U.S. patent application number 10/017828 was filed with the patent office on 2002-08-29 for methods for treating or identifying a subject at risk for a neurological disease by determining the presence of a variant gpiiia and/or variant gpiib allele.
Invention is credited to Schappert, Keith.
Application Number | 20020119479 10/017828 |
Document ID | / |
Family ID | 22290812 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119479 |
Kind Code |
A1 |
Schappert, Keith |
August 29, 2002 |
Methods for treating or identifying a subject at risk for a
neurological disease by determining the presence of a variant
GPIIIa and/or variant GPIIb allele
Abstract
The invention provides methods for treating or identifying
subjects having a neurological disease or at risk for a
neurological disease by determining the presence of a variant
GPIIIa and/or GPIIb allele.
Inventors: |
Schappert, Keith; (Montreal,
CA) |
Correspondence
Address: |
CLARK & ELBING LLP
176 FEDERAL STREET
BOSTON
MA
02110-2214
US
|
Family ID: |
22290812 |
Appl. No.: |
10/017828 |
Filed: |
December 7, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10017828 |
Dec 7, 2001 |
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09409648 |
Oct 1, 1999 |
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60102624 |
Oct 1, 1998 |
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Current U.S.
Class: |
435/6.11 ;
435/6.16 |
Current CPC
Class: |
A61P 25/14 20180101;
C12Q 1/6883 20130101; A61P 9/10 20180101; C12Q 2600/156 20130101;
A61P 25/24 20180101; A61P 25/16 20180101; A61P 35/00 20180101; A61P
25/28 20180101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method for identifying a subject at risk for a non-Alzheimer's
neurological disease consisting of Huntington's disease,
Parkinson's disease, amyotrophic lateral sclerosis,
neurofibromatosis, depression, multiple sclerosis, stroke, and
multi-infarct dementia, comprising determining the genotype at
nucleotide 192 of the GPIIIa gene of SEQ ID NO.: 2, wherein the
gene encodes a polypeptide with a proline at amino acid position 33
of SEQ ID NO.: 4, and/or at nucleotide 2622 of GPIIb of gene of SEQ
ID NO.: 6, wherein the gene encodes a polypeptide with a serine at
amino acid position 843 of SEQ ID NO.: 8, of said subject, wherein
said genotype is indicative of said subject having an increased
risk for said non-Alzheimer's neurological disease.
2. A method for diagnosing a subject with a non-Alzheimer's
neurological disease consisting of Huntington's disease,
Parkinson's disease, amyotrophic lateral sclerosis,
neurofibromatosis, depression, multiple sclerosis, stroke, and
multi-infarct dementia, comprising determining the genotype at
nucleotide 192 of the GPIIIa gene of SEQ ID NO.: 2, wherein the
gene encodes a polypeptide with a proline at amino acid position 33
of SEQ ID NO.: 4, and/or at nucleotide 2622 of the GPIIb gene of
SEQ ID NO.: 6, wherein the gene encodes a polypeptide with a serine
at amino acid position 843 of SEQ ID NO.: 8, of said subject,
wherein said genotype is indicative of said subject having said
non-Alzheimer's neurological disease.
3. A method for characterizing the genotype of at least one subject
involved in a clinical trial of a therapy for the treatment of a
non-Alzheimer's neurological disease consisting of Huntington's
disease, Parkinson's disease, amyotrophic lateral sclerosis,
neurofibromatosis, depression, multiple sclerosis, stroke, and
multi-infarct dementia, comprising determining the genotype at
nucleotide 192 of the GPIIIa gene of SEQ ID NO.: 2, wherein the
gene encodes a polypeptide with a proline at amino acid position 33
of SEQ ID NO.: 4, and/or at nucleotide 2622 of the GPIIb gene of
SEQ ID NO.: 6, wherein the gene encodes a polypeptide with a serine
at amino acid position 843 of SEQ ID NO.: 8, of said subject.
4. The method of claim 1, 2, or 3, wherein said method comprises
determining said genotype at nucleotide 192 of the GPIIIa gene and
at nucleotide 2622 of the GPIIb gene of said subject and said
genotype places said subject into a subgroup for said clinical
trial.
5. The method of claim 1, 2, or 3, wherein said determining is
performed using a nucleic acid molecule that specifically binds a
GPIIIa nucleic acid molecule.
6. The method of claim 1, 2, or 3, wherein said determining is
performed using a nucleic acid molecule that specifically binds a
GPIIb nucleic acid molecule.
7. The method of claim 1, 2, or 3, wherein said genotype is T/C at
nucleotide 192 of SEQ ID NO:2.
8. The method of claim 1, 2, or 3, wherein said genotype is T/G at
nucleotide 2622 of SEQ ID NO: 6.
9. The method of claim 1, 2, or 3, wherein said GPIIIa gene encodes
a polypeptide with a proline at amino acid position 33 of SEQ ID
NO: 4.
10. The method of claim 1, 2, or 3, wherein said GPIIb gene encodes
a polypeptide with a serine at amino acid position 843 of SEQ ID
NO: 8.
11. The method of claim 3, wherein said genotype is indicative of
the efficacy or therapeutic benefits of said therapy.
12. The method of claim 1, 2, or 3, wherein said determining the
genotype at nucleotide 192 of the GPIIIa gene comprises performing
restriction enzyme digestion of an amplified product of a GPIIIa
nucleic acid molecule using the enzyme MspI.
13. The method of claim 12, wherein said amplified product is a
polymerase chain reaction product and said GPIIIa nucleic acid
molecule is a GPIIIa gene or a GPIIIa cDNA.
14. The method of claim 1, 2, or 3, wherein said determining the
genotype at nucleotide 2622 of the GPIIb gene comprises performing
restriction enzyme digestion of an amplified product of a GPIIb
nucleic acid molecule using the enzyme HaeII.
15. The method of claim 14, wherein said amplified product is a
polymerase chain reaction product and said GPIIb nucleic acid
molecule is a GPIIb gene or a GPIIb cDNA.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of and claims priority from
U.S. patent application Ser. No. 09/409,648, filed Oct. 1, 1999
which claims priority from U.S. Provisional Application Ser. No.
60/102,624, filed Oct. 1, 1998.
BACKGROUND OF THE INVENTION
[0002] In general, the invention relates to methods for treating a
neurological disease.
[0003] Neurological diseases, for example, Alzheimer's disease, are
often difficult to diagnose and occur in the population in a manner
which is difficult to predict. A method 1 that would allow one to
identify subjects having a neurological disease, or being at risk
for developing a neurological disease, would allow for the more
timely administration of an appropriate therapy.
[0004] The GPIIIa gene encodes a 788 amino acid polypeptide with a
26-residue signal peptide, a 29-residue transmembrane domain near
the carboxy terminus, and four cysteine-rich domains of 33-38
residues each (Zimrin et al., J. Clin. Invest. 81:1470-1475
(1988)). Two different antigenic forms of GPIIIa, alloantigens PlA1
and PlA2 (for Platelet Antigen 1 and 2), have been described and
can be distinguished using a monoclonal antibody (Weiss et al.,
Tissue Antigens 46:374-381 (1995)). The most predominant form of
GPIIIa, PlA2, is carried by 98% of the Caucasian population. The
rarer form of GPIIIa, PlA2, has sustained a point mutation at base
192 that causes a nucleotide change from a T to a C and thus a
leucine to proline (CTG>CCG) amino acid substitution at residue
position 33 (Newman et al., J. Clin. Invest. 83:1778-1781
(1989)).
[0005] The GPIIb polypeptide is the larger component of the
GPIIIa/GPIIb complex and comprises two disulfide-linked subunits of
137 amino acids and 871 amino acids each. The larger GPIIb
polypeptide has a 26 amino acid signal sequence, a potential
transmembrane domain, and four stretches of 12 amino acids each
that are homologous to the calcium binding sites of calmodulin and
troponin C (Poncz et al., J. Biol. Chem. 262(18):8476-8482 (1987)).
Mutational analysis of these domains has indicated that these
calcium-binding domains are required for the correct folding and
transport of the GPIIb polypeptide to the cell surface (Basani et
al., Blood 88:167-173 (1996)). Two antigenic forms of GPIIb,
Bak.sup.a and Bak.sup.b, have been described and can be
distinguished using specific antisera. The less common form of
GPIIb (i.e., Bak.sup.b) was determined to have a T to G point
mutation that results in an isoleucine to serine substitution at
amino acid position 843 (Lyman et al., Blood 75:2343-2348
(1990)).
SUMMARY OF THE INVENTION
[0006] The present invention provides methods for identifying or
treating a subject at risk for, or diagnosed with, a neurological
disease.
[0007] In the first aspect, the invention provides a method for
identifying a subject at risk for a neurological disease by:
identifying the subject; determining the genotype or phenotype of
the GPIIIa or GPIIb locus of the subject; and determining the
presence of a variant GPIIIa or a variant GPIIb allele or isoform,
where the presence of the variant GPIIIa allele or isoform or the
variant GPIIb allele or isoform is indicative of the subject having
an increased risk of the neurological disease. Preferably, the
neurological disease is Alzheimer's Disease (AD).
[0008] In the second aspect, the invention provides a method for
diagnosing a subject with a neurological disease by: identifying
the subject; determining the genotype or phenotype of the GPIIIa or
GPIIb locus of the subject; and determining the presence of a
variant GPIIIa or a variant GPIIb allele or isoform, where the
presence of the variant GPIIIa allele or isoform or the variant
GPIIb allele or isoform is indicative of the subject having a
likelihood of the neurological disease.
[0009] In the third aspect, the invention provides a method for
characterizing the genotype of at least one subject involved in a
clinical trial of a therapy for the treatment of a neurological
disease by: identifying the subject; determining the genotype or
phenotype of the GPIIIa or GPIIb locus of the subject before,
during, or after the clinical trial; and determining the presence
of a variant GPIIIa or a variant GPIIb allele or isoform, where the
presence of the variant GPIIIa allele or isoform or the variant
GPIIb allele or isoform places the subject into a subgroup for the
clinical trial. Preferably, the genotype or phenotype is indexed
against the efficacy or side-effects of the therapy.
[0010] In the fourth aspect, the invention provides a method for
treating a subject with a neurological disease by: identifying the
subject; determining the genotype or phenotype of the GPIIIa or
GPIIb locus of the subject; determining the presence of a variant
GPIIIa or a variant GPIIb allele or isoform; and determining the
preferred therapy for the treatment of the neurological
disease.
[0011] In the fifth aspect, the invention provides a method for
treating a subject at risk for a neurological disease by:
identifying the subject; determining the genotype or phenotype of
the GPIIIa or GPIIb locus of the subject; determining the presence
of a variant GPIIIa or a variant GPIIb allele or isoform;
determining the GPIIIa or GPIIb allele status of the subject, where
the allele status is predictive of patient outcome or drug
efficacy.
[0012] In a preferred embodiment of the above aspects, the method
includes determining the presence of both the variant GPIIIa allele
or isoform and the variant GPIIb allele or isoform.
[0013] In other preferred embodiments of the above aspects, the
neurological disease may be Alzheimer's disease (AD), a non-AD
neurological disease, or a neurological disease selected from the
group consisting of Alzheimer's disease, neurofibromatosis,
Huntington's disease, depression, amyotrophic lateral sclerosis,
multiple sclerosis, stroke, Parkinson's disease, and multi-infarct
dementia.
[0014] In other preferred embodiments of the above aspects, the
determining may be performed using a nucleic acid that specifically
binds a nucleic acid encoded by the variant GPIIIa allele or the
variant GPIIb allele. In other preferred embodiments of the above
aspects, the determining may be performed using an antibody that
specifically binds a polypeptide encoded by the variant GPIIIa
allele or the variant GPIIb allele, but does not bind a polypeptide
encoded by a wild-type GPIIIa allele or a wild-type GPIIb
allele.
[0015] In other preferred embodiments of the above aspects, the
variant GPIIIa allele may have a point mutation at nucleotide base
192 of SEQ ID NO: 2 or encode a polypeptide with a proline at amino
acid position 33 of SEQ ID NO: 4. In other preferred embodiments of
the above aspects, the variant GPIIb allele may have a point
mutation at nucleotide base 2622 of SEQ ID NO: 6 or encode a
polypeptide with a serine at amino acid position 843 of SEQ ID NO:
8.
[0016] The presence of a variant allele may be determined by
genotyping nucleic acids from the subject or by assaying for the
presence of a protein having alterations encoded by the variant
nucleic acid.
[0017] By "neurological disease" is meant a disease, which involves
the neuronal cells of the nervous system. Specifically included
are: prion diseases (e.g, Creutzfeldt-Jakob disease); pathologies
of the developing brain (e.g., congenital defects in amino acid
metabolism, such as argininosuccinicaciduria, cystathioninuria,
histidinemia, homocystinuria, hyperammonemia, phenylketonuria,
tyrosinemia, and fragile X syndrome); pathologies of the mature
brain (e.g., neurofibromatosis, Huntington's disease, depression,
amyotrophic lateral sclerosis, multiple sclerosis); conditions that
strike in adulthood (e.g. Alzheimer's disease, Creutzfeldt-Jakob
disease, Lewy body disease, Parkinson's disease, Pick's disease);
and other pathologies of the brain (e.g., brain mishaps, brain
injury, coma, infections by various agents, dietary deficiencies,
stroke, multiple infarct dementia, and cardiovascular
accidents).
[0018] By "cognitive enhancers" is meant drugs which enhance a)
memory performance, whether it is verbal memory, spatial memory, or
factual memory and b) learning capacity.
[0019] By "cholinomimetic therapy" is meant any drug that mimics
the function of acetylcholine or enhances the activity of
acetylcholine synthesizing cells. These drugs include, but are not
limited to, inhibitors of acetylcholine degradation (acetylcholine
esterase inhibitors such as tacrine), drugs that mimic
acetylcholine structure and function, drugs that block
acetylcholine uptake by neurons, and drugs that interact with
pre-synaptic receptors to induce acetylcholine release from
cholinergic neurons.
[0020] By "non-cholinomimetic vasopressinergic therapy" is meant a
therapy that utilizes a vasopressinergic modulator such as, for
example, S12024 (provided by Servier, Les Laboratoires Servier, 22
rue Garnier, 92200 Neuilly sur Seine, France).
[0021] By "already diagnosed" is meant already diagnosed as having
the neurological disease, having a genetic predisposition to the
disease, or both.
[0022] By "patient profile" is meant data pertaining to the patient
for whom the pharmacogenetic analysis is being performed. Data may
include information on the patient's diagnosis, age, sex, and
genotype. The patient's profile may also include materials from the
patient such as blood or purified RNA or DNA.
[0023] By "prognosis protocol" is meant a therapy plan provided to
the clinician or patient using the pharmacogenetic method. The
prognosis protocol includes an indication of whether or not the
patient is likely to respond positively to a cholinomimetic
therapeutic. In preferred embodiments, the protocol also includes
an indication of the drug dose to which the patient is most likely
to respond. The "pharmacogenetic method" is a method whereby
genetic and diagnostic data, including the patient's neurological
diagnosis and the patient's GPIIIa and/or GPIIb genotype are
processed to provide therapeutic options and prognoses.
[0024] By "non-AD neurological disease" is meant a disease other
than Alzheimer's disease, which involves the neuronal cells of the
nervous system. Specifically included are: prion diseases (e.g,
Creutzfeldt-Jakob disease); pathologies of the developing brain
(e.g., congenital defects in amino acid metabolism, such as
argininosuccinicaciduria, cystathioninuria, histidinemia,
homocystinuria, hyperammonemia, phenylketonuria, tyrosinemia, and
fragile X syndrome); pathologies of the mature brain (e.g.,
neurofibromatosis, Huntington's disease, depression, amyotrophic
lateral sclerosis, multiple sclerosis); conditions that strike in
adulthood (e.g. Creutzfeldt-Jakob disease, Lewy body disease,
Parkinson's disease, Pick's disease); and other pathologies of the
brain (e.g., brain mishaps, brain injury, coma, infections by
various agents, dietary deficiencies, stroke, multi-infarct
dementia, and cardiovascular accidents).
[0025] By "Alzheimer's Disease" is meant a pathology characterized
by an early and extensive loss of entorhinal cortex neurons.
Alzheimer's disease subjects may be identified by progressive and
degenerative effects on the brain which are not attributable to
other causes. A diagnosis of Alzheimer's disease is made using
clinical-neuropathological correlations known in the art (see e.g.,
Arch. Neurology 51(9):888-896 (1994)). Post-mortem, the disease may
be diagnosed by the presence of amyloid plaques and fibrils.
[0026] As used herein, by "therapy for the treatment of a
neurological disease" is meant any therapy suitable for treating a
neurological disease. A suitable therapy can be a pharmacological
agent or drug that may enhance or slow the loss of cognitive
function, motor function, or neuronal activity of the central
nervous system, peripheral nervous system, or inhibit the further
deterioration of any of these faculties. In addition, the term
therapy may also include the close monitoring of an asymptomatic
patient for the appearance of any symptoms of a neurological
disease.
[0027] By "determining the presence of a variant GPIIIa and/or
variant GPIIb allele" is meant subjecting a nucleic acid sample to
any of a variety of detection techniques know in the art for
elucidating a point mutation in a nucleic acid (e.g., polymerase
chain reaction (PCR), reverse transcriptase-PCR (RT-PCR),
ligase-mediated chain reaction step, chip hybridization methods, or
restriction enzyme-mediated digestion). For example, in the
presence of appropriately designed primers, a nucleic acid fragment
can be amplified using PCR and analyzed by restriction enzyme
digestion that can reveal the presence of a variant allelic
sequence. In addition, DNA sequencing may be employed using
techniques known in the art. These nucleic acid techniques allow
for a genotype determination of the GPIIIa or GPIIb locus.
Alternatively, phenotyping of the locus may be performed (and a
genotype thus inferred) by using standard techniques for detecting
the presence of a polypeptide having a particular amino acid change
(e.g., antibodies, isoelectric focusing, and 2-D PAGE). For
example, the presence of a variant GPIIIa polypeptide (e.g., PlA2;
LEU33PRO) can be distinguished from a wild-type GPIIIa polypeptide
(i.e., PlA1) using epitope specific antibodies available in the art
(Weiss et al., Tissue Antigens 46:374-381 (1995)). Antibodies for
detecting different polymorphisms of the GPIIb polypeptide have
also been described (Lyman et al., Blood 75:2343-2348 (1990)).
[0028] By "variant GPIIIa allele" is meant any sequence mutation of
the glycoprotein integrin beta-3 subunit (GPIIIa) gene, that
differs from the predominant wild-type allelic sequence (e.g.,
variant GPIIIa allele (LEU33PRO)) and which is associated with
neurological disease. By "associated" is meant associated with an
altered risk of disease incidence, drug efficacy, or disease
prognosis. Variant GPIIIa alleles not specifically described to be
associated with neurological disease herein can be tested for
association using the techniques provided herein and those known in
the art. Specifically excluded are GPIIIa variants that have an
A>C mutation at nucleotide base 1159, and A>G mutation at
nucleotide base 1549, or a G>C mutation at nucleotide base
1161.
[0029] By "variant GPIIb allele" is meant any sequence mutation of
the glycoprotein integrin alpha-2 subunit (GPIIb) gene that differs
from the predominant wild-type allelic sequence (e.g., variant
GPIIb allele (ILE843SER)) and which is associated with neurological
disease. By "associated" is meant associated with an altered risk
of disease incidence, drug efficacy, or disease prognosis. Variant
GPIIb alleles not specifically described to be associated with
neurological disease herein can be tested for association using the
techniques provided herein and those known in the art. Specifically
excluded are GPIIIa variants that have an A>C mutation at
nucleotide base 1159, and A>G mutation at nucleotide base 1549,
or a G>C mutation at nucleotide base 1161.
[0030] By "risk factor associated with a disease" is meant any risk
factor for a disease known in the art. Examples of risk factors
commonly associated with diseases include age, gender, diet,
exercise, weight, the presence of another disease, and the
occurrence of a specific genotype. Risk factors associated with a
neurological disease in particular may include advanced age, lower
intelligence, smaller head size, history of head trauma, mutations
on chromosomes 1, 14, and 21, or the presence of a variant GPIIIa
and/or variant GPIIb allele (see e.g., Cummings et al., Neurology
(1 Supp.1):S2-S17, 1998).
[0031] By "subject at risk for a neurological disease" is meant a
subject identified or diagnosed as having a neurological disease or
having a genetic predisposition or risk for acquiring a
neurological disease using the methods of the invention and
techniques available to those skilled in the art.
[0032] By "wild-type" is meant any allele, or polypeptide encoded
by such an allele, that is present in that part of the population
considered free of disease.
[0033] By "PCR, RT-PCR, or ligase chain reaction amplification" is
meant subjecting a DNA sample to a Polymerase Chain Reaction step
or ligase-mediated chain reaction step, or RNA to a RT-PCR step,
such that, in the presence of appropriately designed primers, a
nucleic acid fragment is synthesized or fails to be synthesized,
thereby revealing the allele status of a patient. The nucleic acid
may be further analyzed by DNA sequencing using techniques known in
the art.
[0034] The present invention provides a number of advantages. For
example, the methods described herein allow for a determination of
a subject's GPIIIa and/or GPIIb genotype for the timely
administration of a prophylactic therapy for the treatment of a
neurological disease.
[0035] Other features and advantages of the invention will be
apparent from the following detailed description and from the
claims.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The drawings will first be described.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 is a depiction of the cDNA sequence encoding the wild
type human GPIIIa polypeptide (SEQ ID NO: 1).
[0038] FIG. 2 is a depiction of the cDNA sequence encoding the
variant human GPIIIa polypeptide (SEQ ID NO: 2) which has a
nucleotide point mutation at base 192. The T to C point mutation
creates a new Msp I restriction site (underlined) and results in a
codon that encodes a proline at position 33 (indicated in bold and
offset by spaces).
[0039] FIG. 3 is a depiction of the amino acid sequence of the wild
type human GPIIIa polypeptide (SEQ ID NO: 3). The 26 amino acid
signal sequence is underlined and the wild type leucine residue at
position 33 is indicated in bold.
[0040] FIG. 4 is a depiction of the amino acid sequence of the
human GPIIIa polypeptide (SEQ ID NO: 4) with a single amino acid
residue change, from an leucine (L) to a proline (P) at position
33, indicated in bold. The 26 amino acid signal sequence is
underlined.
[0041] FIG. 5 is a depiction of the cDNA sequence encoding the
wild-type human GPIIb polypeptide (SEQ ID NO: 5). The codon
encoding the wild-type isoleucine residue at position 843 is
indicated in bold.
[0042] FIG. 6 represents the cDNA sequence encoding the variant
human glycoprotein IIb polypeptide (SEQ ID NO: 6) which has a point
mutation (T to G) at nucleotide base 2622. The point mutation
creates a new Hae II restriction site (underlined) and a codon
(indicated in bold and offset by spaces) that encodes a serine at
position 843.
[0043] FIG. 7 shows the amino acid sequence of the wild-type human
glycoprotein IIb polypeptide (SEQ ID NO: 7). The wild-type
isoleucine residue at position 843 is indicated in bold.
[0044] FIG. 8 shows the amino acid sequence of the variant human
glycoprotein IIb polypeptide (SEQ ID NO: 8). The single amino acid
residue change, from an isoleucine (I) to a serine (S) at position
843, is indicated in bold.
[0045] The invention described herein features methods for treating
or identifying a subject at risk for a neurological disease, such
as Alzheimer's disease (AD), by determining the presence of a
variant GPIIIa or variant GPIIb allele. The invention also provides
a method for forecasting patient outcome and the suitability of the
patient for entering a clinical drug trial for the testing of a
therapy for a neurological disease.
[0046] Normally, these alleles encode glycoproteins IIIa and IIb of
the GPIIIa/GPIIb complex that belongs to a class of multi-subunit
integrin receptors that bind cell adhesion molecules. These
receptors are composed of alpha and beta subunits referred to,
counter intuitively, as GPIIb and GPIIIa, respectively. Together,
the GPIIIa beta and GPIIb alpha subunits form part of the platelet
complex receptor, fibronectin receptor, and vitronectin receptor,
and play a role in clotting. As expected, these polypeptides are
expressed in platelets and endothelial cells (Hynes et al., Cell
48: 549-554 (1987)).
[0047] We have discovered that GPIIb and GPIIIa alleles are
associated with the occurrence of neurological disease. For
example, the presence of a particular variant GPIIIa allele that
results in a single amino acid change from a leucine to a proline
at residue 33 (LEU33PRO) indicates, with a high probability, that a
subject is at risk for a neurological disease such as Alzheimer's
disease (AD). In addition, we have also observed that the presence
of a variant GPIIb allele (ILE843SER) indicates, with a similar
probability, that a subject may be at risk for acquiring a
neurological disease, such as AD. Importantly, these genes may act
in synergy and when used together as a prognostic tool, predict,
with even greater probability, a subject's risk for a neurological
disease, such as AD.
[0048] One of the advantages of the invention is that a subject at
risk for a neurological disease may be identified and, if
appropriate, administered therapeutics without waiting for
debilitating symptoms of them required for definitive diagnosis to
occur. Initially, treatment of a subject having a variant allele
described herein may involve monitoring of the subject for other
risk factors and/or symptoms. Alternatively, a subject at high risk
for a neurological disease may be treated prophylactically, with
therapies known in the art, in order to delay, inhibit, or prevent
the onset of disease. In one approach, the presence of a variant
GPIIIa and/or variant GPIIb allele is rapidly determined using a
sensitive PCR assay and, alone or in combination with a
determination of other risk factors associated with a neurological
disease, this determination is used to determine if a prophylactic
treatment therapy should be invoked.
[0049] The prediction of drug efficacy may involve cholinomimetic
therapies, for example, tacrine, or non-cholinomimetic therapies,
for example, a vasopressinergic drug. The invention provides a
treatment protocol that utilizes one of the following therapies for
a neurological disease: probucol, a monoamine oxidase inhibitor,
muscarinic agonist, neurotrophic factor, noradrenergic factor,
antioxidant, anti-inflammatory, corticotrophin-releasing hormone
(CRH), somatostatin, substance P, neuropeptide Y, or
thyrotrophin-releasing hormone (TRH).
[0050] The findings described herein indicate the predictive value
of a variant GPIIIa and/or variant GPIIb allele in treating
patients at risk for a neurological disease, such as Alzheimer's
disease (AD). In addition, because the underlying mechanism
influenced by the variant GPIIIa and/or variant GPIIb allele status
is not disease-specific, the GPIIIa and/or GPIIb allele-status is
suitable for making patient predictions for non-AD neurological
diseases as well.
[0051] The following examples, which describe preferred techniques
and experimental results, are provided for the purpose of
illustrating the invention, and should not be construed as
limiting.
EXAMPLE 1
[0052] Methods for Determining the Presence of a Variant GPIIIa
Allele or Variant GPIIb Allele
[0053] We have found that both the variant GPIIIa allele and GPIIb
allele have strong predictive value for identifying a subject at
risk for a neurological disease (e.g., Alzheimer's disease). This
predictive value is even stronger when these variant alleles in
both genes occur together in a given subject. To demonstrate the
effectiveness of the variant GPIIIa and/or variant GPIIb allele for
identifying subjects with such a disease risk, we determined the
allele frequency of either variant allele in a large number of
subjects diagnosed with Alzheimer's disease (N=136) as compared to
age-matched healthy controls (N=70).
[0054] GPIIIa Genotyping
[0055] We genotyped each of the above patients for the presence of
a variant GPIIIa allele using the polymerase chain reaction method
(PCR). In particular, genotyping was carried out by subjecting
nucleic acid samples encoding the GPIIIa gene to a polymerase chain
reaction (PCR) amplification step followed by another round of PCR
amplification using a nested PCR protocol. The first round of PCR
amplification was conducted using outside primers
1 P1A2-4 (5'-AGA CTT CCT CCT CAG ACC TCC ACC T-3' (SEQ ID NO:9))
and P1A2-5 (5'-TAA ACT CTT AGC TAT TGG GAA GTG GTA-3' (SEQ ID
NO:10))
[0056] and using reaction conditions that included a heating step
at 90.degree. C. for 1 min., followed by another heating step at
95.degree. C. for 1 min., followed by 45 cycles of 94.degree. C.
for 25 sec., 45.degree. C. for 55 sec., 72.degree. C. for 45 sec.,
and a final extension step at 72.degree. C. for 3 min. Next, a 1
.mu.l aliquot of the first PCR reaction was used for conducting the
subsequent nested PCR reaction under the same conditions except
that the amplification step performed at 45.degree. C. was changed
to 48.degree. C. and the oligonucleotides
2 P1A2-1 (5'-TTC TGA TTG CTG GAC TTC TCT T-3' (SEQ ID NO:11)) and
P1A2-2 (5'-TCT CTC CCC ATG GCA AAG AGT-3' (SEQ ID NO:12))
[0057] were used.
[0058] When amplified GPIIIa DNA isolated from the subjects
described above was analyzed, we observed a C nucleotide at base
position 192 only in nucleic acids encoded by the variant GPIIIa
allele (or PlA2 form) and this created a new Msp I restriction site
(see FIGS. 1 and 2). Subsequent restriction enzyme analysis of
nucleic acids generated by PCR showed that Msp I digestion
permitted clear discrimination between the type (PlA1) and mutant
form (PlA2) of GPIIIa and individuals could thus be genotyped.
[0059] Specifically, a 5 .mu.l aliquot of the resultant amplified
PCR reaction product was digested with 5 units of the restriction
enzyme Msp I and the resultant DNA products were analyzed using
agarose gel electrophoresis and visualized by ethidium bromide
staining. Another Msp I site, common to both wild-type and variant
GPIIIa alleles, was used as an internal control to insure the
completion of Msp I digestion. Using this protocol, three banding
patterns were observed based on whether the subject was homozygous
wild-type (T/T), heterozygous mutant (C/T), or homozygous mutant
(C/C) for the variant GPIIIa allele (LEU33PRO). The banding pattern
for the homozygous wild-type consisted of two DNA fragments of 222
bp and 38 bp in length. The banding pattern for the homozygous
mutant genotype consisted of three fragments of 175 bp, 49 bp, and
38 bp in length. Accordingly, the banding pattern for the
heterozygous mutant genotype consisted of four fragments of 224 bp,
175 bp, 49 bp, and 38 bp in length. A GPIIIa genotype (LEU33PRO)
was determined for each subject in the study and analyzed for its
predictive value (Examples 2 and 3).
[0060] GPIIb Genotyping
[0061] Each of the above the samples from the patients described
above were genotyped for the presence of a variant GPIIb allele
using the conditions above with the following modifications.
Genotyping was carried out using the same PCR conditions above
except that primers
3 A (5'-CTG TCA ACC CTC TCA AGG TAA (SEQ ID NO:13)) and B (5'-GCC
GGG TGA ATG GGG GAG GGG CTG GCG (SEQ ID NO:14))
[0062] were used.
[0063] Following the PCR amplification reaction, DNA products were
digested with the restriction enzyme Hae II (according to the
manufacturer) and resultant products were resolved using 3%
Nusieve.TM. gel electrophoresis followed by ethidium bromide
staining. As the variant GPIIb nucleic acid encodes an additional
Hae II site, distinctive banding patterns were observed based on
whether the subject was wild-type (T/T; 180 bp only), heterozygous
mutant (C/T; 180, 155, and 25 bp), or homozygous mutant (C/C; 155
and 25 bp) for the GPIIb allele (ILE843SER).
[0064] GPIIIa and GPIIB Phenotyping
[0065] For either the GPIIIa (LEU33PRO) or GPIIb (ILE843SER) gene
product, detection of the variant polypeptide may be performed (and
a genotype thus inferred) using variant polypeptide specific
antibodies as described in the art (see, e.g., Weiss et al., Tissue
Antigens 46:374-381 (1995); Lyman et al., Blood 75:2343-2348
(1990)).
[0066] In addition to the above-mentioned methods, the methods
provided in U.S. Pat. No. 5,935,781 and U.S. Pat. No. 6,022,683;
any of the pending applications (Ser. Nos. US97/22699; 09/160,462;
08/991,850; 09/334,489; 09/616,506) and following references
(Brindle N. et al., Hum. Mol. Genet. 7:933-935 (1998); Singleton et
al., Hum Mol Genet 7:937-939 (1998); Lehmann et al., Hum. Mol.
Genet. 6:1933-1936 (1997); Richard et al., Lancet 349:539 (1997);
and Gustincich S, et al., Biotechniques 11(3):298-300 (1998)) may
also be used.
EXAMPLE 2
[0067] Use of the Variant GPIIIa Allele in Determining a Subjects's
Risk for Alzheimer's Disease
[0068] We have discovered that the presence of a variant GPIIIa
allele (LEU33PRO) contributes an individual's risk for the
development of Alzheimer's disease. To reach this conclusion, we
compiled the GPIIIa genotypes for 135 Alzheimer's disease subjects
and 69 age-matched healthy controls (Table 1) and analyzed the
distribution of variant GPIIIa alleles in control subjects versus
subjects with disease. As shown in Table 1, a significant number of
subjects diagnosed with Alzheimer's disease had at least one mutant
GPIIIa allele.
4TABLE 1 GPIIIa Nucleotide Dimorphism in Controls vs. Subjects with
Alzheimer's Disease (AD) C/C C/T T/T Genotype (homozygous mt)
(heterozygous mt) (wild-type) Control 1 13 55 AD 2 46 87
[0069] In Table 2 we present the total number of subjects having at
least one variant GPIIIa allele as a function of the subject's
disease status. This data shows that the occurrence of a variant
GPIIIa allele in a subject with Alzheimer's disease is more than
twice as high as in age-matched healthy controls (the odds ratio
(O.R.) is 2.17). The Yates value calculated for this data set
indicates that this distribution occurring by chance alone is
remote (4%). These data predict a strong correlation between the
presence of the variant GPIIIa allele and the occurrence of
Alzheimer's disease in a given subject.
5TABLE 2 Chi Square for GPIIIa Allelic Frequency in Controls vs.
Subjects with Alzheimer's Disease (AD) AD Control C/C or C/T
(Mutant 48 14 Genotypes) T/T (Wild-Type 87 55 Genotype) Yates =
0.037 O.R. = 2.17
[0070] In Table 3 the data is shown as the total number of mutant
alleles (a C at base position 192) versus wild-type alleles (a T at
position 192) occurring in subjects of each health group (i.e.,
control vs. AD). Stated in another way, each mutant allele is
counted and a frequency of occurrence (ranging from 0-1.0) is
calculated for the likely appearance of this allele in either a
healthy subject or a subject with Alzheimer's disease. A percent
occurrence is obtained by multiplying the frequency factor by 100.
Thus, the frequency of the variant GPIIIa allele occurring in
subjects diagnosed with Alzheimer's disease was 18.5%
(0.185.times.100) as compared to only 11% in healthy age-matched
controls.
6TABLE 3 Variant GPIIIa Allele Frequency in Controls vs. Subjects
with Alzheimer's Disease (AD) C (Mutant Alleles) T (Wild-Type
Alleles) Control 0.11 (15/138) 0.89 (123/138) AD 0.185 (50/270)
0.815 (220/270)
[0071] Finally, as shown in Tables 4-9, we examined a number of
silent mutations (i.e., a wild-type protein is encoded from a
mutated nucleic acid) found within the coding region of the GPIIIa
gene and found no correlation (the odds ratios are all around 1)
between AD and the presence of these mutations. These studies
indicate that it is likely that the GPIIIa polypeptide, and not the
nucleic acid, plays a possible role in AD. Accordingly, nucleic
acid changes that result in amino acid alterations are more likely
to be predictive of neurological disease or a predisposition to
neurological disease.
7TABLE 4 Val 381 Val Silent Mutation (A-C at base 1159) Genotype of
Normal Subjects vs. Patients with AD AA AC CC wild-type
heterozygous mt homozygous mt Alzheimer's 51 62 23 cases Control 26
35 9
[0072]
8TABLE 5 Odds Ratio and Chi-Square Analysis of the Val 381 Val
Silent Mutation Occurring in Patients with AD as Compared to
Controls AD Control CX 85 44 AA 51 26 Chi-square = 0.92 O.R. =
0.98
[0073]
9TABLE 6 Glu 511 Glu Silent Mutation (A-G at base 1549) Genotype of
Normal Subjects vs. Patients with AD AG AA heterozygous GG
wild-type mutant homozygous mt Alzheimer's 0 36 33 cases Control 0
66 69
[0074]
10TABLE 7 Odds Ratio and Chi-Square Analysis of the Glu 511 Glu
Silent Mutation Occurring in Patients with AD as Compared to
Controls AD Control CX 66 36 AA 69 33 Chi-square = 0.76 O.R. =
0.88
[0075]
11TABLE 8 Arg 515 Arg Silent Mutation (G-C at base 1161) Genotype
of Normal Subjects vs. Patients with AD AA AG GG wild-type
heterozygous mt homozygous mt Alzheimer's 5 28 34 cases Control 19
50 64
[0076]
12TABLE 9 Odds Ratio and Chi-Square Analysis of the Arg 515 Arg
Silent Mutation Occurring in Patients with AD as Compared to
Controls AD Control CX 69 33 AA 64 34 Chi-square = 0.84 OR. =
1.11
EXAMPLE 3
[0077] Use of the Variant GPIIb Allele Alone and in Combination
with the Variant GPIIIa Allele in Determining a Subject's Risk for
Alzheimer's Disease
[0078] Using the techniques presented in Example 1, we determined
the GPIIb genotype of patients with AD and normal control subjects
(Table 10).
13TABLE 10 Variant GPIIb Genotype in Normal Subjects vs. Patients
with AD GT GG (heterozygous TT Genotype (homozygous mt.) mt.)
(wild-type) Alzheimer's 15 71 50 Cases Control 8 28 34
[0079] We observed that a significant number of subjects with AD
had at least one mutant GPIIb allele. A chi-square and odds ratio
analysis was performed on this data set (Table 11). A significative
increase in the odds ratio (p value of 0.10) was seen in patients
with AD as compared to age-matched healthy control subjects. This
supports the notion that the GPIIb gene may be involved in the
development of neurological disease such as AD.
14TABLE 1 Odds Ratio and Chi-Square Analysis of the GPllb Allele
Occurring in Normal Subjects vs. Patients with AD CX versus TT
Genotypes Alzheimer's Cases Control GX (mutant genotypes) 86 36
TT(wild-type) 50 34 O.R. = 1.62 (C.I. 0.91 to 2.91) Chi-square p =
0.10
[0080] Given these findings we decided to explore the possibility
that the predictive value of the variant GPIIIa allele and the
variant GPIIb allele could be used together in predicting a
neurological disease risk. The occurrence of these alleles
appearing individually or together in normal subjects versus
patients with AD is presented below (Table 12).
15TABLE 12 GPIIIa (Leu33Pro)/GPIIb (IIe843Ser) Genotypes in Normal
Subjects vs. Patients with AD GPIIIa (L33P) GPIIb (I843S) Control
AD O.R. P value - - 27 34 Ref -- - + 29 53 1.45 0.28 + - 7 16 1.82
0.25 + + 7 33 3.74 0.005
[0081] Importantly, we found that in addition to the GPIIIa or
GPIIb variant alleles being present at high levels in patients with
AD (with an odds ratio of 1.82 and 1.45, respectively), together
these alleles were present at an even higher level (with an odds
ratio of 3.74). Stated another way, patients with AD are almost
4-fold more likely to have mutations in both the GPIIIa and GPIIb
allele than normal control subjects. Thus, we have determined that
there is an added predictive value or synergy in using both of
these alleles when evaluating a subject for a neurological disease
risk.
EXAMPLE 4
[0082] Use of the Variant GPIIIa and GPIIb Alleles for Prognosis in
Alzheimer's Disease
[0083] We believe that the method of the invention can be used as a
powerful prognostic tool for the treatment of Alzheimer's disease.
For example, subjects can be tested at an early asymptomatic age
for the presence of a variant GPIIIa and/or GPIIb allele and
administered an appropriate prophylactic therapy. Initially, for
asymptomatic subjects, this may involve a characterization of other
risk factors associated with Alzheimer's disease, avoidance of
environmental risk factors, and/or close monitoring. Accordingly, a
subject may be characterized as a candidate for prophylactic
therapies that can delay, inhibit, or prevent degenerative
neurological symptoms. Further, either alone or in combination with
other health data, the variant GPIIIa and GPIIb alleles can be used
to predict a subject's outcome by comparing the subjects GPIIIa and
GPIIb genotypes (and other health data) to a patient database
containing the GPIIIa and GPIIb genotypes (and other health data)
of similarly afflicted subjects. Based on this database comparison,
a subject's likely outcome, i.e., progression of disease, cure
rate, response to therapy, morbidity and mortality, can be
statistically assessed.
[0084] Thus, our results demonstrate that the presence of the
variant GPIIIa and/or GPIIb alleles can afford subjects at risk for
a neurological disease (e.g., Alzheimer's disease) the ability to
start prophylactic therapies before disease strikes. Ideally, the
risk of Alzheimer's disease is calculated for all individuals when
they are asymptomatic, young adults and well before the onset of
measurable symptoms. Then preventive therapies are invoked, as the
individual ages, in order to stop or lessen the progression of
Alzheimer's disease later in life.
Other Embodiments
[0085] The invention described herein provides a method for
treating subjects with a neurological disease risk by determining a
subject's GPIIIa and/or GPIIb genotype and providing an appropriate
therapy based on that determination. We believe that the predictive
value of these alleles may also include other variant GPIIIa or
GPIIb alleles associated with a neurological disease (e.g.,
Alzheimer's disease) and this may be readily determined using the
methods of the invention. For example, any other variant GPIIIa
allele may be detected using the methods described in Example 1.
Known polymorphisms in GPIIIa that may be determined to be variants
using the methods of the invention are: GPIIIa (ARG62Term), GPIIIa
(LEU117TRP), GPIIIa (ASP119TYR), GPIIIa (SER162LEU), GPIIIa
(ARG214GLN), GPIIIa (ARG214TRP), GPIIIa (CYS374TYR), GPIIIa
(PRO407ALA), GPIIIa (ARG636CYS), and GPIIIA (SER752PRO). Using the
guidance provided in Example 2, one can calculate the allelic
frequency of the variant GPIIIa allele/s in patients diagnosed with
Alzheimer's disease, as compared to healthy control subjects, and
determine if the particular variant GPIIIa allele is over
represented in patients with disease. Likewise, known polymorphisms
in GPIIb may also be exploited, alone, or in combination with the
above GPIIIa mutations. GPIIb variants which may be tested are:
GPIIb (LEU183PRO), GPIIb (GLY242ASP), GPIIb (PHE289SER), GPIIb
(GLU324LYS), GPIIb (ARG327HIS), GPIIb (GLY418ASP), GPIIb
(ARG553TERM), GPIIb (ILE565THR), GPIIb (GLN747PRO), and GPIIb
(SER870TERM). Furthermore, the predictive value of these alleles
can then be assessed and, if appropriate, used alone or in
combination with other risk factors for the treatment of
Alzheimer's disease.
[0086] In addition, while the methods described herein are
preferably used for the treatment of human subjects. Non-human
animals (e.g., pets and livestock) may also be treated using the
methods of the invention.
[0087] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each independent publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0088] Other embodiments are within the claims.
* * * * *