Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
CHROMOSOME 12 GENE AND GENE PRODUCTS
RELATED TO ALZHEIMER'S DISEASE
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 common 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
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 visuospatial and attention problems.
To date, three genes have been identified that, when
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 precursor protein
The major protein of the senile plaques is ~-amyloid
(A~), a 39 to 43 amino acid peptide (Glenner and Wong, 1984; Masters
et a/., 1985; ) derived from the ,B-amyloid precursor protein (APP).
Plaques are found mainly in the hippocampus and in the temporal lobe
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cortex. APP was the first gene in which mutations were found to cause
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 a/., 1991; Fidani et al., 1992; Goate et
a/., 1991; Karlinsky et al., 1992; Mullan et al., 1992; Murrell et a/., 1991;
Naruse et al., 1991; but they account for only 5% of published early-onset
FAD .
Presenilins
0 In 1992, Schellenberg etal (Schellenberg eta/., 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 ~rans,ne,nbrane
clo,na;ns. More than 35 different mutations have been found in the PS1
gene in over 50 families of difreren~ 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 pc I ~ ion
with familial early-onset AD indicated another locus on chromosome 1
(Levy-Lahad etal., 1995a). The chromoso,ne 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
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rare cause of FAD protein (Levy-Lahad et a/., 1995b; Rogaev et al.,
1 995).
APO e4
The apolipoprotein E (APOE) gene located on
5 c;h,o",osome 19q13.2 has been identified as a susceptibilty factor for AD
by genetic analysis of late-onset FAD pedigrees (Pericak-Vance et al.,
1991). APO Eis 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 dependenl 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 dcvelop AD.
APOE testing is therefore not useful for predicting whether someone will
develop AD.
Research on the molecular ethiology of the a co",plex
disease such as Alzheimer dise~se 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 complex disease. This markers can be
used eventually to provide genetic counselling in some affected families.
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-
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markers will also make it possible to evaluate the role of gene(s) in this
chromosomal region in the dirrerent 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 Alzheime~s disease (AD). Herein the
term AD-related nucleic acid is not meant to be restrictiv eto AD only
10 since other CNS disol-ders are herein shown to share common 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 sy, IU ~esi~ed chemically. Having the AD-
related nucleic acid segments of the present invention parts thereof or
oligos derived ll,e~ef,u"" 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 variety of purposes such as in vitro
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
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a cell, e~ressing 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 afffinity of the protein or
al(er"dli~ely, 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 ",onoclonal can be prepared
from the protein encoded by the Ad-related nucleic acid segment of the
invention. Such antibod:es can be used for a variety of purposes
including afffinity 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 lheref~o",, 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
predisposilion 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.
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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 inçludes, as a final s~, inge,)c~ wash, an incubation of the blot at 65~C
in 0.1 X SSPE, 0.1% SDS for 1 hour.
In the specirica~ions 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 fo",~ed, is
suitable. Since the formation of a stable duplex depends on the
sequence and length of the oligonucleotide and its complementarity to
25 the template it hybridizes to, as well as the hybridization conditions, one
skilled in the art may readily determine the degree of mismatching that
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can be tolerated betw~cn 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 seg" ,e"ts. Such mo il ~'~tors 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 conce"~s 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) compari,)g the level of activity of the product thereof or the
level of expression of the AD related sequence. It is herein
contemplated to use the control regions of AD-related nucleic acids
20 hooked to heterologous genes such as any appropriate reporter gene
(i.e. Iuciferase, chlora,nphenicol acetyl t,dns~erase, green fluorescent
protein or ,B-gAl~Gtosidase)
The invention is based on the results of an Associ~tion
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
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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.
0 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
presenoe 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 poly",o".his-"s or the like linked to Alzheimer's disease.
The use RFLP's is only one preferled embodiment of
detecting the polymorphisms. The most common methodology for
det~ the presence of RLFP is to carry out restriction analysis using
a given enzyme, pe,rullll a Southern procedure with a desired probe and
identify a given RFLP or RFLPs. The use RLFPs in linkage analysis and
genelic 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
dirrere,1ces 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
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be used. Techniques such as amplification of the desired regional
chromoso",e coupled with direct sequencing, a location of polymorphisms
and the c~" o" ,osome by radio-labelling, fluorescent-labelling and
enzyme-labelling can also be utilized.
DNA and/or RNA can be amplified using an amplificable
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
commonly known in the art.
Comparison of the RLFP or RLFPs for affected and
u"drre~ed individuals in the family line of the subject, with the RLFP or
RLFPs (or other ,neU lods) for the subject under investigation will quickly
reveal the presence or absence of the Alzheimer disease gene(s) in the
subject. Results of this expresses in terms of probability of presence of
the Alzheimer disease gene(s) in the subject.
A number of ~ thods 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
include random DNA sequences which can be tested for their specificity,
construction of DNA libraries and isolation of clones tl,eref,om. The
results of such methods is to identify a probe which can detect a
poly"~ol ~hisl-l useful for testing for Alzheimer disease. The polymorphism
must be found to be linked to Alzheimer disease or the other useful
markers in families studies, all to be adjacent to preexisting markers.
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A particular probe can have any desired sequence as long
as its is capable of identifying the polymorphism in the involved DNA
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
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
which can utilized in diagnosis.
Having now generally described the invention, the same will
be under~tood by re~erence to oertain specific eAdillFl~s that are provided
here in exemplary form only and are not intended to be limiting unless
otherwise specified.
DESCRIPTION OF THE PREFERRED EMBODIMENT
GENETIC ANALYSIS
The study of genetic diseases in families by linkage
analysis has been very useful to find the genes involved in simple genetic
disorders. But for complex disorders in which genetic factors may be
numerous and may be only part of the cause, family studies have given
only modest results. Methods based on drre~ed sib pairs which do not
necessitate knowledge of the familial inheritance pattern were successful
in a few cases. Finally, association studies which are designed as
case-control studies to compare unrelated affected and unaffected
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individuals in a pop~ ion are widely used to search for genes or genetic
markers that can be ~ssor,i~t~ with a di~se. In some cases, a positive
association can be found bec~use some patients in the sample are
distant relatives and thus share a specific variation in or around the
5 disease gene which is not widely present in the general population
(referred to as linkage disequilibrium). A systematic search of the
genome for such ~ssor,i~tions has been proposed, but this would require
a large number of DNA markers if done on a normal pop~ 'ion. However,
it was thought that this would be feasible in recenlly founded populations
10 because seemingly unrelated patients are in fact related close enough
that they share large sey,llents of DNA, i,lheriled with their disease gene
from corr""on ar,cestors (Houwen et al., 1995). This was recently
confirmed by the localization of the benign recurrent int, ahepatic
cholestasis (BRIC) gene in only three patients from an isolated
15 community in The Netherlands as well as for the infantile-onset
sp .,oo~r~LP"~~ataxia (IOSCA) gene in the Finnish population (Houwen
et al., 1995; Nikali et al., 1995).
One of the pr~clical adva. ,lages of this approach is that
there is no need to collect families as for linkage analysis or a large
20 number of ~f~t~ and urldl~;t3d individuals as for an ~-ss~ 'ion study.
All that is necessary is to find disla, Illy related affected individuals in an
appropriale popl ~ ion, that is, one which is relatively young, descended
from a relatively small number of fourlders, and which growth has
occurred primarily via reproduction and not by immigration.
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The Saguenay - Lac-Saint~ean population
The population of the Saguenay - Lac-Saint-Jean
(SLSJ) region in Quebec (Canada) is a founder population which has the
characteristics previously described. It is homogeneous from a
sociocultural point of view, being 95% francophone and of catholic
tradition. This is also true at the genetic level: some diseases show
relatively high or low incidences in SLSJ. The reasons for the genetic
homogeneity can be traced back to recent waves of immigration. SLSJ
was first opened to settlement around 1840. From this time until the
beginning of the 20th century, the neighboring region of Charlevoix -
which itself was relatively homogeneous - provided for the majority of
immigrants who settled in SLSJ. Moreover, the "familial nature" of this
immigration contributed to a more favorable implantation of the people
originating from Charlevoix as co",pared to other isolated immigrants
coming from other parts of Quebec. The rapid increase of this population
by natural reprodl ~ction all through the 1 9th century and the early part of
the 20th century also con~ iL,uted to its establishment as the main core of
the population of the SLSJ region (Bouchard and De Braekeleer, 1991;
Heyer and Tremblay, 1995).
We have conrir",ed that the SLSJ population is a
suitable population for linkage disequilibrium mapping by searching for
ancestral founder haplotypes around the genes of two single-gene
disorders which had been previously mapped: Steinert myotonic
dyslrophy and pseudo-vitamin D-deficient rickets (Bétard et al., 1995).
The results showed that we could have localized the appropriate genes
by doing a genome-wide screen with 10 cM- or 20 cM-spaced markers on
only ten patients taken randomly from the SLSJ population. Thus, the
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SLSJ population seems to meet the requirement necessary for applying
this method, that is, it is shallow in terms of genealogical distances
between patients.
5 Application of linkage disequilibrium mapping to Alzheimer disease
(AD)
Late-onset Alzheimer's disease has all the
chara~,te, islics which make it difficult to apply traditional linkage analysis
to find its genetic component or components: incomplete penetrance,
10 heterogeneity, phenocopies, etc. It is difficult to propose a model of
inheritance for this disease and to define the parameters necessary for
linkage analysis. Also the late age-of-onset precludes the collection of
families with many living patients over several generations. Linkage to
chromosome 19 has been reported, followed by evidence of an
15 association with the E4 allele of the apolipoprotein E gene on this
chromosome (Strittmatter et al., 1993; Poirier et a/., 1993; Rebeck et a/.,
1993; Saunders et a/., 1993). The apoE4 allele may be a major risk factor
for the late-onset form of the ~;ceA-se, but many patients do not carry this
allele. Thus, other genes are probably involved as well. To circumvent
20 the problems associated with traditional genetic studies in AD families,
we have applied the linkage disequilibrium approach on distantly related
AD cases from the population of SLSJ.
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METHODS
Sel~lion of a sample of late-onset Alzheimer patients
Selection of a sample of Alzheimer patients was done
by means of genealogical analysis. Sixty-three
5 neuropathologically-coi)ri""ed late-onset Alzheimer cases that is
defined as senile dementia of the Alzheimer type a (SDAT) were taken
from our brain bank. The SDAT diagnosis was established according to
a modified Khatchaturian scale (Khachaturian et al. 1985). Genealogical
data for these SDAT cases was obtained from IREP (Institut
10 Interuniversitaire de Recherches sur les Populations Chicoutimi
Québec). Ascending pedigrees were reconsl, ucted and analyzed in order
to select patients who were related through a limited number of common
ancestors at a distance of approximately six generations. First the
minimum number of generations connecting each of the 63 patients with
15 each of the others was determined. Cluster-type analysis provided a
dendrogram which summarized genetic distances between groups of
patients. Patients too closely or too dislanlly related were discar~led.
Genetic contribution of ancestor~ was also dete",~ined in order to identify
anoeslo, ~ who counted among their descendants a high number of SDAT
20 cases (Heyer and Trei"blay 1995). Only descend~n~s from these
souroes were selected We obtained a sa~ le of 23 SDAT cases who are
all related to each other at an average distance of 5.33 generations. The
average age of onset for AD in this sample is 73.7 + 6.4 years.
25 Linkage disequilibrium mapping
The 23 selected SDAT cases and two family members
were genotyped at more than 600 microsatelite markers (an average
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di~tarlce of app, OA;II ,ately 7 cM). A denser map of markers was analyzed
in the regions of the presenilin-1 gene (PS-1 ) on chromosome 14 which
is linked to early-onset AD (She~ington et al. 1995) and of the Apo E
gene on chromosome 19. Two types of pedigrees were studied: 1 ) the
5 case his or her spouse and one orr~pring (n=10); and 2) the case and
two ~rspring (n=13). The spouses' average age when the study began
was approximately 84 years and they are all related to each other
through their ascending pedigrees at an average distance of 6.25
generations.
An association-type analysis was done on the tested
markers by esti,nating the linkage disequilibrium parameter / (Terwilliger
1995) a measure of the degree of association or di~erence in allele
frequencies between a group of dise~se affected persons and a
non~ise~se control group at specific markers. This parameter is
15 mathematically defined in terms of conditional probabilities for allelic
frequencies given the absence or prese"ce of a dise~se chro",osome
and is esli")ated using a ",axi",um likelihood approach derived from
multinomial probability theory. Dr Lodewijk Sandkuijl ( Leiden and
Erasmus University The Nell,erlands) has modified the LINKAGE ILINK
20 program (Jurg Ott Columbia University N.Y. N.Y.) to calculate a
maximum likelihood e~limale of I from LINKAGE format pedigree data.
This modification performs a two-point analysis (marker and r~ise~se
locus) for any specified marker. It is capable of deducing
non~lise~se~arrying chromosomes to construct a control group. The
25 analysis was done under the dominance model and the frequency of the
disease chromosome was set at 0.01.
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Haplotypes were reco"s(, ucted and the 46 case
chr~r"osomes were searched for sharing of multiple successive markers;
co",pa, isons were made with the 20 spouse ~uo",osomes and - from the
orrspri,1g in the type-2 pedigrees - the 13 chro",osomes which were
transmitted by the non-dise~sed parent.
DATA ANALYSIS
Data analysis from a genome wide screening of
Alzheimers patients (23) using 700 microsatellites (positioned at an
average of 4 to 7 cM) reveals seven (excluding ApoE) difrerenl regions
in the genome which seem to be i,nplicaled in the physiopathology of AD.
Genetic markers represe,lting these regions have been sorted with
relative P values and are ordered from greatest impGI lance as follows:
D10S212 > D6S273 > D1S228 > D1S232 > Gata89a1 > D2S126 > ApoE
> 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 regions however were found to be weaker than those observed
for the microsalellites listed above.
The microsatellite D10S212 coincides with the region of
principal interest as revealed by fine mapping and is found to be
adjacent to an intron of the inositol polyphosphate-5-pl ,osphalase 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
and complex responses that often depend on the mobilization of Ca2+
CA 02203069 1997-04-18
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 reoeptors (reviewed by Golovina and Blaustein, 1997), and
5 release of Ca2 from one of the stores requires my~inositol 1,4,5-
llispl1osphate (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
10 by protein kinase C, while Type ll is not phosphorylated by this kinase.
5~hosphatase enzymes hydrolyze three subslrates 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).
Several studies suggest that alterations in the receptor-
1s mediated phosphoinositide cascade and cytosolic free calciumconcentration [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
phosphatidylinositol-specific phospholipase C on phosphalidylinosilol
20 4,5-bispl~ospl,ale in ,~spo"se 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 adenylate cyclase/cyclic nucleotide phosphodiesterase
25 system (Ross and Gilman, 1990), phospholipase C forms the active
signalling molecules, while the 5-phosphatase acts to degrade them.
CA 02203069 1997-04-18
18
Changes in the activity of either of these enzymes may alter cellular
responses to agonists.
Three inositol 1,4,5-trisphosphate receptors have so far
been cloned in humans. They mapped to three different chror"osoinal
regions: the Types 2 and 3 r~specti~ely in chromosome 1 2p1 1 and 6p21,
respectively, and the Type 1 in chromosome 1p. The inositol 1,4,5-
triphosphate ~eceptor~ (IP3R) act as IP3-gated Ca2 release channels in
a variety of cell types. The Type 1 receptor (IP3R1 ) is the major neuronal
member of the IP3R family in the central nervous system. It is
predominantly enriched in cerebellar Purkinje cells, but is also
concentrated in neurons of the hippocampal CA1 region, GAud~te-
putamen, and cerebral cortex. We have shown recently (unpublished
results), that Type 2 and Type 3 receptors are also expressed in specific
regions of the brain. Matsumoto et al.(1996) have shown that IP3R1-
deficient mice exhibit severe ataxia and tonic or tonic clonic seizures, and
die by the weaning period. Ele~t,oencepl1alog,dms de",onsl,ate that
such mice suffer from epilepsy, indicating that IP3R1 is esse,ltial for
proper brain function. Liu et al. (1995), in studies on juvenile myoclon--c
epilepsy (JME) in human families with cl~ssirvAl JME, shown that in a
region of about 7cM on chr~"~osor"e 6p21.2~11 an epilepsy locus exists
whose mutated phenotype consists of classic JME with convulsions
and/or elect~oencephalographic (EEG) rapid multispike wave complexes.
Again our marker D6S273 is within this interval.
IP3R binding sites were studied in autopsiecJ brains from
subjects with dementia of the Alzheimer type (DAT) and, in the parietal
cortex and hippocampus, there was a 50-70% loss of (3H( IP3 binding,
CA 02203069 1997-04-18
19
whereas no significant cl ,anges were observed in frontal occipilal and
te",poral cortices caudate or amygdala (L.Trevor Young et al. 1988).
Strikingly chro,noso",e 12p11 is not identified as a hot
spot by our genetic analysis. This could be explainable by the fact that
5 the founding population did not have a defect on chromosome 12p11.
Since a type ll inositol 1 4 5-lliphosphate receptor maps to the
chromosome 12p11 IP3R2 should also act as an important target in AD
diagnosis and/or treatment. Cloning and analysis of the IP3R2 gene will
be carried out to identify mutations or markers associated with AD and
10 CNS diso,der~ in general. The genomic DNA cor,esponding to the exon
and intron/exon junctions of the gene could be amplified using PCR and
screened for mutations by the method of single strand confol",alion
pol~,nol~his", (SSCP) from which some nucleotide changes have been
observed. Experi,nents employing RT-PCR to analyze this polymorphism
15 on the basis of dirrerential e~ression levels within a set of patient
samples shall also be pe, ro""ed
All of this data s~ron!Jly suggesls that one or more
col"pol-ents of the inositol pathway are considered as exoellent
cancJidates for the developl"ent of a physiopdtl,olog:cal model of
20 Alzheimer disease. In light of the fact that the IP3R1-/- (from human
chromosome 1) transgenic mice dcvelo~ epilepsy and that studies on
human families affected by the JME reveal that the arrect~ loci
cosegregate with chr~"~oso,ne 6p21 where the homolog gene (IP3R3) is
lo~ted it apped,~ highly probable that alterations in this pathway could
25 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
CA 02203069 1997-04-18
epilepsy in our examined population is significantly higher than that
normally e)~l~ected. These observations point tantilizingly towards the
hypothesis that various alteralions within the inositol biochemical
pathway may result in vastly differing phenotypic manifestations,
5 including epilepsy and Alzheimer's disease.
Having now identified the inositol phosphate pathway
and more specifically the IP3R2 gene as a key player in CNS disorders
and especially in AD, the present invention now permits a biochemical
~1isse~tjon of these ~~ise~ses. Further, genetic analysis can now be more
10 focussed, and should enable the identificalion of other genes or products
thereof which are part of the pathway or which affect it indirectly. Such
analyses should also enable the identification of the critical role of the
inositol pathway in other CNS disorders.
The present desu i~"ion refers to a number of documents, the conle"ts
15 of which are inco",oraled by re~erence.
CA 02203069 1997-04-18
REFERENCES
M.J.Berridge, R.F.lrvine. Inositol phosphates and cell signalling. Nature
341: 197-205, 1989.
V.A. Golovina, M.P.Blaustein. Spatially and functionally distinct Ca+
stores in sarcoplasmic and endoplasmic reticulum. Science 275:1643-
1648,1997.
10 K.M. Laxminarayan, B.K. Chan,T.Tetaz, P.l. Bird, C.A. Mitchell.
Chara~leri~dtion of a cDNA encoding the 43-kDa membrane-associated
inositol-poly~l ,ospha~e 5-phosphatase. J. Biol. Chem. 269: 17305-17310,
1994.
15 A.W. Liu, A.V. Delgado-Escueta, J.M. Ser,atosa, M.E. Alonso, M.T.
Medina, M.N. Gee, S. Cordova, H.Z. Zhao, J.M. Spelln~an, J.R. Ramos
Peek, F. Rubio Donnadieu, R.S.Sparkes. Juvenile Myoclonic Epilepsy
locus in ~;hro,l,osome 6p21.2-p11: Linkage to convulsions and
electroencephaloy, aph~ trait. Am.J.Hum.Genet. 57:368-381, 1995.
A.R. Maranto. Primary structure, ligand binding, and loG~ Ation of the
human type 3 Inositol 1,4,5-Trisphosphate Receptor expressed in
inles(inal epithelium. J. Biol. Chem. 269:1222-1230,1994.
25 M. Matsumoto, T. Nakagawa, T. Inoue, E. Nagata, lC Tanaka, H. Takano,
O. Minowa, J. Kuno, S. Sakakibara, M. Yamada, H. Yoneshima, A.
Miyawaki, Y. Fukuuchi, T. Furuichi, H. Okano, K Mikoshiba, T. Noda.
Ataxia and epileptic seizures in mice lacking type 1 inositol 1,4,5-
trisphosphate receptor. Nature 379; 168-171,1996.
T.S. Ross, A.B. Jefferson, C.A. Mitchell, P.W. Majerus. Cloning and
e~ression of human 75-kDa Inositol Poly~l)osphate-5-phosphatase. J.
Biol. Chem. 266:20283-20289,1991.
35 M. Ya",a",oto-Hino, T. Sugiyama, K. Hikichi, M.G. Mattei, K. Hasegawa,
S. Sekine, K. Sakurada,-A. Miyawaki, T. Furuichi, M. Hasegawa,
K.Mikoshiba. Cloning and characterization of human type 2 and type 3
Inositol 1,4,5-Trisphosphate receptors. Receptors and Channels 2:9-22,
1994.
CA 02203069 1997-04-18
L.T. Young, S.J. Kish,P.P. Li, J.J. Warsh. Decreased brain H3 inositol
1,4,5-trisphosphate binding in Alzheimer's disease. Neuroscience
Letters, 94: 198-202, 1988.
The present description refers to a number of
documents, the conten~s of which are incor~orated by referei ,ce.
M. C. Chartier-Harlin, F. Crawford, H. Houlden, A. Warren, D. Hughes,
L. Fidani, A. Goate, M. Rossor, P. Roques, J. Hardy et al. Early-onset
Alzheimer's disease caused by mutations at codon 717 of the beta-
amyloid precursor protein gene. Nature 353: 844 846 (1991).
E. H. Corder, A. M. Saunders, W. J. St,i(l"~dller, D. E. Schmechel, P. C.
Gaskell, G. W. Small, A. D. Roses, J. L. Haines, M. A. Pericak-Vance.
Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's
disease in late onset families. Science 261: 921-923 (1993).
L. Fidani, ~ Rooke, M. C. Chartier-Harlin, D. Hughes, R. Tanzi, M.
Mullan, P. Roques, M. Rossor, J. Hardy, A. Goate. Screening for
mutations in the open reading frame and promoter of the beta-amyloid
precursor protein gene in familial Alzheimer's disease: identification of a
further family with APP717 Val >lle. Hum. Mol. Genet. 1: 165-168
(1992).
G. G. Glenner, C. W. Wong. Alzheime~s dise~se: initial report of the
purification and chara~;teri~alion of a novel cerebr~/As~la- amyloid
protein. Biochem. Biophys. Res. Commun. 120: 885 890 (1984).
A. Goate, M. C. Chartier-Harlin, M. Mullan, J. Brown, F. Crawford, L.
Fidani, L. Giuffra, A. Haynes, N. Irving, L. James et al. Segregation of a
missense mutation in the amyloid precursor protein gene with familial
Alzheimer's disease. Nature 349: 704-706 (1991).
J. Kang, H. G. Lemaire, A. Unter6eck, J. M. Salbaum, C. L.1~12sters, K.
H. Grzeschik, G. Multhaup, K. Beyreuther, B. Muller-Hill. The precursor
of Alzheimer's disease amyloid A4 protein resembles a cell-surface
receptor. Nature 325: 733-736 (1987).
H. Karlinsky, G. Vaula, J. L. Haines, J. Ridgley, C. Bergero,1, M. Mortilla,
R. G. Tupler, M. E. Percy, Y. Robitaille, N. E. Noldy et al. Molecular and
prospective phenotypic characterization of a pedigree with familial
' CA 02203069 1997-04-18
Ald,ei."e,'s ~iseAse and a missense mutation in codon 717 of the beta-
amyloid precursor protein gene. Neurology 42: 1445-1453 (1992).
H. G. Lemaire, J. M. Salbaum, G. Multhaup, J. Kang, R. M. Bayney, A.
Unterbeck, K Beyreuther, B. Muller-Hill. The PreA4(695) precursor
protein of Alzheimer's diseAse A4 amyloid is encoded by 16 exons.
NucleicAcids Res. 17: 517-522 (1989).
E. Levy-Lahad, E. M. Wijsman, E. Nemens, L. Anderson, K. A. Goddard,
J. L. Weber, T. D. Bird and G. D. Schellenberg. A familial Alzheimer's
~liseAse locus on chromosome 1. Science 269: 970-973 (1995a)
E. Levy-Lahad, W. Wasco, P. Poorkaj, D. M. Romano, J. Oshima, W. H.
Pettingell, C. E. Yu, P. D. Jondro, S. D. Schmidt, K. Wang et al.
Candidate gene for the chromoso,. e 1 familial Ald ,e.me,'s disease locus.
Science 269: 973-977 (1995b).
C. L. Master~, G. Simms, N. A. Weinman, G. Multhaup, B. L. McDonald,
K. Beyreuther. Amyloid plaque core protein in Akl ~imer ~ise~se and
Down syndrome. Proc. Natl. Acad. Sci. U S A 82: 424~9. (1985).
M. Mullan, H. Houlden, M. Wi,l~el~ )l, L. Fidani, C. Lombardi, P. Diaz,
M. Rossor, R. Crook, J. Hardy, K. Duff et a/. A locus for familial early-
onset Ald ,eim~i 's ~lise~se on the long arm of ~I ,ro",osome 14, proximal
to the alpha 1-antichymotrypsin gene. Nat. Genet. 2: 34~342 (1992).
J. Murrell, M. Farlow, B. Ghetti, M. D. Benson. A mutation in the amyloid
precursor protein ~ssoci~ed with hereditary Alzheimer's ~;seAse.
Science 254: 9799 (1991).
S. Naruse, S. lgarashi, H. Kobayashi, K. Aoki, T. Inuzuka, K. Kaneko, T.
Shimizu, K. Iihara, T. Kojima, T. Miyatake et a/. Mis-sense mutation Val--
lle in exon 17 of amyloid precursor protein gene in Japanese familial
Alzheimer's ~;se~se. Lancet 337: 978-979 (1991).
M. A Pericak-Vance, J. L. Bebout, P. C. Gaskell, L. H. Yamaoka, W. Y.
Hung, M. J. Alberts, A. P. Walker, R. J. Bartlett, C. A. Haynes, K. A.
Welsh et a/. Linkage studies in familial Alzheimer disease: evidence for
chromosome19 linkage. Am J Hum Genet 48: 1034-50 (1991).
CA 02203069 1997-04-18
24
E. l. Rogaev, R. She"in!Jton, E. A. Rogaeva, G. Levesque, M. Ikeda, Y.
Liang, H. Chi, C. Lin, K. Holman, T. Tsuda et a/. Familial Alzheimer's
~~ise~se in kindreds with missense mutations in a gene on chromosome
1 related to the Alzheimer's disease type 3 gene. Nature 376: 775-778
(1995).
G. D. Schellenberg, T. D. Bird, E. M. Wijsman, H. T. Orr, L. Anderson, E.
Nemens, J. A. White, L. Bonnycastle, J. L. Weber, M. E. Alonso et a/.
Genetic linkage evidence for a familial Alzheimer's disease locus on
chromosome
14. Science 258: 668~71 (1992).
R. Sherrington, E. l. Rogaev, Y. Liang, E. A. Rogaeva, G. Levesque, M.
Ikeda, H. Chi, C. Lin, G. Li, K. Holman et a/. Cloning of a gene bearing
missense mutations in early-onset familial Alzheimer's dise~se. Nature
375: 754-760 (1995).
C. van Broeckhoven. Presenilins and Alzheimer's disease. Nat. Genet.
11: 230-232 (1995).
1. Houwen RHJ, Bai)a,loo S, Blankenship K, et a/. Genome screening by
searching for shared seg"~enls: mapping a gene for benign recurrent
i~ ,lral ,epatic cholest~sis, Nature Genet (1994) 8:380-386.
2. Nikali K, Suomalainen A, Terwilliger J, et al. Random search for
shared ~;I"-omos~"al regions in four dr~cted individuals: the assignment
of a new heredila,~ ataxia locus, Am J Hum Gen (1995) 56:1088-1095.
3. Bouchard G, De Braekeleer M, ed. Histoire d'un géno,ne: Population
et génétique dans l'est du Québec. Sillery, Québec: Presses de
l'Université du Québec, 1991.
4. Heyer E, Tremblay M.1995. Variability of the genetic contribution of
Quebec pop~ ~'~'ion founders associated to some deleterious genes. Am
J Hum Genet. 56:970-978
5. Bétard C, Raeymaeker, Ouellette G, et al. 1995. The validation of a
novel linkage disequilibrium mapping technique on Steinert myotonic
dystrophy and pse~ ~dovitamin D deficient rickets in a founder population.
40 Abstract. European Society of Human Genetics. Berlin, May 1995.
Medizinische Genetik.
CA 02203069 1997-04-18
6. St,it~matler WJ, Saunders AM, Schmechel D, et al. Apolipoprotein E:
high avidity binding to beta-amyloid and increased frequency of type 4
allele in late-onset familial Alzhemier's disease. Proc Natl Acad Sci USA
1993;90: 1977-81.
7. Poirier J, Davignon J, Bouthillier D, et al. Apolipoprotein E
polymorphism and Alzheimer's disease. Lancet 1993;342:697-9.
8. RebeckGW, ReiterJS, Strickland, et al. Neuron 1993;11:575~0.
9. Saunders AM, Strittmatter WJ, Schmechel D, et al. Association of
apolipotrotein E allele E4 with late-onset familial and sporadic
Alzheimer's disease. Neurology 1993;43: 1467-72.
10. Khachaturian ZS. Diagnosis of Alzheimer=s disease. Arch Neurol
1985;42: 1097-1105.
11. She" in~yto,l R, Rogaev El, Liang Y, et al. 1995. Cloning of a gene
bearing ",issense mutations in early-onset familial Alzhei,ne(-s disease.
Nature 375:754-760.
Te~illiger JD. 1995. A powerful likelihood method for the analysis of
linkage disequilibrium between trait loci and one or more polymorphic
marker loci. Am. J. Hum. Genet. 56:777-87.
Bétard C, Robitaille Y, Gee M, et al. 1994. Apo E allele frequencies in
AlzheimeGs dise~se, Lewy body dementia, AlzheimeGs ~ise~se with
cerebr~/ascular ~ se and vascular der"entia. Neuroreport. 5: 1893-95.
Schellenber GD, Payami H, Wijsman EM, ef al. 1993. Chromosome 14
and late-onset familial Alzheimer disease (FAD). Am. J. Hum. Genet.
53:619-28.
15. Wragg M, Hutton M, Talbot C et al. 1996. Genetic ~ssoci~'ion
between an intronic polymorphism in presenilin-1 gene and late-onset
Alzheimer's disease. The Lancet. 347:509-12.
