WO1996032502A1 - Reagent specific for apolipoprotein-j polymorphisms and uses thereof - Google Patents

Abstract

This invention provides a method for detecting in a suitable nucleic acid containing sample the presence of a polymorphism associated with an allelic variation in an apolipoprotein-J gene. The sample is contacted with an oligonucleotide capable of detecting the allelic variation under conditions such that the oligonucleotide hybridizes with nucleic acid contained in the sample, if such allelic variation is present in the nucleic acid. The presence of any oligonucleotide hybridized to the nucleic acid is detected and thereby the presence of the polymorphism in the apolipoprotein-J gene is detected in the sample. This invention also provides a method for detecting in a nucleic acid containing sample the presence of a polymorphism associated with an allelic variation in an apolipoprotein-J gene. This invention is suited for determining the probability of a subject developing Alzheimer's Disease.

Description

REAGENT SPECIFIC FOR APOLIPQPRQTEIN J POLYMORPHISMS AND USES THEREOF

This application is a continuation of U.S. Serial No. 08/420,291, filed April 11, 1995, the contents of which are hereby incorporated by reference into the present application. The invention disclosed herein was made with Government support under NIH Grants No. R35-AGIO963, POl- AG07232, RR00645 and P50-AG-08702 from the Department of Health and Human Services. Accordingly, the U.S. Government has certain rights in this invention.

Background of the Invention

Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

As a condition which affects about 10 percent of people over the age of 65 and which shows a definite but limited familial component, Alzheimer's Disease (AD) is a complex genetic disease with major public health importance. Several genetic loci have been associated with AD: the amyloid precursor protein (APP) gene on chromosome 21, the APOE gene on chromosome 19 and an incompletely characterized locus on chromosome 14 (reviewed in Poirier, 1994; Strittmatter et al . , 1994) . The chromosome 21 and 14 mutations are apparently rare and are associated with familial AD with onset of symptoms before age 50. In contrast, as first shown by Roses and coworkers (Strittmatter et al . , 1992) , the APOE gene, in particular the e4 allelic variant, is associated with sporadic and familial AD cf both late and earlv onset wit:. -.:. attributable risk in some populations estimated to be as high as 50 percent. In cross-sectional and case-control studies e4/e4 homozygosity is associated with a nearly 10- fold increase in risk of AD and 4/e3 heterozygosity is associated on average with an approximate 4-fold increase in risk. The association between e4 and AD has been investigated mostly in North American and European Caucasian populations (reviewed in Strittmatter et al . , 1994; Harrington et al . , 1994) , and to a lesser extent in other populations including Japanese (Noguchi et al . , 1993) , Finnish (Kuusisto et al . , 1994) and African-Americans (Mayeux et al . , 1993; Hendrie et al . , 1995; Maestre et al . , 1995) . In a community-based study of Caucasians, Hi panics and African-Americans in northern Manhattan a significant association was found between e4 homozygosity and AD in all three ethnic groups, but the association between e4 heterozygosity and AD was attenuated in African-Americans relative to Caucasians (Mayeux, et al., 1993; Hendrie et al . , 1995; Maestre et al . , 1995) , suggesting both similarities and differences in the genetic factors underlying AD in these ethnic groups.

The ApoJ protein (also referred to as clusterin, SGP-2, TRPM-2, AR, SP-40,40 and GpIII) can regulate complement function (Jenne et al . , 1989; Kirszbaum et al . , 1989) , and in addition shares certain interesting properties with ApoE, including cholesterol binding in apolipoprotein complexes (Kirszbaum et al . , 1989; de Silva et al., 1990) , production by astrocytes in response to inflammatory cytokines (Zwain et al . , 1994), increased expression after experimental brain injury in animals (May et al . , 1990; Pasinetti and Finch, 1991; Laping et al. , 1991), localization to the pathological amyloid-core plaques of AD (Choi-Miura et al . , 1992; McGeer et al., 1992) binding to soluble amyloid beta peptide (Ghiso et al. , 1993; Zlokovic et al . , 1994) and increased expression in AD brains (May et al . , 1990; Duguid et al . , 19891. Early work using isoelectric focusing (IEF) noted polymorphisms in serum ApoJ protein to be present in an African population and Americans of African descent but absent in Caucasians (Kamboh et al . , 1991) .

Summarv of the Invention

This invention provides a method for detecting in a suitable nucleic acid containing sample the presence of a polymorphism associated with an allelic variation in an apolipoprotein-J gene. The sample is contacted with an oligonucleotide capable of detecting the allelic variation under conditions such that the oligonucleotide hybridizes with nucleic acid contained in the sample, if such allelic variation is present in the nucleic acid. The presence of any oligonucleotide hybridized to the nucleic acid is detected and thereby the presence of the polymorphism in the apolipoprotein-J gene is detected in the sample. This invention also provides a method for detecting in a nucleic acid containing sample the presence of a polymorphism associated with an allelic variation in an apolipoprotein-J gene. This invention is suited for determining the probability of a subject developing Alzheimer's Disease.

Brief Description of the Fijures

Figures LA, IB and 1C: Detection and sequencing of APOJ coding polymorphisms.

(A) SSCP analysis of exon 7 PCR products. The diagnostic bands for the allelic variants are indicated by the arrowheads. Genotypes, confirmed by sequencing, are indicated above the lanes. The exon 7 3' (b) primer was used as the downstream primer. (B) Direct sequencing of exon 7 PCR products. The allelic nucleotide substitutions are indicated by the brackets. Genotypes are indicated below each sequence set. (C) Slot-blot analysis of exon 7 PCR products with ASOs. Genotypes, confirmed by SSCP and direct PCR sequencing, are indicated on the left.

Figure 2: Alignment of human ApoJ protein polymorphisms (SEQ ID NO: 21) with the rat ApoJ sequence (SEQ ID NO: 20) .

Amino acids are numbered according to de Silva et al . , 1990. Amino acid identities are indicated by the asterisks, conserved cysteines are shown by the dots and potential glycosylation sites are indicated by the rectangles with the variable potential glycosylation sites cross-hatched. The human polymorphisms are indicated by the arrows.

Figure 3: Nucleotide sequence of the J2 allele-speci ic oligonucleotide (SEQ ID NO: 17) .

Figure 4: Nucleotide sequence of the J3 allele-specific oligonucleotide (SEQ ID NO: 18) . Detailed Description of the Invention

This invention provides a method for detecting in a suitable nucleic acid containing sample the presence of a polymorphism associated with an allelic variation in an apolipoprotein-J gene. The sample is contacted with an oligonucleotide capable of detecting the allelic variation under conditions such that the oligonucleotide hybridizes with nucleic acid contained in the sample if such allelic variation is present in the nucleic acid. The presence of any oligonucleotide hybridized to the nucleic acid is detected and thereby the presence of the polymorphism in the apolipoprotein-J gene is detected in the sample.

As used herein, the allelic variation may be a J2 variant, a J3 variant or a neutral polymorphism of the apolipoprotein-J gene. The sample may be cDNA, a cloned human genomic library, blood, urine, plasma, serum or tissue. The oligonucleotide may be labeled with a detectable moiety including a florescent label, a radioactive atom, a chemiluminescent label, a paramagnetic ion, biotin or a label which can be detected through a secondary enzymatic or binding step. The secondary nonradioactive enzymatic or binding step may utilize digoxigenm, alkaline phosphatase, horseradish perox dase, /^β-galactosidase, fluorescein or streptavidin/biotm (e.g. , Boehringer Mannheim, Genius_* Systems) . The subject may be of African or Hispanic descent.

This invention also provides a method for detecting in a nucleic acid containing sample the presence of a polymorphism associated with an allelic variation m ar. apolipoprotein-J gene. The sample is contacted with a pair of polymerase chain reaction oligonucleotide primers capable of hybridizing with nucleic acid sequences encoding tne apolipoprotein-J gene under conditions allowing for hybridization. This mixture is contacted with DNA poly erase under conditions allowing for amplification to form an amplified polymerase chain reaction product which is then denatured so as to form a single-stranded polymerase chain reaction product. This product is contacted with an oligonucleotide capable of detecting the allelic variation under conditions such that the oligonucleotide hybridizes with the single-stranded polymerase chain reaction product if such allelic variation is present in the nucleic acid. The presence of any oligonucleotide hybridized to the single-stranded polymerase chain reaction product is detected and thereby the presence of the polymorphism in the apolipoprotein-J gene is detected in the sample.

One embodiment of this invention is a method of determining the probability of a subject developing Alzheimer's Disease.

A suitable biological sample is obtained from the subject and contacted with an oligonucleotide capable of detecting an allelic variation in the apolipoprotein-J gene under conditions such that the oligonucleotide hybridizes with nucleic acid contained in the sample, if such an allelic variation is present in the nucleic acid. The presence of any oligonucleotide hybridized to the allelic variation present in the apolipoprotem-J gene is detected and thereby the probability of a subject developing Alzheimer's Disease is determined.

This invention provides a method for detecting in a subject the presence of a polymorphism associated with an allelic variation in an apolipoprotein-J gene. A suitable biological sample containing nucleic acid is obtained from the subject and contacted with a pair of polymerase chain reaction oligonucleotide primers capable of hybridizing with nucleic acid sequences encoding the apolipoprotein-J gene under conditions allowing for hybridization. This mixture is contacted with DNA polymerase under conditions allowing for amplification to form an amplified polymerase chain reaction product and the product is denatured so as to form a single-stranded polymerase chain reaction product. The product is contacted with an oligonucleotide capable of detecting the allelic variation under conditions such that the oligonucleotide hybridizes with the single-stranded polymerase chain reaction product if such allelic variation is present in the nucleic acid. The presence of any oligonucleotide hybridized to the single-stranded polymerase chain reaction product is detected and thereby the presence of the polymorphism in the apolipoprotein-J gene of the subject is detected.

Another embodiment of this invention is a method of determining the probability of a subject developing a cognitive disorder. A suitable biological sample is obtained from the subject and contacted with an oligonucleotide capable of detecting an allelic variation in the apolipoprotein-J gene under conditions such that the oligonucleotide hybridizes with nucleic acid contained in the sample, if such an allelic variation is present in the nucleic acid. The presence of any oligonucleotide hybridized to the allelic variation present m the apolipoprotein-J gene is detected and thereby the probability of a subject developing a cognitive disorder is determined.

Another embodiment of this invention is a method of determining the probability of a subject developing prostatic carcinoma. A suitable biological sample is obtained from the subject and contacted with an oligonucleotide capable of detecting an allelic variation in the apolipoprotein-J gene under conditions such that the oligonucleotide hybridizes with nucleic acid contained in the sample, if such an allelic variation is present m the nucleic acid. The presence of any oligonucleotide hybridized to the allelic variation present in the apolipoprotein-J gene is detected and thereby the probability of a subject developing prostatic carcinoma is determined.

This invention provides a reagent capable of detecting the presence of a polymorphism associated with an allelic variation in an apolipoprotein-J gene or gene product. The reagent may detect the J2 or J3 allelic variant and may be an oligonucleotide encoding a polypeptide having at least a portion of the sequence shown in Figure 2 (SEQ ID NO: 20 or 21) . This oligonucleotide may be the sequence TGTTCCACCAACCACCC (SEQ ID NO: 17) or the sequence GAGCTCGACGAATCCCT (SEQ ID NO: 18) . The reagent may be an oligonucleotide which is a complementary sequence of SEQ ID NO: 17 or 18. The reagent may be an antibody specific for a polypeptide associated with the J2 or J3 variant of apolipoprotein-J having at least a portion of the polypeptide sequence shown in Figure 2 (SEQ ID NO: 20 or 21) . This polypeptide preferably has about 8 to about 20 amino acids.

Another embodiment of this invention is a method for determining whether a compound is capable of interfering with the formation of a complex between a polymorphic apolipoprotein-J protein and an amyloid _/S-protein. The compound is incubated with an appropriate apolipoprotein-J protein affinity derivative and an amyloid -protein or with an appropriate amyloid _/S-protein affinity derivative and an apolipoprotein-J protein. This incubate is contacted with an appropriate affinity medium under conditions so as to bind the amyloid S-protein-apolipoprotein-J-affinity derivative-protein complex or the apolipoprotein-J-amyloid -protein-affinity-derivative-protein complex, if such a complex forms. The amount of the affinity protein complex formed is measured so as to determine whether the compound is capable of interfering with the formation of the complex between the apolipoprotein-J protein and the amyloid β - protein. In one embodiment of this invention, one could utilize the method of Fields and Sonα (see U. S. Patent Kc. 5,283,173) and covalently link either apolipoprotein-J or the amyloid S-protein to the Gal4 DNA binding domain and the other to the Gal4 activation domain in order to analyze the protein-protein interaction (see also, Clontech Matchmaker Two Hybrid System., Clontech® 1995 Catalog) .

Another embodiment of this invention is a method for determining the concentration of a polymorphic apolipoprotein-J protein in a biological fluid. The fluid is contacted with a measured amount of a soluble first monoclonal antibody to the polymorphic apolipoprotein-J protein in order to form a soluble complex of the antibody and the polymorphic apolipoprotein-J protein present m the fluid, the first monoclonal antibody being labeled The soluble complex is contacted with a second monoclonal antibody to the polymorphic apolipoprotein-J protein, the second monoclonal antibody being bound to a solid carrier, the solid carrier being insoluble in the fluid, in order to form an insoluble complex of the first monoclonal antibody, the polymorphic apolipoprotein-J protein and the second monoclonal antibody bound to the solid carrier. The solid carrier is separated from the fluid sample and unreacted labeled antibody and either the amount of labeled antibody associated with the solid carrier or the amount of unreacteα labeled antibody is measured. The amount of labeled antibody measured is compared with the amount of labeled antibody measured for a control sample prepared in the same manner described above, to determine the presence of the polymorphic apolipoprotein-J protein in the fluid sample. Alternatively, the amount of labeled antibody measured is related with the amount of labeled antibody measured foi samples containing known amounts of the polymorphic apolipoprotein-J protein prepared in the same manner as described above, to determine the concentration of tne polymorphic apolipoprotein-J protein m the fluid sample.

This invention provides an antibody immunoreac ive with ar. allele-specific antigen associated with a specific apolipoprotein-J polymorphism. The antibody may be specific for an antigen which may be a polypeptide from at least a portion of the sequence shown in Figure 2.

This invention provides a transgenic nonhuman mammal whose somatic and germ cells contain and express a gene coding for an allelic variant of an apolipoprotein-J gene. The gene, having been introduced into the nonhuman mammal, or an ancestor of the nonhuman mammal at the single cell stage or an embryonic stage, is operably linked to a promoter and integrated into the genome of the nonhuman mammal. One skilled in the art would be familiar with the experimental methods necessary to produce a transgenic mammal, e.g. Leder et al., U. S. Patent No. 4,736,866 and Krimpenfort and Berns, U. S. Patent No. 5,175,384 and Wagner and Chen, U. S. Patent No. 5,175,385. Preferably, the nonhuman mammal may be a mouse. The gene may be a combination of human apolipoprotein-J nucleic acid sequences and adjacent, homologous nonhuman mammal apolipoprotein-J nucleic acid sequences. The promoter may be a nerve tissue specific promoter such as the mouse neurofilament-light gene promoter or the rat neuronal specific enolase promoter (Forss-Petter et al . , 1990) , which is effective for the expression of the gene in neuronal cells of the brain. The human platelet- derived growth factor-/? gene promoter, which is effective for the expression of the gene in cells of the brain may also be utilized. Other nerve tissue specific promoters which may be used are rat sodium channel gene promoter (Maue et al . , 1990) , the human APP gene promoter (Wirak et al . , 1991) and mouse mylein basic protein gene promoter (Readhead et al . , 1987) . A yeast artificial chromosome construct containing the human apolipoprotein-J gene may also be utilized.

This invention provides a nonhuman mammal whose neuronal cells or glial cells or both, express an allelic variant of an apolipoprotein-J gene. Preferably, the nonhuman mammal may be a mouse. The gene, having been introduced into the mouse by localized infection with retrovirus, is operably linked to a promoter. The retrovirus has an inducible retroviral vector consisting of a marker gene, a constitutive promoter and an inducible promoter. Retroviral-mediated gene transfer is a procedure known to individuals skilled in the art. Procedures for the infection of neuronal progenitor cells have been established, see, for example, Levison and Goldman (1993) .

ApoJ-containing retroviral expression constructs may be introduced into fetal and neonatal animals by direct viral infection of subventricular zone (primitive neuronal and glial precursor) cells (see Levison and Goldman, 1993) . In this protocol, the ApoJ constructs may be cloned downstream of a constitutive promoter (e.g. SV40) in tandem with a beta-galactosidase gene under the control of the retroviral long terminal repeat (LTR) promoter. Thus, ApoJ-producing retrovirally infected cells will be specifically marked by /S-galactosidase enzymatic activity (i.e. blue stain in tissue sections) . It would then be possible to search for effects of local ApoJ protein overexpression in the intact animal brain on neuronal morphology, amyloid deposition, tau protein phosphorylation and determine whether these effects differ for each ApoJ isoform. If pathological changes are observed, then these animals would serve as a useful in vi vo assay system for pharmacological agents with activity against AD pathology.

The transgenic nonhuman mammals may provide an experimental medium for elucidating aspects of the molecular pathogenesis of AD and to serve as tools for screening drugs that may have potential application as therapeutic agents to prevent or limit plaque formation. Transgenic nonhuman mammals provide both a prognostic and diagnostic means for the study of AD, in particular for determining the efficacy of pharmaceutical drugs in treating a subject.

A search for DNA sequence polymorphisms in the APOJ/CLI gene (on chromosome 8, Fink et al. , 1993; Minoshi a et al . , 1991) was carried out and the frequencies of these polymorphisms were calculated and their possible association with AD in expanded versions of the original African-American and Hispanic cohorts was determined.

Expression of ApoJ protein has been shown to be associated with chemically-induced carcinogenesis in rat prostate and seminal vesicle (Kadomatsu, K. et al . , 1993) . ApoJ mRNA and protein are overexpressed in both prostatic carcinoma and in the regressing prostate gland in androgen-depπved rats. This is evidence for a possible association of this protein with prostatic carcinoma. A particular variant of the ApoJ protein may more efficiently protect prostatic cells from cell death and therefore lead to an increased susceptibility to the subsequent development of malignant clones. Direct evidence may come from a genetic epidemiological study using the APOJ polymorphisms. A molecular marker such as a particular ApoJ allele would be useful m the early determination of prostatic carcinoma. Prostatic carcinoma shows a reproducibly higher incidence in African-Americans than in American Caucasians. The genetic basis for the higher incidence is not known.

Attempts have been made to study a number of different types of genetic diseases utilizing transgenic animals. Methods to produce transgenic animals are well established and familiar to one skilled in the art. Transgenic animal models to study AD have been made using amyloιd-_/3-proteιn constructs. U.S. Patent No. 5,387,742 discloses transgenic mice which express the amyloιd- ?-protem m neuronal tissues. PCT International Publication No. WO 93/14200 discloses transgenic animal models for AD which express the amyloιd-3-proteιn. WO 93/14200 identifies several tissue specific promoters which may be utilized, e.g. the mouse neurofilament-light gene promoter, the human platelet- derived growth factor- β gene promoter, the rat neuronal specific enolase promoter, the mouse mylein basic protein gene promoter, and the human APP gene promoter. PCT International Publication No. WO 95/05466 discloses transgenic cell and animal models for AD that express a kinase that is capable of modulating the phosphorylation of the microtubule-forming protein, tau. These patent publications are incorporated herein by reference to disclose protocols for neuronal tissue specific expression of a transgenic construct and the production of transgenic mice. Allelic variants of the ApoJ gene are disclosed herein as being associated with AD, thus transgenic animal models based on these polymorphisms may be useful tools in the research and discovery of therapeutics.

This invention provides a catalytic mRNA or ribozyme which is capable of cleaving the mRNA encoded by the sequence shown in either Figure 3 or Figure 4 or both. For Group I introns, see Cech, U. S. Patent No. 4,987,071; for RNase P see Altman, U. S. Patent No. 5,168,053; and for hammerhead ribozymes see Hazeloff, U. S. Patent No. 5,254,678. This invention also provides the antisense to the sequence shown in either Figure 3 or Figure 4 or both. For methods for the production of antisense nucleic acid molecules see Inoue U. S. Patent No. 5,208,149 and U. S. Patent No. 5,190,931 and Schewmaker, U. S. Patent No. 5,107,065.

The coding polymorphisms in APOJ can be conveniently assessed by direct PCR sequencing, single-strand conformation polymorphism (SSCP) analysis and slot-blot hybridization with allele-specific oligonucleotides (ASO) . One of these polymorphisms eliminates a signal sequence for enzymatic post-translational glycosylation of ApoJ protem and appears to be associated with an increased risk for AΓ in homczvσotes. Sequence variants were identified by screening for polymorphisms using SSCP. APOJ exons 2 - 8, encoding all but the last two amino acids, were initially amplified from DNAs of ten African-American subjects by PCR using flanking intronic primers. SSCP variants were observed for exons 2, 5, 6 and 7. Direct sequencing of the PCR products showed that the exon 6 variant was due to a single nucleotide insertion in the intron sequence upstream of the 3' primer and that the exon 2 and exon 5 variants were due to nucleotide substitutions in exon sequences which were neutral with respect to amino acid coding potential (data not shown) . In contrast, two of the exon 7 SSCP variants corresponded to nucleotide substitutions which altered the predicted amino acid sequence (Figures 1 and 2) . The most common allele, corresponding to the published sequence (de Silva et al . , 1990) , was designated as Jl, the next most common exon 7 allele as J2 and the more rare allele as J3. The J2 allele is an A to C replacement at nucleotide position 865 according to the numbering system of de Silva et al . and corresponds to a substitution of histidine for asparagine at amino acid position 295; the J3 allele is a G to A replacement at nucleotide position 898 and corresponds to a substitution of asparagine for aspartate at amino acid position 306. A third rare sequence variant, a C to G replacement at nucleotide position 870 which is neutral with respect to amino acid coding potential, was also observed in exon 7. Each of these exon 7 sequence variants could be reproducibly distinguished by their characteristic SSCP band patterns (Figure la and data not shown) . It is likely that all of the major sequence variants in APOJ exon 7 were identified, but since the SSCP analysis of the other exons involved fewer individuals and since fewer of the PCR products from these exons were sequenced, there may be additional APOJ sequence variants.

Allele-specific oligonucleotides (ASOs) were designed as 17- ers which differed at a single position corresponding to the allelic nucleotide substitutions. When applied as hybridization probes to slot-blots of the exon 7 PCR products, the ASOs allowed rapid and unambiguous scoring of APOJ coding polymorphism genotypes (Figure lc) .

The human ApoJ protein is subject to enzymatic glycosylation, including the addition of negatively charged sialic acid, at seven asparagine residues roughly evenly distributed through the primary sequence and located within asparagine-X-threonine/serine (N-X-S/T) consensus glycosylation signal sequences. Both of the allelic variants which have been identified are potentially associated with altered glycosylation: the J2 allele disrupts a N-P-S sequence and the J3 allele creates a new potential glycosylation site with the sequence N-E-S. An interesting evolutionary comparison can be made between these human sequence variants and the mouse (Jordan-Stark et al., 1994) and rat (Collard et al. , 1987) ApoJ/SGP2 sequences (Figure 2) . The human J2 allele deletes a glycosylation site which is not present at the corresponding position in the rodent sequence, but which appears to have its rodent counterpart at a slightly more C-terminal position; the human J3 allele produces a potential glycosylation site at precisely this position. This suggests that both positions in the protein might be accessible to glycosylation in vivo .

This invention is illustrated in the Experimental Detail section which follows. These sections are set forth to aid in an understanding of the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which fellow thereafter. EXPERIMENTAL DETAILS EXAMPLE 1: APOJ Allele Frequencies.

Genotyping was carried out on DNA from 72 African-American patients with AD and 85 healthy elderly unrelated controls (our previous series (Maestre et al . , 1995) augmented by 59 additional subjects) , 78 Hispanic patients and 83 healthy elderly unrelated controls and 24 patients and 27 controls who were identified as Caucasian non-Hispanic. Cases and controls did not differ by gender. Patients were older than controls (AD 77.1 ±8.4; controls 73.0 ±6.3, p<.05) and had less education (AD 6.0±4.2 vs. controls 8.3±4.5, p<.05) . None of the controls were spouses or relatives of cases. All DNA samples were analyzed by both SSCP and slot-blotting and all results were concordant by the two independent methods. There was a significant difference among the three ethnic groups in the distribution of APOJ exon 7 polymorphisms, with both variants showing the highest frequency in African-Americans (X²=43.2, df(4) , p< .00001; Table 1) .

Table 1. Frequency of the APOJ Exon 7 Polymorphisms Among African-Americans, Hispanics and Caucasians.

APOJ Polymorphisms

Exon 7 African- Hispanic Caucasian

American (N=161) (N=51)

(N=157)

Jl .76 .90 .98

J2 .21 .09 .02

J3 .03 .01 0

Please note. Differences were statistically significant. X- = 43.2, df (4) , pc.OOOl.

Overall, the frequencies of both the J2 and J3 variants were slightly but not significantly higher among patients with AD compared tc controls (J2 : AD 14.3% vs. controls 12.1%, J3 : AD 1.8% v≤ , controls 2.3%.. This difference was αreatest for African-Americans (J2 AD 23.3% vs. controls 20%; J3 AD 4.1% vs. controls 2.4%; X² = 1.5; df(2) , p=.4) . The J2 variant was observed in only 2.1% of Caucasians; no J3 variant was found in this group. Differences in the frequency of J2 and J3 variants were not related to age or gender in any group.

While APOJ^" DNA polymorphisms have not been previously reported, in an IEF analysis of serum ApoJ protein Kamboh et al. found a single major variant, designated "J*2", at an allele frequency of 0.237 in a series of 158 African- Americans and this isoform was not detected in a series of 240 Caucasians (Kamboh et al . , 1991) . The "J*2" \ ariant carried an increased net positive charge relative to the default "J*l" isoform, both before and after desialation with neuraminidase. A single African-American individual carried a second more positively charged IEF variant, which was designated "J*3". Since the J2 DNA allele is found at a similar frequency to the "J*2" isoform in African- Americans, is very rare in Caucasians and is predicted to increase the net positive charge of the ApoJ protein, both by replacing asparagine with histidine and by eliminating a consensus recognition site for post-translational addition of negatively charged sialic acid residues, it is likely that it corresponds to the this IEF variant. Similarly, the J3 DNA allele may correspond to the "J*3" IEF variant since it is predicted to produce a greater increase in net positive charge of the desialated protein. The J3 allele frequency for African-Americans is higher than the "J*3" IEF allele frequency observed by Kamboh et al . (current study .03 vs. Kamboh et al 0.003) and if the new potential glycosylation site created by the J3 DNA polymorphism is utilized for addition of sialic acid then the predicted IEF behavior of this variant might not be consistent with the pattern observed by Kamboh et al. for "J*3". IEF analysis of serum ApoJ from genotyped individuals in this series is in progress. PCR and SSCP. Genomic DNA, 100 ng, was subjected to PCR with upstream and downstream intronic primers flanking individual APOJ coding exons in standard PCR buffer (Perkin- Elmer, Branchburg, NJ) with 1.25 mM of each dNTP and 1 U of Taq polymerase in a volume of 50 μL. Thermal cycling consisted of initial denaturation for 4 minutes at 94 °C followed by 30 cycles of annealing at 54 °C for 30 seconds, extension at 72 °C for 45 seconds and denaturation at 94 °C for one minute, with a final extension at 72 °C for 5 minutes. Aliquots of the PCR products were visualized on ethidium-stained 1.4% agarose gels to confirm successful amplification and lack of extraneous products. SSCP analysis was a modification of the procedure of Orita et al. (Orita et al. , 1989) . An aliquot of PCR product was diluted 1:20 into water and then 1:10 into fresh PCR reagents containing an 80-fold reduced concentration of dNTPs and including cv³²P-dCTP (1 μCi/10 μL) . Radiolabeling was carried out for 6 PCR cycles and the radiolabeled product was diluted 1:20 into 0.1% SDS/10 mM EDTA, heated to 65 °C for 5 minutes, diluted 1:1 into standard sequencing stop solution containing 50% formamide, heated to 75 °C for 3 minutes and loaded on a non-denaturing 6% acrylamide gel maintained at 4 °C. Electrophoresis was at 400 V for 16 - 20 hours.

PCR sequencing. PCR products were gel-isolated using GlassPacs (National Scientific, San Rafael, CA) and subjected to cycle-sequencing using reagents and Taq polymerase from the fmols system (Promega, Madison, WI) with appropriate ³²P- end-labeled primers. Cycling conditions were as above except that annealing was at 52 °C and extension was at 70 ^CC. After 30 PCR cycles the sequence reactions were analyzed on 6% acrylamide/ 7 M urea gels.

Slot-blotting with ASOs. PCR products, 6 μL, were denatured by addition of 1 μL of 4N NaOH/10 mM EDTA and incubation for 10 minutes at room temperature, neutralized by addition cf 150 μL of ice-cold 1 M ammonium acetate and subjected to duplicate transfer (50 μL/slot) to nylon membrane using a vacuum manifold apparatus. The membrane was rinsed briefly in 2X SSC, UV cross-linked at 0.3 J/cm³, baked at 80 °C for one hour in a vacuum oven and then cut into strips corresponding to the duplicate transfers for hybridization with two allelic oligonucleotide probes. Oligonucleotides, 150 ng, were end-labeled in reaction volume of 10 μL using 10 U of T4 polynucleotide kinase and 5 μCi of γ³²P-ATP. Prehybridization and hybridization were carried out for 6 - 16 hours each in 6X SSC containing 0.1% SDS, 3% formamide, 5 mM sodium pyrophosphate and 1% blocking reagent from the Genius. Kit (Boerhinger-Mannheim, Indianapolis, IN) at 41 °C. Blots were washed for 15 minutes at room temperature in two changes of 6X SSC and then at 50 °C for 5 minutes in 6X SSC. Autoradiograms were exposed for 2 - 6 hours at -80 °C with intensifying screens.

Primers and ASOs. APOJ PCR primers were: exon 2: 5', CGTGCAAAGACTCCAGAA (SEQ ID NO: 1]

3', TGGCCAGAGGAACATCAT (SEQ ID NO: 2 exon 3 : 5' , CTCTTGCACTTCTCTTGC SEQ ID NO: 3)

3 ' , TCCAGTGGGATGGTCAAG SEQ ID NO: 4) exon 4 : 5' , AGCCTTGTGTCTTCCTGT SEQ ID NO: 5) 3' , GCATATTTCACTAGGCTC SEQ ID NO: 6) exon 5 : 5' , GAGCTTCTCCTAACTGTG SEQ ID NO: 7)

3 ' , AAAGGCCATGAGCTTCCA SEQ ID NO: 8) exon 6 : 5' , CTGGATGACTGACTCTTC SEQ ID NO: 9)

3 ' , TCCATAAAGGCAGCACCA SEQ ID NO: 10 exon 7: 5' , CTTCCCTTCACACTTCTC SEQ ID NO: 11

3' (a) , TCCATAAAGGCAGCACCA (SEQ ID NO: 12) ;

3' (b) , GACTTTAGCAGCTCGTTG (SEQ ID NO: 13) ; exon 8: 5' , CCACAGTGTTTCAGCTCT (SEQ ID NO: 14) ;

3', TTTTGTGGCTCCCAGAGA (SEQ ID NO: 15) . The exon 7 3' (a) primer was used in the initial screening for polymorphisms by SSCP and direct sequencing and the exon 7 3' (b) primer, which brackets the coding polymorphisms more closely, was used for subsequent PCR, SSCP and slot- blotting. ASOs were:

J7.1 (Jl-specific in "J2 region") : TGTTCCACCAACAACCC (SEQ ID NO: 16) J7.2 (J2-specific) : TGTTCCACCAACCACCC (SEQ ID NO: 17) ; J7.5 (J3-specific) : GAGCTCGACGAATCCCT (SEQ ID NO: 18) ; J7.6 (Jl-specific in "J3 region") : GAGCTCAACGAATCCCT (SEQ ID NO: 19) .

Genotyping for APOE was as previously described (Mayeux et al. , 1993) .

Subjects, diagnosis and ethnicity.

Patients and controls were identified from a community-based study of dementia. Cases were part of a registry for Alzheimer's disease based on data from a number of sources: regional hospitals (including inpatient and outpatient services) , private practitioners in the community, federal and state health agencies, health maintenance organizations and senior centers. Controls were recruited from the same sources as cases and from a random sample of Medicare recipients identified in a health survey. All cases and controls received identical interviews and clinical assessments (described below) which included a structured interview of family history. The development of these diagnostic methods and the relationship to the cultural and educational demographics of this community were previously reported (Stern et al . , 1992; Pittman et al . , 1992) . A physician elicited the medical and neurological history and conducted a standardized physical and neurological examination. All ancillary information, including medical charts and reports of laboratory studies, were included in the evaluation, but data regarding APOE or APOJ genotypes were shielded from the clinical diagnostic process A standardized neuropsychological battery that measured performance in memory, orientation, abstract reasoning, language, and construction and measurement of activities of daily living were used to determine if subjects met cognitive and functional criteria for dementia. All clinical information was reviewed at a diagnostic conference of physicians and neuropsychologists to arrive at consensus diagnosis. The diagnosis of AD was based on criteria from the Diagnostic and Statistical Manual (3rd Edition

Revised) and the National Institute of Neurological and Communicative Disorders and Stroke - Alzheimer's Disease and Related Disorders Association. For ethnic group classification, the format suggested by the 1990 United States Census Bureau was used. The 1990 census allows for the identification of Hispanics as a cultural group with further designation of African-American or black, white and other. Subjects were separated into 3 ethnic groups according to self-report: African-American, Hispanic and white (non-Hispanic) , based on direct interview with the subjects or a family member. The majority of patients were alive at the time of this investigation, but data were also available on 16 patients with postmortem confirmation of diagnoses identified in the same registry.

Data Analysis. Allele frequencies for patients with AD and controls were determined by counting alleles and calculating sample proportions. Frequencies of APOJ and APOE alleles in patients and controls were compared using the chi square test and the approximate test based on the normal approximation to the binomial distribution. Both simple and stratified (by ethnic group) odds ratios were estimated for AD associated with the presence of the J2 allele (homozygous and heterozygous) and the J3 allele, using subjects with the Jl genotype as the reference group. For AD associated with the presence of the ApoE-e4 allele (homozygous and heterozygous) subjects with the e3/e3 genotype were used as the reference group. The frequencies for the demographic categories, including ethnic groups, were compared among cases and controls using chi-square analyses and Fisher's exact tests. Both univariate and multivariate odds ratios for AD with particular genotypes were also calculated from logistic regression adjusting for age.

EXAMPLE 2: APOJ GENOTYPE - CASE-CONTROL COMPARISONS

For all ethnic groups combined, the age-adjusted odds ratio (OR) for AD associated with homozygosity for J2 was 4.4 (95% confidence interval {ci}1.2-16.6, p<.01) . No association between AD and J2 or J3 heterozygosity was observed (J2 heterozygosity: OR= 0.8; 95% ci 0.5-1.4, p<.5; J3 heterozygosity: OR=1.5; 95% ci 0.5-4.8, p<.3) . The statistical effect of J2 homozygosity was most pronounced among African-Americans (OR=9.3; 95% ci 1.6-52.1, p<.01) ; the effect was homogenous for Hispanics but did not achieve statistical significance, perhaps because of the small number of Hispanic subjects homozygous for this allele (Table 2) . No association between AD and J2 heterozygosity was observed in African-Americans (OR= 0.8; 95% ci 0.4-1.7, p<.9) , and while the risk of AD associated with J3 heterozygosity appeared to be slightly increased in this group, this was not statistically significant and was not reflected in the Hispanic sample (Table 2) . Thus, the only significant and consistent disease association was with J2 homozygosity.

Table 2. Age-Adjusted Odds Ratios for AD associated with APOJ (Exon 7) Genotypes Among African-Americans, Hispanics and Caucasians.

Ethnic Groups Jl/Jl J1/J2 J1/J3 J2/2 J2/J3 African-American

AD (n=72) 41 17 5 8 1

Control (n=85) 51 28 2 2 2

Odds ratio 1.0 0.8 3.5 9.3* 0.7

(reference) (0.4-1.7) (0.5-23.0) (1.7-52.1) (0.1-9.5) Hispanic

AD (n=78) 63 11 2 2 0

Control (n=83) 69 10 2 2 0

Odds ratio 1.0 1.1 0.6 2.1

(reference) (0.4-2.9) (0.1- 3.9) (0.2-25.7) Caucasian

AD (n=24) 22 1 0 0 0

Control (n=27) 27 1 0 0 0

Odds ratio 1.0 1.5

(reference) (0.4-4.9) - Please note: Numbers in parentheses represent the 95% confidence interval. * denotes statistical significance, p<.01.

Because of the age difference m cases and controls the analyses were repeated in African-Americans using cases and controls matched to within 5 years of age. This reduced the sample size of this ethnic group to 52 cases and 52 controls, but the outcome for the analysis concerning J2 homozygosity was similar (OR= 7.9; 95% cι= 1.1-66.8, p<.05^

APOE Genotype - Case-Control Comparisons. The APOE allele frequency differed significantly by ethnic group (e2, e3, e4: African-Americans: .08, .65, .22; Caucasians: .05, .77, .18; Hispanics: .04, .78, .18; X'= 15.1, df(4) , p<.001) . The allele frequency differences between Caucasian and Hispanic cases and controls were similar to previous observations (Caucasian AD: .03, .68, .29 vs. control .08, .85, .07; Hispanic AD: .05, .71, . .24 vs. control .03, .85, .13; p<.01 for both) . Among African-American cases and controls the frequencies were distinctly different from the other ethnic groups (AD: .11, .58, .31 vs. control .05, .71, .24; p<.08) . The age-adjusted OR for AD in African-Americans associated with e4 homozygosity was 16.7 (95% ci 2.1-135.8, p<.001) while that for heterozygosity was 1.0 (95% ci 0.6-1.8, p<.5) consistent with our previous observations (Mayeux et al . , 1993; Maestre et al . , 1995) .

Among the combined African-American and Hispanic subjects 3 patients and no controls were doubly homozygous for J2 at the APOJ locus and e4 at the APOE locus. A model postulating no interaction between the two loci in terms of disease association would predict fewer double homozygotes (predicted value=l, X= 5.3, df(l), P<.02) . While the number of subjects is too low to allow a strong conclusion this suggests the possibility of an additive or synergistic effect of the two variant proteins on AD susceptibility. Two additional J2-homozygous patients were also e3/e4 heterozygous as was a single control and the remaining 5 J2- homozygous patients were e3/e3, as was a single control. Including J2 homozygosity and adjusting for the presence of an e4 allele in the overall logistic regression model minimally changed the magnitude of the association of J2 homozygosity with AD (OR=4.1, 95% c. i .1.1-15.3 ; p<.04) . This analysis was repeated using the age-matched African- American cases and controls described above and adjusted for the presence of an APOE e4 allele, but this did not alter the association with J2 homozygosity (OR= 9.0; 95% ci= 1.1- 78.5, pc.05) in this group.

EXAMPLE 3 : APOJ AND ALZHEIMER'S DISEASE AGE-AT-ONSET

The estimated distributions of age-at-onset for African- American and Hispanic patients with and without J2 homozygosity were compared. There was nearly a six year difference m the median aαe of onset ''"5 vs. 81. . T.ne relative risk of AD associated with J2 homozygosity was estimated based on a Cox proportional hazard model. This compared the cumulative risk of AD among those homozygous for the J2 variant to those individuals with other APOJ genotypes, and further, double censoring was applied to adjust for using prevalent and incident cases in this analysis. The relative risk of AD associated with homozygosity of the J2 variant was 2.5 (95% ci. 1.5-4.9, p<.05), consistent with calculations based on the odds ratio predicted from logistic regression (above) .

While in principle the association of AD with a oding polymorphism in the APOJ gene could reflect linkage disequilibrium with a nearby AD susceptibility gene, given the highly reproducible association of AD with a coding polymorphism in a second unlinked but functionally related gene, APOE, it seems likely that the findings presented herein reflect an underlying shared biological influence of these two genes on neuronal survival in the aging brain. There is a large literature concerning multiple proposed functions of the ApoJ/clusterin protein (reviewed in Rosenberg et al . , 1993) . Of seemingly greatest relevance to the current study, this protein is expressed and secreted by astrocytes in response to interleukins 1 and 2 in vi tro (Zwain et al . , 1994) and in a delayed response to experimental brain injury in rats there is induction of high ApoJ mRNA and protein expression both in astrocytes and in neurons located in areas of secondary axonal sprouting and synaptic regeneration (May et al . , 1990; Pasinetti et al . , 1991; Laping et al . , 1991) . Certain neurons also express ApoJ mRNA under "basal" conditions, suggesting a role in membrane and synaptic maintenance (Danik et al , 1993; 0'Bryan et al, 1993) . Furthermore, ApoJ has been identified in amyloid-core plaques of AD (Choi-Miura et al . , 1992; McGeer et al . , 1992) and exists in a complex with soluble amyloid beta peptide in cerebrσspinal fluid, where it may bind to this peptide with a greater affinity than does ApcE (Ghiso et al . , 1993; Zlokovic et al, 1994) .

Two possible scenarios for the role of ApoJ protein in AD can be envisioned: either this protein has a protective role, i.e. as has been suggested for ApoE (Poirier et al . , 1993) its cholesterol transport function is necessary for the efficient maintenance and/or local regeneration of neuronal processes and synapses and its affinity for soluble amyloid beta peptide prevents the deposition of insoluble amyloid fibrils or, alternatively, it has a deleterious role, i.e. its affinity for soluble amyloid beta peptide eventually leads to the nucleation of extracellular amyloid fibrils which exert direct or indirect toxic effects on neurons. The available experimental evidence seems consistent with either or both of these possibilities. In the context of the findings presented herein, models which invoke amyloid in AD pathogenesis would predict differences in the relative affinities of the Jl and J2 ApoJ protein isoforms for soluble amyloid beta peptide. Since ApoJ also binds and inhibits the complement membrane attack complex and since complement components have been detected in senile plaques of AD (McGeer et al . , 1989) , a role in pathogenesis related to potentially differing affinities of the Jl and J2 variants for complement components is another possibility. While amyloid binding is a property which is shared by ApoJ and ApoE, complement inhibition does not appear to be a shared property. Finally a completely different model, involving interactions with the microtubule-associated tau protein, has been proposed by Strittmatter et al . to explain the association of ApoE variants with AD (Poirier, 1994) . The possibility of an interaction of ApoJ with tau has not yet been tested.

Despite the known increase in Apo-E-e4 allele frequency among persons of African descent relative to those of

European descent, the frequency of AD does not appear to be increased relative to other ethnic groups. An attenuation in the estimated risk of AD associated with heterozygosity for e4 in African-Americans compared to Caucasians was reported and this was attributed either to linkage disequilibrium of APOE with a nearby AD locus in Caucasians, with attenuated linkage disequilibrium in African-Americans, or to unique genetic or environmental modifying factors which protect e4-heterozygous African-Americans against AD. Given the findings presented here concerning APOJ, the first possibility seems less likely. Rather, the data emphasize differences in the genetic influences on AD susceptibility in different ethnic groups and therefore indirectly support the second explanation. But since even among African- Americans J2 homozygosity is rare, the lack of association between e4 heterozygosity and AD in this population is not directly explained by the presence of APOJ polymorphisms and must reflect other as yet undefined genetic or environmental factors .

As in all cross-sectional, case-control studies, genotype- disease associations may reflect differential survival of cases and controls. It has been attractive to suggest that the lack of an association between e4 heterozygosity and AD in African-Americans might be explained by selective mortality due to fatal myocardial infarction (Eichner et al . , 1993; Wilson et al . , 1994) . In a cross-sectional study, this point cannot directly be addressed. However, the frequency of e4 in African-Americans remains elevated relative to other populations into the eight decade of life (Maestre et al . , 1995) . Moreover, a history of non-fatal cardiovascular disease does not account for the attenuated association between e4 heterozygosity and AD in African- Americans. The frequency of e4 is also increased among persons of Finland compared with other Caucasian populations (0.22 vs. 0.12 world wide, Hallman et al . , 1991; Gerdes et al . , 1992) . While the frequency of e4 decreases with advancing age due to cardiovascular disease in that population, a significant association between this allele and AD was found in a population-based study in Kuopio (Kuusisto et al., 1994) . While some population samples have shown an inverse association of AD with the APOE e 2 allele, researchers (Corder et al., 1994; Maestre et al. , 1995 and van Duijn et al . , 1995) have found that AD can be directly associated with APOE genotypes that included both e2 and e4 alleles. However, survival for AD patients identified as e 2 carriers was significantly reduced compared with AD patients with e4 in the Dutch study (van Duijn et al . , 1995) . In view of these uncertainties concerning differential survival, until prospective, population based studies are completed interpretations of the precise biological significance of associations between AD and both APOE and APOJ must be considered tentative.

From a technical point of view, the APOJ DNA polymorphisms described here should be a useful addition to the growing panel of available genetic markers which show markedly different allele frequencies in genetically distinct racial/ethnic groups (Dean et al . , 1994) . Such markers have been used for anthropogenetic studies and more recently have shown theoretical promise for application in mapping by the admixture linkage disequilibrium method, which seeks to capitalize on linkage disequilibrium of markers with genetic traits or diseases in recently racially-admixed populations such as Hispanics and African-Americans (Stephens et al . , 1994) .

REFERENCES

Anderton, B.H. and Miller, C.C., PCT International Publication No. WO 95/05466, International Publication date: February 23, 1995, International Filing date: August 1, 1994.

Choi-Miura, N.H., et al . SP-40,40 is a constituent of Alzheimer's amyloid. Acta Neuropa thol . 83, 260-264 (1992) .

Collard, M.W. & Griswold, M.D. Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat Sertoli cells. Biochemis try 26, 3297-3303 (1987) .

Cordell, B. U. S. Patent No. 5,387,742, issued February 7, 1995, filed June 17, 1995.

Corder, E.H., et al . Apolipoprotein E type 2 allele decreases the risk for late onset Alzheimer's disease. Na ture Genet. 7, 180-184 (1994) .

Danik, M., Chabot, J.G., Hassan-Gonzalez, D. , Suh, M. ,

Quirion, R. Localization of sulfated glycoprotein-

2/clusterin mRΝA in the rat brain by in situ hybridization. J. Compar . Neurol . 334, 209-227 (1993) .

Dean, M., et al . Polymorphic admixture typing in human ethnic populations. Am. J. Hu . Genet . 55, 788-808 (1994) .

de Silva, H.V. Harmony, J.A.K., Stuart, W.D., Gil, CM. & Robbins, J. Apolipoprotein J: structure and tissue distribution. Biochemis try 29, 5380-5389 (1990) .

de Silva, H.V., et al . A 70-KDa apolipoprotein designated apo J is a marker for subclasses of human plasma high density lipoproteins . J. Bid . Chem . 265, 13240-13247 (1990) . Duguid, J.R., Bohmont, C.W. , Ningai, L. & Tourtellotte, W.W. Changes in brain gene expression shared by scrapie and Alzheimer's disease. Proc . Natl . Acad . Sci . USA 86: 7260- 7264 (1989) .

Eichner, J.E., et al. Relation of apolipoprotein E phenotype to myocardial infarction and mortality from coronary artery disease. Am. J. Cardiol . 71, 160-165 (1993) .

Fields, S. and Song, 0., U. S. Patent No. 5,283,173, filed January 24, 1990, issued February 1, 1994.

Fink, T.M., et al . Human clusterin (CLI) maps to 8p21 in proximity to lipoprotein lipase (LPL) gene. Genomi cs 16, 526-528 (1993) .

Forss-Petter et al . Neuron 5: 197-198 (1990) .

Gerdes, L.U., Klausen, I.C., Sihm, I. & Faergeman, O. Apolipoprotein E polymorphism in a Danish population compared to findings in 45 other study populations around the world. Genet. Epidemiol . 9, 155-167 (1992) .

Ghiso, J., et al . The cerebrospinal-fluid soluble form of Alzheimer's amyloid beta is complexed to SP-40,40 (apolipoprotein J) , an inhibitor of the complement membrane- attack complex. Biochem . J. 293, 27-30 (1993) .

Hallman, D.M., et al . The apolipoprotein E polymorphism: A comparison of allele frequencies and effects in nine populations. Am. J. Hum . Gene t . 49, 338-349 (1991) .

Harrington, C.R. et al . Influence of apolipoprotein E genotype on senile dementia of the Alzheimer and Lewy body types. Am. J. Pa thol . 145, 1472-1484 (1994) .

Hendrie, H. C, et al . Apolipoprotein Ξ genotypes and Alzheimer's disease in a community study of elderly African- Americans. Ann. Neurol . 37, 118-120 (1995) .

Jenne, D.E. £_ Tschopp, J. Molecular structure and functional characterization of a human complement cytolysis inhibitor found in blood and seminal plasma: identity to sulfated glycoprotein 2, a constituent of rat testis fluid. Proc . Natl . Acad. Sci . USA 86, 7123-7127 (1989) .

Jenne, D.E., et al . Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-l in human plasma. J. Biol . Chem . 266, 11030-11036 (1991) .

Jordan-Stark, T.C., et al . Mouse apolipoprotein J: characterization of a gene implicated in atherosclerosis. J. Lipid Res . 35, 194-210 (1994) .

Kadomatsu, K. , Anzano, M.A., Slayter, M.V. , Winokur, T.S. , Smith, J.M. and Sporn, M.B. Expression of sulfated Glycoprotein 2 is associated with carcinogenesis induced by Ν-Νitroso-Ν-methylurea in rat prostate and seminal vesicle. Cancer Res . 53: 1480-1483 (1993) .

Kamboh, M.I., Harmony, J.A.K., Sepehrnia, B. , Νwankwo, M. & Ferrell, R.E. Genetic studies of human apolipoproteins . XX. Genetic polymorphism of apolipoprotein J and its impact on quantitative lipid traits in normolipidemic subjects. Am. J. Hum . Genet . 49, 1167-1173 (1991) .

Kirszbaum, L., et al . Molecular cloning and characterization of the novel, human complement-associated protein, SP-40,40: a link between the complement and reproductive systems. EMBO J. 8, 711-718 (1989) .

Krimpenfort, P. J. A. and Berns, A. J. M., U. S. Patent No. 5,175,384, filed December 5, 1988, issued December 29, 1992. Kuusisto, J. , et al . Association of apolipoprotein E phenotypes with late onset Alzheimer's disease: population based study. Brit. Med. J. 309, 636-638 (1994) .

Laping, N.J., Nichols, N.R., Day, J.R. & Finch, C.E. Corticosterone differentially regulates the bilateral response of astrocyte mRNAs in the hippocampus to entorhinal cortex lesions in male rats. Mol . Brain Res . 10: 291-297 (1991) .

Leder, P. and Stewart, T.A. , U. S. Patent No. 4,736,866, issued April 12, 1988.

Levison, S.W. and Goldman, J.E. Both oligodendrocytes and astrocytes develop from progenitors in the subventricular zone of postnatal rat forebrain. Neuron 10: 201-212 (1993) .

Maestre, G., et al . Apolipoprotein-E and Alzheimer's Disease: Ethnic variation in genotypic risks. Ann. Neurol . 37, 254-259 (1995) .

Maue et al . Neuron 5: 223-231 (1990) .

May, P.C., et al. Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer's disease and in response to experimental lesions in rat. Neuron 5, 831- 839 (1990) .

Mayeux, R., et al. The apolipoprotein E4 allele in patients with Alzheimer's disease. Ann. Neurol . 34, 752-754 (1993) .

McGeer, P.L., Akiyama, H., Itagaki, S. & McGeer, E.G.

Activation cf the classical complement pathway in brain tissue of Alzheimer patients. Neurosci . Le t t . 107, 341-346 (1989) .

McGeer, P., Kawarrata, T. & Walker, D clusterin in Alzheimer brain tissue. Brain Res . 579, 337-341 (1992) .

Minoshima, S., et al . Assignment of human SP-40,40 gene to chromosome 8. Cytogenet . Cell Genet . 58, 1929 (1991) .

Noguchi, S., Murukami, K. & Yamada, N. Apolipoprotein E genotype and Alzheimer's disease. Lancet 342, 737 (1993)

O'Bryan, M.K. , Cheema, S.S., Bartlett, P.F., Murphy, B.F. & Pearse, M.J. Clusterin levels increase during neuronal development. J. Neurobiol . 24, 421-432 (1993) .

Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K. & Sekiya, T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc . Na tl . Acad . Sci . USA 86, 2766-2770 (1989) .

Pasinetti, G.M. & Finch, C.E. Sulfated glycoprotein-2 (SGP- 2) mRNA is expressed in rat striatal astrocytes following ibotenic acid lesions. Neurosci . Le t t . 130, 1-4 (1991) .

Pittman, J., et al . Diagnosis of dementia in a heterogenous population: A comparison of paradigm based diagnosis and physician's diagnosis. Arch Neurol 1992; 49: 461-467.

Poirier, J. , Baccichet, A., Dea, D. & Gauthier, S.

Cholesterol synthesis and lipoprotein reuptake during synaptic remodelling in hippocampus in adult rats. Neuroscience 55, 81-90 (1993) .

Poirier, J. Apolipoprotein E in animal models of CNS injury and in Alzheimer's disease. TINS 17, 525-530 (1994) .

Readhead, et al . Cel l 48: 703-712 (1987) .

Rosenberg, M.E., Dvergsten, J. & Corea-Rotter, R. Clusterin: an enigmatic protein recruited by diverse stimuli. J. Lab . Clin . Med. 121, 205-214 (1993) .

Stephens, J.C., Briscoe, D. &. O'Brien, S.J. Mapping by admixture linkage disequilibrium in human populations: limits and guidelines. Am. J. Hum. Genet . 55, 809-824 (1994) .

Stern, Y. , et al . Diagnosis of dementia in a heterogenous population: Development of a neuropsychological paradigm and quantified correction for education. Arch . Neurol . 49, 453- 460 (1992) .

Strittmatter, W.J. et al . Hypothesis: Microtubule instability and paired helical filament formation in Alzheimer's disease brain are related to apolipoprotein E genotype. Exp . Neurol . 125, 163-171 (1994) .

Strittmatter, W.J., et al . Apolipoprotein E: High-avidity binding to _/S-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc . Na t . Acad . Sci . USA 90, 1977-1981 (1992) .

Wagner, T.E. and Chen, X., U. S. Patent No. 5,175,385, filed September 3, 1987, issued December 29, 1992.

Wilson, W.F., et al. Apolipoprotein E alleles, dyslipidemia, and coronary artery disease. JAMA 272, 1666-1671 (1994) .

Wirak, D. 0. et al . The EMBO J. 10: 289-296 (1991) .

Zlokovic, B.V., et al . Biochem . Bi ophyε . Res . Com . 205, 1431-1437 (1994) .

Zwain, I.H., Grima, J. & Cheng, C.Y. Regulation of clusterin secretion and mRNA expression in astrocytes by cytokines. Mol . Cel l . Neuroεci . 5, 229-237 (1994) . -36- SEQUENCE LISTING

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(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 GTTGCTCGAC GATTTCAG (2) INFORMATION FOR SEQ ID NO: 14:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14 : CCACAGTGTT TCAGCTCT 18

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: AGAGACCCTC GGTGTTTT 18

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TGTTCCACCA ACAACCC 17

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TGTTCCACCA ACCACCC 1

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GAGCTCGACG AATCCCT 17

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GAGCTCAACG AATCCCT 17

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 90 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: PEPTIDE

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Val Asp Phe Leu Lys Glu Gly Glu Asp Asp Pro Thr Val Cys Lys Glu 1 5 10 15 lie Arg His Asn Ser Thr Gly Cys Leu Lys Met Lys Gly Gin Cys Glu 20 25 30

Lys Cys Gin Glu lie Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ala 35 40 45

Gin Ala Asn Leu Arg Gin Glu Leu Asn Asp Ser Leu Gin Val Ala Glu 50 55 60

Arg Leu Thr Gin Gin Tyr Asn Glu Leu Leu His Ser Leu Gin Ser Lys 65 70 75 80

Met Leu Asn Thr Ser Ser Leu Leu Glu Gin 85 90

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 90 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Thr Glu Phe He Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu 1 5 10 15

He Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gin Cys Asp 20 25 30

Lys Cys Arg Glu He Leu Ser Val Asp Cys Ser Thr Asn His Pro Ser 35 40 45

Gin Ala Lys Leu Arg Arg Glu Leu Asn Glu Ser Leu Gin Val Ala Glu 50 55 60

Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gin Trp Lys 65 70 75 80

Met Leu Asn Thr Ser Ser Leu Leu Glu Gin 85 90

Claims

What is claimed is:

1. A method for detecting in a suitable nucleic acid containing sample the presence of a polymorphism associated with an allelic variation in an apolipoprotein-J gene which comprises:

(a) contacting the sample with an oligonucleotide capable of detecting the allelic variation under conditions such that the oligonucleotide hybridizes with nucleic acid contained in the sample if such allelic variation is present in the nucleic acid; and

(b) detecting the presence of any oligonucleotide hybridized to the nucleic acid and thereby detecting the presence of the polymorphism in the apolipoprotein-J gene in the sample.

2. The method of claim 1, wherein the allelic variation is a J2 variant and the oligonucleotide comprises at least a portion of the sequence shown in Figure 2.

3. The method of claim 1, wherein the allelic variation is a J3 variant and the oligonucleotide comprises at least a portion of the sequence shown m Figure 2.

4. The method of claim 1, wherein the allelic variation is a neutral polymorphism.

5. The method of claim 1, wherein the sample comprises cDNA.

6. The method of claim 5, wherein the cDNA comprises a cloned human genomic library.

7. The method of claim 1, wherem the sample comprises blood, urine, plasma, serum or tissue.

8. The method of claim 1, wherein the oligonucleotide is labeled with a detectable moiety.

9. The method of claim 8, wherein the detectable moiety is a florescent label, a radioactive atom, a chemiluminescent label, a paramagnetic ion, biotin or a label which can be detected through a secondary enzymatic or binding step.

10. A method for detecting in a nucleic acid containing sample the presence of a polymorphism associated with an allelic variation in an apolipoprotein-J gene which comprises:

(a) contacting the sample with a pair of polymerase chain reaction oligonucleotide primers capable of hybridizing with nucleic acid sequences encoding the apolipoprotein-J gene under conditions allowing for hybridization; and

(b) contacting the mixture from step (a) with DNA polymerase under conditions allowing for amplification to form an amplified polymerase chain reaction product; and

(c) denaturing the product from step (b) so as to form a single-stranded polymerase chain reaction product;

(d) contacting the single-stranded polymerase chain reaction product with an oligonucleotide capable of detecting the allelic variation under conditions such that the oligonucleotide hybridizes with the smgie-stranded polymerase chair. reaction product if such allelic variation is present in the nucleic acid; and

(e) detecting the presence of any oligonucleotide hybridized to the single-stranded polymerase chain reaction product and thereby detecting the presence of the polymorphism in the apolipoprotein-J gene in the sample.

11. A method of determining the probability of a subject developing Alzheimer's Disease which comprises:

(a) obtaining a suitable biological sample from the subject;

(b) contacting the sample from step (a) with an oligonucleotide capable of detecting an allelic variation in the apolipoprotein-J gene under conditions such that the oligonucleotide hybridizes with nucleic acid contained in the sample, if such an allelic variation is present in the nucleic acid; and

(ci detecting the presence of any oligonucleotide hybridized to the allelic variation present in the apolipoprotein-J gene and thereby determining the probability of a subject developing Alzheimer's Disease.

12. The method of claim 11, wherein the oligonucleotide comprises the J2 allelic variant which comprises at least a portion of the sequence shown in Figure 2.

13. The method of claim 11, wherein the oligonucleotide comprises the J3 allelic variant which comprises at least a portion of the sequence shown m Figure 2.

14. The method of claim 11, wherein the εuriect is of African descent .

15. The method of claim 11, wherein the subject is of Hispanic descent .

16. The method of claim 11, wherein the biological sample is a blood, urine, serum, plasma or tissue sample.

17. The method of claim 11, wherein the oligonucleotide is labeled with a detectable moiety.

18. The method of claim 17, wherein the detectable moiety is a florescent label, a radioactive atom, a chemiluminescent label, a paramagnetic ion, biotin or a label which can be detected through a secondary enzymatic or binding step.

19. A method for detecting in a subject the presence of a polymorphism associated with an allelic variation in an apolipoprotein-J gene which comprises:

(a) obtaining a suitable biological sample containing nucleic acid from the subject;

(b) contacting the sample with a pair of polymerase chain reaction oligonucleotide primers capable of hybridizing with nucleic acid sequences encoding the apolipoprotein-J gene under conditions allowing for hybridization; and

(c) contacting the mixture from step (b) with DNA polymerase under conditions allowing for amplification to form an amplified polymerase chain reaction product; and

(dl denaturing the product from step (c) so as to form a single-stranded polymerase chain reaction product;

(e) contacting the single-stranded polymerase -chain reaction product with an oligonucleotide capable of detecting the allelic variation under conditions such that the oligonucleotide hybridizes with the single-stranded polymerase chain reaction product if such allelic variation is present in the nucleic acid; and

(f) detecting the presence of any oligonucleotide hybridized to the single-stranded polymerase chain reaction product and thereby detecting the presence of the polymorphism in the apolipoprotein-J gene of the subject.

20. The method of claim 19, wherein the sample comprises cDNA.

21. The method of claim 20, wherein the cDNA comprises a cloned human genomic library.

22. The method of claim 19, wherein the sample comprises blood, urine, plasma, serum or tissue.

23. A method of determining the probability of a subject developing a cognitive disorder which comprises :

(a) obtaining a suitable biological sample from the subject;

(b) contacting the sample from step (a) with an oligonucleotide capable of detecting an allelic variation in the apolipoprotein-J gene under conditions such that the oligonucleotide hybridizes with nucleic acid contained m tr.e sample, if such an allelic variation is present in the nucleic acid; and

(c) detecting the presence of any oligonucleotide hybridized to the allelic variation present in the apolipoprotein-J gene and thereby determining the probability of a subject developing the cognitive disorder.

24. A method of determining the probability of a subject developing a prostatic carcinoma which comprises:

(a) obtaining a suitable biological sample from the subject;

(b) contacting the sample from step (a) with an oligonucleotide capable of detecting an allelic variation in the apolipoprotein-J gene under conditions such that the oligonucleotide hybridizes with nucleic acid contained in the sample, if such an allelic variation is present in the nucleic acid; and

(c) detecting the presence of any oligonucleotide hybridized to the allelic variation present in the apolipoprotein-J gene and thereby determining the probability of a subject developing prostatic carcinoma.

25. A reagent capable of detecting the presence of a polymorphism associated with an allelic variation in an apolipoprotein-J gene or gene product.

26. The reagent of claim 25, wherein the allelic variation is a J2 variant and the reagent is an oligonucleotide encoding a polypeptide having at least a portion of the sequence shown in Figure 2.

27. The oligonucleotide of claim 26, wherein the oligonucleotide comprises the sequence TGTTCCACCAACCACCC (SEQ ID NO: 17) .

28. The reagent of claim 25, wherein the allelic variation is a J3 variant and the reagent is an oligonucleotide encoding a polypeptide having at least a portion of the sequence shown in Figure 2.

29. The oligonucleotide of claim 28, wherein the oligonucleotide comprises the sequence GAGCTCGACGAATCCCT (SEQ ID NO: 18) .

30. The reagent of claim 25, wherein the reagent is an antibody specific for a polypeptide associated with the J2 variant of apolipoprotein-J which comprises at least a portion of the polypeptide sequence shown in Figure 2.

31. The polypeptide of claim 30, comprising about 8 to about 20 amino acids.

32. The reagent of claim 25, wherein the reagent is an antibody specific for a polypeptide associated with the J3 variant of apolipoprotein-J which comprises at least a portion of the polypeptide sequence shown in Figure 2.

33. The polypeptide of claim 32, comprising about 8 to about 20 amino acids.

34. A method for determining whether a compound is capable of interfering with the formation of a complex between a polymorphic apolipoprotein-J protein and an amyloid _/S-protein, which comprises:

(a¹ incubating the compound with an appropriate apolipoprotein-J protein affinity derivative and an amyloid β-protein;

(b) contacting the incubate of step (a) with an appropriate affinity medium under conditions so as to bind the amyloid S-protein- apolipoprotein-J-affinity derivative-protein complex, if such a complex forms; and

(c) measuring the amount of the affinity protein complex formed in step (b) so as to determine whether the compound is capable of interfering with the formation of the complex between the apolipoprotein-J protein and the amyloid β- protein.

35. A method for determining whether a compound is capable of interfering with the formation of a complex between an amyloid -protein and a polymorphic apolipoprotein-J protein which comprises :

(a) incubating the compound with an appropriate amyloid /S-protein affinity derivative and an apolipoprotein-J protein;

(b) contacting the incubate of step (a) with an appropriate affinity medium under conditions so as to bind the amyloid _/S-protem- apolipoprotein-J-affinity derivative-protein complex, if such a complex forms; and

(c) measuring the amount of the affinity protein complex formed in step (b) so as to determine whether the compound is capable of interfering with the formation of the complex between the apolipoprotein- protein and the amyloid β - protein .

36. A method for determining the concentration of a polymorphic apolipoprotein-J protein in a biological fluid which comprises:

(a) contacting the fluid with a measured amount of a soluble first monoclonal antibody to the polymorphic apolipoprotein-J protein in order to form a soluble complex of the antibody and the polymorphic apolipoprotein-J protein present in the fluid, the first monoclonal antibody being labeled;

(b) contacting the soluble complex with a second monoclonal antibody to the polymorphic apolipoprotein-J protein, the second monoclonal antibody being bound to a solid carrier, the solid carrier being insoluble in the fluid, in order to form an insoluble complex of the first monoclonal antibody, the polymorphic apolipoprotein-J protein and the second monoclonal antibody bound to the solid carrier;

(c) separating the solid carrier from the fluid sample and unreacted labeled antibody;

(d) measuring either the amount of labeled antibody associated with the solid carrier or the amount of unreacted labeled antibody; and

(e) comparing the amount of labeled antibody measured in step (d) with the amount of labeled antibody measured for a control sample prepared in accordance with steps (a) - (d) , to determine the presence of the polymorphic apolipoprotein- J protein in the fluid sample, or relating the amount of labeled antibody measured with the amount of labeled antibody measured for samples containing known amounts of the polymorphic apolipoprotein-J protein prepared in accordance with steps (a) - (d) to determine the concentration of the polymorphic apolipoprotein-J protein in the fluid sample.

37. An antibody immunoreactive with an allele-specific antigen associated with a specific apolipoprotein-J polymorphism.

38. A transgenic nonhuman mammal whose somatic and germ cells contain and express a gene coding for an allelic variant of an apolipoprotein-J gene, the gene having been stably introduced into the nonhuman mammal, or an ancestor of the nonhuman mammal at the single cell stage or an embryonic stage, and wherein the gene is operably linked to a promoter and integrated into the genome of the nonhuman mammal .

39. The transgenic nonhuman mammal of claim 38, wherein the promoter is effective for directing gene expression to neuronal cells of the brain.

40. A nonhuman mammal whose neuronal cells or glial cells or both express an allelic variant of an apolipoprotein-J gene, the gene having been introduced into the mammal by localized infection with retrovirus, and wherein the gene is operably linked to a promoter.

41. The nonhuman mammal of claim 40, wherein the retrovirus is comprised of an inducible retroviral vector.

42. The nonhuman mammal of claim 40, wherein the retroviral vector comprises a marker gene, a constitutive promoter and an inducible promoter.