Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
5-PHOSPHATASE T~PE-II AND IMPLICATION IN
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 t,eat,nent of Alzheimer's disease.
BACKGROUND OF THE INVENTION
Ald~oimer's d:ceAse (AD) is the most COlllillGi I cause of
progressive cognitive decline in the aged population. It ~l~ses 100 000
deaths each year in the United States where it is the fourth leading cause
of death. Al l,ei."er described amyloid plaques neurofibrillary tangles
and dementia that characteri~e AD in 1907. The usual prese.~ting
sy""~toms are deficits of recent ",e,nory often in associdtion with with
language and vis~ ~osp~ti~l and attention problems.
T~ date three genes have been ide, lliried that when
ml It::~ted, can lead to early onset forms of AD and variation in a fourth one
has been implicated as a risk or susceplibility factor for AD.
~amyloid precursorprotein
The major protein of the senile plaques is ~-amyloid
(AO a 39 to 43 amino acid peptide (Glenner and Wong 1984; Masters
et al., 1985; ) derived from the ~-amyloid precursor protein (APP).
Plaques are found mainly in the hippocampus and in the temporal lobe
CA 0220308~ 1997-04-18
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 a/., 1989). Four mutations in the APP gene have
been described (Chartier-Harlin et a/., 1991; Fidani et al., 1992; Goate et
al., 1991; Karlinskyetal., 1992; Mullaneta/., 1992; Murrelletal., 1991;
Naruse et a/., 1991; but they account for only 5% of published early~nset
FAD .
Presenilins
In 1992, Scl,ellenberg eta/ (Schellenberg eta/., 1992)
reported a second locus causing early-onset AD on chro",os~me
14q24.3. A positional cloning strategy permitted the identification of a
candidate gene, the S182 gene (Sherrington et a/., 1995) later renamed
presenilin-1 or PS1, that carried coding region mutations in families
multiply affected by early-onset AD. The PS1 gene, co""~osed of 10
exons, encodes a 467 amino acids protein with 7 to 10 transmelnbrane
domains. More than 35 dirrerent mutatiGns have been found in the PS1
gene in over 50 fa,nil;es of different ethnic origins (see van Broechl~ven,
1995 for review). The proportion of early-onset familial AD cases due to
mutations in the PS1 gene is around 50%.
A geno",e -wide search conduGted on ~,~Uler polulation
with familial early-onset AD indicated another locus on chromosome 1
(Levy-Lahad et a/., 1995a). The cl ,romoso",e 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 a/., 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 a/.,
1 995).
APO e4
The apolipoprotein E (APOE) gene, located on
5 chromosome 1 9q13.2 has been ider,liried as a susceplibilty factor for AD
by genetic analysis of late-onset FAD pedigrees (Pericak-Vance et a/.,
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 dise~se
(Corder et a/., 1993).
Not everyone having the susceptibility e4 allele will
develop illness and many who lackcthe allele will also develop AD.
15 APOE testing is U ,erefore not useful for predicting whether someone will
develop AD.
Research on the ",o!ecul- ethiology of the a complex
disease such as Alzheimer ~iseAse has been confounded by the large
number of heredita,~ and enviror""elnal factors involved and by the
20 paucity of neu~op~tl,ological and neu,~lle",i -' 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
dirrere,~t genes underlying this complex ~ise~se. This markers can be
used eventually to provide genetic counselling in some affected families.
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-
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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 dise~se.
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 tl ,e~apeutic tools for Ald ,ei, ner's disease (AD). Herein, the
term AD-related nucleic acid is not meant to be restrictiv eto AD only,
10 sinoe other CNS ~Jisorder~ are herein shown to share con""on genes and
products thereof.
The present invention seeks to provide a nucleic acid
segment isol?ted from human co"~p,ising at least a portion of a gene
responsible for CNS disorder~ and particularly to AD. The AD-related
15 nucleic acid segment can be isol-'ed using conventional methods which
include for exa,nple YAC and BAC cloning, exon trapping and the like.
Such nucleic acids could also be s~l ~U ~si~ chemically. Having the AD-
related nucleic acid sey",enls of the present invention, parts thereof or
oligos derived lherer,om, other AD-related sequences using ",etl,ocls
20 described herein or other well known methods.
The invention also seeks to provide prokaryotic and
eukaryotic ex~"ession vectors l)a,L,o,ing the AD-related nucleic acid
segment of the invention in an e~ressible from, and cells l,ansro""ed
with same. Such cells can serve a varietv of purposes such as fn 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 pha",)aceutical compounds that regulate neural function or
inositol pl ,osphate 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 purifled by standard biochemical methods based on
charge, molecular weight, solubility or affinity of the protein or
~Ite,nali~/ely, the protein can be purified by using gene fusion techniques
such as GST fusion, which permits the purification of the protein of
i"leresl on a gluthathion column. Other types of purification methods or
fusion proteins could also be used.
Antibodies both polyclonal and " ,onoclol ,al can be p, ~par~
from the protein encoded by the Ad-related nucleic acid segment of the
invention. Such anlibocJias can be used for a variety of purposes
including affinity pu, iricaliGn of the AD-related protein and diagnosis of
a predisposilion 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 betv/een AD affected individuals and non AD-affected
individuals. Similarly such segments can be used to identify a
;sposition to AD in individulas. The AD-related sequenoes 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 diay, ~slic 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
indude the use of for",a~r,ide. An example of washing conditions for the
blot incl~des, as a final s~ ingenc~ wash, an in~ ~h~tion 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 ul~r~lood that absolute complementarity between the pri,ners and the
template is not required. Any oligonucleotide having a sufficient
co""~lementarity with the template, so that a stable duplex is for",ed, is
suitable. Since the formation of a stable duplex clepenJs on the
sequence and length of the oligonucleotide and its complementarity to
25 the ten,plale 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 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 ll,ereto, other kinases, pl,osp;)alases,
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,nents. Such modulators can be used as lead
compounds to design or search drugs that can modul~te the level of
expression of these genes or the activity of their products.
Further, the present invention concems a method for
measuring the ability of a compound to act as an agonisl or ~, Itagonist of
15 AD-related gene products co,n,c,rising (a) contacting the cG",pound with
a transfe~ed host cell expressing an AD-related sequence or mutant
threof, and (b) c~"pa, ing the level of activity of the product thereof or the
level of expression of the AD related sequence. It is herein
cGntel"plated to use the control regions of AD-related nucleic acids
20 hooked to heterologous genes such as any appro~riate rep~,ler gene
(i.e. Iucirerase, chloramphenicol acetyl transferase, green fluorescent
protein or ~ ctosid~se).
The invention is based on the results of an ~ssoci-'ion
study in recently founded populations in which a linkage dise~uilil~rium
25 mapping of Alzheimer's disease was carried out. This analysis perrnitted
the construction of haplotypes and enabled the identification of additional
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markers in the vicinity of the most significant markers idenliried by the
~ssoci~tion analysis.
From these data, it was i,lfer,~d that the Alzheimer's
~lise~se loci comprise D10S212, D6S273, D1S228, D1S232, Gata89a1,
D2S126, and D8S552.
Now that the localion of Alzheimer's dise~se markers have
been identified, other markers can be found using methods known in the
art. Generally, p(imer~ 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
preset~e of genes involved in Al~l ,ei...~, 's disease by analysing human
chromosomes, particularly ~ro",osG",e 10, 6, 1, 9, 2 and 8 for further
markers or DNA poly",~,ul ,;sms or the like linked to Ald)eime, 's d;seAse.
The use RFLP's is only one prefel,e~l e"lbo~i,nent of
detecting the poly",o"vl,is",s. The most common metllodolo~y for
detecting the presence of RLFP is to carry out resl, i~;tiOI I analysis using
a given enzyme, ~,rO"" a Southem pr~ceJure with a desired probe and
identify a given RFLP or RFLPs. The use RLFPs in linkage analysis and
gen~tic testing is well known in the art (for example, see ~ ~sell~ US 4,
666,828 i, ~rporaled herein by reference in Donnus-Keller et al., 1987,
Cell. 51:319-337). It should be clear that other ",etl~ds to identify
dirrerences 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
cl)ro"~osG",e coupled with direct sequen~ng a location of polymorphisms
and the chromosome by radio-labelling fluorescenl-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 fluorescenl~abel biotin-avidin
label and the like which allow for the detection after hybridization as
co"~ri~GI~ly known in the art.
Compa,ison of the RLFP or RLFPs for affected and
u, l~f~t~J individuals in the family line of the subject with the RLFP or
RLFPs (or other "n:thods) for the subject under invesligalion will quickly
reveal the pr~sence or absence of the Alzheimer diseAse gene(s) in the
subject. Results of this expresses in terms of probability of presence of
the Al~l ,ei."er d;seAse gene(s) in the subject.
A number of ll~tllods 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
indude ,a, ~" DNA sequences which can be tested for their specificity
construction of DNA libraries and isol-'ion of clones therehu",. The
results of such "~etl,ods is to identify a probe which can detect a
poly"~o,~l,i;"" useful fortesting forAlzheimer (li~A.se. 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. H a polymo(~ is", 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 iclenlified 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 desu il,ecl the invention, the same will
be unde, ~lo~ by r~ferenoe to oertain specific examples that are provided
here in exemplary form only and are not intended to be limiting unless
otherwise specified.
DESCRIPTION OF THE PRt~tKkED EMBODIMENT
GENETIC ANALYSIS
The study of genetic ~ise~ses in fa,nilies 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 affected 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~ ~'~tion are widely used to search for genes or ge"elic
markers that can be ~SSQC; ~'~ with a ~ise~se. In some cases, a positive
association can be found because 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 pop~ tion
(referred to as linkage disequilibrium). A systematic search of the
geno" ,e for such ~ssoci~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 recently founded populations
10 because seemingly unrelated patients are in fact related close enough
that they share large segments of DNA, inherited with their ~ise~-se gene
from common ancestors (Houwen et a/., 1995). This was recently
confirmed by the localkalion of the benign recurrent inbdhepalic
cholestasis (BRIC) gene in only three palie, Its from an isol-'ed
15 community in The Netherlands as well as for the infantile-onset
spinocerebellar ataxia (IOSCA) gene in the Finnish poru ~~tion (Houwen
et a/., 1995; Nikali et al., 1995).
One of the pr~ical advantages of this appr~a~ is that
there is no need to collect families as for linkage analysis or a large
20 number of ~ected and un~f~;ted individuals as for an ~ssori~'ion study.-
All that is necessary is to find distanlly related arreclecl individuals in an
approp, idle po~ ion, that is, one which is relatively young, descend~J
from a relatively small number of founders, and which growth has
occurred prima,ily via reproduction and not by i".r"ig(ation.
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12
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
characte, istics previously described. It is homogeneous from a
sociocultural point of view, being 95% r,anco,cl,one and of catholic
tradition. This is also true at the genetic level: some dise~ses 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 ho,l,ogeneous - provided for the majority of
immigrants who settled in SLSJ. Moreover, the "familial nature" of this
immigration cont,ibuted to a more favorable i,~"~lantation of the people
originating from Charlevoix as compared to other isol~ted immigrants
ooming from other parts of Quebec. The rapid increase of this population
by natural reprod~.tion all through the 1 9th century and the early part of
the 20th century also contributed to its esPhlisl"l,ent as the main core of
the popu~-tion of the SLSJ region (Bouchard and De Brael~eleer, 1991;
Heyer and Tr~ lay, 1995).
We have confi",)ed that the SLSJ population is a
suitable population for linkage disequilibrium mapping by searching for
ancestral founder haplotypes around the genes of two sillyle gene
disorders which had been previously marPe ~ Steinert myotonic
d~slro~l ,y and pseudo-vitamin D~icient rickets (Bétard et a/., 1995).
The results showed that we could have localized the appropriale 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 Applicaffon of linkage disequilibnium mapping to Alzheimer disease
~AD)
Late-onset Alzheimer's disease has all the
chara~,1eristics which make it difficult to apply traditional linkage analysis
to find its genetic component or co,nponents: incomplete penetrance,
10 heterogeneity, phenocopies, etc. It is difficult to propose a model of
inheritance for this disease and to define the parameters neoessary for
linkage analysis. Also the late age-of-onset precludes the collection of
families with many living patients over several ge"erations. Linkage to
chro,.,osome 19 has been reported, followed by evidence of an
15 ~ssooi-tion with the E4 allele of the apolipoprotein E gene on this
cl)~mosome(Sbit~nal~ereta/., 1993; Poiriereta/., 1993; Rebecketa/.,
1993; Saunders etal., 1993). The apoE4 allele may be a major risk factor
for the late onset form of the disease, but many patients do not carry this
allele. Thus, other genes are probably involved as well. To circumvent
20 the problems associaled with traditional genetic studies in AD families,
we have applied the linkage ~ise~uilibrium approach on dislantly related
AD cases from the population of SLSJ.
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METHODS
Selection of a sample of late~nset Alzheimer patients
Selection of a sample of Alzheimer patients was done
by means of genealogical analysis. Sixty-three
5 neuropall,ologically~"r,r",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 Kl~at~)at-Jrian scale (Khachaturian et a/. 1985). Genealogical
data for these SDAT cases was obtained from IREP (Institut
10 Interuniversitaire de Recherches sur les Populations Chicoutimi
Québec). Asoending pedigrees were reconstructed and analyzed in order
to select p~tients who were related through a limited number of common
ancestors at a distance of approximately six generatiGns. First the
minimum number of generations connecting each of the 63 patients with
15 each of the others was deterl"ined. Cluster-type analysis provided a
dendrograr" which summarized genetic distances between groups of
patients. Patients too closely or too distautly related were discar~ed.
Genetic c~r,b il ution of anoeslo~s was also determined in order to identify
a, ~ , who counted among their desoen~lants a high number of SDAT
20 cases (Heyer and Tremblay 1995). Only desce"Jants from b'lese
souroes were 5~1e~ ~ 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 miuosatelite markers (an average
CA 0220308~ 1997-04-18
di;,la"oe of approAi",ately 7 cM). A denser map of markers was analyzed
in the regions of the presenilin-1 gene (PS-1) on chroi,losome 14 which
is linked to early-onset AD (Sherrington 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 vrr~pring (n=10); and 2) the case and
two offspring (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 es~i,nating the linkage disequilibrium parameter I (Terwilliger
1995) a measure of the degree of associ~tion or dirrerence in allele
frequencies l)etv/een a group of disease drre~1ed persons and a
non-dise~se control group at specific markers. This para,neter is
15 mathematically defined in terms of conditional probabilities for allelic
frequencies given the absence or presence of a dise~se chromosome
and is esli"~ated using a maximum likelihood approach derived from
multinomial pr~bability theory. Dr Lodewijk Sandkuijl ( Leiden and
Erasmus University The Netherlands) has modified the LINKAGE ILINK
20 program (Jurg Ott Columbia University N.Y. N.Y.) to c~lcll~te a
maximum likelihood e:jlir,~ale of / from LINKAGE format pedigree data.
This modification pe,ror",s a two~oint analysis (marker and di~e~se
locus) for any specified marker. It is capatle of deducing
non-disease carrying 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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16
Haplotypes were reconsl, ucted and the 46 case
cl "~moso",es were searched for shal ing of multiple successive markers;
c~",pa(isons were made with the 20 spouse ~ "omosomes and - from the
offspring in the type-2 pedigrees - the 13 chror"osomes which were
s transmitted by the non-diseased 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) dirrerent regions
in the genome which seem to be i" ,plicaled in the physiopathol~gy of AD.
Genetic markers representing these regions have been sorted with
relative P values and are ordered from greatest i",pollance as follows:
D10S212 ~ D6S273 > D1S228 > D1S232 ~ ~t~89~1 > D2S126 ~ ApoE
~ D8S552. Other potential sites of inlerest have also been detected in
the genomic regiGI)s conlaining the rreseni1i., gene which have
previously been shown to be implicated in AD paU loloy~. The P values
for these regions however were found to be weaker than those observed
for the ",icrosatellites listed above.
The microsatellite D10S212 coincides with the region of
principal interesl as rcve~'ed by fine mapping and is found to be
a~jac~nt to an intron of the inositol poly~hospl)ate-5-,cl,ospl)alase gene
(IPP1). This gene encodes a 43-Kda pr~tein involved in the inositol
phosphate pathway its role being that of a downregu~tor within the
2s 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 0220308~ 1997-04-18
17
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
ibn of ,eceptor~ (reviewed by Ga'~v;.,a and Bl~ustein, 1997), and
5 release of Ca2+ from one of the stores requires my~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
pr~tei"s respecti~ely. The Type I protein is phospl ,o, ~lated and activated
10 by protein kinase C, while Type ll is not phospl,o,~lated by this kinase.
5 pl ,osphatase enzymes hydrolyze three su~,st, ates involved in calcium
mobilization: inositol 1,4,5-triphosphate (IP3), inositol cyclic 1:2,4,5-
tetrakisphosphate and inosilol 1,3,4,5-tetrakisphospllate (IP4).
Several studies s~ ~gest that alterations in the receptor-
15 mediated phosp;,oinositide G~scAde and cytosolic free calciumconce,It,dtion [Ca2 ]j are involved in the patl,opiIysiology of aging, and
in AW.ei.ne~'s diseAse. Cellular calcium ion signall;ng is induced by
inositol pl,os~ ates fo",~ed directly or indirectly by the action of
phospl,dtidylinositol-spe~lic phospholipase C on pl)osphdlidylinositol
20 4,5-bisphospl)~te in response to extr~r~ll~ agGIlis~s (Berridge and
Irvine, 1989; Bansal and Majerusl 1990; Rana and Hokin, 1990). These
inositol ,chospl~ale signaling molecules are inactivated by inositol
poly~hospl ,ale-5~1 ,ospl)dtase enzymes (5~hospl ,alase). Thus, by
analogy with the adenylate cyclase/cyclic nucleotide phosphou:eslerase
25 system (Ross and Gilman, 1990), phospholipase C forms the active
signalling molecules, while the 5-phosphatase acts to degrade them.
CA 0220308~ 1997-04-18
Changes in the activity of either of these enzymes may alter cellular
respo"ses to agonists.
Three inositol 1,4,5-trisphosphate receptors have so far
been cloned in humans. They mapped to three dirrerei-t ch,~",oso",al
regions: the Types 2 and 3 respectively in chromosome 12p11 and 6p21,
respectively, and the Type 1 in chromosome 1p. The inositol 1,4,5-
lli,,~i~osphate r~ceptor~ (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, caudate-
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-
cl~,ciant mice exhibit severe ataxia and tonic or tonic clonic seizures, and
die by the weaning period. Elect,oe,1cepl,alograrns d~",onst,ate that
such mice suffer from epilepsy, indicating that IP3R1 is essenlial for
proper brain function. Liu et al. (1995), in studies on juvenile myoclonic
epilepsy (JME) in human families with classical JME, shown that in a
region of about 7cM on cl"omosorne 6p21 .2-p11 an epilepsy locus exists
whose mutated phenotype consisls of classic JME with convulsions
and/or eleclr~l cephalographic (EEG) rapid multispike wave complexes.
Again our marker D6S273 is within this interval.
IP3R binding sites were studied in ~u top~ied 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 0220308~ 1997-04-18
19
whereas no significant changes were observed in frontal, occipital and
temporal cortices, caudate or amygdala (L.Trevor Young et al., 1988).
The third most promising region maps to a region of
approximately 6 cM surrounding the microsatellite D1S228. This gene,
5 the FK506 binding protein ripamycin Associ~ted proteinl (FRAP) whose
precise function is unknown, shows a homology with the C-terminal
regions ( 21% identity on average) of several phosphatidylinositol
kinases (Brown et al., 1994; Moore et al., 1996). In addition, and perhaps
of more relevance is the fact that the Type ll phosphatase (IPP-2,
10 75kDa), maps to this region of chro",osome 1. Thus once again the
genetic analysis reinforces the inositol pathway as a critical element in
AD. Based on clinical and genetic data, epilepsy and other CNS
disorders are likely to share at least some of the genes and gene
products involved in this pathway.
Cloning and analysis of the IPP-2 gene will be carried
out to identify mutations or markers ~ssoci~ted with AD and CNS
~isolJ~s in general. The geno,nic DNA co"esponding to the exons and
intron/exon junctions of the gene could be amplified using PCR and
screened for mutations by the ,nelhod of single strand confoll,~dtion
20 pol~",~,~)his", (SSCP), from which some nucleotide chan~es have been
observed. E~erin~enls employing RT-PCR to analyze this poly"~o" hism
on the basis of dirrerential e~ression levels within a set of patient
salllples shall also be pelror"~ed.
All of this data strongly suggesls that one or more
25 components of the inositol pathway are considered as excellent
candidates for the development of a physiopathological model of
Alzheimer disease. In light of the fact that the IP3R1-/- (from human
CA 0220308~ 1997-04-18
chromosome 1) l,ansgenic mice develop epilepsy, and that studies on
human families affected by the JME reveal that the arrected loci
cosegr~gale with .;hr~mosGme 6p21 where the homolog gene (IP3R3) is
located, it appears highly probable that alterations in this pathway could
5 be shared by dfflerent forms of genetic neurodisorders. If this proposed
scena, io 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 significantly higher than that
normally expected. These observations point tantilizingly towards the
10 hypothesis that various allerations within the inosilol biochemical
pathway may result in vastly differing phenotypic rnar,i~estatiGns,
including epilepsy and Alzheimer's dise~se.
Having now identified the inositol pl,osphate pathway
and more specifically the IPP-2 gene as a key player in CNS diso,de,s
15 and especially in AD, the present invention now pe".)its a bioche",i~al
.lissecti~ of these ~ e~ Es. Further, genetic analysis can now be more
fo~ Issed, and should enable the idel Iti~i~tion of other genes or products
thereof which are part of the pathway or which affect it indirectly. Such
analyses should also enable the identiricatiGn of the critical role of the
20 inositol pathway in other CNS d;sorders.
The present des~ i~tion refers to a number of
documents, the contenls of which are inc~ ,..oraled by refere,)ce.
CA 02203085 1997-04-18
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