4 (if
present)
occurs, and detecting the presence or absence of an amplification product, or
detecting the size of the amplification product and comparing the length to a
control sample. PCR and/or LCR may be desirable to use as a preliminary
amplification step in conjunction with some of the techniques used for
detecting a
mutation, an associated allele, a particular aliele of a polymorphic locus, or
the
like described in the above sections. Other mutation detection and mapping
methods are described in previous sections of the detailed description of the
present invention.
[000260] The present invention also relates to further methods for diagnosing
ENDOMETRIOSIS or a related disorder or subtype, a predisposition to such a
disorder and/or disorder progression. In some methods, the steps comprise
contacting a target sample with (a) nucleic molecule(s) or fragments thereof
and
determining the presence or absence of a particular aliele of a polymorphism
that
confers a disorder-related phenotype (e.g., predisposition to such a disorder
and/or disorder progression). The presence of at least one allele from Tables
5-
16 that is associated with ENDOMETRIOSIS ("associated allele"), at least 5 or
10
associated alleles from Tables 5-16, at least 50 associated alleles from
Tables 5-
16 at least 100 associated alleles from Tables 5-16, or at least 200
associated
alleles from Tables 5-16 determined in the sample is an indication of
ENDOMETRIOSIS disease or a related disorder, a disposition or predisposition
to such kinds of disorders, or a prognosis for such disorder progression. Such
samples and cells can be obtained from any parts of the body such as the hair,
mouth, rectum, scalp, blood, dermis, epidermis, skin cells, cutaneous
surfaces,
intertrigious areas, genitalia and fluids, vessels and endothelium. Some non-
limiting examples of cells that can be used are: ovarian cells, uterus cells
and
other cells of the reproductive system, muscle cells, nervous cells, blood and
vessels cells, T cell, mast cell, lymphocyte, monocyte, macrophage, and
epithelial cells.
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[000261] In other embodiments, alterations in a gene from Tables 2-4 can be
identified by hybridizing sample and control nucleic acids, e.g., DNA or RNA,
to
high density arrays or bead arrays containing tens to thousands of
oligonucleotide probes (Cronin et al., 1996; Kozal et al., 1996). For example,
alterations in a gene from Tables 2-4 can be identified in two dimensional
arrays
containing light-generated DNA probes as described in Cronin et al., (1996).
Briefly, a first hybridization array of probes can be used to scan through
long
stretches of DNA in a sample and control to identify base changes between the
sequences by making linear arrays of sequential overlapping probes. This step
allows the identification of point mutations, associated alleles, particular
alleles of
a polymorphic locus, or the like. This step is followed by a second
hybridization
array that allows the characterization of specific mutations by using smaller,
specialized probe arrays complementary to all variants, mutations, alleles
detected. Each mutation array is composed of parallel probe sets, one
complementary to the wild-type gene and the other complementary to the mutant
gene.
[000262] In yet another embodiment, any of a variety of sequencing reactions
known in the art can be used to directly sequence a gene from Tables 2-4 and
detect an associated allele, a particular allele of a polymorphic locus, or
the like
by comparing the sequence of the sample gene from Tables 2-4 with the
corresponding wild-type (control) sequence (see text described in previous
sections for various sequencing techniques and other methods of detecting an
associated allele, a particular allele of a polymorphic locus, or the likes in
a gene
from Tables 2-4. Such methods include methods in which protection from
cleavage agents is used to detect mismatched bases in RNA/RNA, DNA/DNA or
RNA/DNA heteroduplexes (Myers et al., 1985) and alterations in electrophoretic
mobility. Examples of other techniques for detecting point mutations, an
associated allele, a particular allele of a polymorphic locus, or the like
include, but
are not limited to, selective oligonucleotide hybridization, selective
amplification,
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selective primer extension, selective ligation, single-base extension,
selective
termination of extension or invasive cleavage assay.
[000263] Other types of markers can also be used for diagnostic purposes. For
example, microsatellites can also be useful to detect the genetic
predisposition of
an individual to a given disorder. Microsatellites consist of short sequence
motifs
of one or a few nucleotides repeated in tandem. The most common motifs are
polynucleotide runs, dinucleotide repeats (particularly the CA repeats) and
trinucleotide repeats. However, other types of repeats can also be used. The
microsatellites are very useful for genetic mapping because they are highly
polymorphic in their length. Microsatellite markers can be typed by various
means, including but not limited to DNA fragment sizing, oligonucleotide
ligation
assay and mass spectrometry. For example, the locus of the microsatellite is
amplified by PCR and the size of the PCR fragment will be directly correlated
to
the length of the microsatellite repeat. The size of the PCR fragment can be
detected by regular means of gel electrophoresis. The fragment can be labeled
internally during PCR or by using end-labeled oligonucleotides in the PCR
reaction (e.g. Mansfield et al., 1996). Alternatively, the size of the PCR
fragment
is determined by mass spectrometry. In another alternative, an oligonucleotide
ligation assay can be performed. The microsatellite locus is first amplified
by
PCR. Then, different oligonucleotides can be submitted to ligation at the
center of
the repeat with a set of oligonucleotides covering all the possible lengths of
the
marker at a given locus (Zirvi et al., 1999). Another example of design of an
oligonucleotide assay comprises the ligation of three oligonucleotides; a 5'
oligonucleotide hybridizing to the 5' flanking sequence, a repeat
oligonucleotide
of the length of the shortest allele of the marker hybridizing to the repeated
region
and a set of 3' oligonucleotides covering all the existing alleles hybridizing
to the
3' flanking sequence and a portion of the repeated region for all the alleles
longer
than the shortest one. For the shortest allele, the 3' oligonucleotide
exclusively
hybridizes to the 3' flanking sequence (U.S. Pat. No. 6,479,244).
[000264] The methods described herein may be performed, for example, by
utilizing pre-packaged diagnostic kits comprising at least one probe nucleic
acid
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selected from the SEQ ID of Tables 5-16, or antibody reagent described herein,
which may be conveniently used, for example, in a clinical setting to diagnose
patient exhibiting symptoms or a family history of a disorder or disorder
involving
abnormal activity of genes from Tables 2-4.
Method to treat an animal suspected of having ENDOMETRIOSIS
[000265] The present invention provides methods of treating a disease
associated with ENDOMETRIOSIS disease by expressing in vivo the nucleic
acids of at least one gene from Tables 2-4. These nucleic acids can be
inserted
into any of a number of well-known vectors for the transfection of target
cells and
organisms as described below. The nucleic acids are transfected into cells, ex
vivo or in vivo, through the interaction of the vector and the target cell.
The
nucleic acids encoding a gene from Tables 2-4, under the control of a
promoter,
then express the encoded protein, thereby mitigating the effects of absent,
partial
inactivation, or abnormal expression of a gene from Tables 2-4.
[000266] Such gene therapy procedures have been used to correct acquired
and inherited genetic defects, cancer, and viral infection in a number of
contexts.
The ability to express artificial genes in humans facilitates the prevention
and/or
cure of many important human disorders, including many disorders which are not
amenable to treatment by other therapies (for a review of gene therapy
procedures, see Anderson, 1992; Nabel & Felgner, 1993; Mitani & Caskey, 1993;
Mulligan, 1993; Dillon, 1993; Miller, 1992; Van Brunt, 1998; Vigne, 1995;
Kremer
& Perricaudet 1995; Doerfler & Bohm 1995; and Yu et al., 1994).
[000267] Delivery of the gene or genetic material into the cell is the first
critical
step in gene therapy treatment of a disorder. A large number of delivery
methods
are well known to those of skill in the art. Preferably, the nucleic acids are
administered for in vivo or ex vivo gene therapy uses. Non-viral vector
delivery
systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed
with a delivery vehicle such as a liposome. Viral vector delivery systems
include
DNA and RNA viruses, which have either episomal or integrated genomes after
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delivery to the cell. For a review of gene therapy procedures, see the
references
included in the above section.
[000268] The use of RNA or DNA based viral systems for the delivery of
nucleic acids take advantage of highly evolved processes for targeting a virus
to
specific cells in the body and trafficking the viral payload to the nucleus.
Viral
vectors can be administered directly to patients (in vivo) or they can be used
to
treat cells in vitro and the modified cells are administered to patients (ex
vivo).
Conventional viral based systems for the delivery of nucleic acids could
include
retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus
vectors for gene transfer. Viral vectors are currently the most efficient and
versatile method of gene transfer in target cells and tissues. Integration in
the
host genome is possible with the retrovirus, lentivirus, and adeno-associated
virus gene transfer methods, often resulting in long term expression of the
inserted transgene. Additionally, high transduction efficiencies have been
observed in many different cell types and target tissues.
[000269] The tropism of a retrovirus can be altered by incorporating foreign
envelope proteins, expanding the potential target population of target cells.
Lentiviral vectors are retroviral vectors that are able to transduce or infect
non-
dividing cells and typically produce high viral titers. Selection of a
retroviral gene
transfer system would therefore depend on the target tissue. Retroviral
vectors
are comprised of cis-acting long terminal repeats with packaging capacity for
up
to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for
replication and packaging of the vectors, which are then used to integrate the
therapeutic gene into the target cell to provide permanent transgene
expression.
Widely used retroviral vectors include those based upon murine leukemia virus
(MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus
(SIV), human immuno deficiency virus (HIV), and combinations thereof (see,
e.g.,
Buchscher et al., 1992; Johann et al., 1992; Sommerfelt et al., 1990; Wilson
et
a/., 1989; Miller et a/.,1999;and PCT/US94/05700).
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[000270] In applications where transient expression of the nucleic acid is
preferred, adenoviral based systems are typically used. Adenoviral based
vectors
are capable of very high transduction efficiency in many cell types and do not
require cell division. With such vectors, high titer and levels of expression
have
been obtained. This vector can be produced in large quantities in a relatively
simple system. Adeno-associated virus ("AAV") vectors are also used to
transduce cells with target nucleic acids, e.g., in the in vitro production of
nucleic
acids and peptides, and for in vivo and ex vivo gene therapy procedures (see,
e.g., West et al., 1987; U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, 1994;
Muzyczka, 1994). Construction of recombinant AAV vectors is described in a
number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al.,
1985;
Tratschin, et al., 1984; Hermonat & Muzyczka, 1984; and Samulski et al., 1989.
[000271] In particular, numerous viral vector approaches are currently
available for gene transfer in clinical trials, with retroviral vectors by far
the most
frequently used system. All of these viral vectors utilize approaches that
involve
complementation of defective vectors by genes inserted into helper cell lines
to
generate the transducing agent. pLASN and MFG-S are examples are retroviral
vectors that have been used in clinical trials (Dunbar et al., 1995; Kohn et
al.,
1995; Malech et al., 1997). PA317/pLASN was the first therapeutic vector used
in
a gene therapy trial (Blaese et al., 1995). Transduction efficiencies of 50%
or
greater have been observed for MFG-S packaged vectors (Ellem et al., 1997;
and Dranoff et al., 1997).
[000272] Recombinant adeno-associated virus vectors (rAAV) are a promising
alternative gene delivery systems based on the defective and nonpathogenic
parvovirus adeno-associated type 2 virus. All vectors are derived from a
plasmid
that retains only the AAV 145 bp inverted terminal repeats flanking the
transgene
expression cassette. Efficient gene transfer and stable transgene delivery due
to
integration into the genomes of the transduced cell are key features for this
vector system (Wagner et al., 1998, Kearns et al., 1996).
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[000273] Replication-deficient recombinant adenoviral vectors (Ad) are
predominantly used in transient expression gene therapy; because they can be
produced at high titer and they readily infect a number of different cell
types. Most
adenovirus vectors are engineered such that a transgene replaces the Ad Ela,
E1 b, and E3 genes; subsequently the replication defector vector is propagated
in
human 293 cells that supply the deleted gene function in trans. Ad vectors can
transduce multiple types of tissues in vivo, including nondividing,
differentiated
cells such as those found in the liver, kidney and muscle tissues.
Conventional
Ad vectors have a large carrying capacity. An example of the use of an Ad
vector
in a clinical trial involved polynucleotide therapy for antitumor immunization
with
intramuscular injection (Sterman et al., 1998). Additional examples of the use
of
adenovirus vectors for gene transfer in clinical trials include Rosenecker et
al.,
1996; Sterman et al., 1998; Welsh et al., 1995; Alvarez et al., 1997; Topf et
al.,
1998.
[000274] Packaging cells are used to form virus particles that are capable of
infecting a host cell. Such cells include 293 cells, which package adenovirus,
and
yr2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene
therapy are usually generated by a producer cell line that packages a nucleic
acid
vector into a viral particle. The vectors typically contain the minimal viral
sequences required for packaging and subsequent integration into a host, other
viral sequences being replaced by an expression cassette for the protein to be
expressed. The missing viral functions are supplied in trans by the packaging
cell
line. For example, AAV vectors used in gene therapy typically only possess ITR
sequences from the AAV genome which are required for packaging and
integration into the host genome. Viral DNA is packaged in a cell line, which
contains a helper plasmid encoding the other AAV genes, namely rep and cap,
but lacking ITR sequences. The cell line is also infected with adenovirus as a
helper. The helper virus promotes replication of the AAV vector and expression
of
AAV genes from the helper plasmid. The helper plasmid is not packaged in
significant amounts due to a lack of ITR sequences. Contamination with
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adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more
sensitive than AAV.
[000275] In many gene therapy applications, it is desirable that the gene
therapy vector be delivered with a high degree of specificity to a particular
tissue
type. A viral vector is typically modified to have specificity for a given
cell type by
expressing a ligand as a fusion protein with a viral coat protein on the
viruses
outer surface. The ligand is chosen to have affinity for a receptor known to
be
present on the cell type of interest. For example, Han et al., 1995, reported
that
Moloney murine leukemia virus can be modified to express human heregulin
fused to gp70, and the recombinant virus infects certain human breast cancer
cells expressing human epidermal growth factor receptor. This principle can be
extended to other pairs of viruses expressing a ligand fusion protein and
target
cells expressing a receptor. For example, filamentous phage can be engineered
to display antibody fragments (e.g., Fab or Fv) having specific binding
affinity for
virtually any chosen cellular receptor. Although the above description applies
primarily to viral vectors, the same principles can be applied to nonviral
vectors.
Such vectors can be engineered to contain specific uptake sequences thought to
favor uptake by specific target cells.
[000276] Gene therapy vectors can be delivered in vivo by administration to an
individual patient, typically by systemic administration (e.g., intravenous,
intraperitoneal, intramuscular, subdermal, or intracranial infusion) or
topical
application. Alternatively, vectors can be delivered to cells ex vivo, such as
cells
explanted from an individual patient (e.g., lymphocytes, bone marrow
aspirates,
and tissue biopsy) or universal donor hematopoietic stem cells, followed by
reimplantation of the cells into a patient, usually after selection for cells
which
have incorporated the vector.
[000277] Ex vivo cell transfection for diagnostics, research, or for gene
therapy
(e.g., via re-infusion of the transfected cells into the host organism) is
well known
to those of skill in the art. In a preferred embodiment, cells are isolated
from the
subject organism, transfected with a nucleic acid (gene or cDNA), and re-
infused
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back into the subject organism (e.g., patient). Various cell types suitable
for ex
vivo transfection are well known to those of skill in the art (see, e.g.,
Freshney et
al., 1994; and the references cited therein for a discussion of how to isolate
and
culture cells from patients).
[000278] In one embodiment, stem cells are used in ex vivo procedures for cell
transfection and gene therapy. The advantage to using stem cells is that they
can
be differentiated into other cell types in vitro, or can be introduced into a
mammal
(such as the donor of the cells) where they will engraft in the bone marrow.
Methods for differentiating CD34+ cells in vitro into clinically important
immune
cell types using cytokines such a GM-CSF, IFN-y and TNF-a are known (see
Inaba et al., 1992).
[000279] Stem cells are isolated for transduction and differentiation using
known methods. For example, stem cells are isolated from bone marrow cells by
panning the bone marrow cells with antibodies which bind unwanted cells, such
as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and lad
(differentiated antigen presenting cells).
[000280] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)
containing
therapeutic nucleic acids can be also administered directly to the organism
for
transduction of cells in vivo. Alternatively, naked DNA can be administered.
[000281] Administration is by any of the routes normally used for introducing
a
molecule into ultimate contact with blood or tissue cells, as described above.
The
nucleic acids from Tables 2-4 are administered in any suitable manner,
preferably
with the pharmaceutically acceptable carriers described above. Suitable
methods
of administering such nucleic acids are available and well known to those of
skill
in the art, and, although more than one route can be used to administer a
particular composition, a particular route can often provide a more immediate
and
more effective reaction than another route (see Samulski et al., 1989). The
present invention is not limited to any method of administering such nucleic
acids,
but preferentially uses the methods described herein.
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[000282] The present invention further provides other methods of treating
ENDOMETRIOSIS disease such as administering to an individual having
ENDOMETRIOSIS disease an effective amount of an agent that regulates the
expression, activity or physical state of at least one gene from Tables 2-4.
An
"effective amount" of an agent is an amount that modulates a level of
expression
or activity of a gene from Tables 2-4, in a cell in the individual at least
about 10%,
at least about 20%, at least about 30%, at least about 40%, at least about
50%,
at least about 60%, at least about 70%, at least about 80% or more, compared
to
a level of the respective gene from Tables 2-4 in a cell in the individual in
the
absence of the compound. The preventive or therapeutic agents of the present
invention may be administered, either orally or parenterally, systemically or
locally. For example, intravenous injection such as drip infusion,
intramuscular
injection, intraperitoneal injection, subcutaneous injection, suppositories,
intestinal lavage, oral enteric coated tablets, and the like can be selected,
and the
method of administration may be chosen, as appropriate, depending on the age
and the conditions of the patient. The effective dosage is chosen from the
range
of 0.01 mg to 100 mg per kg of body weight per administration. Alternatively,
the
dosage in the range of 1 to 1000 mg, preferably 5 to 50 mg per patient may be
chosen. The therapeutic efficacy of the treatment may be monitored by
observing
various parts of the reproductive system and other body parts, or any other
monitoring methods known in the art. Other ways of monitoring efficacy can be,
but are not limited to monitoring pelvic pain, fertility, ovarian cysts
formation and
progression, or any other ENDOMETRIOSIS related symptom.
[000283] The present invention further provides a method of treating an
individual clinically diagnosed with ENDOMETRIOSISs' disease. The methods
generally comprises analyzing a biological sample that includes a cell, in
some
cases, a cell, from an individual clinically diagnosed with ENDOMETRIOSIS
disease for the presence of modified levels of expression of at least 1 gene,
at
least 10 genes, at least 50 genes, at least 100 genes, or at least 200 genes
from
Tables 2-4. A treatment plan that is most effective for individuals clinically
diagnosed as having a condition associated with ENDOMETRIOSIS disease is
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then selected on the basis of the detected expression of such genes in a cell.
Treatment may include administering a composition that includes an agent that
modulates the expression or activity of a protein from Tables 2-4 in the cell.
Information obtained as described in the methods above can also be used to
predict the response of the individual to a particular agent. Thus, the
invention
further provides a method for predicting a patient's likelihood to respond to
a drug
treatment for a condition associated with ENDOMETRIOSIS disease, comprising
determining whether modified levels of a gene from Tables 2-4 is present in a
cell, wherein the presence of protein is predictive of the patient's
likelihood to
respond to a drug treatment for the condition. Examples of the prevention or
improvement of symptoms accompanied by ENDOMETRIOSIS disease that can
monitored for effectiveness include prevention or improvement of pelvic pain,
infertility, or any other ENDOMETRIOSIS related symptom.
[000284] The invention also provides a method of predicting a response to
therapy in a subject having ENDOMETRIOSIS disease by determining the
presence or absence in the subject of one or more markers associated with
ENDOMETRIOSIS disease described in Tables 5-16, diagnosing the subject in
which the one or more markers are present as having ENDOMETRIOSIS
disease, and predicting a response to a therapy based on the diagnosis e.g.,
response to therapy may include an efficacious response and/or one or more
adverse events. The invention also provides a method of optimizing therapy in
a
subject having ENDOMETRIOSIS disease by determining the presence or
absence in the subject of one or more markers associated with a clinical
subtype
of ENDOMETRIOSIS disease, diagnosing the subject in which the one or more
markers are present as having a particular clinical subtype of ENDOMETRIOSIS
disease, and treating the subject having a particular clinical subtype of
ENDOMETRIOSIS disease based on the diagnosis. As an example, treatment for
the pelvic pain or infertility subtypes of ENDOMETRIOSIS.
[000285] Thus, while there are a number of available treatments to relieve the
symptoms of ENDOMETRIOSIS, they all are accompanied by various side
effects, high costs, and long complicated treatment protocols, which are often
not
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available and effective in a large number of individuals. Symptoms also often
come back shortly after treatments are stopped. Accordingly, there remains a
need in the art for more effective and otherwise improved methods for
diagnosing, treating and preventing ENDOMETRIOSIS. Thus, there is a
continuing need in the medical arts for genetic markers of ENDOMETRIOSIS
disease and guidance for the use of such markers. The present invention
fulfills
this need and provides further related advantages.
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EXAMPLES
Example 1: Identification of cases and controls
[000286] All individuals were sampled from the Quebec founder population
(QFP). Membership in the founder population was defined as having four
grandparents of the affected child having French Canadian family names and
being born in the Province of Quebec, Canada or in adjacent areas of the
Provinces of New Brunswick and Ontario or in New England or New York State.
The Quebec founder population is expected to have two distinct advantages over
general populations for LD mapping: 1) increased LD resulting from a limited
number of generations since the founding of the population and 2) increased
genetic alleic homogeneity because of the restricted number of founders
(estited
2600 effective founders, Charbonneau et al. 1987). Reduced allelic
heterogeneity
will act to increase relative risk imparted by the remaining alleles and so
increase
the power of case/control studies to detect genes and gene alleles involved in
complex disorders within the Quebec population. The specific combination of
age in generations, optimal number of founders and large present population
size
makes the QFP optimal for LD-based gene mapping.
[000287] All enrolled QFP subjects (patients and controls) provided a 20 ml
blood sample (2 barcoded tubes of 10 ml). Samples were processed
immediately upon arrival at the laboratory. All samples were scanned and
logged
into a LabVantage Laboratory Information Management System (LIMS), which
served as a hub between the clinical data management system and the genetic
analysis system. Following centrifugation, the buffy coat containing the white
blood cells was isolated from each tube. Genomic DNA was extracted from the
buffy coat from one of the tubes, and stored at 4 C until required for
genotyping.
DNA extraction was performed with a commercial kit using a guanidine
hydrochloride based method (FlexiGene, Qiagen) according to the
manufacturer's instructions. The extraction method yielded high molecular
weight
DNA, and the quality of every DNA sample was verified by agarose gel
electrophoresis. Genomic DNA appeared on the gel as a large band of very high
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molecular weight. The remaining two buffy coats were stored at -80 C as
backups.
[000288] The QFP samples were collected as cases and controls consisting of
ENDOMETRIOSIS disease subjects and controls. 511 cases and 511 controls
were used for the analysis reported here.
[000289] The cases had a diagnosis of ENDOMETRIOSIS confirmed by the
typical black lesions observed during surgery or laparoscopy, or confirmed by
a
pathology report. The controls were minimally phenotyped and met the following
criteria:
- Female
- 40 years of age or older
- Must have at least one child
- No self-declared ENDOMETRIOSIS
- No other self-declared uterine disease
Example 2: Genome Wide Association
[000290] Genotyping was performed using the QLDM-Max SNP map using
Illumina's Infinium-II technology Single Sample Beadchips. The QLDM-Max map
contains 374,187 SNPs. The SNPs are contained in the Illumina HumanHap-300
arrays plus two custom SNP sets of approximately 30,000 markers each. The
HumanHap-300 chip includes 317,503 tag SNPs derived from the Phase I
HapMap data. The additional (approx.) 60,000 SNPs were selected by to
optimize the density of the marker map across the genome matching the LD
pattern in the Quebec Founder Population, as established from previous studies
at Genizon, and to fill gaps in the Illumina HumanHap-300 map. The SNPs were
genotyped on the 459 trios for a total of -515,255,499 genotypes.
[000291] The genotyping information was entered into a Unified Genotype
Database (a proprietary database under development) from which it was
accessed using custom-built programs for export to the genetic analysis
pipeline.
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Analyses of these genotypes were performed with the statistical tools
described
in Example 3. The GWS and the different analyses permitted the identification
of
candidate chromosomal regions linked to ENDOMETRIOSIS disease (Table 1).
[000292] Example 3: Genetic Analysis
1. Dataset quality assessment
[000293] Prior to performing any analysis, the sample was examined to
ascertain that no subjects were related more closely than 5 meiotic steps.
[000294] The data were then subjected to a cleaning step. The program,
DataStats was used to calculate the following statistics per marker or per
<individual>:
^ Minor allele frequency (MAF) for each marker
^ Number of markers with MAF < 5%, < 4%,< 3%,< 2%,< 1%
^ Number of missing values for each marker and individual
^ Monomorphic markers
^ Departure from Hardy-Weinberg equilibrium within control
individuals for each marker
The following acceptance criteria were required for further analysis:
^ Missing values per marker or individual < 1%
^ Minor allele frequency per marker > 4 %,
^ Allele frequencies for controls in Hardy-Weinberg equilibrium
Markers and individuals not meeting criteria were removed from the
dataset using DataPullPC. If a case or a control was removed by the
cleaning process, its region and gender matched case or control were also
be removed from the analysis.
2. Phase Determination
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[000295] Haplotypes will were estimated from the case/control genotype data
using ggplem a modified version of the PL-EM algorithm. The programs
geno2patctr and tagger determined case and control genotypes and prepared the
data in the input format for PL-EM. An EM algorithm module consisting of
several applications was used to resolve phase ambiguities. PLEMPre first
recoded the genotypes for input into the PL-EM algorithm which used an 11-
marker sliding block for haplotype estimation and deposited the constructed
haplotypes into a file, happatctr which was the input file for haplotype
association
analysis performed by the program, LDSTATS.
[000296] The program GeneWriter was used to create a case-control
genotype file, .penopatctr, which was the input for the program, SINGLETYPE,
which was used to perform single marker case-control association analysis.
3. Haplotype association analysis
[000297] Haplotype association analysis was performed using the program
LDSTATS. LDSTATS tests for association of haplotypes with the disease
phenotype. The algorithms LDSTATS (v2.0) and LDSTATS (v4.0) define
haplotypes using multi-marker windows that advance across the marker map in
one-marker increments. Windows of size 1, 3, 5, 7, and 9 were analyzed. At
each position the frequency of haplotypes in cases and controls was determined
and a chi-square statistic was calculated from case control frequency tables.
For
LDSTATS v2.0, the significance of the chi-square for single marker and 3-
marker
windows was calculated as Pearson's chi-square with degrees of freedom.
Larger windows of multi-allelic haplotype association were tested using
Smith's
normalization of the square root of Pearson's Chi-square.
[000298] LDSTATS v4.0 calculates significance of chi-square values using a
permutation test in which case-control status is randomly permuted until 350
permuted chi-square values are observed that are greater than or equal to chi-
square value of the actual data. The P value is then calculated as 350 / the
number of permutations required.
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[000299] Tables 5.1-16.1 lists the results for association analysis using
LDSTATs (v2.0 and v4.0) for the candidate regions described in Table 1 based
on the genome wide scan genotype data for various subphenotypes from the
QFP cases and controls. For each one of these regions, we report in Tables 5.2-
16.2 the allele frequencies and the relative risk (RR) for the haplotypes
contributing to the best signal at each SNP in the region.
4. Singletype analysis
[000300] The program SINGLETYPE was used to calculate both allelic and
genotype association for each single marker, one at a time using the genotype
data in the file, genopatctr as input. Allelic association was tested using a
2 X 2
contingency table comparing allele 1 in cases and controls and allele 2 in
cases
and controls and genotype association was tested using a 2 X 3 contingency
table comparing genotype 11 in cases and controls, genotype 12 in cases and
controls and genotype 22 in cases and controls. SINGLETYPE was also used to
test dominant and recessive models (11 and 12 genotypes combined vs. 22; or
22 and 12 genotypes combined vs. 11).
5. Conditional analysis
[000301] Conditional analyses were performed on subsets of the original set of
511 cases using the program LDSTATS (v2.0). The selection of a subset of
cases and their matched controls was based on the carrier status of cases at a
gene or locus of interest. We selected genes PRKCE on chromosome 2, RAF1
on chromosome 3, DNAH5 on chromosome 5 and SYNE1 on chromosome 6,
based on our association findings using LDSTAT (v2.0). The most significant
single SNP association signal in PRKCE, using build 36, was obtained with a
SNP corresponding to SEQ ID 4519 (see Table below for conversion to the
specific DNA genotypes used). We selected a set of risk genotypes for
conditional analyses. The set consisted of genotypes 1/2 and 2/2. Using this
set,
we partitioned the cases into two groups; the first group consisting of those
cases
that were carrier of a risk genotype and the second group consisting of the
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remaining cases, the non-carriers. The resulting sample sizes were
respectively
329 and 181. LDSTAT (v2.0) was run in each group and regions showing
association with endometriosis are reported in Table 12.1. Regions associated
with endometriosis in the group of carriers (has_PRKCE-1_cr) indicate the
presence of an epistatic interaction between risk factors in those regions and
risk
factors in PRKCE (Table 12.2).
[000302] A second conditional analysis was performed using gene RAF1 on
chromosome 3. The most significant association in RAF1, using build 36, was
obtained with a SNP corresponding to SEQ ID 4676 (see Table below for
conversion to the specific DNA genotypes used). We selected a set of risk
genotypes for conditional analyses. The set consisted of genotypes 1/2 and
2/2.
Using this risk set, we partitioned the cases into two groups; the first group
consisting of those cases that were carrier of a risk genotype and the second
group consisting of the remaining cases, the non-carriers. The resulting
sample
sizes were respectively 222 and 289. LDSTAT (v2.0) was run in each group and
regions showing association with endometriosis are reported in Table 13.1.
Regions associated with endometriosis in the group of non-carriers (not_RAF1-
1_cr) indicate the existence of risk factors acting independently of RAF1
(Table
13.2).
[000303] A third conditional analysis was performed using gene DNAH5 on
chromosome 5. The most significant association in DNAH5, using build 36, was
obtained with a SNP corresponding to SEQ ID 5001 (see Table below for
conversion to the specific DNA genotypes used). We selected a set of risk
genotypes for conditional analyses. The set consisted of genotypes 1/2 and
2/2.
Using this risk set, we partitioned the cases into two groups; the first group
consisting of those cases that were carrier of a risk genotype and the second
group consisting of the remaining cases, the non-carriers. The resulting
sample
sizes were respectively 461 and 50. LDSTAT (v2.0) was run in each group and a
region showing association with endometriosis is reported in Table 14.1. A
region associated with endometriosis in the group of carriers (has_DNAH5-1_cr)
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indicates the presence of an epistatic interaction between risk factors in the
region and risk factors in DNAH5 (Table 14.2).
[000304] A fourth conditional analysis was performed using gene SYNE1 on
chromosome 6. The most significant association signal in SYNE1, using build
36, was obtained with a SNP corresponding to SEQ ID 5106 (see Table below for
conversion to the specific DNA alleles used). We selected a set of risk
genotypes
for conditional analyses. The set consisted of genotypes 1/1 and 1/2. Using
this
risk set, we partitioned the cases into two groups; the first group consisting
of
those cases that were carrier of a risk genotype and the second group
consisting
of the remaining cases, the non-carriers. The resulting sample sizes were
respectively 214 and 297. LDSTAT (v2.0) was run in each group and regions
showing association with endometriosis are reported in Table 15.1 for the
group
of carriers and in Table 16.1 for the group of non-carriers. Regions
associated
with endometriosis in the group of carriers (has_SYNE1-1_cr) indicate the
presence of an epistatic interaction between risk factors in the region and
risk
factors in SYNE1 (Table 15.2). A region associated with endometriosis in the
group of non-carriers (not_SYNE1-1_cr) indicates the existence of risk factors
acting independently of SYNE1 (Table 16.2).
[000305] For each region that was associated with endometriosis in the
conditional analyses, we report in Tables 12.2, 13.2, 14.2, 15.2 and 16.2 the
allele frequency and the relative risk (RR) for each SNP in the region. For a
given SNP, the association with endometriosis was evaluated with a Chi-Square
test by comparing the allele frequency in the cases with the allele frequency
in
the controls. Alleles with a relative risk greater than one increase the risk
of
developing endometriosis while alleles with a relative risk less than one are
protective and decrease the risk.
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DNA alieles used in haplotypes (PRKCE)
Se ID 4519
Genotypes A/C
12 AC
22 CC
DNA alleles used in haplotypes (RAF1)
Se ID 4676
Genotypes T/G
12 TG
22 GG
DNA alleles used in haplotypes (DNAHS)
Se ID 5001
Genotypes A/G
12 AG
22 GG
DNA alleles used in haplotypes (SYNE1)
Se ID 5106
Genotypes T/C
12 TC
22 CC
6. Sub-phenotype analysis
[000306] Association analysis was performed on the full cohort and on
specific subsets (6 subphenotype analyses) of the dataset corresponding to
specific subtypes. The subtypes used are listed below along with the number of
samples used for the analysis:
= Stage III and Stage IV combined
= Women with history of infertility
= Women with no history of infertility
= Absence of chronic pelvic pain
= Presence of ovarian cysts
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= Absence of ovarian cysts
[000307] Stage of disease progression was determined during the course of
surgical procedures using the American Society for Reproductive Medicine
(ASRM) standards. A history of infertility was defined as failure to conceive
after
attempting to conceive for 12 months or longer. Chronic pain and the presence
of ovarian cysts were diagnosed by medical clinicians. Tables 6.1-11.1 lists
the
results for association analysis using LDSTATs (v2.0 and v4.0) for the regions
described above based on the genome wide scan genotype data from the various
subtypes. For each one of these regions, we report in Tables 6.2-11.2 the
aliele
frequencies and the relative risk (RR) for the haplotypes contributing to the
best
signal at each SNP in the region.
Example 4: Gene identification and characterization
[000308] A series of gene characterization was performed for each candidate
region described in Table 1. Any gene or EST mapping to the interval based on
public map data or proprietary map data was considered as a candidate
ENDOMETRIOSIS disease gene. The approach used to identify all genes
located in the critical regions is described below.
Public gene mining
[000309] Once regions were identified using the analyses described above, a
series of public data mining efforts were undertaken, with the aim of
identifying all
genes located within the critical intervals as well as their respective
structural
elements (i.e., promoters and other regulatory elements, UTRs, exons and
splice
sites). The initial analysis relied on annotation information stored in public
databases (e.g. NCBI, UCSC Genome Bioinformatics, Entrez Human Genome
Browser, OMIM - see below for database URL information). Table 2 lists the
genes that have been mapped to the candidate regions.
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[000310] For some genes the available public annotation was extensive,
whereas for others very little was known about a gene's function. Customized
analysis was therefore performed to characterize genes that corresponded to
this
latter class. Importantly, the presence of rare splice variants and
artifactual ESTs
was carefully evaluated. Subsequent cluster analysis of novel ESTs provided an
indication of additional gene content in some cases. The resulting clusters
were
graphically displayed against the genomic sequence, providing indications of
separate clusters that may contribute to the same gene, thereby facilitating
development of confirmatory experiments in the laboratory. While much of this
information was available in the public domain, the customized analysis
performed revealed additional information not immediately apparent from the
public genome browsers.
[000311] A unique consensus sequence was constructed for each splice
variant and a trained reviewer assessed each alignment. This assessment
included examination of all putative splice junctions for consensus splice
donor/acceptor sequences, putative start codons, consensus Kozak sequences
and upstream in-frame stops, and the location of polyadenylation signals. In
addition, conserved noncoding sequences (CNSs) that could potentially be
involved in regulatory functions were included as important information for
each
gene. The genomic reference and exon sequences were then archived for future
reference. A master assembly that included all splice variants, exons and the
genomic structure was used in subsequent analyses (i.e., analysis of
polymorphisms). Table 3 lists gene clusters based on the publicly available
EST
and cDNA clustering algorithm, ECGene.
[000312] An important component of these efforts was the ability to visualize
and store the results of the data mining efforts. A customized version of the
highly
versatile genome browser GBrowse (http://www.gmod.org/) was implemented in
order to permit the visualization of several types of information against the
corresponding genomic sequence. In addition, the results of the statistical
analyses were plotted against the genomic interval, thereby greatly
facilitating
focused analysis of gene content.
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Computational Analysis of Genes and GeneMaps
[000313] In order to assist in the prioritization of candidate genes for which
minimal annotation existed, a series of computational analyses were performed
that included basic BLAST searches and alignments to identify related genes.
In
some cases this provided an indication of potential function. In addition,
protein
domains and motifs were identified that further assisted in the understanding
of
potential function, as well as predicted cellular localization.
[000314] A comprehensive review of the public literature was also performed in
order to facilitate identification of information regarding the potential role
of
candidate genes in the pathophysiology of ENDOMETRIOSIS disease. In
addition to the standard review of the literature, public resources (Medline
and
other online databases) were also mined for information regarding the
involvement of candidate genes in specific signaling pathways. A variety of
pathway and yeast two hybrid databases were mined for information regarding
protein-protein interactions. These included BIND, MINT, DIP, Interdom, and
Reactome, among others. By identifying homologues of genes in the
ENDOMETRIOSIS disease candidate regions and exploring whether interacting
proteins had been identified already, knowledge regarding the GeneMaps for
ENDOMETRIOSIS disease was advanced. The pathway information gained from
the use of these resources was also integrated with the literature review
efforts,
as described above.
[000315] Genes identified in the WGAS and subsequent studies for
ENDOMETRIOSIS disease (ENDOMETRIOSIS) were evaluated using the
Ingenuity Pathway Analysis application (IPA, Ingenuity systems) in order to
identify direct biological interactions between these genes, and also to
identify
molecular regulators acting on those genes (indirect interactions) that could
be
also involved in ENDOMETRIOSIS. The purpose of this effort was to decipher
the molecules involved in contributing to ENDOMETRIOSIS. These gene
interaction networks are very valuable tools in the sense that they facilitate
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extension of the map of gene products that could represent potential drug
targets
for ENDOMETRIOSIS.
ENDOMETRIOSIS Genemap and Pathways
[000316] The GWAS and subsequent data mining analyses resulted in a
GeneMap that contains networks highly relevant to ENDOMETRIOSIS as well as
many genes under hormonal control. The following examples of the emerging
GeneMaps includes signaling pathways in cell proliferation, apoptosis, cell
cycle,
cell communication, cell structure, motility and hormonal regulation. Many of
the
identified regions contain genes involved in biologically relevant pathways,
or
associated conditions such as an oncogenesis-like mechanism, angiogenesis
and infertility.
[000317] Signalinq pathway; It has already been suggested based on
expression studies that the RAS/RAF/MAPK and P13K pathways may be involved
in initial ENDOMETRIOSIS development and pathophysiology. Among genes
discovered in the GWAS that relates and confirms these observations are, RAF1,
PRKCE, PRKD1, RALGPS1 and PIK3C2A (Stage III/IV subphenotype). These
genes play roles in a multitude of pathways including cell proliferation, cell
differentiation, survival/apoptosis, cell cycle, development, cytoskeleton,
angiogenesis, transformation and invasion/locomotion. Moreover, the PRKCE
gene has been shown to be over-expressed in ectopic endometrium as
compared to eutopic tissue. All of these are biologically relevant for
ENDOMETRIOSIS.
[000318] Cell proliferation / Angiogenesis: Angiogenesis might also play an
important role in the pathogenesis of ENDOMETRIOSIS. It is viewed as a major
prerequisite for the initiation and progression of the disease: known role in
the
survival of the implants and the development of ENDOMETRIOSIS. Anti-
angiogenic agents may provide a novel therapeutic approach for the treatment
of
ENDOMETRIOSIS. The genes from the observed GWAS results herein that may
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explain the Angiogenesis connection are RAF1, PPARG, PRKCE, PRKD1,
PIK3C2A (Stage III/IV subphenotype) and SMOC2 (Not Infertlity subphenotype).
[000319] Hormonal regulation; ENDOMETRIOSIS is an estrogen-dependent
disease and it is known that treatments tend to suppress estrogen synthesis.
Several of the identified pathways include genes that are regulated by or
involved
in the regulation of estrogen signaling: RAF1, PRKCE, KCNQ3, AVPR2 (from
conditional, epistatic to SYNE1) and ACE2 (from conditional, heterogeneity to
SYNE1).
[000320] Cell Structure and Motility (Cytoskeleton): Active cell proliferation
necessitates constant reorganization of the cytoskeleton. In ENDOMETRIOSIS
there is adherence of endometrial cells to ectopic locations. Cell motility in
also
involved in ENDOMETRIOSIS because of migration and transport of endometrial
tissue to ectopic locations. We have identified several cell adhesion
molecules
that could be involved in cell-cell or cell-matrix interaction necessitated in
adherence to ectopic locations. RALGPS1, PRKD1 and PACSIN2 (from
conditional, epistatic to SYNE1) are GWAS genes that are known to have a role
in cell proliferation and cytoskeleton remodeling. SYNE1, KCNQ3, PRICKLEI
and SLC8A1 (from conditional, epistatic to SYNE1) are GWAS genes involved in
cell structure. PPFIBP1 (Stage III/IV subphenotype), is involved in focal
adhesions, tumor invasiveness and metastasis.
[000321] Infertility: Kinetics between endometrial/fallopian ciliated cells
and
uterine contractions may be important for normal function of fertilization and
menstruation cycle. Two of the genes, DNAH5 and DNAHL1 code for cilia motor
proteins. A study on ultrastructural aspects of endometrium in infertile women
with septate uterus have shown irregular nonciliated cells with rare
microvilli,
incomplete ciliogenesis on ciliated cells, and decrease in the
ciliated:nonciliated
cell ratio.
[000322] Oncogenesis-like mechanism: Although ENDOMETRIOSIS is not a
cancer, molecular and/or regulatory mechanisms responsible for the
development of the disease may be similar. From the analyses of the GWAS
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data, it was found that the downstream genes that may explain the oncogenesis
connection of ENDOMETRIOSIS include RAF1, MCM3AP, MAD2L2 and H2AFY.
RAF1 is an oncogene while the other genes are respectively implicated in DNA
replication, cell division and gene silencing, all activities that are known
to be
important in the development of tumorogenesis.
ENDOMETRIOSIS and drug targets
[000323] Considering the fact that angiogenesis might play an important role
in the pathogenesis of ENDOMETRIOSIS, anti-angiogenic factors are used as an
experimental treatment in animal models. They have been shown to cause
regression and/or inhibition of the growth of endometriotic lesions. Most
antiangiogenic agents have been discovered by identifying endogenous
molecules that inhibit endothelial cells growth. This traditional approach has
produced a number of anti-angiogenics; platelet factor-4 (PF4),
thrombospondin,
tumour necrosis factor (TNF)-a, interferon-c-inducible protein-10 (IP-10),
angiostatin, endostatin, vasostatin, bactericidal-permeability increasing
protein
(BPI). About 40 anti-angiogenic agents, identified using various approaches,
are
currently known. These drugs are relatively safe; some compounds affect normal
reproductive functions but most are safe. VEGF has a known role in
angiogenesis. Formation of endometriotic lesions is significantly impaired
with
anti-hVEGF antibody. Anti-angiogenic agents may provide a novel therapeutic
approach for the treatment of ENDOMETRIOSIS.
[000324] PPARG is one of the genes identified on our GWAS study. PPARG
is a member of the peroxisome proliferator-activated receptor subfamily of
nuclear receptors. PPARG is a regulator of adipocyte differentiation and has
been implicated in the pathology of numerous diseases including obesity,
diabetes, atherosclerosis and cancer. Thiazolidinediones (TZD) are artificial
ligands of PPARs, and are used clinically as anti-diabetic drugs. It has been
shown that PPAR-a and -y are expressed by peritoneal macrophages isolated
from ENDOMETRIOSIS patients. Also, the PPARG ligand rosiglitazone inhibit
angiogenesis in tumors. This is relevant for ENDOMETRIOSIS.
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[000325] ENDOMETRIOSIS is an estrogen-dependent disease.
Endometriotic implants are dependent on estrogen for their maintenance and
growth. Treatments tend to suppress estrogen synthesis. In the examples of
Genemaps described above, several of the identified pathways include genes
that are regulated by or involved in the regulation of estrogen signaling.
Examples of such genes are PRKCE, RAF1, KCNQ3 and AVPR2 (from
conditional analysis herein). Also numerous network genes in the described
Genemaps are targeted by estrogen. These are IQGAP1, SRC, EP300, SP1,
E2F1, MAP2K1, HEXIM1, NTS, OXT and OXTR.
[000326] In the signaling pathway of the described Genemap, many
compounds target GWAS identified genes or network genes. Recent
breakthroughs in molecular oncology have identified a number of critical
downstream signaling cascades that are activated by tyrosine kinases
implicated
in the development of cancer. Compounds are focused on inhibiting these
critical
downstream pathways, which include the P13K, RAS/RAF/MAPK cascades (all
genes identified in the GWA study, are also found in the described Genemap).
For example, sorafenib (Nexavar) is a known drug that target one our top hits,
RAF1. Sorafenib is an anticancer medicine used to treat adults with kidney
cancer called advanced renal carcinoma. Another compound that specifically
targets RAF1 is XL281. Phase 1 trial in patients with advanced solid tumors is
ongoing. Similar compound is XL147, which selectively targets P13K. A Phase 1
trial in patients with solid tumors is ongoing. Also drugs that are PKC
inhibitor,
which inhibit PRKCE and / or PRKD1 (both genes identified in the GWA study),
are used for various indications. MAPK inhibitors (MAP2K1 is a network gene)
are also drugs tested for multiple tumors and advanced cancers.
[000327] Another network gene in the described Genemap is EP300, a
histone acetyltransferase that regulates transcription via chromatin
remodeling
and is important in the processes of cell proliferation and differentiation.
EP300
is a target for histone deacetylase inhibitors, and is used in cancer therapy.
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[000328] Thus, in the described genome wide association study (GWAS),
many of the genes are druggable and biologically relevant for ENDOMETRIOSIS.
Expression Studies
[000329] In order to determine the expression patterns for genes, relevant
information was first extracted from public databases. The UniGene database,
for
example, contains information regarding the tissue source for ESTs and cDNAs
contributing to individual clusters. This information was extracted and
summarized to provide an indication in which tissues the gene was expressed.
Particular emphasis was placed on annotating the tissue source for bona fide
ESTs, since many ESTs mapped to Unigene clusters are artifactual. In addition,
SAGE and microarray data, also curated at NCBI (Gene Expression Omnibus),
provided information on expression profiles for individual genes. Particular
emphasis was placed on identifying genes that were expressed in tissues known
to be involved in the pathophysiology of endometriosis. To complement
available
information about the expression pattern of candidate disease genes, two
experimental approaches were used. The first one was a RT-PCR based semi-
quantitative gene expression profiling method that could be applied to a large
number of target sequences (genes, transcripts, ESTs) over a panel of 24
selected tissues. The second was to map expression sites of mouse transcripts
orthologous to a small set of human disease candidate genes in the mouse
embryo (day 10.5, 12.5 and 15.5), in the postnatal stages (day 1 and 10) and
at
adulthood using in situ hybridization (ISH) method.
Semi-quantitative gene expression profiling by RT-PCR
[000330] Total human RNA samples from 24 different tissues Total RNA
sample were purchased from commercial sources (Clontech, Stratagene) and
used as templates for first-strand cDNA synthesis with the High-Capacity cDNA
Archive kit (Applied Biosystems) according to the manufacturer's instructions.
A
standard PCR protocol was used to amplify genes of interest from the original
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sample (50 ng cDNA); three serial dilutions of the cDNA samples corresponding
to 5, 0.5 and 0.05 ng of cDNA were also tested. PCR products were separated by
electrophoresis on a 96-well agarose gel containing ethidium bromide followed
by
UV imaging. The serial dilutions of the cDNA provided semi-quantitative
determination of relative mRNA abundance. Tissue expression profiles were
analyzed using standard gel imaging software (Alphalmager 2200); mRNA
abundance was interpreted according to the presence of a PCR product in one or
more of the cDNA sample dilutions used for amplification. For example, a PCR
product present in all the cDNA dilutions (i.e. from 50 to 0.05 ng cDNA) was
designated ++++ while a PCR product only detectable in the original undiluted
cDNA sample (i.e., 50 ng cDNA) was designated as + or +/-, for barely
detectable
PCR products (see Table 17). For each target gene, one or more gene-specific
primer pairs were designed to span at least one intron when possible. Multiple
primer-pairs targeting the same gene allowed comparison of the tissue
expression profiles and controlled for cases of poor amplification.
in situ hybridization (ISH) study
General procedure:
[000331] 4 genes, highlighted in the GWAS study (full sample and has
ovarian cyct subphenotype dataset), namely H2afy, Mad212, Mcm3ap and Nrxnl,
were selected for further characterization by ISH in mouse. For each gene, a
fragment of the mouse ortholog cDNA was use for the synthesis of cRNA probes
(Table 18). To maximally preserve the integrity of tissue in its environment,
mouse whole-body sections were used (Figure D). Whole bodies were frozen cut
into 10- m sections. To complement the whole-body sections, tissue arrays
including reproductive organs (RO), general tissue array (TA) and brain array
(BA) were used (Figure D). Tissue slices were mounted on glass microscope
slides, fixed in formaldehyde and hybridized with 35S-labeled cRNA probes.
Antisense cRNA generated positive signals whereas sense cRNA (identical to
mRNAs) generated negative (control) signals. Prior to gene-specific ISH, the
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tissues were validated with riboprobes to LDL receptor mRNA (data not shown).
Following ISH, gene expression patterns were analyzed by both x-ray film
autoradiography and emulsion autoradiography with appropriate exposure times.
Detailed procedure:
Mouse cDNA clone and DNA templates preparation
[000332] cDNA clones of mouse orthologs to human genes H2afy, Mad212,
Mcm3AP and Nrxnl were obtained from commercial source (Open Biosystem).
DNA fragments to be used as templates for the cRNA probes synthesis were
amplified by PCR and cloned into pGEM-7Zf(+)/LIC-F (ATCC #87048). After
sequence validation, the templates for the antisense cRNA probes synthesis
were generated by PCR using forward primers located at the 5' end of the
cloned
DNA fragments and a reverse primer located upstream of the SP6 polymerase
promoter (in the vector). Similarly, the templates for the sense (control)
cRNA
probes synthesis were generated by PCR using a forward primer located
upstream of the T7 promoter (in the vector) and reverse primers located at the
3'
end of the cloned DNA fragments.
cRNA probe preparation
[000333] cRNA transcripts were synthesized in vitro from linear DNA
fragments by run-off transcription with the SP6 or T7 RNA Polymerase from
their
respective promoters. Cold probe synthesis proved that DNA templates are
functional and, hence, applied to radioactive probe synthesis labeled with 35S-
UTP (>1,000 Ci/mmol; Amersham).
Tissues preparation.
[000334] Tissues were frozen-cut into 10- m sections, mounted on gelatin-
coated slides and stored at -80 C. Before ISH, they were fixed in 4%
formaldehyde (freshly made from paraformaldehyde) in phosphate-buffered
saline (PBS), treated with triethanolamine/acetic anhydride, washed and
dehydrated with a series of ethanol.
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Hybridization and washing procedures.
[000335] Sections were hybridized overnight at 55 C in 50% deionized
formamide, 0.3 M NaCI, 20 mM Tris-HCI, pH 7.4, 5 mM EDTA, 10 nM NaPO4,
10% dextran sulfate, 1 x Denhardt's, 50 g/ml total yeast RNA, and 50-80,000
cpm/ l 35S-labeled cRNA probe. The tissue was subjected to stringent washing
at 65 C in 50% formamide, 2 x SSC, and 10 mM DTT, followed by washing in
PBS before treatment with 20 g/ml RNAse A at 37 C for 30 minutes. After
washes in 2 x SSC and 0.1 x SSC for 10 minutes at 37 C, the slides were
dehydrated, apposed to X-ray film for 5 days, then dipped in Kodak NTB nuclear
track emulsion, and exposed for 12 days in light-tight boxes with desiccant at
4 C.
Imaging.
[000336] Photographic development was undertaken with Kodak D-19. The
slides were lightly counterstained with cresyl violet and analyzed under both
light-
and darkfield optics. Sense control cRNA probes (identical to mRNAs) always
gave background levels of the hybridization signal.
Storage and rehydration
[000337] "Crystallization" of any section could be repaired by allowing the
coverslips to fall off after soaking in xylene for 24-48 hours. The slides
were
rehydrated to 70% EtOH and then re-dehydrated again in a series of ethanol
(80%, 96% and 2 x 100% for 2 minutes each). After 3 changes with xylene, the
coverslips were mounted with Cytoseal (VWR Scientific) or other comparable
mounting medium. Using the same method, the coverslips were removed for
histological staining to take brightfield micrographs. Histological stains
that
require acidic conditions could dissolve silver grains. Overstaining could
obscure
the silver grains. Any excess mounting medium or residual emulsion on the back
of the slides was removed with a single-edged razor. The re-coverslipped
slides
were dried flat for 24 hours, and stored indefinitely at room temperature.
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Viewing original slides
[000338] The results are best viewed by darkfield illumination, with x2.5, x4,
x10, x25 and 40x objectives; the silver grains can be localized over
particular
cells. The antisense probe detects mRNA, and the sense control probe shows
the background level of silver grains for the experiments.
Results:
H2afy
[000339] Following ISH, H2afy gene expression patterns were analyzed by
both x-ray film autoradiography and emulsion autoradiography with exposure
times of 3 days and 12 days, respectively. Results are presented in Tables 19
and 20 and Figures E to N.
[000340] Analysis of ISH results provide evidence for H2afy mRNA presence
in all embryonic stages studied (Figure E). H2afy displays a widespread if not
ubiquitous distribution pattern in the midgestation stages e10.5, e12.5 and
e15.5.
At birth, H2afy mRNA distribution pattern shows differentiation in high and
low
expression sites, to form a mosaic like pattern later at adulthood. More or
less
pronounced hybridization labeling occurs in the central nervous system,
pituitary
gland, adrenal gland, thymus, spleen, lymph nodes, testis, ovary and uterus.
The
later, in pregnant female displays hybridization in the endometrium and
decidua
containing embryonic origin trophoblasts. Complete picture of H2afy mRNA
distribution in the adult mouse is shown in Table 19.
[000341] In conclusion, H2afy belongs to a class of ubiquitously expressed
genes in the embryonic mouse which over a postnatal developmental
differentiation process, acquire a cell and tissue specific pattern of
distribution.
This expression profil suggest that H2afy may play a role in both
developmental
and adulthood functions, including nervous, endocrine, immune and reproductive
functions.
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Table 19: Detection of H2AFY mRNA in whole body sections from 3
different mouse ontogeny stages, 2 postnatal stages and adulthood
Stage Whole body CNS Comments
or
Tissue array
e10.5 Embryo, mid-gestation +++++ Ubiquitous Expression
e12.5 Embryo, mid-gestation +++++ Ubiquitous Expression
e15.5 Embryo, late-gestation ++++ Ubiquitous Expression
P1 Newborn + to +++ Heterogeneous
Expression
P10 Postnatal + to ++++ Heterogeneous
Expression
P56-77 Adulthood - to +++ Heterogeneous
Expression
Average labeling level: - = not detectable; + = very weak; ++ = weak; +++ =
medium;
and ++++ = high and +++++ = very high H2AFY mRNA concentration. Abbreviation
CNS = central nervous system; PNS = peripheral nervous system.
Table 20: H2AFY mRNA tissue distribution in the adult mouse
No Tissue Labeling
I Central nervous system:
1 White matter
2 Oligodendrocytes .............................................. -
3 Grey matter
4 Cerebral cortex :...... ... ... ......... ............................. + to
+++
Neurons ......................................................... - to +++
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6 Neuroblasts ..................................................... -
7 Glial cel Is ... . . . . . . . . . ... . . . ... . . . . . . . . . . . . ...
. .. ... . . . .. . .. . .. . . -
8 Astrocytes .................................................... -
9 Microglial cells.............................................. -
Circumventricular organs :... ... ... ... ... ... ... ... ... ... ... ... ++
11 Ependymocytes .............................................. ++
12 Tanycytes ..................................................... +
13 Choroid plexus ............................................... ++
14 Hippocampus :......... ...... ........................ ...... ....... + to
+++
CAl area pyramidal neurons .............................. ++
16 CA2 area pyramidal neurons .............................. ++
17 CA3 area pyramidal neurons ............................. +
18 Dentate gyrus granules .................................... +
19 Stratum moleculare .... ... ... ... ........ ... ... ... ... ... ... . ++
Hypothalamus :................................................... ++
21 Thalamus : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . .. ++
22 Epithalamus :.................................................... ++
23 Cerebellum :... ......... ... ...... ...... ...... ... ......... ... ...
... + to +++
24 Purkinje cells .................................................. +++
Granules ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
....... . +
26 Deep cerebellar nuclei ........................................ -
27 Medulla oblongata :............................................... ++
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28 Spinal cord......................................................... +
II Peripheral nervous system:
29 Cranial ganglia :... ......... ........................ ... .......... ++
30 Trigeminal ganglion ... ... ... ... ... ... ... ... ... ... ... ... ... ...
++
31 Spinal ganglia :................................................... ++
32 Dorsal root ganglia ............................................ ++
33 Neurons...................................................... ++
34 Satelite cells ................................................. -
35 Sympathetic ganglia :............................................ NE
36 Paravertebral ganglia .........................................
37 Previsceral ganglia ............................................
38 Visceral ganglia ................................................
39 Peripheral nerves :............................................... -
40 Schwann cells ... ... .. . ... ... ... . .. ... ... ... ... ... ... ... ...
...
41 Macrophages ... ... ... ... ... ... .. . ... ... ... ... ... ... ... ... .
..
42 Fibroblasts... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .. .
...
43 Olfactory neuroepithelium :... ... ... ... ... ... ... ... ... ... ... ...
. +++
44 Pseudostratified ciliated columnar epithelium......... +++
45 Olfactory cells ................................................ CI
46 Sustencular cells ...... ...... ... ... ................. .......... +++
47 Mucus glands (Bowman's glands) ........................ +++
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48 Eye ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
... ... .... + to +++
49 Cornea ........................................................... +++
50 Choroid ......................................................... -
51 Retina ............................................................ ++
52 Ganglion cells ..........:........................................ ++
53 Lens ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
... ... ... .. -
54 Ciliary body .................................................... -
55 Iris ............ ........................ ... ............ ... ... ......
NE
56 Conjuctiva ...................................................... ++
57 Harderian gland ................................................ ++
58 Lacrimal gland ................................................. ++
59 Ear ............................................................... +
60 Cochlear duct ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
..... NE
61 Corti organ ..................................................... +
III Circulatory system:
62 Heart :.............................................................. -
63 Myocardium ...................................................
64 Va Ive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . .
65 Endocardium ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
...
66 Epicardium ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
.... .
67 Purkinje fibres ..................................................
68 Vessels :............................................................ -
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69 Aorta . . . . . . ... . . . .. . . .. ... . . . . . . ... . . . . . . . ..
. .. ... ... .. . . . . . . . .. .
70 Tunica intima endothelial cells ...........................
71 Tunica media smooth muscle cells .......................
72 Tunica adventitia fibroblasts ..............................
73 Coronary artery .................................................
74 Capillaries .......................................................
75 Venules ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
... ... ..
IV Respiratory System:
76 Nasa/ passage :... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
... ... . N E
77 Ciliate columnar epithelium ................................
78 Hyaline cartilage ... ... ... ... ... ... ... ... ... ... ... ... ... ...
...
79 Serous and mucous glands ..................................
Nasal mucosa: (see 43)
Pseudostratified ciliated columnar epith. (see 44)
Olfactory cells (see 45)
Sustencular cells (see 46)
Mucus glands (see 47)
80 Trachea : ... ... .. . ... ... ... . .. ... . .. ... ... ... ... ... ...
... ... ... ... . N E
81 Ciliated columnar cells .....................................
82 Serous glands ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
...
83 Mucous glands ...... ... ........ ... ... ... ... ... ... ... ... ......
84 Hyaline cartilage .............................................
85 Goblet cells ...................................................
86 Lung :.............................................................
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87 Columnar epithelium ... ... ... ... ....... ... ... ........ ... ...
88 Intermediate cells ... ... ... ... ... ... ... ... ... ... ... ... ... ....
89 Basal cells ...................................................
90 Smooth muscle cells ........................................
V Gastrointestinal system:
91 Tongue : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . N E
92 Oesophagus :...................................................... ++
93 Stratified squamous epithelium............................ +
94 Basal layer dividing cells .................................. ++
95 Oesophageal mucus cells ................................... -
96 Stomach :........................................................... ++
97 Non-glandular region .......................................... ++
98 Glandular region ............................................... ++
99 Small intestine :................................................... ++
100 Villus columnar epithelium ................................ +
101 Goblet cells ...... ...... ............ ... ... ... ... ... ......... ... -
102 Endocrine cells ............................................... CI
103 Brunner glands (duodenum region) ....................... NE
104 Crypt of Lieberkuhn ........................................ ++
105 Lamina propria .............................................. ++
106 Lymphoid nodule (Peyer's patch) ........................
107 Large intestine :......... ... ...... ...... ...... ... ...... ... ... ...
... ++
108 Mucosa ........................................................ ++
109 Submucosa .................................................... -
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VI Gut associated tissues:
110 Salivary glands :... ... ... ... ... ... ... ... ... ... ... ... ... ...
... ... ... +
111 Pancreas :........................................................... -
112 Acini ... ... ... ... ... ... ... ... ..... ... ... ... ... ... ... ...
... ... ... .
113 Islet cells ........................................................
114 Liver :............................................................... +
115 Hepatocyte .................................................... +
116 Kupffer cell ................................................... -
117 Gallbladder :....................................................... -
118 Columnar epithelium........................................ -
119 Lamina propria............................................... -
120 Smooth muscle ................................................ -
Vil Lymphatic tissues:
121 Thymus ..................... ........... ............................. ++
122 Cortex ........................................................... ++
123 Medulla ......................................................... +
124 Hassall's corpuscle ............................................ +
125 Fat ................................................................ -
126 Artery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . .. ... . . . . . . . .. -
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127 Spleen :............................................................. +
128 Trabeculum ..................................................... -
129 White pulp ...................................................... -
130 Red pulp ......................................................... +
131 Lymphatic nodes :...... ... ..................... ............... .... +
132 Cortex ............................................................ +
133 Medulla .......................................................... -
VIII Endocrine System:
134 Pituitary gland : ... ... ... ... ... ... ... ... ... ... ... ... ... ...
... ... .... . ++
135 Anterior pituitary lobe cells ................................... ++
136 Intermediate pituitary lobe cells ............................. ++
137 Posterior pituitary lobe cells .................................. -
138 Thyroid :........................................................... +
139 Follicular epithelial cells .................................... +
140 Thyroid C-cells ............................................... -
141 Parathyroid :....................................................... NE
142 Adrenals : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . ++
143 Cortex ............................................................ ++
144 Medulla .......................................................... +
Endocrine pancreas: (see 111, islets) -
Ovary: (see 161, reproductive system) ++++
Testis: (see 172, reproductive system) ++
IX Exocrine System:
Lacrimal gland: (see 58, eye)
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Hardenia gland: (see 57, eye)
145 Mammillary glands :... ... ... ... ... ... ... ... ... ... ... ... ... ...
.... -
146 Subaceus glands :...... ... ...... .............................. ..... -
147 Sweet glands :... ......... ... ...... ............ ... ... ......
......... NE
X Urinary system:
148 Kidney................ .............................................. +
149 Cortex ............................................................ +
150 Tubules (proximal and/or distal) ......................... +
151 Glomeruli ................................................... -
152 Juxtaglomerular apparatus ...............................
153 Medulla .......................................................... +
154 Outer medulla ................................................. +
155 Inner medulla .................................................. +
156 Bladder : ... ... .. . . .. ... ... ... ... ... ... ... ... ... ... ...
... ... . .. ... .. N E
157 Transitional epithelium ....................................
158 Lamina propria .............................................
159 Smooth muscle cells ..................................... ...
XI Reproductive system: a
160 Ovary :... ......... ......... ... ... ................. ... ... ... ...
....... + to ++++
161 Germinal epithelial cells ...................................... -
162 Granulosa cells ................................................. ++
163 Ovum ........................................................... +
164 Thecal cells ..................................................... -
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165 Corpora lutea cells ............................................. ++++
166 Uterus :... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
... ... ... ... . - to +++
167 Endometrium luminal epithelium ........................ +++
168 Endometrium glandular epithelium ....................... +++
169 Endometrium stroma ....................................... +
170 Myometriu m .... .. . ... . . . ..... .. . .. . ... . . . . . . . . . . .
. . .. . . . . . . .
171 Testis :............................................................... ++
172 Seminiferous tubules ........................................... ++
173 Sertoli cells ................................................... CI
174 Spermatogonium ........................................... ++
175 Spermatocyte ................................................ ++
176 Spermatozoa................................................. -
177 Interstitium ...................................................... -
178 Leydig cells ................................................ -
179 Epididymis :........................................................ -
180 Seminal vesicle: ................................................
181 Prostate :........................................................... -
182 Stroma cells .................................................... -
183 Epithelial cells.............................................. -
184 Urethra :..........................................................
185 Epithelial cells ... ... ... ... ... ... ... ... ... ... ... ... ... ...
...
186 Mucosal gland epithelial cells ............................
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XII Skin:
187 Derma :............................................................. ++
188 Hear bulb ................................................... ++
189 Epidermis :.........................................................
190 Hypodermis :.....................................................
191 Adipocyte ...................................................
Bone and Cartilage:
192 Bone
:..............................................................................
..... -
-
193 Osteocyte ................
................................................
194 Osteoblast ................................................... -
195 Osteoclast .................................................... -
196 Bone marrow cells ... ............ ................ ........... ++
197 Cartilage :.........................................................
198 Osteoblasts ... ... ... ... ... ... ... ... ... ... ... ... ... ... ....
.....
199 Fibrocartilage ... ... ... ... ... ... ... ... ... ... ... ... ... .....
...
XIV Tooth: (Observed in p10 postnatal mouse)
200 Enamel ........................................................... -
201 Dentine .......................................................... -
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202 Odontoblasts ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
... ... +
203 Dental pulp ......................... ..................... +
204 Ameloblast layer ... ... ... ... ... ... ... ... ... ... ... ... ... ...
... .. +++
205 Enamel pulp ................................................... ++
Scale: NE = not determined; NE = not examined; - = negative; + weak; ++ _
=
intermediate; +++ = medium; ++++ = strong and +++++ = very strong labelling;
Cl
criteria insufficient to identify cell type at present condition.* As the cell
types were solely
established based on their topography and morphology they are considered as
presumptive only. Specific phenotype markers are required to identify cell
type
unambiguously.
Mad212
[000342] Following ISH, Mad212 gene expression patterns were analyzed by
both x-ray film autoradiography and emulsion autoradiography with exposure
times of 4 days and 14 days, respectively. Results are presented in Tables 21
and 22 and Figures 0 to S.
[000343] Analysis of ISH results provide evidence for Mad212 mRNA
presence in all embryonic stages studied, including e10.5, e12.5, and e15.5
(Figure 0). Mad212 seems to be mostly expressed in the primordium of the
nervous system, where neuronal expression patterns remain elevated until the
end of the intrauterine life, followed by dramatic decline on postnatal day 10
and
adulthood. Exception is the cerebellum, which continues its development after
birth, displaying postnatal pattern of hybridization. Still, Mad212 mRNA
concentration declines at adult stage. Whenever embryonic in majority, or
postnatal in the cerebellar region particularly, Mad212 activity seems likely
to
relate to the formation of the embryonic nervous system. Once formed, the
adult
brain and spinal cord synthesize much less Mad212 mRNA. Similar process has
been observed in the peripheral nervous system sensory ganglia (dorsal root
ganglia), ortosympathetic ganglia (paravertebral ganglia), olfactory
neuroepithelium, retina in the eye and the organ of Corti in the ear, all
displaying
strong hybridization signal in p1 mice but weak in the adulthood.
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[000344] In contrast to the developmental mice, in the adulthood, low Mad212
expression levels were evident in most tissues, as shown in Table 21. Highest
Mad212 mRNA levels were detected in the male testis seminiferous tubules. In
the female, the ovary and uterus contained low concentrations Mad212 mRNA. In
contrast, pregnant mouse uterine tissue examined on day 5.5 and 7.5 post-
coitum, displayed increased levels of Mad212 mRNA around the sites of the
conceptus implantation, where a decidua is formed. This observation suggests
an
induction of Mad212 expression specifically involved in the implantation or
post-
implantation processes. Finally, the liver, spleen and kidney displayed low
Mad212 levels.
[000345] Comparison of Mad212 ontogeny expression patterns with that of
PCNA provides, evidence of their partial overlapping. Thus, in p1 mice high
concentrations of Mad212 mRNA occur in the olfactory neuroepithelium,
cerebellum, dorsal root ganglia, paravertebral ganglia, kidney marginal zone
and
intestine this correlation is evident (Table 22). In the adult mice, high
level
overlapping occurs in the testis and a pregnant mice uterus. Mad212 / PCNA
overlapping expression patterns suggests a link with cell proliferative /
periproliferative events (proliferation arrest, apoptosis, cell migration).
Still, in
some regions of the embryonic and newborn mice, this correlation appears of a
low degree, suggesting selectivity. For example, low correlation appears to
occur
between the brain (high Mad212 and low PCNA mRNA levels) or liver (low
Mad212 and high PCNA mRNA levels). It is, thus, possible that Mad212 brings
the
specificity to the tissue and cell peri-proliferative processes.
[000346] In conclusion high level Mad212 expression was documented in the
developing central and peripheral nervous system, but down-regulated to the
adulthood. In the late stage, following a postnatal up-regulation, high mad212
mRNA concentration occurs in the testis, whereas following gene induction,
increased Mad212 expression levels are evident in the pregnant mouse uterus,
both processes suggesting Mad212 role in the reproductive events. Partially
overlapping ontogeny pafterns with that of PCNA suggest mad212 functional
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involvement in the tissue- and cell-specific peri-proliferative events in the
mouse
development and reproduction.
Table 21: Detection of MAD2L2 mRNA in whole body sections from 3
different mouse ontogeny stages, 2 postnatal stages and adulthood
Stage Whole body or Tissue array CNS Comments
e10.5 Embryo, mid-gestation ++ Dominant Expression in CNS
e12.5 Embryo, mid-gestation ++++ Dominant Expression in CNS
e15.5 Embryo, late-gestation ++++ Dominant Expression in CNS
P1 Newborn ++++ Dominant Expression in CNS and
PNS
P10 Postnatal ++ Overall low to medium expression
levels, high expression in the
cerebellum (++++)
P56-77 Adulthood + Overall low expression levels,
except the testis (+++++), ovary
to (+), and liver (+); induction in the
+++++ pregnant female uteri (+++).
Average labeling level: - = not detectable; + very weak; ++ = weak; +++ _
medium; and ++++ = high and +++++ = very high MAD2L2 mRNA concentration.
Abbreviations: CNS = central nervous system; PNS = peripheral nervous
system.
Table 22: Relative Correlation Between MAD2L2 and PCNA mRNA
Ontogeny Distribution Patterns. Scale as in Table 21.
TISSUE MAD2L2 PCNA
P1
Liver - +++
Brain +++ -
Cerebellum ++++ ++++
O N E ++++ ++++
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DRG ++++ +++
PVG +++ +++
Kidney ++ +++
Intestine ++ +++
ADULT
Liver + +
Brain + +
+
Cerebellum + +
+
ONE + +
+
DRG + +
PVG + +++++
++++
Kidney +
Intestine +
Testis +++++
Uterus 5.5 and 7.5 +++
Mcm3ap
[000347] Following ISH, Mcm3ap gene expression patterns were analyzed
by both x-ray film autoradiography and emulsion autoradiography with exposure
times of 5 days and 17 days, respectively. Results are presented in Tables 23
and 24 and Figures T to W.
[000348] Analysis of ISH results provide evidence for a Mcm3ap expression
at low-level in the embryonic stages studied (Figure T). ISH results were
readable
following 5-day exposure of X-Ray Films, which is a limit of mRNA detection by
a
technique. Mcm3ap displays a widespread if not ubiquitous distribution pattern
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observed from the midgestation stages e10.5, e12.5 and e15.5 to the adulthood
with no significant changes in the pattern of tissue specificity and mRNA
concentration. Although slightly elevated hybridization levels were observed
in
some tissues such as the thymus and brain regions such as cerebellum and
hippocampus of the newborn, postnatal and adult mice, these tissues are
characterized by locally high density of cells. Thus, mcm3ap concentration in
these structures reflected rather the increasing cell density than gene
expression
regulation mechanism. The overview of mcm3ap mRNA distribution pattern in the
adult mouse is shown in Table 24.
[000349] In conclusion, Mcm3ap belongs to a class of low-level ubiquitously
expressed genes that maintain their mRNA distribution pattern and
concentration
level spanning over a life, in the mouse from the embryonic stages to the
adulthood. Mcm3ap represents likely a housekeeping class of the genes.
Table 23: Detection of MCM3AP mRNA in whole body sections from 3
different mouse ontogeny stages, 2 postnatal stages and adulthood
Stage Whole body or Tissue array CNS Comments
e10.5 Embryo, mid-gestation + Low-level ubiquitous expression
e12.5 Embryo, mid-gestation + Low-level ubiquitous expression
e15.5 Embryo, late-gestation + Low-level ubiquitous expression
P1 Newborn + Low-level ubiquitous expression
P10 Postnatal + Low-level ubiquitous expression
P56-77 Adulthood + Low-level ubiquitous expression
Average labeling level: -= not detectable; + = very weak; ++ = weak; +++ =
medium;
and ++++ = high and +++++ = very high GENE7 mRNA concentration.
Table 24: MCM3AP mRNA tissue distribution in the adult mouse
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STRUCTURE Labeling
Central nervous system:
White matter -
Grey matter +
Cerebral cortex: +
Neurons +
Neuroblasts CI
Glial cells -
Circumventricular organs: +
Ependymocytes -
Tanycytes
Choroid plexus +
Striatum: +
Hippocampus: +
Hypothalamus: +
Thalamus: +
Epithalamus: +
Cerebellum: +
Medulla oblongata: +
Spinal cord +
Peripheral nervous system:
Cranial ganglia: +
Spinal ganglia: +
Neurons +
Satelite cells -
Sympathetic ganglia: -
Paravertebral ganglia -
Previsceral ganglia -
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Visceral plexus -
Peripheral nerves: -
Olfactory euroepithelium: +
Eye +
Retina +
Lens NE
Ear +
Corti organ NE
Circulatory system:
Heart -
Blood Vessels -
Respiratory System: -
Nasal passage -
Nasal mucosa +
Trachea -
Lung -
Gastrointestinal system:
Tongue -
Oesophagus -
Stomach +
Small intestine +
Large intestine
+
Gut associated tissues:
Salivary gland
+
Exocrine pancreas
Liver
Gallbladder
Lymphatic tissues:
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Thymus
Spleen +
Lymphatic nodes +
Endocrine System: +
Pituitary gland +
Thyroid -
Parathyroid -
Endocrine pancreas NE
Adrenals -
Exocrine System: +
Olfactory Bowman's glands
Lacrimal gland +
Hardenia gland +
Mammillary glands +
Subaceus glands -
Sweet glands -
Urinary system: -
Kidney
Cortex +
Medulla +
Urinary bladder +
Reproductive system: -
Ovary
Uterus +
Testis -
Epididymis -
Seminal vesicle -
Prostate -
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U reth ra -
Skin: NE
Derma
Epidermis +
Hypodermis -
Bone, Cartilage and Tooth: +
Bone
Bone marrow -
Cartilage: +
Tooth NE
Scale: - = not detectable; + = weak; ++ = intermediate; +++ = medium; ++++ =
strong
and +++++ = very strong labelling; Cl = criteria insufficient to identify cell
type at present
condition.*; NE = not examined. *As the cell types were solely established
based on their
topography and morphology they are considered as presumptive only. Specific
phenotype markers are required to identify cell type unambiguously.
Nrxnl
[000350] Following ISH, Nrxnl gene expression patterns were analyzed by
both x-ray film autoradiography and emulsion autoradiography with exposure
times of 2 days and 10 days, respectively. Results are presented in Tables 25
and 26 and Figures X to CC.
[000351] Analysis of ISH results provide evidence for a Nrxnl expression
generally at high-level in the embryonic, newborn postnatal and adult mouse
stages Table 25 and Figure X. Not detectable on day 10.5, ISH signal was
evident on day 12.5 in the rudimental central (CNS) and peripheral (PNS)
nervous system, persisting elevated along further developmental stages. The
highest level of expression was noted to occur in CNS and PNS on postnatal day
10, followed by decline in the adult stage. Nrxnl distribution in the adult
stage is
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summarized in the Table 26. Briefly, presence of Nrxnl mRNA was confined to
neuronal cells of the grey matter. There was not detectable Nrxnl mRNA in the
white matter cells with oligodendrocyte topography. Important to note is that
the
majority, but not all neuronal cells displayed Nrxnl mRNA labeling. For
example,
there was not labeling in the Purkinje cells of the cerebellar folia and few
other
discreet regions but not shown here. For the above reason, Nrxnl distribution
cannot be defined as pan-neuronal, but rather neuron-specific.
[000352] To scrutinize Nrxnl mRNA-labeled neurons in PNS ganglia the
newborn and postnatal stages (p1 and p10) appeared especially useful when
compared to adult stage: (1) there were higher gene expression levels evident
in
ppl and p10 ganglia and (2) higher choice of sections that passed throughout
relevant regions in comparison to low choice in the adult stage sections
selection.
A list of Nrxnl-labeled PNS ganglia include the sensory cranial ganglia such
as
the trigeminal ganglion as well as dorsal root ganglia. The ganglia of the
sympathetic nervous system and visceral microganglia contributing to the
plexus
Auerbach expressed Nrxnl mRNA. In addition to CNS and PNS, the endocrine
glands such as the pituitary gland and adrenal medulla displayed a low to
medium Nrxnl mRNA concentrations.
[000353] In conclusion, Nrxnl belongs to a class of high-level neuronal-
specific genes with distribution pattern following most CNS and PNS regions
and
two endocrine glands. In CNS and PNS, Nrxnl occurs at concentrations that are
up-regulated postnatally to a maximal levels measured on day 10. Nrxnl
represents likely a good neuronal marker, especially to the plexus Auerbach in
the gut.
Table 25. Detection of NRXN1 mRNA in whole body sections from 3
different mouse ontogeny stages, 2 postnatal stages and adulthood
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Stage Whole body or Tissue CNS Comments
array
e 10.5 Embryo, mid-gestation - -
e12.5 Embryo, mid-gestation ++ Low-level expression CNS and
PNS
e15.5 Embryo, late-gestation +++ Medium-level expression CNS and
PNS
P1 Newborn +++ High-level expression CNS and
+ PNS
P10 Postnatal +++ Very high-level expression CNS
++ and PNS
P56- Adulthood +++ High-level expression CNS and
77 PNS
Average labeling level: - = not detectable; + = very weak; ++ = weak; +++ _
medium; and ++++ = high and +++++ = very high GENE9 mRNA concentration.
Table 26. NRXN1 mRNA tissue distribution in the adult mouse
1) STRUCTURE Labeling COMMENTS
Section 1.02 Central nervous
system: -
WHITE MATTER
+++++
GREY MATTER
++ to +++++
Cerebral cortex:
+ to +++++
Neurons
ci
Neuroblasts
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Glial cells -
Circumventricular organs:
Ependymocytes -
Tanycytes -
Choroid plexus -
Striatum: +
Hippocampus: ++
Hypothalamus: ++
Thalamus: +++
Epithalamus: +++
Cerebellum: ++
Medulla oblongata: ++
Spinal cord ++
Section 1.03 Peripheral nervous
system: +++ +++++ in plO
Cranial ganglia:
+++ +++++ in plO
Spinal ganglia:
+++ +++++ in plO
Neurons
Satelite cells
NE ++++ in p1 & p10
Paravertebral ganglia
NE NE
Previsceral ganglia
++ +++++ in p10
Visceral plexus
Peripheral nerves:
+
Olfactory euroepithelium:
+
Retina
Lens
NE - in p1
Corti organ
Section 1.04 Circulatory system: -
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Section 1.05 Heart
Section 1.06 Blood Vessels
Respiratory System:
Nasal passage
Nasal mucosa
Trachea
Lung
Section 1.07 Gastrointestinal _
system:
Tongue _
Oesophagus _
Stomach -
Small intestine
Large intestine -
Section 1.08 Gut associated
tissues:
Salivary gland
Exocrine pancreas
Liver
Gallbladder
Section 1.09 Lymphatic tissues: -
Thymus
Spleen
+
Lymphatic nodes
Section 1.10 Endocrine System: NE
Pituitary gland
Thyroid
+
Parathyroid
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Endocrine pancreas -
Adrenals -
Section 1.11 Exocrine System:
Article II. Olfactory Bowman's -
glands
Lacrimal gland -
Hardenia gland -
Mammillary glands
Subaceus glands -
Sweet glands -
Section 2.01 Urinary system:
Kidney
Cortex
Medulla
Urinary bladder
Section 2.02 Reproductive system:
Ovary
Uterus
Testis
NE
Epididymis
Seminal vesicle
Prostate
U reth ra
Skin:
Derma
Epidermis
Hypodermis
Bone, Cartilage and Tooth:
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Bone
Bone marrow
Cartilage:
Tooth
Scale: - = not detectable; + = weak; ++ = intermediate; +++ = medium; ++++ =
strong
and +++++ = very strong labelling; Cl = criteria insufficient to identify cell
type at present
condition.*; NE = not examined.
*As the cell types were solely established based on their topography and
morphology they are considered as presumptive only. Specific phenotype
markers are required to identify cell type unambiguously.
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