Note: Descriptions are shown in the official language in which they were submitted.
' CA 02203083 1997-04-18
TITLE OF THE INVENTION
GENE AND cDNA INVOLVED IN ALZHEIMER'S
DISEASE
5 FIELD OF THE INVENTION
This invention relates generally to central nervous
system (CNS) disorders. More particularly, this invention relates to
Alzheimer's disease. In addition the invention relates to the diagnosis
and treatment of Alzheimer's disease.
BACKGROUND OF THE INVENTION
Alzheimer's disease (AD) is the most con"non cause of
progressive cognitive decline in the aged population. It causes 100 000
deaths each year in the United States where it is the fourth leading cause
15 of death. Alzheimer described amyloid plaques, neurofibrillary tangles
and dementia that characterize AD in 1907. The usual presenting
symptoms are deficits of recent memory often in association with with
language and visuospati~l and attention problems.
To date, three genes have been identified that, when
20 mutated, can lead to early onset forms of AD and variation in a fourth one
has been implicated as a risk or susceptibility factor for AD.
~amyloid precursorprotein
The major protein of the senile plaques is ~amyloid
(A~), a 39 to 43 amino acid peptide (Glenner and Wong, 1984; Masters
25 et al., 1985; ) derived from the ~-amyloid precursor protein (APP).
Plaques are found mainly in the hippocampus and in the temporal lobe
cortex. APP was the first gene in which mutations were found to cause
CA 02203083 1997-04-18
familial Alzheimer's disease (FAD). The APP gene, located on
chromosome 21, has 19 exons and A~ is encoded by parts of exons 16
and 17 (Lemaire et al., 1989). Four mutations in the APP gene have
been described (Chartier-Harlin et al., 1991; Fidani et al., 1992; Goate et
a/., 1991; Karlinsky et al., 1992; Mullan et a/., 1992; Murrell et al., 1991;
Naruse et al., 1991; but they account for only 5% of published early-onset
FAD .
Presenilins
In 1992, Schellenberg etal (Schellenberg etal., 1992)
reported a second locus causing early-onset AD on chromosome
14q24.3. A positional cloning strategy permitted the identification of a
candidate gene, the S182 gene (Sherrington et al., 1995) later renamed
presenilin-1 or PS1, that carried coding region mutations in families
multiply affected by early-onset AD. The PS1 gene, composed of 10
exons, encodes a 467 amino acids protein with 7 to 10 transmembrane
domains. More than 35 different mutations have been found in the PS1
gene in over 50 families of different ethnic origins (see van Broeckhoven,
1995 for review). The proportion of early-onset familial AD cases due to
mutations in the PS1 gene is around 50%.
A genome -wide search conducted on another polulation
with familial early-onset AD indicated another locus on chromosome 1
(Levy-Lahad et al.,1995a). The chromosome 1 FAD gene was cloned by
virtue of its homology to PS1. The PS2 gene is composed of 12 exons
and encodes a 448 amino acids protein (Levy-Lahad et al., 1995b). It
shows 67% identity with the PS1 protein. Only two mutations have been
identified in the PS2 gene suggesting that mutations in this gene are a
CA 02203083 1997-04-18
rare cause of FAD protein (Levy-Lahad et al., 1995b; Rogaev et al.,
1 995).
APO e4
The apolipoprotein E (APOE) gene, located on
5 chromosome 1 9q13.2 has been identified as a susceptibilty factor for AD
by genetic analysis of late-onset FAD pedigrees (Pericak-Vance et al.,
1991). APO E is a major serum lipoprotein involved in cholesterol
metabolism. Three common isoforms of APOE are encoded by alleles
e2, e3, and e4 as a result of amino acids changes at codons 112 and
10 158. The APO e4 allele shows a dose dependent increase in risk for AD,
apparently mediated through a decrease in the age of onset of disease
(Corder et a/., 1993).
Not everyone having the susceptibility e4 allele will
develop illness and many who lackcthe allele will also develop AD. APOE
15 testing is therefore not useful for predicting whether someone will dcvclop
AD.
Research on the molecular ethiology of the a complex
disease such as Alzheimer disease has been confounded by the large
number of hereditary and environmental factors involved and by the
20 paucity of neuropathological and neurochemical studies on brains for
affected individual. The finding of a linked marker involved in one
hereditary form of Alzheimer disease will help to resolve the number of
different genes underlying this corl,plex disease. This markers can be
used eventually to provide genetic counselling in some affected fa,.,ilies.
25 Most importantly, the delineation of the genomic region containing
Alzheimer disease gene will provide a mean to eventually discover and
characterize this gene(s) in its encoded protein(s). The finding of link-
CA 02203083 1997-04-18
markers will also make it possible to evaluate the role of gene(s) in this
chromosomal region in the different levels of severity and onset of
Alzheimer's disease.
5 SUMMARY OF THE INVENTION
The invention seeks to provide diagnosis and therapeutic
tools for CNS disorders. Particularly the inventio seeks to provide
diagnosis and therapeutic tools for Alzheimer's disease (AD). Herein, the
term AD-related nucleic acid is not meant to be restrictiv eto AD only,
10 since other CNS disorders are herein shown to share co" "1)on genes and
products thereof.
The present invention seeks to provide a nucleic acid
segment isolated from human comprising at least a portion of a gene
responsible for CNS disorders and particularly to AD. The AD-related
15 nucleic acid segment can be isolated using conventional methods which
include for example YAC and BAC cloning, exon trapping and the like.
Such nucleic acids could also be synthesized chen, - ~"y. Having the AD-
related nucleic acid segments of the present invention, parts thereof or
oligos derived therefrom, other AD-related sequences using methods
20 described herein or other well known methods.
The invention also seeks to provide prokaryotic and
eukaryotic expression vectors harboring the AD-related nucleic acid
segment of the invention in an expressible from, and cells transformed
with same. Such cells can serve a variet,v of purposes such as in vifro
25 models for the function of AD-related gene as well as for screening
pharmaceutical compounds that could regulate the expression of the
gene or the activity of the protein encoded therefrom. For example, such
CA 022030X3 1997-04-18
a cell, expressing a DNA sequence encoding a protein involved in proper
neural function through the inositol phosphate pathway could serve to
screen for pharmaceutical compounds that regulate neural function or
inositol phosphate pathway.
An expression vector harboring AD-related nucleic acid
segment or part thereof, can be used to obtain substantially pure protein.
Well-known vectors can be used to obtain large amounts of the protein
which can then be purified by standard biochemical methods based on
charge, molecular weight, solubility or affinity of the protein or
alternatively, the protein can be purified by using gene fusion techniques
such as GST fusion, which permits the purification of the protein of
interest on a gluthathion column. Other types of purification methods or
fusion proteins could also be used.
Antibodies both polyclonal and monoclonal can be prepared
from the protein encoded by the Ad-related nucleic acid segment of the
invention. Such antibodies can be used for a variety of purposes including
affinity purification of the AD-related protein and diagnosis of a
predisposition to AD or othre CNS disorders.
The AD-related nucleic acid segment, parts thereof or
oligonucleotides derived therefrom, can further be used to identify
differences between AD affected individuals and non AD-affected
individuals. Similarly such segments can be used to identify a
predisposiVon to AD in individulas. The AD-related sequences can further
be used to obtain animal models for the study of CNS disorders.
Transgenic animals can be obtained. The functional activity of the AD
protein encoded by these nucleic acids, whether native or mutated, can
be tested in in vitro or in vivo models.
CA 02203083 1997-04-18
The human AD-related sequences can be used in a DNA-
based diagnostic assay to identify these individuals in the population who
are at risk for the above mentioned types of diseases.
Further, the present invention seeks to provide the use of
5 the AD-related protein as a pharmacological target for modulating
neuronal function and the like.
As used herein in the specifications and appended claims,
the term "oligonucleotide" includes both oligomers of ribonucleotides and
oligomers of deoxyribonucleotides.
The term high stringency hybridization conditions, as used
herein and well known in the art, includes, for example: 5 X SSPE (1 X
SSPE is 10 mM Na-phosphate, pH 7.0; 0.18 M NaCI; 1 mM Na2 EDTA),
5 x Denhardt's solution (from a 100 X solution containing 2% BSA, 2%
Ficoll, 2% polyvinyl pyrollidone), 0.1% SDS, and 0,5 mg/ml denatured
15 salmon sperm DNA, at 65~C. Other conditions considered stringent
include the use of formamide. An example of washing conditions for the
blot includes, as a hnal stringency wash, an incubation of the blot at 65~C
in 0.1 X SSPE, 0.1% SDS for 1 hour.
In the specifications and appended claims, it is to be
20 understood that absolute complementarity between the primers and the
template is not required. Any oligonucleotide having a sufficient
complementarity with the template, so that a stable duplex is formed, is
suitable. Since the formation of a stable duplex depends on the
sequence and length of the oligonucleotide and its complementarity to the
25 template it hybridizes to, as well as the hybridization conditions, one
skilled in the art may readily determine the degree of mismatching that
. CA 02203083 1997-04-18
can be tolerated between the oligonucleotide and its target sequence for
any given hybridization condition.
The invention features the means to identify factors that
modulate the transcriptional activity of AD-related genes. Such factors
5 include, without being limited thereto, other kinases, phosphatases,
nuclear receptors and transcriptionally regulatory proteins.
The present invention is also related to the use of AD-
realted sequences of the present invention and functional derivatives
thereof to screen for agents that modulate gene expression or the actity
10 of the products of these segments. Such modulators can be used as lead
compounds to design or search drugs that can modulate the level of
expression of these genes or the activity of their products.
Further, the present invention concerns a method for
measuring the ability of a compound to act as an agonist or antagonist of
15 AD-related gene products comprising (a) contacting the compound with
a transfected host cell expressing an AD-related sequence or mutant
threof, and (b) comparing the level of activity of the product thereof or the
level of expression of the AD related sequence. It is herein colltemplated
to use the control regions of AD-related nucleic acids hooked to
20 heterologous genes such as any appropriate reporter gene (i.e.
Iuciferase, chloramphenicol acetyl transferase, green fluorescent protein
or ~-galactosidase).
The invention is based on the results of an association
study in recently founded populations in which a linkage disequilibrium
25 mapping of Alzheimer's disease was carried out. This analysis permitted
the construction of haplotypes and enabled the identification of additional
. CA 02203083 1997-04-18
markers in the vicinity of the most significant markers identified by the
association analysis.
From these data, it was inferred that the Alzheimer~s
disease loci comprise D10S212, D6S273, D1S228, D1S232, Gata89a1,
D2S126, and D8S552.
Now that the location of Alzheimer's disease markers have
been identified, other markers can be found using methods known in the
art. Generally, primers are utilized which will identify markers associated
with Alzheimer disease, for example (GD)n and RFLP markers.
The invention also extents to products useful for carrying out
the assay, such as DNA probes (labelled or unlabelled), kits, and the like.
As broadest, the invention comprises detecting: the
presence of genes involved in Alzheimer's disease by analysing human
chromosomes, particularly chromosome 10, 6, 1, 9, 2 and 8 for further
markers or DNA polymorphisms or the like linked to Alzheimer's dise~se.
The use RFLP's is only one preferred embodiment of
detecting the polymorphisms. The most common methodology for
detecting the presence of RLFP is to carry out resl-i~tion analysis using
a given enzyme, perforrn a Southern procedure wHh a desiled probe and
identify a given RFLP or RFLPs. The use RLFPs in linkage analysis and
genetic testing is well known in the art (for example, see Gusella, US 4,
666,828 incorporated herein by reference in Donnus-Keller et al., 1987,
Cell. 51:319-337). It should be clear that other methods to identify
differences at the DNA level, or RNA level which are not related to RFLPs
can also be used. These methods are well known in the art of human
genetics. Any method capable of directing the polymorphisms can also
be used. Techniques such as amplification of the desired regional
, CA 02203083 1997-04-18
chro",osome coupled with direct sequencing, a location of polymorphisms
and the chromosome by radio-labelling, fluorescent-labelling and enzyme-
labelling can also be utilized.
DNA and/or RNA can be amplified using an amplificable
5 RNA sequence as a probe and q~-replicas.
The polynucleotide probes may be RNA or DNA and
preferably DNA, and can be labelled by standard labelling techniques
such as with the radio-label, enzyme-label, fluorescent-label, biotin-avidin
label and the like, which allow for the detection after hybridization as
10 commonly known in the art.
Comparison of the RLFP or RLFPs for affected and
unaffected individuals in the family line of the subject, with the RLFP or
RLFPs (or other methods) for the subject under investigation will quickly
reveal the presence or absence of the A!zheimer disease gene(s) in the
15 subject. Results of this expresses in terms of probability of presence of
the Alzheimer disease gene(s) in the subject.
A number of methods are available to the person of ordinary
skill to obtain other genetic sequences useful for probes in accordance
with the present invention. Non limiting examples of such methods
20 include random DNA sequences which can be tested for their specificity,
construction of DNA libraries and isolation of clones therefrom. The
results of such methods is to identify a probe which can detect a
polymorphism useful for testing for Alzheimer dise~se. The polymorphism
must be found to be linked to Alzheimer disease or the other useful
25 markers in families studies, all to be adjacent to preexisting markers.
A particular probe can have any desired sequence as long
as its is capable of identifying the polymorphism in the involved DNA
, CA 02203083 1997-04-18
regional or locus, it can be a DNA or RNA fragment, maybe synthesized
chemical, enzymatically or isolated from a plasmid as well known to the
person of ordinary skill. If a polymorphism is found in a gene product,
such as a mRNA, the presence of that polymorphic mRNA may be
5 assayed directly with the probe, especially with antisense RNA probe.
Now that chromosomal location of the Alzheimer disease
genes have been identified and defined to a small region, the region can
be cloned and characterized by general methods known in the art.
The method lends itself readily to the formulation of kits
10 which can utilized in diagnosis.
Having now generally described the invention, the same will
be understood by reference to certain specific examples that are provided
here in exemplary form only and are not intended to be limiting unless
othen,vise specified.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The human Type I phospl.atase, a critical determinant in AD
Data analysis from a genome wide screening of Al~l ,ei. ner's
paliel1ls (23), using 700 microsatellites (positioned at an average of 4 to
20 7 cM), reveals seven (excluding ApoE) different regions in the genome
which seem to be implicated in the physiopathology of AD. Genetic
markers representing these regions have been sorted with relative P
values, and are ordered from greatest importance as follows: D10S212
> D6S273 > D1S228 > D1S232 > Gata89a1 > D2S126 > ApoE >
25 D8S552. Other potential sites of interest have also been detected in the
genomic regions containing the Presenilin gene which have previously
been shown to be implicated in AD pathology. The P values for these
CA 02203083 1997-04-18
regions, however, were found to be weaker than those observed for the
microsatellites listed above.
The microsatellite D10S212 coincides with the region of
principal interest as revealed by fine mapping, and is found to be adjacent
5 to an intron of the inositol polyphosphate-5-phosphatase gene (IPP1).
This gene encodes a 43-Kda protein involved in the inositol phosphate
pathway, its role being that of a downregulator within the cascade by
inactivating inositol phosphate signalling molecules.
Biochemical messengers within most cells effect diverse
10 and complex responses that often depend on the mobilization of Ca2
from intracellular stores within the sarcoplasmic (in muscle) or
endoplasmic reticulum (S-ER). Two types of S-ER Ca2 stores have
been functionally characterized and identified by immunocyto-chemical
localization of receptors (reviewed by Golovina and Blaustein, 1997), and
15 release of Ca2 from one of the stores requires myo-inositol 1,4,5-
trisphosphate (IP3).
Two distinct human genes coding for 5-phosphatase (Types
I and ll) have been cloned, and encode for 43-kDa and 75-kDa proteins
respectively. The Type I protein is phosphorylated and activated by
20 protein kinase C, while Type ll is not phosphorylated by this kinase. 5-
phosphatase enzymes hydrolyze three substrates involved in calcium
mobilization: inositol 1,4,5-triphosphate (IP3), inositol cyclic 1:2,4,5-
tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate (IP4).
The published cDNA of the inositol polyphosphate-5-
phosphatase (IPP-1 ) is not complete, but we have cloned the full length
cDNA from a humain brain cDNA library as well as part of the promoter
~ CA 02203083 1997-04-18
region, and have determined the intron/exon junction sequences. The
genomic DNA corresponding to the exons and intron/exon junctions of the
gene have been amplified using PCR and screened for mutations by the
method of single strand conformation polymorphism (SSCP), from which
5 some nucleotide changes have been observed. All the polymorphisms
except one are found within non-coding sequences, or in the third codon
position in exons, which do not alter the amino acid residue. The only
non- conservative mutation found is within the third exon of the gene,
which changes the amino acid Iysine (MG) to arginine (AGG). However,
10 this transition is conservative if we consider the presence of a putative
phosphorylation consensus sequence serine/threonine-X-lysine/arginine
at that site. Experiments employing RT-PCR to analyze this
polymorphism on the basis of differential expressio" levels within a set of
patient samples did not show any differences.
Several studies suggest that alterations in the receptor-
mediated phosphoinositide casc~de and cytosolic free calcium
conceril,dlion [Ca2+]j are involved in the pathophysiology of aging, and in
Alzheimer's disease. Cellular calcium ion signalling is induced by inositol
phosphates formed directly or indirectly by the action of
20 phosphatidylinositol-specific phospholipase C on phosphdlidylinositol 4,5-
bisphosphate in response to extracellular agonists (Berridge and Irvine,
1989; Bansal and Majerus, 1990; Rana and Hokin, 1990). These inositol
phosphate signaling molecules are inactivated by inositol polyphosphate-
5-phosphatase enzymes (5-phosphatase). Thus, by analogy with the
25 adenylate cyclase/cyclic nucleotide phosphodiesterase system (Ross and
Gilman, 1990), phospholipase C forms the active signalling molecules,
CA 02203083 1997-04-18
while the 5-phosphatase acts to degrade them. Changes in the activity
of either of these enzymes may alter cellular responses to agonists.
One or more components of the inositol pathway are thus
considered as excellent candidates for the development of a
physiopathological model of Alzheimer disease. In light of the fact that
the IP3R1-/- (from human chromosome 1) transgenic mice develop
epilepsy, and that studies on human families affected by the JME reveal
that the affected loci cosegregate with chromosome 6p21 where the
homolog gene (IP3R3) is located, it appears highly probable that
alterations in this pathway could be shared by different forms of genetic
neurodisorders. If this proposed scenario is correct, we would expect to
find in our population of AD some incidence of epilepsy, and this is indeed
the case; the incidence of epilepsy in our examined population is
signihcantly higher than that normally expected. These observations
point tantilizingly towards the hypothesis that various alterations within the
inositol biochemical pathway may result in vastly differing phenotypic
manifestations, including epilepsy and Alzheimer's disease.
The 5-phospl1atase gene present on chromosome 10 was
cloned and its cDNA and gene sequence is shown in Fig. 1.
The present invention therefore provides sequences of the
human Type 1 5-phosphatase (IPP-1) and products thereof for diagnosis
and l,eal"~e"l of CNS disorders such as AD. Having identified this gene
from human, homologs thereof can be identified and isolated from other
animals. Such homologs could also be used in accordance with thye
present invention.
The present description refers to a number of documents,
the contents of which are incorporated by reference.
~ CA 02203083 1997-04-18
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