CA2203070A1 - Chromosome 6 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 ner~ous 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 dise~se.

BACKGROUND OF THE INVENTION
A~d ,ei,ne~s ~Jisease (AD) is the most common cause of pr~gressive 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 characteri~e AD in 1907. The usual presenting s~ pt~ms are deficits of recent memory often in association with with language and visllosp~ti~l and attention problems.
To date, three genes have been identified that, when m~ted, can lead to early onset forms of AD and variation in a fourth one has been implicated as a risk or s~lsceplibility factor for AD.
~amyloid precursorprotein The major protein of the senile plaques is ~-amyloid (A~), a 39 to 43 amino acid peptide (Glenner and Wong, 1984; Masters et al., 1985; ) derived from the ~-amyloid precursor protein (APP).
Plaques are found mainly in the hippocampus and in the temporal lobe cortex. APP was the first gene in which mutations were found to cause 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 des~ il,ed (Chartier-Harlin et a/., 1991; Fidani et a/., 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 In 1992, Schellenberg eta/ (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 arrecled by early-onset AD. The PS1 gene, composed of 10 exons, encodes a 467 amino acids protein with 7 to 10 transmembrane domains. More than 35 different mutations have been found in the PS1 gene in over 50 families of difrerent 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 anvtl ,er polulation with familial early-onset AD indicated another locus on chror"osome 1 (Levy-Lahad eta/., 1995a). The chro",osome 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 rare cause of FAD protein (Levy-Lahad et a/., 1995b; Rogaev et a/., 1995).
APO e4 The apolipoprotein E (APOE) gene, located on 5 c;hro"loson)e 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 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 therefore not useful for predicting whether someone will develop AD.
Research on the molecular ethiology of the a complex disease such as Al,heil,~er disease has been confounded by the large number of heredilary and env;ronr,le,ltal factors involved and by the 20 paucity of neuropa~l)ological 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.
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-markers will also make it possible to evaluate the role of gene(s) in this chromosomal region in the different levels of severity and onset of Alzheimer's disease.

The invention seeks to provide diagnosis and therapeutic tools for CNS disorders. Particularly the inventio seeks to provide diagnosis and therapeutic tools for Alzheimer's disease (AD). Herein, the term AD-related nucleic acid is not meant to be restrictiv eto AD only, 10 since other CNS disor~ers are herein shown to share co""non 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 add segment can be isolated using conventional methods which include for example YAC and BAC cloning, exon trapping and the like.
Such nudeic acids could also be s~, IU ,esi~ chemically. Having the AD-related nucleic acid segments of the present invention, parts U ,ereof or oligos derived thereflu"" other AD~elated sequenoes using ",etl,ods 20 described herein or other well known methods.
The invention also seeks to provide prokaryotic and eukaryotic expression vectors ha~t~ri,)g the AD-related nucleic acid segment of the invention in an ex~ressible from, and cells llansrû""ed with same. Such cells can serve a variety of purposes sudh as in vitro 25 models for the function of AD-related gene as weli 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 oell eA~ressi"g a DNA sequenoe 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.
s An expression vector harboring AD-related nucleic acid se~""ent or part thereof can be used to obtain substantially pure protein.
Well-known vectors can be used to obtain large amounts of the protein which can then be purified by standard biochemical methods based on charge molecular weight solubility or affinity of the protein or alle"lali~/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 antibodies can be used for a variety of purposes including affinity purification of the AD-related protein and diagnosis of a predisposition to AD or othre CNS disorders.
The AD-related nucleic acid segment parts thereof or oligonucleotides derived theref,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 predisposition 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 diay, loslic 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 for,na",ide. An example of washing conditions for the blot incl~des, as a final s~ ingency wash, an incubation of the blot at 65~C
in 0.1 X SSPE, 0.1% SDS for 1 hour.
In the specifications and appended claims, it is to be 20 ~ "der~tood that absolute complementarity between the primers and the template is not required. Any oligonucleotide having a sufricient complementarity with the template, so that a stable duplex is formed, is suitable. Since the formation of a stable duplex depends on the sequence and length of the oligonucleotide and its complell,entalily 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 between the oligonucleotide and its target sequence for any given hybridization condition.
The invention features the means to identify factors that modulate the transcriptional activity of AD-related genes. Such factors 5 include, without being limited thereto, other kinases, phosph~t~ses, nuclear receptors and transcriptionally regulatory proteins.
The present invention is also related to the use of AD-realted sequences of the present invention and functional derivatives thereof to screen for agents that modulate gene expression or the actity 10 of the products of these segments. Such modulators can be used as lead cG"~pounds to design or search drugs that can modulate the level of expression of these genes or the activity of their products.
Further, the presènt invention concerns a method for measuring the ability of a compound to act as an agonist or antagonisl 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) co",pa, ing the level of activity of the product ll ,ereof 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, chlorampl1enicol acetyl transferase, green fluorescent protein or ~-g~ctosidase).
The invention is based on the results of an ~ssoci~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 infer,a-l that the Alzheimer's disease loci comprise D10S212, D6S273, D1S228, D1S232, ~ta89~1, 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. Ge"erally, primers are utilized which will identify markers ~ssoci~ted 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 presence of genes involved in Alzheimer's dise~se by analysing human chromosomes, particularly chromosome 10, 6, 1, 9, 2 and 8 for further markers or DNA pol~",o"vhis,ns or the like linked to Alzheimer's disease.
The use RFLP's is only one prefer,ed embodi."ent of detecting the polymorphisms. The most common ",etl,odology for dete~Aing the presence of RLFP is to carry out restriction analysis using a given enzyme, perform a Southem procedure with a desired probe and identify a given RFLP or RFLPs. The use RLFPs in linkage analysis and genetic testing is well known in the art (for example, see ~US~"~, US 4, 666,828 incG" orated herein by reference in Donnus-Keller et al., 1987, Cell. 51:319-337). It should be clear that other methods to identify differences at the DNA level, or RNA level which are not related to RFLPs can also be used. These methods are well known in the art of human genetics. Any method capable of directing the polymorphisms can also be used. Techniques such as amplification of the desired regional ro,noso,ne coupled with direct sequencing, a location of polymorphisms and the chromosome by radio-labelling, fluorescent-labelling and enzyme-labelling can also be utilized.
5DNA 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 10label 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 unaffected individuals in the family line of the subject, with the RLFP or RLFPs (or other methods) for the subject under investigation will quickly 15reveal 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 ~lise~se gene(s) in the subject.
A number of methods are available to the person of ordinary skill to obtain other genetic sequences useful for probes in accordance 20with the present invention. Non limiting examples of such methods include randorn DNA sequences which can be tested for their specificity, construction of DNA libraries and isolation of clones ther~,om. The results of such methods is to identify a probe which can detect a pol~",G"~his,n useful for testing for Alzheimer rliseAse. The polymorphism 25must 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 c~p~hle 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 u. ,der~lood by reference to certain specific examples that are provided here in exemplary form only and are not intended to be limiting unless othelwise 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 disor~ers.
But for complex diso~lers in which genetic factors may be numerous and may be only part of the cause, family studies have given only l.lodesl 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 individuals in a population are widely used to search for genes or genetic markers that can be ~sso~'ed with a disease. In some cases, a positive ~ssoci~tion can be found because some patients in the sample are distant relatives and thus share a specific variation in or around the disease gene which 5 is not widely present in the general population (referred to as linkage disequilibrium). A syste,na(ic search of the genome for such associations has been proposed, but this would require a large number of DNA
markers if done on a normal population. However, it was thought that this would be feasible in recently founded populations because seemingly 10 unrelated patients are in fact related close enough that they share large segments of DNA, inherited with their disease gene from cor"",o"
ancestors (Houwen et a/., 1995). This was recenlly conri""ed by the localization of the benign recurrent intrahepatic cholestasis (BRIC) gene in only three patients from an isolated community in The Netherlands as 15 well as for the infantile-onset spinoc~rebellar ataxia (IOSCA) gene in the Finnish population (Houwen et al., 1995; Nikali et al., 1995).

One of the practical advantages of this approacl ~ is that there is no need to collect families as for linkage analysis or a large number of 20 affected and unaffected individuals as for an association study. All that is necessary is to find distantly related affected individuals in an appropriate po~ ~'ntion, that is, one which is relatively young, descencl6J
from a relatively small number of founders, and which growth has occurred primarily via reproduction and not by im,1,igralion.

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 5 view, being 95~h francophone and of catholic tradition. This is also true at the genelic level: some ~~iseases 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 10 neighboring region of Charlevoix - which itself was relatively homogeneous - provided for the majority of i"~ igrants who settled in SLSJ. Moreover, the '~amilial nature" of this immigration contributed to a more favorable implantation of the people originating from Charlevoix as compared to other isolated immigrants coming from other parts of 15 Quebec. The rapid ina-ease of this population by natural reprod.lction all through the 19th century and the early part of the 20th century also contributed to its establishmenl as the main core of the pOp ul~tion of the SLSJ region (Bouchard and De Braekeleer, 1991; Heyer and Tremblay, 1 995).
We have cGnfi",~ed that the SLSJ population is a suitable pOp~ ion for linkage disequilibrium mapping by searching for ancest, al founder haplotypes around the genes of two single-gene disorder~ which had been previously ",ar.ped Steinert myotonic dyslrophy and 25 pseudo-vitamin D-deficient rickets (Betard 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
popul~tion seems to meet the requirement necessary for applying this eUlocl, that is, it is shallow in terms of genealogical distances between patients.

Application of linkage disequilibrium mapping to Alzheimer ~lic~se (AD) Late-onset Alzheimer's disease has all the characteristics which make it difficult to apply traditional linkage analysis to find its genetic 10 component or cG")ponents: inco,nplete penetrance, heterogeneity, phe,)ocopies, 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 patie, It~ over several generations. Linkage to chromosome 19 has 15 been repo,l~l, followed by evidence of an association with the E4 allele of the apolipoprotein E gene on this chromoso,ne (St,ill",dtler et a/., 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 ~lise~se, but many patients do not carry this allele. Thus, other genes are 20 probably involved as well. To circumvent 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
5el~ction of a sample of late-onset Alzheimer patients Selection of a sample of Alzheimer patients was done by means 5 of genealogical analysis. Sixty-three neuropathologically conri,l"ed late-onset Alzheimer cases that is defined as senile dementia of the Alzheimer type ' (SDAT) were taken from our brain bank. The SDAT
diagnosis was established according to a modified Khatchaturian scale (~achaturian et al. 1985). Genealogical data for these SDAT cases was 10 obtained from IREP (Institut Interuniversitaire de Recherches sur les Populations Chicoutimi Québec). Ascending pedigrees were reconsln~cted and analyzed in order to select patients who were related through a limited number of common ancestors at a distance of approxi"~lely six generalions. First the minimum number of generations 15 connecting each of the 63 patients with each of the others was determined. Cluster-type analysis provided a dendrogram which s~J"""a,i~ed genetic distances between groups of patients. Patients too closely or too dislar,lly related were d;scarded. Genetic conl. ibution of ancesl~ was also determined in order to identify anceslors who counted 20 among their descendants a high number of SDAT cases (Heyer and Tremblay 1995). Only descendants from these sources were selected.
We obtained a sample 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.

Linkage disequilibrium mapping The 23 selected SDAT cases and two family rne"lbers were genotyped at more than 600 microsatelite markers (an average distanoe of approximately 7 cM). A denser map of markers was analyzed in the 5 regions of the presenilin-1 gene (PS-1) on chromosome 14 which is linked to early~nset AD (Sherrington et al. 1995) and of the Apo E gene on chromosome 19. Two types of pedigrees were studied: 1 ) the case his or her spouse and one offspring (n=10); and 2) the case and two offspring (n=13). The spouses average age when the study began was 10 approximately 84 years and they are all related to each other through their ascending pedigrees at an average distance of 6.25 generations.

An ~ssoci~tion-type analysis was done on the tested markers by estimating the linkage disequilibrium para"~eter I (TenNilliger 1995) a 15 measure of the degree of association or dirrerence in allele frequencies between a group of disease affected persons and a non~ l;seAse control group at specific markers. This parameter is mathematically defined in terms of conditional pr~babilities for allelic frequencies given the absenoe or pr~senoe of a ~E~e~se chro"~osG",e and is ~s~ t~J using a ,.,~imum 20 likelihood approach derived from mulLino,l,ial probAhility theory. Dr Lodewijk Sandkuijl ( Leiden and Erasmus University The Netherlands) has modified the LINKAGE ILINK prograln (Jurg Ott Columbia University N.Y. N.Y.) to calculate a maximum likelihood esli,nale of / from LINKAGE
format pedigree data. This modification performs a two-point analysis 25 (marker and disease iocus) for any specified marker. It is capable of deducing non-disease-carrying chromosomes to construct a control group. The analysis was done under the dominance model and the frequency of the dise~se chromosome was set at 0.01.

Haplotypes were reconstructed and the 46 case chromosomes 5 were searched for sharing of multiple successive markers; coi"pa,isons were made with the 20 spouse chromosomes and - from the orr~pri,19 in the type-2 pedigrees - the 13 chromosomes which were transmitted by the non-diseased parent.

Data analysis from a genome wide screening of Alzheimer s patients (23) using 700 microsatellites (positioned at an average of 4 to 7 cM) reveals seven (excluding ApoE) different regions in the geno,ne which seem to be i",plica(ed in the physiopatl,ology of AD. Genetic 15 markers representing these regions have been sorted with relative P
values and are ordered from greatest importance as follows: D10S212 D6S273 ~ D1S228 ~ D1S232 ~ G~t~89~1 ~ D2S126 ~ ApoE ~
D8S552. Other poterdial sites of interest have also been detected in the genomic reg:~os containing the Presenilin gene which have previously 20 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 micros~tellites listed above.
The microsatellite D10S212 coincides with the region of principal inleresl as revealed by fine mapping and is found to be 25 adjace,~t to an intron of the inositol polyphosphate-5-phosphatase gene (IPP1). This gene encodes a 43-Kda protein involved in the inositol phosphate pathway, its role being that of a downregulator within the ~scAde 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~hemical iiol) of recepto,~ (reviewed by Golovina and Blaustein, 1997), and release of Ca2 from one of the stores requires my~inositol 1,4,5-trisphosphate (IP3).
Two distinct human genes coding for 5~hosphatase (Types I and ll) have been cloned, and encode for 43-kDa and 75-kDa proteins respectively. The Type I protein is phosphorylated and activated by protein kinase C, while Type ll is not phosphorylated by this kinase. 5-phosphatase enzymes hydrolyze three substrates involved in calcium mobilization: inositol 1,4,5-triphosphate (IP3), inositol cyclic 1:2,4,5-tetrakispl,osphate and inositol 1,3,4,5-tetrakisphosphale (IP4).
Several shldies sl~ggest that alterations in the receptor-mediated phospl)c.nositide cAscade and cytosolic free calcium concenl, ation [Ca2 ]j are involved in the pathophysiology of aging, and in Alzheimer's ~ise~se. Cellular calcium ion signalling is induced by inositol phosphates formed directly or indirectly by the action of phosphatidylinositol-specific phospholipase C on phospl,alidylinositol 4,5-bisphosphate in response to extracellular agonists (Berridge and Irvine, 1989; Bansal and Majerus, 1990; Rana and Hokin, 1990). These inositol phosphate signaling molecules are inactivated by inositol polyphosphate-5-phosphatase enzymes (5-phosphatase). Thus, by analogy with the adenylate cyclase/cyclic nucleotide phosphodiesterase 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 5 responses to agonists.
The second-most promising region, represented by the microsatellite D6S273, is in the vicinity (at a distance of about 2.7 cM) of a gene functionaly related to the IPP1, namely: the inositol 1,4,5-trisphosphate receptor type 3 gene (IP3R3). There is also on chromosome 12 another member of the inositol 1,4,5-trisphosphate receptor gene family, namely the type 2 receptor gene (IP3R2).
Three inositol 1,4,5-trisphosphate receptors have so far been cloned in humans. They mapped to three dirrerent chro",oso,nal regions: the Types 2 and 3 respectively in chr~moson)e 12p11 and 6p21, respectively, and the Type 1 in chromosome 1p. The inositol 1,4,5-triphospha~e recepto,~ (IP3R) act as IP3-gated Ca2 release channels in a variety of oell types. The Type 1 receplor (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 20 concentrated in neurons of the hippocampal CA1 region, ~d~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-d~:rlcient mice exhibit severe ataxia and tonic or tonic-clonic seizures, and 25 die by the weaning period. Electroencephalograms demonstrate that such mice suffer from epilepsy, indicating that IP3R1 is essential for proper brain function. Liu et al. (1995), in studies on juvenile myoclonic epilepsy (JME) in human families with cl ~ssic~l JME, shown that in a region of about 7cM on cluomosome 6p21.2-p11 an epilepsy locus exists whose mutated phenotype consists of classic JME with convulsions and/or electroencephalographic (EEG) rapid multispike wave complexes.
5Again our marker D6S273 is within this interval.
IP3R binding sites were studied in autopsied 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 changes were observed in frontal, occipital and 10temporal cortices, caudate or amygdala (L.TrevorYoung et al., 1988).
Cloning and analysis of IP3R3 gene will be carried out to identify mutations or markers ~ssociqted with CNS disorders. The genomic DNA corresponding to the exons and intronlexon junctions of the gene could be amplified using PCR and screened for mutations by the 15method of single strand confor",ation polymorphism (SSCP), from which some nucleotide changes have been observed. Ex~.eri~,ents employing RT-PCR to analyze this polymorphism on the basis of dirrare"tial e~ressio" levels within a set of patient samples shall also be pe, rO",~.
All of this data strongly sUggest-C that one or more 20co",ponents of the inositol pathway are considered as e~ll~t candidates for the development of a physiopathological model of Alzheimer disease. In light of the fact that the IP3R1~- (from human chromosome 1) transgenic mice develop epilepsy, and that studies on human families affected by the JME reveal that the affected loci 25cosegregate with ~romoso"1e 6p21 where the homolog gene (IP3R3) is localed, it appears highly probable that alterations in this pathway could be shared by different forms of genetic neurodisorders. If this proposed scenafio 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 5 hypothesis that various alterations within the inositol biochemical pathway may result in vastly differing phenotypic manifestations, including epilepsy and Alzheimer's disease.
Having now identified the inositol phosphate pathway and more specifically the IPP-1 gene as a key player in CNS disorders and 10 especially in AD, the present invention now permits a biochemical ~~issection of these ~;se~ses. Further, genetic analysis can now be more fo~ Isse~l and should enable the idefil;fic~tion of other genes or products thereof which are part of the pathway or which affect it indirectly. Such analyses should also enable the idenliricalion of the uitical role of the 15 inositol pathway in other CNS disorders.
The present desuiption refers to a number of documents, the conlents of which are incG",orated by reference.

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Claims (2)

1. Chromosome 6p21 gene and gene products related to AD
encode a member of the inositol phosphate pathway and use of said chromosome 6p21 gene and gene products for diagnosis and/or treatment of AD.

2. The Chromosome 6p21 gene and gene products related to AD wherein said gene and said gene products is IP3R3.