CA2203069A1 - Chromosome 12 gene and gene products related to alzheimer's disease - Google Patents

Abstract

The present invention discloses nucleic acid molecules encoding genes involved in CNS disorders such as Alzheimer's disease and epilepsy. Also disclosed are gene products, vectors and host cells suitable for expression of such gene products. Methods are providing for detecting the presence of a gene involved in Alzheimer's disease in human subjects and for treating humans suffering from Alzheimer's disease. Furthermore, markers which are associated with Alzheimer's disease are disclosed. Methods in kits for the detection of Alzheimer disease in a subject are additionally provided.

Description

TITLE OF THE INVENTION

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 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 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-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.

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 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.

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 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 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 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.

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 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.

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 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.

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 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.

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+

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.

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, 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 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.

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Claims

WHAT IS CLAIMED IS:

1. Chromosome 12p11 gene and gene products related to AD encode a member of the inositol phosphate pathway and use of said chromosome 12p11 gene and gene products for diagnosis and/or treatment of AD.