METHODS OF DETECTING ALZHEIMER'S DISEASE

Abstract: Methods of diagnosing or prognosing Alzheimer's disease in a subject are disclosed. The method involves detecting the presence or absence of an apolipoprotein E type 4 (ApoE4) isoform or DNA encoding ApoE4 in the subject. The presence of ApoE4 indicates the subject is afflicted with Alzheimer's disease or at risk of developing Alzheimer's disease.

Description: METHODS OF DETECTING ALZHEIMER'S DISEASE This invention was made with Government support under NIH LEAD Award 5R35 AGO 7922 and NIH Alzheimer's Disease Research Center 5P50 AGO 5128. The Government has certain rights to this invention. Related Applications This application is a Continuation-in-Part of commonly owned, copending application Serial Number 07/959,992, filed October 13, 1992, the disclosure of which is incorporated by reference herein in its entirety. Field ofthe Invention The present invention relates to methods of diagnosing and prognosing Alzheimer's disease. Background ofthe Invention The senile plaque and congophilic angiopathy are abnormal extracellular structures found in abundance in brain of patients with Alzheimer's disease. The biochemical composition of these structures has been extensively studied to better understand their possible role in the pathogeneεis of this dementing disease. The mature senile plaque is a complex structure, consisting of a central core of amyloid fibrils surrounded by dystrophic neurites, axonal terminals and dendriteε, icroglia and fibrous astrocytes. See D. Selkoe Neuron 6, 4 87- 4 98 (1991) . The amyloid core of the senile plaque surrounding blood vessels, producing the congophilic angiopathy, is a peptide of 39 to 43 amino acids termed the β-Amyloid (AB) peptide. G. Glenner and C. Wong, Biochem. Biophys . Res . Comm. 120, 885-890 (1984) . AB peptide is found in brain in Alzheimer's disease, Down's syndrome, hereditary cerebral hemorrhage of the Dutch type, and in old age. K. Kosik, Science 256, 780-783 (1992) . Kβ is produced by abnormal proteolytic processing of a larger protein, the amyloid precursor protein (APP) . See K. Beyreuther and C. Masters, Brain Path. 1, 241-251 (1991). The senile plaque and congophilic angiopathy contain proteins in addition to BA peptide. APP itself, among others, has been identified in the senile plaque by histochemical studies employing antibodies recognizing either the amino- and carboxy-termini of the precursor protein. See, e. g. , F. Tagliavine et al., Neurosci. Lett. 128, 117-120 (1991); C. Joachim et al. , Amer. Jour. Path. 138, 373-384 (1991); The mechanisms by which these proteins aggregate in the extracellular space to associate with the senile plaque and congophilic angiopathy are not known. While there has been considerable research into the mechanisms underlying Alzheimer's disease, there continues to be an ongoing need for new ways to investigate and combat this disorder. Summary ofthe Invention Methods of diagnosing or prognosing Alzheimer's disease in a subject are disclosed. One embodiment comprises detecting the presence or absence of DNA encoding an apolipoprotein E type 4 (ApoE4) isoform in the subject. Another embodiment comprises detecting the presence or absence of an ApoE4 isoform. The presence of an ApoE4 isoform or of DNA encoding an ApoE4 isoform indicates that the subject is afflicted with Alzheimer's disease or at risk of developing Alzheimer's disease. The foregoing and other objects and aspects of the instant invention are explained in detail in the drawings herein and the specification set forth below. Brief Description of the Drawings Figure 1, top panel, -shows proteins in cerebrospinal fluid which bind to immobilized BA peptide, and to a control peptide (the "even-hydro peptide", or "E.H.") after incubation and: elution with phosphate buffered saline, column 1; elution with 5% sodium dodecyl sulfate, column 2; with 4 molar urea, column 3; or elution with 6 molar guanidine hydrochloride, column 4. Binding of Apolipoprotein E is shown by immunohistochemical staining in the bottom panel. Figure 2, top panel, shows the binding of proteins in cerebrospinal fluid to various immobilized peptideε. Binding of Apolipoprotein E is again shown by immunohistochemical staining in the bottom panel. Figure 3 shows the age at onset for subjects with 0, 1, and 2 ApoE4 alleles. Each curve is labeled by the number of ApoE4 alleles. The symbol '*• indicates multiple diagnoses within a short interval. Onset curves were estimated by Kaplan-Meier product limit distributions and were clearly distinct (logrank chi- square=53.8, 2 degrees of freedom, p < 0.0001). Detailed Description of the Invention As noted above, the present invention provides a method of screening (e.g., diagnosing or prognosing) for Alzheimer's disease in a subject. The method comprises detecting the presence or absence of ApoE4 isoform or of DNA encoding an ApoE4 isoform in the subject. The presence of such isoform or DNA indicates that the subject is afflicted with Alzheimer's disease or at risk of developing Alzheimer's disease. Suitable subjects include those which have not previously been diagnosed as afflicted with Alzheimer's disease, those which have previously been determined to be at risk of developing Alzheimer's disease, and those who have been initially diagnosed as being afflicted with Alzheimer's disease where confirming information is desired. For example, patients diagnosed or determined to be afflicted with dementia, particularly patients who had previously been clinically normal who are determined to be afflicted with a progressive dementia, are suitable subjects. Thus, the present invention may be employed in detecting both familial Alzheimer's disease (late onset and early onset) as well as sporadic Alzheimer's disease. The step of detecting the presence or absence of ApoE4 or of DNA encoding such isoform may be carried out either directly or indirectly by any suitable means. A variety of techniques are known to those skilled in the art. All generally involve the step of collecting a sample of biological material containing either DNA or ApoE from the subject, and then detecting whether or not the subject possesses ApoE4 or DNA encoding such isoform from that sample. For example, the detecting step may be carried out by collecting an ApoE sample from the subject (for example, from cerebrospinal fluid, or any other fluid or tissue containing ApoE) , and then determining the presence or absence of an ApoE4 4 isoform in the ApoE sample (e.g., by isoelectric focusing or immunoassay) . In the alternative, the detecting step may be carried out by collecting a biological sample containing DNA from the subject, and then determining the presence or absence of DNA encoding an ApoE4 isoform in the biological sample. Any biological sample which contains the DNA of that subject may be employed, including tissue samples and blood samples, with blood cells being a particularly convenient source. Determining the presence or absence of DNA encoding an ApoE4 4 isoform may be carried out with an oligonucleotide probe labelled with a suitable detectable group, or by means of an amplification reaction such as a polymerase chain reaction or ligase chain reaction (the product of which amplification reaction may then be detected with a labelled oligonucleotide probe) . Further, the detecting step may include the step of detecting whether the subject is heterozygous or homozygous for the gene encoding an ApoE4 isoform. Numerous different oligonucleotide probe assay formats are known which may be employed to carry out the present invention. See, e.g., U.S. Patent No. 4,302,204 to Wahl et al.; U.S. Patent No. 4,358,535 to Falkow et al.; U.S. Patent No. 4,563,419 to Ranki et al.; and U.S. Patent No. 4,994,373 to Stavrianopoulos et al. (applicants specifically intend that the disclosures of all U.S. Patent references cited herein be incorporated herein by reference) . Amplification of a selected, or target, nucleic acid sequence may be carried out by any suitable means. See generally D. Kwoh and T. Kwoh, Am. Biotechnol . Lab. 8, 14-25 (1990). Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction, ligase chain reaction, strand displacement amplification(see generally G. Walker et al., Proc. Natl . Acad. Sci . USA 89, 392-396 (1992); G. Walker et al.. Nucleic Acids Res. 20, 1691-1696 (1992)), transcription-based amplification (see D. Kwoh et al., Proc. Natl . Acad Sci . USA 86, 1173-1177 (1989)), self- sustained sequence replication (or "3SR") (see J. Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874-1878 (1990)), the Qβ replicase system (see P. Lizardi et al., BioTechnology 6, 1197-1202 (1988)), nucleic acid sequence-based amplification (or "NASBA") (see R. Lewis, Genetic Engineering News 12 (9), 1 (1992)), the repair chain reaction (or "RCR") (see R. Lewis, supra) , and boomerang DNA amplification (or "BDA") (see R. Lewis, supra) . Polymerase chain reaction is currently preferred. In general, DNA amplification techniques such as the foregoing involve the use of a probe, a pair of probes, or two pairs of probes which specifically bind to DNA encoding ApoE4, but do not bind to DNA encoding ApoE2 or ApoE3 under the same hybridization conditions, and which serve as the primer or primers for the amplification of the ApoE4 DNA or a portion thereof in the amplification reaction (likewise, one may use a probe, a pair of probes, or two pairs of probes which specifically bind to DNA encoding ApoE2, but do not bind to DNA encoding ApoE3 or ApoE4 under the same hybridization conditions, and which serve as the primer or primers for the amplification of the ApoE2 DNA or a portion thereof in the amplification reaction; and one may use a probe, a pair of probes, or two pairs of probes which specifically bind to DNA encoding ApoE3, but do not bind to DNA encoding ApoE2 or ApoE4 under the same hybridization conditions, and which serve as the primer or primers for the amplification of the ApoE3 DNA or a portion thereof in the amplification reaction) . In general, an oligonucleotide probe which is used to detect DNA encoding ApoE4 is an oligonucleotide probe which binds to DNA encoding ApoE4, but does not bind to DNA encoding ApoE2 or ApoE3 under the same hybridization conditions. The oligonucleotide probe is labelled with a suitable detectable group, such as those set forth below in connection with antibodies. Likewise, an oligonucleotide probe which is used to detect DNA encoding ApoE2 is an oligonucleotide probe which binds to DNA encoding ApoE2 but does not bind to DNA encoding ApoE3 or ApoE4 under the same hybridization conditions, and an oligonucleotide probe which is used to detect DNA encoding ApoE3 is an oligonucleotide probe which binds to DNA encoding ApoE3 but does not bind to DNA encoding ApoE2 or ApoE4 under the same hybridization conditions. Polymerase chain reaction (PCR) may be carried out in accordance with known techniques. See, e.g., U.S. Patents Noε. 4,683,195; 4,683,202; 4,800,159; and 4,965,188. In general, PCR involves, first, treating a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) with one oligonucleotide primer for each strand of the specific sequence to be detected under hybridizing conditions so that an extension product of each primer is synthesized which is complementary to each nucleic acid- strand, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith so that the extension product synthesized from each primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer, and then treating the sample under denaturing conditions to separate the primer extenεion productε from their templates if the sequence or sequences to be detected are present. These steps are cyclically repeated until the desired degree of amplification is obtained. Detection of the amplified sequence may be carried out by adding to the reaction product an oligonucleotide probe capable of hybridizing to the reaction product (e.g., an oligonucleotide probe of the present invention) , the probe carrying a detectable label, and then detecting the label in accordance with known techniques. Ligase chain reaction (LCR) is also carried out in accordance with known techniques. See, e.g., R. Weiss, Science 254, 1292 (1991). In general, the reaction is carried out with two pairs of oligonucleotide probes: one pair binds to one strand of the sequence to be detected; the other pair binds to the other strand of the sequence to be detected. Each pair together completely overlapε the εtrand to which it correεpondε. The reaction iε carried out by, firεt, denaturing (e.g., separating) the strands of the sequence to be detected, then reacting the εtrandε with the two pairε of oligonucleotide probes in the preεence of a heat stable ligase so that each pair of oligonucleotide probeε is ligated together, then separating the reaction product, and then cyclically repeating the process until the sequence has been amplified to the desired degree. Detection may then be carried out in like manner as described above with respect to PCR. It will be readily appreciated that the detecting steps described herein may be carried out directly or indirectly. Thus, for example, if either ApoE2 or ApoE3 is also detected in the subject, then it is determined that the subject is not homozygouε for ApoE4; and if both ApoE2 and ApoE3 are detected in the subject, then it is determined that the subject is neither homozygous nor heterozygous for ApoE4. As an alternative to isoelectric focusing and techniques for allele detection, the step of determining the presence or absence of the ApoE4 isoform in a εample may be carried out by an antibody assay with an antibody which selectively binds to ApoE4 (i.e., an antibody which binds to ApoE4 but exhibits esεentially no binding to ApoE2 or ApoE3 in the same binding conditions) . When one wiεheε to determine the preciεe ApoE complement of a patient and whether or not that patient iε homozygous or heterozygous for ApoE4, then antibodies which selectively bind to ApoE2 and ApoE3 may also be employed (i.e., an antibody which binds to ApoE2 but exhibits essentially no binding to ApoE3 or ApoE4 in the same binding conditions; an antibody which binds to ApoE3 but exhibits essentially no binding to ApoE2 or ApoE4 in the same binding conditions) . Antibodies used to selectively bind ApoE2, ApoE3, and ApoE4 can be produced by any suitable technique. For example, monoclonal antibodies may be produced in a hybrido a cell line according to the techniques of Kohler and Milstein, Nature 265, 495-97 (1975). ApoE2, ApoE3, or ApoE4 may be obtained from a human patient determined to be homozygous therefore, then purified by the technique described in S. Rail et al.. Methods in Enzymol . 128, 273 (1986), and used as the immunogen for the production of monoclonal or polyclonal antibodies. Purified ApoE isoforms may be produced by recombinant means to express a biologically active isoform, or even an immunogenic fragment thereof may be used as an immunogen. Monoclonal Fab fragments may be produced in Eεcherichia coli from the known sequences by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246, 1275-81 (1989) (recombinant Fab techniques); P. Wenham et al.. Lancet 337, 1158 (1991) (Apo PCR primers). The DNA encoding one subtype of ApoE can be obtained and converted to the other by cite-directed mutagenesis. See, e.g., T. Kunkel et al., Methods in Enzymol . 154, 367-382 (1987); T. Kunkel, U.S. Patent No. 4,873,192. The term "antibodieε" aε used herein refers to all typeε of i munoglobulinε, including IgG, IgM, IgA, IgD, and IgE. The antibodies may be monoclonal or polyclonal and may be of any specieε of origin, including (for example) mouεe, rat, rabbit, horse, or human, or may be chimeric antibodieε, and include antibody frag entε such as, for example, Fab, F(ab') 2 , and Fv fragmentε, and the corresponding fragments obtained from antibodieε other than IgG. Antibody aεεays may, in general, be homogeneous assays or heterogeneous. In a homogeneouε aεεay the immunological reaction uεually involveε the specific antibody a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution. Immunochemical labelε which may be employed include free radicals, radioisotopeε, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth. In a heterogeneous assay approach, the reagents are usually the specimen, the antibody of the invention and means for producing a detectable signal. Similar specimens as described above may be used. The antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase. The support iε then εeparated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the presence of the analyte in the specimen. Meanε for producing a detectable εignal include the use of radioactive labelε, fluoreεcent labels, enzyme labelε, and so forth. For example, if the antigen to be detected contains a second binding site, an antibody which bindε to that εite can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The preεence of the detectable group on the solid support indicateε the preεence of the antigen in the teεt εample. Examples of suitable immunoasεayε are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoasεayε, and the like. Thoεe εkilled in the art will be familiar with numerous specific immunoasεay formatε and variations thereof which may be useful for carrying out the method disclosed herein. See generally E. Maggio, Enzyme- Immunoasεay, (1980) (CRC Press, Inc., Boca Raton, FL) ; see also U.S. Patent No. 4,727,022 to Skold et al. titled "Methods for Modulating Ligand-Receptor Interactions and their Application," U.S. Patent No. 4,659,678 to Forrest et al. , U.S. Patent No. 4,376,110 to David et al. , U.S. Patent No. 4,275,149 to Litman et al. , U.S. Patent No. 4,233,402 to Maggio et al. , and U.S. Patent No. 4,230,767 to Boguslaεki et al. Antibodieε which εelectively bind an ApoE isoform (i.e., bind to one of ApoE2, ApoE3 or ApoE4 while showing essentially no binding to the other under the same binding conditions) may be conjugated to a εolid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polyεtyrene) in accordance with known techniqueε, εuch aε precipitation. Antibodies which bind an ApoE isoform may likewise be conjugated to detectable groups such aε radiolabelε (e.g., 35 S, 125 I, 131 I) , enzyme labelε (e.g., horεeradiεh peroxidaεe, alkaline phoεphataεe) , and fluorescent labels (e.g., fluorescein) in accordance with known techniques. Diagnostic kits for carrying out antibody assays may be produced in a number of ways. In one embodiment, the diagnostic kit compriεeε (a) an antibody which bindε ApoE2, ApoE3, or ApoE4 conjugated to a εolid εupport and (b) a εecond antibody which binds ApoE2, ApoE3, or ApoE4 conjugated to a detectable group. The reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharideε and the like. The diagnostic kit may further include, where necesεary, other members of the signal-producing system of which syεtem the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like. A second embodiment of a test kit compriseε (a) an antibody as above, and (b) a specific binding partner for the antibody conjugated to a detectable group. Ancillary agents as described above may likewise be included. The test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructionε for carrying out the teεt. The preεent invention iε explained in greater detail in the following non-limiting Exampleε. EXAMPUE 1 Binding of Cerebrospinal Apolipoprotein E to Immobilized Beta-Amyloid Precursor Protein Fragments BA peptides (Bachem) , other peptides, or ethanolamine were covalently immobilized to Immobilon AV affinity membrane (Millipore) . The membrane is a chemically activated hydrophilic microporous membrane which covalently immobilizes peptides and proteinε through amino and thiol groupε. BA peptideε, or control peptides, -were dissolved in distilled water at one mg peptide/100 μL. Ten microliters (containing one hundred microgramε peptide) were applied to a 13 mm diameter Immobilon diεc, and incubated to dryneεε overnight at room temperature. Peptide was in large excess to the number of functional binding groups on the membrane. Control membranes were prepared by incubating the membrane in 2.0 M ethanolamine in 1.0 M NaHC0 3 , pH 9.5, to block the reactive groups on the Immobilon AV membrane. Membranes were stored at -20° C in a desiccator. Prior to use the membranes were washed with phoεphate buffered εaline. The 12-28 Hydropathic Mimic Peptide and the Even-Hydro Peptide were εynthesized uεing εtandard f-Moc εyntheεiε on an Applied Bio-Science 430A peptide εyntheεizer. Amino acids were protected with blocking groupε, and were deprotected and washed prior to manual cleavage. Purity of the peptides was verified by reverse phase high performance liquid chromatography. Binding of CSF proteins to immobilized peptideε. Immobilon AV membraneε previously bound with BA peptides, with ethanolamine, or with the hydromi ic or even-hydro peptides were incubated with 100 μl> cerebrospinal fluid (previously filtered through a 0.22 μ filter) with 50 μL phoεphate-buffered εaline, pH 7.3, for thirty minuteε at room temperature. After incubation the membraneε were placed in a Millipore filter holder (Swinnex) and washed with 3.0 ml phosphate-buffered saline, then washed with 700 μ 5 % sodium dodecylsulfate (SDS) . The membraneε were removed from the filter holder, cut in half and placed in 150 /xL of Laemmli buffer (2% sodium dodecylsulfate, 5% beta- mercaptoethanol, pH 6.8) and boiled five minutes to solubilize retained proteins. Forty-five /xL of Laemmli buffer with εolubilized protein were loaded in each of ten lanes of a Bio-Rad Minigel apparatus, with a stacking gel of 4%, and a separating gel of 12% polyacrylamide with 2% SDS. Immunodetection of APOE. Electrophoresed proteins were transferred to Immobilon P using standard Western transfer techniques. After transfer the membrane was incubated in Blotto (5% dried milk in Tris buffered saline, pH 7.6, with 0.5% Tween-20 (Pierce)) at room temperature for one hour. The membrane was next incubated in rabbit - anti-human apolipoprotein E antibody at 1:1,000 dilution (kindly εupplied by Dr. Joel Morriεett, Dept. of Medicine, Baylor College of Med. , Houεton, TX.) in Blotto overnight at 4° C, then waεhed five timeε in Blotto. The membrane waε expoεed to goat - anti-rabbit secondary antibody conjugated with horse radish peroxidase at 1:10,000 dilution for one hour at room temperature, then waεhed εeven timeε in Blotto. Horεe-radiεh peroxidaεe waε then visualized with Enhanced Chemiluminesce Detection kit (Amerεham) , and exposed to Hyperfilm ECL (Amersham) . Cerebrospinal fluid samples. Cerebrospinal fluid was obtained from clinical diagnostic lumbar punctures performed on human subjects following informed consent, and stored at -80° C. Resultε. Cerebroεpinal fluid containε many proteins which bind to immobilized BA peptide, and to a control peptide ("even-hydro peptide") after incubation and elution with phosphate buffered εaline, Figure 1, top, column 1. Among theεe retained proteinε iε apolipoprotein E, aε εhown in Figure 1, bottom, column 1. Many of theεe proteinε are eluted from both immobilized peptideε by 5% sodium dodecyl sulfate, column 2; by 4 molar urea, column 3; or by 6 molar guanidine hydrochloride; column 4. Guanidine hydrochloride did not however elute apolipoprotein E from the BA (1 _ 28) peptide, but eluted virtually all the apolipoprotein E previously bound to the control peptide, as shown in Figure 1, bottom, column 4. The binding of apolipoprotein E to various immobilized peptides is shown in Figure 2. Apolipoprotein E in cerebrospinal fluid binds with high affinity to immobilized BA (1 . 40) , BA (1 _ 28) and BA (12 . 28) following incubation and washing with PBS and 5% sodium dodecylsulfate. The "12-28 even hydro-peptide" containε the εame amino acidε aε &A {12 . 28) , but has a different hydropathic profile. Apolipoprotein E did not bind to this immobilized peptide, as shown in Figure 2 bottom. The 12-28 hydropathic mimic peptide, contains different amino acids from BA (12 _ 28) , but has a hydropathy profile very similar to A (12 _ 28) , and also bound apolipoprotein E. EXAMPLE2 Tτnττmτ>r>fl g fr.ftction of ApoE in CSF of Alzheimer's and Control Patients Cerebrospinal fluid from both Alzheimer's diεease patients, and control patients, containε immunoreactive apolipoprotein E. Apolipoprotein E in each control CSF bound to the immobilized BA (1 . 28) . Apolipoprotein E in the Alzheimer's diseaεe cerebroεpinal fluid εampleε, however, demonεtrated marked variability of binding to the BA peptide. EXAMPLE3 Identification of ApoE Isoforms in Alzheimer's CSF bv Isoelectric Focusing To determine whether the variation in binding to the βA peptide noted above could be explained by heterogeneity of the apolipoprotein E itεelf, CSF proteinε were resolved by isoelectric focusing and immunoreactive apolipoprotein E was visualized. Cerebrospinal fluid (CSF) proteins were delipidated by agitation of 30 μl CSF with an equal volume of chloroform:methanol :: 2:1 (V/V) . Delipidated CSF was then isoelectric focused using standard techniques on a urea, polacrylamide gel with Biorad ampholytes Ph 3 -10 in a vertical slab gel apparatuε (Biorad Minigel) . Following electrophoreεis, proteins were transferred to Immobilon P using Western technique and detected as described above. Several apolipoprotein E isoforms were detected by isoelectric focusing. EXAMPLE4 Detection of DNA Encoding the ApoE Type 4 Isoform in Blood Cells of Alzheimer's Disease Patients Population asεociation εtudies of apolipoprotein E were carried out with over 85 blood samples from subjects afflicted with familial Alzheimer's diseaεe, wherein the diagnoεiε of Alzheimer's diεeaεe was autopsy-proven. Genomic DNA from the blood samples was extracted in accordance with standard techniques and amplified by polymerase chain reaction in either a Stratagene SCS-96 or Techne MW-2 thermocycler using Techne HI-TEMP 96 well plates and the primers deεcribed by P. Wenham et al., Lancet 337, 1158 (1991). The polymeraεe chain reaction protocol waε eεsentially as described by Wenham et al. supra , and J. Hixson and D. Vernier, J. Lipid Res. 31, 545 (1990) . Each amplification reaction contained 20 ng genomic DNA, 1.0 pmol/μL each primer, 10% dimethylsulfoxide (Sigma) , 200 μM each dNTP (Pharmacia), 2.0 μCi (alpha- 32 P) dCTP (800 Ci /mol in 10 mM Tricine, NEN Reεearch Productε) 0.05 Unitε/μL Tag DNA polymeraεe and supplied IX incubation buffer (Boehringer Mannheim) in a final volume of 15 μL. An initial denaturation at 94° C for 5 minutes waε followed by 35 cycles of annealing at 65° C for 0.5 minuteε, extenεion at 70° C for 1.5 minuteε, denaturation at 94° C for 0.5 minuteε, and a final extension at 70° C for 10 minutes. After amplification, 5 units of H al (Pharmacia) were directly added to each well, and the plates were incubated at least 3 hours at 37° C. Three μL of each reaction were loaded on a 6% nondenaturing gel (0.4 mm thick X 43 cm long) and electrophoresed for one hour under constant current (45mA) . After electrophoresiε, the gel waε transferred to Whatman 3M chromatography paper, dried, and autoradiographed for one hour using Kodak XAR-5 film. Data are given in Table 1 below. The results showed that the otherwise uncommon type 4 isoform waε highly aεεociated with Alzheimer'ε disease, compared to the normal population. The gene frequency of this type 4 isoform of apolipoprotein E in the general population is 16%, while in the Alzheimer's diεeaεe patientε examined the gene frequency waε 51%. Table 1 : ApoE Isotype in Normal and Alzheimer' s Disease Patients. Theεe data indicate that detection of DNA encoding the type 4 isoform of apolipoprotein E is useful as a prognostic and diagnostic test for familial Alzheimer ' ε diεeaεe. EXAMPLE 5 Association of ApoE4 with Both Late-Onset • pjnn-niai nmi sporadic Alzheimer's Disease These data further support the involvement of the ApoE4 allele (ApoE-e4) in the pathogenesis of late- onset familial and sporadic AD. Families. Blood samples for the genomic DNA studies were obtained from families described previously (J. Murrell et al., Science 254, 97-99 (1991); H. Karlinsky et al., Neurology 42, 1445-1453 (1992); P. St. George-Hyslop et al., Nature Genet. 2, 330-334 (1992); M. Pericak-Vance et al., Am. J. Hum. Genet. 48, 1034-1050 (1991); and P. St. George-Hyslop et al., Nature 347, 194- 197 (1990)). All sampled individuals diagnosed aε probable Alzheimer's diεease patients (AD) were examined by a neurologist and associated diagnostic personnel of the Joseph and Kathleen Bryan Alzheimer's Disease Research Center (ADRC) Memory Disorders Clinic at Duke University, the Centre for Neurodengenerative Diseases at the University of Toronto, or the Departments of Neurology at Masεachusetts General Hospital and the Harvard Medical School. The clinical diagnosis waε made according to the NINCDS-ADRDA criteria (C. McKhann et al.. Neurology 34, 939-944 (1984)). The Duke pedigreeε were primarily late-onset AD families with an average age of 66.1±10.3 years in the 35 families. Three of the familieε could be claεεified as early-onset (M<60 years) AD families. One family segregates with the APP717val- ile mutation (A. Goate et al., Nature 349, 704-706 (1991)), a second segregates with the APP717val-phe mutation (J. Murrell et al., Science 254, 97-99 (199 1 )), and the other is linked to chromosome 14 markers with a maximum lod score of 3.5 (G. Schellenberg et al.. Science 258, 668-671 (1992) and P. St. George-Hyslop et al. , Nature Genet 2, 330-334 (1992)). The Toronto pedigrees were clasεified aε primarily early-onεet familieε ( 1 3 o f the 17 familieε) . Five of the familieε were linked to chromosome 14 with lod scores greater than 3.0 in each family (P.H. St. George-Hyslop et al., Nature Genet 2, 330-334 (1992)) while a sixth pedigree had the APP17val- ile mutation (H. Karlinsky et al.. Neurology 42, 1445- 1453 (1992)). The family and genotypic data were processed via the PEDIGENE* system (C. Haynes et al.. Genet. Epidemiol . 3, 235-239 (1986)). Genomic DNA from εome patients diagnosed as sporadic caseε of probable AD at Duke, Toronto, and Boεton have been banked over the past six yearε. Sporadic AD patientε were defined as those without a known family hiεtory of AD or dementia. The sporadic, probable AD patients represented all of the banked DNAs in the Toronto and Duke banks as of November 1992, except for an ongoing prospective series begun in August 1992 at the Bryan ADRC Memory Disorders Clinic. The DNA from these individuals had been collected randomly prior to any interest in ApoE isotyping. Not all sporadic AD patients evaluated in these clinics were banked routinely. The diagnosiε of probable AD in thiε group can be expected to be in the 80-90% accuracy range that iε obεerved in moεt εpecialized AD clinicε. Brain DNA waε obtained from autopεy-confirmed, Caucasian caseε of AD that had been banked in the Kathleen Bryan Brain Bank at Duke, the University of Toronto, and the Harvard Medical School. Six black or American Indian autopsies were eliminated from the series of sporadic AD autopsieε εince the aεεociation analyses are sensitive to the control group. Two setε of controlε were used in the study. The first set waε 91 unrelated grandparentε from the Centre d'Etude du Polymorphiεme Humain (CEPH) reference familieε (J. Dauεett et al., Genomics 6, 575- 577 (1990)). Theεe families were collected for human gene mapping and are characterized by having grandparents, parentε, and many grandchildren available for DNA mapping. The grandparentε repreεent a random group of Caucasian aged-controls of European and American background, similar to the late-onset FAD families and the autopsy-confirmed sporadic AD population. Twenty-one Caucasian spouses of patients participating in an ongoing prospective analysis of probable AD patients and spouεeε were alεo used as control group. Genomic DNA. High molecular weight DNA was obtained from transformed lymphoblasts according to know techniques (M. Pericak-Vance et al.. Neurology 36, 1418- 1423 (1986) and M. Pericak-Vance et al., Exp Neurol 102, 271-279 (1988)) or to the GENEPURE 341~ nucleic acid extractor's supplied protocol (Applied Biosyεtemε) . Genomic DNA from brain tissue was isolated by pulverizing approximately 300 mg of frozen brain tissue under liquid nitrogen, adding 4 ml of lysis buffer (Applied Biosystemε) and 1 mg of proteinaεe K (Applied Biosystems) , and gently rocking overnight at 37°C before extracting on the GENEPURE 341™. Aroplification and restriction isotvpinσ of ApoE. Genomic DNA was amplified by polymeraεe chain reaction (PCR) in a Techne MW-2 thermocycler uεing HI- TEMP 96-well plates (Techne) and the primers described by P. Wenham et al., Lancet 337, 1158-1159 (1991). The PCR protocol was based on those described by Wenham et al. and J. Hixson et al., J. Lipid Res. 31, 545-548 (1990). Each amplification reaction contained 20 ng genomic DNA, 1.0 pmol/μl each primer, 10% dimethylεulfoxide (Sigma), 200 μM each dNTP (Pharmacia), 1.0 μCi (alpha- 32 P)dCTP (800 Ci/mol in 10 M Tricine, NEN Reεearch Products) , 0.05 units/μl Taq DNA polymerase and supplied IX buffer (Boehringer Mannheim) in a final volume of 15 μl. An initial denaturation at 94'C for 5 minutes was followed by 35 cycles of annealing at 65"C for 0.5 minutes, extension at 70 C C for 1.5 minuteε, denaturation at 94°C for 0.5 minutes, and a final extension at 70°C for 10 minuteε. After amplification, 5 units of Hhal (Gibco) were directly added to each well, and the plates were incubated at least 3 hours at 37"C. Fifteen μL of 2X Type III stop dye (J. Sambrook et al.. Cold Spring Harbor Laboratory Press B.24 (1989)) were added to each well, and 3 μL of each reaction were loaded on a 6% nondenaturing polyacrylamide gel (0.4 mm thick X 43 cm long) and electrophoreεed for one hour under conεtant current (45mA) . After electrophoreεis, the gel was tranεferred to Whatman 3M chromatography paper, dried, and autoradiographed for one hour using Kodak XAR-5 film. Each autoradiograph was read independently by two different observers. Statistical analysis. Allele frequencies for the control and AD groups were estimated by counting alleles and calculating sample proportions. Allele frequency eεtimateε for the early- and late-onεet FAD familieε were calculated uεing one randomly εelected affected patient from each family. The Z statistic for comparing two proportions was calculated (R. Elεton and W. Johnεon, Essentials of Bioεtatiεticε, (1987) and G. Schellenberg et al. , J. Neurogenet. 4, 97-108 (1987)). An extreme value of Z compared to the probabilitieε for the εtandard normal diεtribution would suggest rejecting the null hypotheεiε that the allele frequencieε in the two groupε are equal. In order to compare the ApoE allele frequencieε in the different populationε, i . e . , AD versus controls, and between control groups, the following comparisons were made: 1) CEPH controls verεuε εpouεe controlε; 2) CEPH controlε versus literature controls; 3) CEPH controls versus late-onset; 4) CEPH controls versus early-onset; 5) CEPH controls verεuε clinical (probable) sporadic AD; and 6) CEPH controls versus autopsy-confirmed sporadic AD. Affected-pedigree- member (APM) linkage analysiε for ApoE waε analyzed in accordance with known techniqueε (M. Pericak-Vance et al.. Am. J. Hum. Genet. 48, 1034-1050 (1991) and D. Weekε et al., Am. J. Hum. Genet. 42, 315-326 (1988)). Two point lod εcoreε were calculated using the computer program LINKAGE™ Program Package (version 5.0) (G. Lanthrop et al., Am. J. Hum. Genet. 36, 460-465 (1984) and G. Lanthrop et al., Proc. Natl . Acad. Sci . USA 81, 3443-3446 (1984)). The age-of-onset and disease parameters used in the lod score calculations were as formerly outlined (M. Pericak-Vance et al., supra) . Results. Table 2 illustrates the ApoE e4 allele frequency estimates in three control populations: 1) 91 grandparentε from the CEPH reference families; 2) 21 spouses from an ongoing prospective study examining consecutive sporadic, probable AD patientε, and 3) a representative control serieε from a similar population in the literature (G. Lanthrop et al., Proc. Natl . Acad. Sci . USA 81, 3443-3446 (1984)). Also illustrated are the ApoE e4 allele frequency estimateε for εeveral different Alzheimer's disease groupε: 1) one randomly εelected affected individual in the combined Duke and Toronto/Boεton late-onεet FAD εerieε; 2) one randomly selected affected individual in the combined Toronto/Boston and Duke early-onset FAD families; 3) banked DNA εa pleε from sporadic patients carrying the diagnosiε of probable AD from the Duke and Toronto/Boεton clinicε; and 4) DNA from 176 autopεy-confirmed εporadic AD patients from Duke and Toronto/Boston. Consiεtent with Exampleε 1-4 above, the ApoE e4 allele frequency of the randomly εelected affected patients in the predominantly late-onset FAD families was significantly different from that of the CEPH controlε; 0.50±0.06 verεus 0.16±0.027 (allele frequency eεtimate±εtandard error, Z=2.44, P=0.014). Likewise, the combined Duke and Toronto/Boston late-onset FAD serieε preεented here iε εignificantly different from the CEPH controlε (P=0.000017) . The ApoE-e4 frequency of the combined early-onεet FAD series from Toronto/Boston and Duke (0.19±0.069) did not differ εignificantly (P=0.069) from the frequency in the CEPH controls. Statistical analyses demonstrate highly significant differences in the ApoE-e4 allele frequencieε in both sporadic (probable) AD (P=0.00031) and autopsy-confirmed sporadic AD (P<0.00001) when compared to the CEPH controls. Table 2. ApoE-e4 Allele Frequency Estimates 1 Allele frequency estimates ± the standard error; number of chromosomes counted is presented in parentheses. 2 Z values are versus the CEPH control group. 3 91 unrelated grandparents from the Centre D'Etude du Polymorphism Humain (CEPH). ^Spouse controls in the Bryan ADRC Memory Disorders Clinic, Duke University. Population in H. Menzel et al., Apolipoprotein E polymorphism and Coronary Artery Disease, Arteriosclerosis 3, 310-315 (1983). ^LOAD" means Late Onset AD; One randomly selected affected from each of 32 Duke and 4 Toronto/Boston late-onset FAD families. 7 "EOAD" means Early Onset AD; One randomly selected affected from each of 13 Toronto Boston and 3 Duke early-onset FAD families. δ 39 Duke and 30 Toronto/Boston sporadic (probable) AD patients. 9 143 Duke and 33 Toronto Boston autopsy-confirmed sporadic AD subjects. EXAMPLE 6 Relation of Amyloid Beta-Peptide Deposition to Apolipoprotein E Type in Post-Mortem Tissue In this Example, a serieε of brainε from patientε with εporadic late-onset AD of known ApoE genotype was examined to determine whether AD brainε with an ApoE-e4 allele have a diεtinct pattern of amyloid deposition. Brains of patients homozygous for ApoE-e4 were found to contain increased vascular amyloid deposits and number and density of amyloid and neuritic plaques compared to ApoE e3 homozygoteε. In immunocytochemical studies, J-peptide was significantly increased in those patients with one or two ApoE-e4 alleles. The three main ApoE genotypes studied (e3/3, e3/4, and e4/4) showed no statistically significant difference in sex, age of onset or duration of illness. Late-onset Ad cases associated with one or two ApoE-e4 alleles thus have a distinct neuropathological phenotype compared to cases homozygous for the ApoE-e3 allele. Case selection. 143 autopsy-confirmed cases of late-onεet Alzheimer's Disease (AD) meeting NIH and CERAD criteria (C. McKhann et al.. Neurology 34, 939-944 (1984) and S. Mirra et al.. Neurology 41, 479-486 (1991)) and without affected family members or other neurological disease were obtained through the Kathleen Bryan Brain Bank at Duke University and analyzed for ApoE genotype. These caεeε were banked between 1985 and 1992. The average age at death for these 143 cases was 77.2±8.9 years of age with average duration of illness 8.6+4.2 years, and 63% of the patientε were women. ApoE Genotvping. Genomic DNA was obtained by isolating DNA from approximately 300 mg of frozen brain tissue from each case. ApoE genotyping for each patient waε carried out aε described above using amplification by polymerase chain reaction with ApoE primers and Hhal restriction isotyping with autoradiographic detection. ApoE genotyping demonstrated: 47 cases ApoE e3/3, 64 cases ApoE e3/4, 23 cases ApoE e4/4, 3 caseε ApoE e2/3 and 6 cases ApoE e2/4. Neuropathological analysis. Information on age at death, duration of illness, gender, brain weight, preεence of amyloid depoεitε in cerebral veεεelε (using congo red εtain) and description of neuritic plague density and amount of neurofibrillary tangles (modified King'ε silver stain [B. Lloyd et al., J. Hiεtotech 8, 155-156 (1985)]) were taken from the original neuropathological reports. Neuritic plaque counts were mentioned in 100 cases and neurofibrillary tangle counts in 95 cases. Each count represented a single microscopic field felt to be the most affected area in the section of that region. Plaques were counted with a lOx objective (field of 2.92 mm 2 ) while tangles were counted with a 20x object (field of 0.72 mm 2 ) . Plaque countε were truncated at 100 per 10x, field. All of thiε data had been entered into the patientε medical record one to five yearε prior to ApoE genotype analyεiε. Analvεiε of conσo red stained material for vascular amyloid. A subset of 53 patients was chosen by selecting in chronological order those caseε with preexiεting congo red εtained slides. Selection ended when 17 patients with ApoE genotype e4/4 (of the 23 total patientε) ; 20 patients with ApoE genotype e3/3 (of the 49 total patients) ; and 16 patientε with ApoE genotype e3/4 (of the 65 total patientε) had been choεen. Forty of the 53 autopεy reportε εpecifically mentioned vaεcular amyloid and formed one εet of blinded obεervationε completed before genotyping. The preεence of vaεcular amyloid waε graded at three levelε: no evident amyloid depoεitε (grade 0) , trace amyloid depoεitε including one poεitive veεεel (grade 1) , and readily identifiable vaεcular amyloid (grade 2) . In addition, congo red εtained εlideε of frontal cortex and hippocampus including entorhinal cortex from all 53 caseε were examined by three observers for vascular amyloid using the same grading system. These observations were analyzed independently and double-blind with average inter-rater agreement of 8.5%. Immunocvtochemical methods. Paraffin blocks of hippocampal region, frontal lobe and parietal lobe were obtained for 7 caseε of ApoE e4/4, 8 caεes of ApoE e3/3, and 4 caseε of ApoE e3/4. Selection of cases was random without knowledge of neuropathological report. 6-8 micron paraffin sections were cut and mounted on coated slides for immunocytochemistry. Sections were deparaffinized, treated with 90% formic acid for 3 minuteε, waεhed and then incubated with monoclonal antibody for immunolocalization of amyloid ^-peptide (antibody deεcribed in S. Ikeda et al.. Prog. Clin. Biol . Reε . 317, 313-323 (1989), gift of Drε. George Glenner and David Allsop) . Sections were reacted in parallel with identical antibody dilution and enzymatic detection steps using the ABC method (Vectorstain, Burlingame, CA) , and a coverslip was placed over Permount after dehydration. Semi-adjacent sections (untreated with formic acid) were used to demonstrate neuritic plaques with monoclonal antibody (dilution 1:1000) to 164kd-neurofilament protein (SMI-34, Sternberger-Meyer Immunocytochemicalε, Inc) and for ApoE uεing a polyclonal antibody (dilution 1:20,000) to human ApoE which recognizeε ApoE2, ApoE3 and ApoE4 iεoformε on Weεtern blotε (gift of Dr. Joel Morriεett, Baylor College of Medicine) . Parallel controlε were unεtained in theεe experiments. Analysis of immunocvtoche ical material. Regionε of maximum plaque denεity in each εection were εelected and graphical analysiε waε performed by drawing plaque outlines using a camera lucida onto a defined rectangle of 0.25 mm 2 divided into 160 equal grids. Any gridboxes containing all or any portion of an immunoreactive plaque were counted, and the total waε divided to actual genotype. Inter-rate reliability iε 85% with theεe methodε, and variance between separate fields from the same εection 15%. Repreεentative fields were photographed with a Zeiεε photomicroscope. Statistical analvεiε. Data were entered in a Statgraphics package and analyεiε of variance uεed to deεcribe the relationεhipε between variableε. No εignificant differenceε existed between the main set of 143 cases, the εubεet of 53 caεeε, or the smaller subεets above of ApoE e3/3, e3/4, and e4/4 genotypes chosen for congo red or immunocytochemical analysis with regard to age at death, gender ratio or brain weight. For comparison of plaque and neurofibrillary tangle counts, Kruskal-Wallis one-way analysis was employed since observations did not fit a normal distribution. Im unolocalization of g-peptide amyloid depoεitε in brainε of εporadic AD patientε homozyoouε for APOE e3 or e4. Immunocytochemistry for 9-peptide in brains of patients with εporadic late-onset AD demonstrated conεiεtent differences between immunostained sections of cerebral cortex from ApoE e3/3 and ApoE e4/4 cases (data no shown) . 0-peptide immunoreactivity in the εectionε from ApoE e3/3 caεeε revealε minimal to abεent vaεcular staining and faintly immunoreactive plaques while sections from ApoE e4/4 caseε are darkly εtained with abundant immunoreactive veεεelε on the cerebral εurface and εtrongly immunoreactive plaqueε and vesselε in the parenchyma. The difference iε typically of εuch magnitude that β-peptide immunostained sectionε from ApoE e4/4 brainε can be differentiated from εectionε from ApoE e3/3 brainε without a microεcope. Immunolocalization of APOE in brains AD patients homozygous for ApoE e3 or e4. ApoE immunoreactivity was observed in cerebral vesεelε, neuronε, glial cellε, εenile plaques and neurofibrillary tangles as described in previous reports (J. Diedrich et al., J. Virol . 65, 4759-4768 (1991); and Y. Namba et al. , Brain Reε. 541, 163-166 (1991)). Like /3-peptide immunoreactivity, ApoE immunoreactivity is enhanced after formic acid treatment. There were no major differences in the localization or intensity of ApoE immunoreactivity in ApoE e3/3 cases compared to ApoE e4/4 (note that this polyclonal antiserum to ApoE detectε both ApoE 3 and ApoE 4 isoformε) . In particular, moεt larger cerebral veεεelε were ApoE immunoreactive in both ApoE e4/4 cases and in ApoE e3/3 caseε. Extent of vaεcular amyloid in e3/3. e4/4 and e3/4 A P OE genotypeε - retrospective analysis of autopsy reports. In 40 of the 53 caseε, the presence or absence of vascular amyloid detected by congo red staining was clearly noted in the autopsy report. Retrospective grading of these autopsy reports for amount of vascular amyloid deposits revealed a significant asεociation of vaεcular amyloid with number of ApoE e4 alleles (p < .0001). Typically, no vascular amyloid waε mentioned for ApoE e3/3 cases, trace vascular amyloid for e3/4 caseε, and large amountε were obεerved in oεt e4/4 cases (Table 3.A.1 below). Proεpective analvεis of congo red εtained material. To examine proεpectively the extent of vascular amyloidosis in the total serieε of 53 cases, a double-blind review of congo red stained sections of hippocampus and frontal cortex was conducted. This analysis confirmed a highly significant association between the presence of congo red positive amyloid angiopathy and the dose of ApoE e4 allele in the 53 caseε (Table 3.A.1). Such amyloidotic veεεels were not necesεarily preεent throughout a given cortical region in ApoE e4/4 homozygoteε, but often predominated in the depths of sulci εuch aε the hippocampal fissure. Analysiε of g-peptide immunoreactive vessels. When sectionε from a subset of the serieε of 53 patientε were im unoreacted for 3-peptide and εimilarly rated, the same statistically significant association between the amount of vascular amyloid and the dose of ApoE e4 allele waε found (Table 3.C.1). Amyloidotic veεεelε in ApoE e4 homozygoteε included leptomeningeal veεεelε and large and small vesεelε in the cortical plate with εurrounding plaque-like accumulation of amyloid (plaque-like angiopathy of Scholze [W. Scholze, Z. Geεamte Neurol . Psych. 162, 694-715 (1938)]). Neuritic plague number iε increaεed in APOE e4 homozygoteε compared to APOE e3 homozygoteε. In four out of five cortical regions, the average number of neuritic plaques in silver stained material was greater in ApoE e4 ho ozygotes than in ApoE-e3 homozygotes (Table l.A.2) . Kruskal-Wallis one-way analysis revealed that increased average neuritic plaque count was significantly associated with ApoE-e4 allele dose in three regions: frontal, temporal and parietal cortex and that the association bordered on significance in the CA1 subfield of hippocampus. Neuritic plague counts did not correlate with duration of illness. Neurofibrillary tangle counts are mildly increased in APOE e4 compared to ApoE e3 homozygotes. In all five of the cortical regions presented above, the average number of neurofibrillary tangles was greater in ApoE-e4 homozygotes compared to ApoE-e3 homozygotes (Table 3.A.3). The chance that all five regions would have increased average neurofibrillary tangle count in ApoE e4/4 cases compared to ApoE e3/3 caseε is 3% (nonparametric paired εa ple teεt) . The differences in average counts for each area are modest and not significant by Kruskal-Wallis test. However, the average neurofibrillary tangle count in each cortical region varies significantly (p < .01) with duration of illnesε in the whole set of patients and in the ApoE genotype subεets. The increase in average neurofibrillary tangle counts in ApoE-e4 homozygotes is apparently accounted for by the upward trend in duration of illness asεociated with ApoE-e4. Immunocytochemical analysis of amyloid deposition in sporadic AD caseε. The large increase in amyloid depoεition noted in ApoE-e4 homozygoteε compared with ApoE-e3 homozygoteε with εporadic AD includeε both vascular amyloid deposition and deposition in senile plaques. In order to confirm qualitatively and quantitatively these differences, a set of 7 patientε homozygouε for ApoE-e4 waε compared to a set of 8 patients homozygous for ApoE-e3. Four heterozygotes (ApoE-e3/4) were also examined to determine possible ApoE dosage effects. Low power views of cerebral cortex reveal the typical difference in over-all 0-peptide immunoreactivity observed in 0-peptide immunoreacted cortical sections of ApoE e3 homozygotes compared to e4 homozygoteε (date not shown) . Theεe differenceε are consistently found even when ?-peptide immunostained sections from a given cortical region are searched for microscopic fields with maximtim density of immunoreactive plaques. Plaques in ApoE e4/4 cases were more numerous, somewhat larger often including a vessel, and darker than those observed in ApoE e3/3 cases. Quantitative analysis of g-peotide immunoreactivity of plagues. Sections of frontal cortex from fifteen cases of sporadic AD were immunoreacted for 3-peptide localization and quantitative analysis was made of the microscopic fieldε containing maximum denεity of /S-peptide immunoreactive plaqueε for each caεe (Table 3.B.2). There was a highly significant, seven- fold greater average area covered by εtrongly 9-peptide immunoreactive plaqueε in ApoE e4/4 homozygotes compared to ApoE e3/3 homozygotes (p < .002). ApoE e3/4 caseε are intermediate. In εeveral ApoE e3/3 caεeε, weakly immunoreactive plaqueε were alεo preεent and their number was significantly higher than in ApoE e4/4 caseε (p=.04). Nevertheleεε, the average total area covered by all ,6- peptide immunoreactive plaques was two-fold greater (p<.008) in ApoE e4/4 homozygotes compared to ApoE e3/3 homozygotes (Table 3.B.2). able 3. Neuropathological and Xmxnunochemic&l Characteristics of Patients with Sporadic AD Extent of neuritic plagueε. Additional semi- adjacent section of frontal cortex from eight of the above cases were immunostained with an antibody to neurofilament protein (SMI-34) to teεt whether neuritic plaque denεity varied between the ApoE-e3 and ApoE-e4 homozygoteε. SMI-34 immunoreactivity reveals neuritic processes and neurons with neurofibrillary tangles. Neuritic plaqueε can be identified by their content of neuritic processes. Although the number and total area covered by neuritic plaques (as defined by SMI-34 immunoreactivity) is less than that for amyloid plaques (as defined by β- peptide immunoreactivity) (data not shown) , comparison of the plaque area measurements in semi-adjacent sections shows a significant and linear correlation for the two methods of plaque detection in these eight caseε (p<.006, r=0.73). The area covered by neuritic plaqueε is also εignificantly greater in ApoE-e4 homozygoteε than in ApoE-e3 homozygoteε (p<.02). EXAMPLE7 Relationship of ApoE4 Gene Dose and Risk of Alzheimer's Disease Thiε Example quantifieε both the increased risk for AD and earlier age at onset conferred by ApoE4 alleles in late onset AD familieε. Risk increased from 20% to 90% and mean age at onset decreased from 84 to 68 years with increaεing number of ApoE4 alleleε. Theεe data indicate that ApoE4 gene dose is a major risk factor for late onset AD, and that ho ozygosity for ApoE4 iε virtually εufficient to cause AD by age 80. Four of the 46 families tested to date are early onset families; two have chromosome 21 APP mutations, and two are linked to chromosome 14. The frequency of ApoE4 was not elevated in these families. Members of 42 late onset families diagnosed with AD or examined and found to be unaffected after age 60, with known ApoE genotype, were evaluated. Before enrollment in our study, informed consent was obtained from each subject or, when necessary, their legal guardian. All study protocols were approved by the Duke University Medical Center Institutional Review Board. Descriptive statistics for affected and unaffected subjects are given in Table 4. On average, women survived longer than men, whether affected (p=0.02) or unaffected (p=0.13). With regard to table 4, note that more than 90% of clinically diagnosed cases were confirmed at autopsy. The predictive value of clinical examination is nearly 100% when a family member haε an autopεy-confirmed diagnoεiε of AD. Four εubjectε with a diagnosis of AD and 30 subjectε examined before age 60 were excluded from analyεiε. Late onεet families were not linked to chromosome 21 or to chromosome 14 within 19% recombination of the region previously linked to early onset AD. The estimated maximum two-point LOD score for AD and APOE was 2.61 at 6% recombination. Table 4. Descriptive Statistics for Affected and Unaffected Subjects The proportion of affected individuals increased with the number of ApoE4 alleles from 20% of subjects with genotype 2/3 or 3/3, to 47% of subjects with genoty , a 2/4 or 3/4, and to 91% of subjects with the 4 /4 genotype (Table 5) . This additive trend was highly significant (chi-εquared=33.4 with 1 df, p < 0.00001) (G. Koch et al., Analyεiε of Categorical Data (Les Presses de L'Universite de Montreal, Montreal, 1985) and SAS Institute, Inc., SAS/STAT Uεer ' ε Guide , Release 6.03 Edition (SAS Institute, Cary NC, 1988)). More women than men were affected (p=0.04) . This suggestε that women may poεεibly be at greater riεk for AD. Table 5. Percent Affected for Each ApoE Genotype Aε εhown in Table 6, riεk of AD increaεed by a factor of 2.84 (95% confidence interval (Cl) 2.03 to 3.96) for each additional ApoE4 allele (D.R. Cox et al., Analyεiε of Survival Data (Chapman and Hall, London, 1984) and L. Wei et al., JASA 84, 1065 (1989)). Hence, subjects with the 4/4 genotype were more than 8 times as likely to be affected as εubjectε with 2/3 or 3/3 genotypes. When separate estimateε were made for εubjectε with 0, 1, and 2 ApoE4 alleleε, there waε conεistent evidence (which did not reach εtatiεtical εignificance) that women were at higher riεk than men. Riεk for women over men waε 1.33 (95% Cl 0.42 to 4.26), 1.39 (95% Cl 0.76 to 2.55), and 1.30 (95% Cl 0.50 to 3.38) at 0, 1, and 2 ApoE4 gene doses, respectively. TABLE 6. Percentage of Affected Subjects and Relative Hazard According to the Number of ApoE4 Alleles. Estimates of riεk in Table 6 were derived by exponentiation of parameter eεtimates obtained from a Cox proportional hazard model which allowed risk to differ in men and women (SAS Institute Inc., Analyεiε of Survival Data (Chapman and Hall, London, 1984) , and SAS Institute Inc., SAS Technical Report P-217, SAS/STAT Software : The PHREG Procedure , Verεion 6 (SAS Institute Inc., Cary NC, 1991)). The εymbol '+' indicates that hazard waε εignificantly different from the reference value of 1. Information on each εubject between the ageε of 60 and 75 waε uεed aε the aεsumption of proportional hazards did not hold after the diagnoεiε by age 75 of nearly all perεonε with 2 ApoE-e4 alleleε. Statiεtically conεistent, if not fully efficient, estimates of relative hazard result from proportional hazards models even when related individuals are evaluated (L. Wei et al., JASA 84, 1065 (1989)). Thus, estimateε of riεk cloεely approximate eεtimates which would have been found by εampling juεt one perεon from each of a much larger collection of familieε. The Mantel-Haenεzel correlation statistic (SAS Institute, Inc., SAS/STAT ϋεer ' ε Guide , Releaεe 6.03 Edition (SAS Institute, Cary NC, 1988) , and D.R. Cox et al., Analyεiε of Survival Data (Chapman and Hall, London, 1984)), stratified by family, was used to evaluate the additive trend in riεk with increasing ApoE4 gene dose. The proportion of affected subjects increased significantly with ApoE4 gene dose (chi-sq=33.4 with l d.f., p<0.00001) . Next examined was age at onset to determine if it was related to ApoE4 gene dose. Onset distributions were constructed from information on age at onset in affected subjects and age when last examined in unaffected subjects and were distinct -for each gene dose (chi-sq=53.84 with 2 d.f., p<0.00001) (R.G. Miller Jr., Survival Analysiε (Whiley, New York, 1981) , and SAS Institute Inc., SAS ϋser f ε Guide : Statisticε , Version 5 Edition (SAS Institute, Cary NC, 1985)) (Figure 3). Each additional ApoE4 allele shifted onεet to younger age; mean onεet waε 84.3 (SE 1.3) yearε in subjects with 1 ApoE4 allele, and 68.4 (1.2) years in subjects with 2 ApoE4 alleles. Onεet tended to be earlier in women than in men (p=0.04) . Similarly, εurvival distributions were constructed from information on age at death in subjects known to be deceased and from age when last examined in other subjectε, regardleεε of affection status. ApoE4 gene dose was related to survival (p=0.004); mean survival was 84.9 (SE 1.3) years in subjects with 0 ApoE4 alleles, 78.8 (SE 0.8) years in εubjectε with 1 ApoE4 allele, and 78.1 (SE 1.4) yearε in subject with 2 ApoE4 alleles. Earlier death in individuals with 1 or 2 ApoE4 alleles was primarily attributable to more frequent and earlier onset of AD in these subjects (p=0.001). Despite shorter survival in subjects with 1 or 2 ApoE4 alleles, the earlier onεet conferred by each ApoE4 allele leads us to suεpect that most diagnoseε of AD and moεt prevalent caεes are in subjects with 1 or 2 ApoE4 alleles. The difference between mean onset and mean survival was 9.7 years in subjectε with 2 ApoE4 alleleε, 3.1 year in subjectε with 1 ApoE4 allele and 0.6 years in subjects with 0 ApoE4 alleles. Previous reports of linkage of AD to markers near the ApoE locus may have resulted from the allelic association of AD with ApoE4. Although these markers do not appear to be in disequilibrium with ApoE, their segregation could mimic linkage in families segregating at least one ApoE4 allele (D.A. Greenberg, Am. J. Hum. Genet . 52, 135-143 (1993)). The strong but not definitive evidence for linkage of AD to the ApoE locus (2=2.61, 0=0.06) also supports this explanation. It is important to realize that 19 of 95 affected subjects in our cohort of pedigrees and 64 of 176 autopsy confirmed sporadic AD cases described by Saunderε et al. (R.G. Miller Jr., Survival Analyεiε (Wiley, New York, 1981)) had no ApoE4 alleleε. Twelve of 42 late onεet familieε had affected memberε with 0 ApoE4 alleles. The fact that these tended to be the largest and based on simulation studies, the potentially most informative familieε for linkage, εtrongly εuggeεts that other genetic sources of riεk exiεt. Theεe other geneε will only be identified once the effectε of the ApoE4 allele are included in εubεequent analysis. The foregoing examples are illustrative of the present invention, and are not to be construed aε limiting thereof. The invention iε defined by the following claims, with equivalents of the claims to be included therein.

Claims: THAT WHICH IS CLAIMED IS: 1. A method of diagnosing or prognosing Alzheimer's disease in a subject, wherein the presence of an apolipoprotein E type 4 (ApoE4) isoform indicates said subject is afflicted with Alzheimer's diseaεe or at risk of developing Alzheimer's disease, said method comprising: detecting the presence or absence of ApoE4 in said subject. 2. A method according to claim 1, wherein said detecting step iε carried out by collecting a biological sample containing DNA from said subject, and then determining the presence or absence of DNA encoding ApoE4 in said biological sample. 3. A method according to claim 2, wherein said determining εtep iε carried out by amplifying DNA encoding ApoE4. 4. A method according to claim 3, wherein εaid amplifying εtep iε carried out by polymeraεe chain reaction. 5. A method according to claim 3, wherein εaid amplifying εtep iε carried out by ligaεe chain reaction. 6. A method according to claim 1, wherein εaid detecting εtep iε carried out by collecting an ApoE sample from said subject, and then determining the presence or absence of the ApoE4 isoform in said ApoE sample. 7. A method according to claim 6, wherein said determining εtep is carried out by isoelectric focusing. 8. A method according to claim 6, wherein said determining step is carried out by immunoasεay. 9. A method according to claim 6, wherein said determining step is carried out by immunoassay with an antibody that selectively binds to the ApoE4 isoform. 10. A method according to claim 1, wherein said subject has previously been determined to be at risk of developing Alzheimer's diseaεe. 11. A method according to claim 1, wherein εaid detecting εtep comprises detecting whether said subject is homozygouε for the gene encoding ApoE4. 12. A method of prognosing Alzheimer's disease in a subject, which subject has not previously been diagnosed as afflicted with Alzheimer's disease, wherein the presence of an apolipoprotein E type 4 (ApoE4) isoform indicates said subject is at riεk of developing Alzheimer'ε disease, said method comprising: detecting the presence or absence of ApoE4 in said subject. 13. A method according to claim 12, wherein said detecting step iε carried out by collecting a biological εample containing DNA from εaid εubject, and then determining the presence or absence of DNA encoding ApoE4 in εaid biological εample. 14. A method according to claim 13, wherein εaid determining step is carried out by amplifying DNA encoding ApoE4. 15. A method according to claim 14, wherein said amplifying εtep iε carried out by polymeraεe chain reaction. 16. A method according to claim 14, wherein said amplifying step is carried out by ligase chain reaction. 17. A method according to claim 13, wherein biological sample comprises a blood sample. 18. A method according to claim 12, wherein said detecting step is carried out by collecting an ApoE sample from said subject, and then determining the presence or abεence of the ApoE4 isoform in said ApoE sample. 19. A method according to claim 18, wherein said determining step iε carried out by iεoelectric focuεing. 20. A method according to claim 18, wherein εaid determining εtep iε carried out by immunoaεεay. 21. A method according to claim 18, wherein εaid determining εtep iε carried out by immunoaεεay with an antibody that selectively binds to the ApoE4 isoform. 22. A method according to claim 18, wherein said ApoE εample compriεeε a blood sample. 23. A method according to claim 12, wherein said subject has previously been determined to be at risk of developing Alzheimer'ε diεeaεe. 24. A method according to claim 12, wherein εaid detecting εtep compriεeε detecting whether said subject is homozygous for the gene encoding the ApoE type 4 isoform. 25. A method of screening a subject afflicted with dementia for Alzheimer's disease, wherein the presence of an apolipoprotein E type 4 (ApoE4) isoform indicates said subject is afflicted with Alzheimer's disease, said method comprising: detecting the presence of ApoE4 in said subject. 26. A method according to claim 25, wherein said detecting step is carried out by collecting a biological sample containing DNA from said subject, and then determining the presence or absence of DNA encoding ApoE4 in said biological sample. 27. A method according to claim 26, wherein said determining εtep iε carried out by amplifying DNA encoding ApoE4. 28. A method according to claim 26, wherein εaid amplifying step is carried out by polymerase chain reaction. 29. A method according to claim 26, wherein said amplifying εtep iε carried out by ligaεe chain reaction. 30. A method according to claim 25, wherein εaid detecting step is carried out by collecting an ApoE sample from said subject, and then determining the presence or absence of the ApoE4 isoform in εaid ApoE εample. 31. A method according to claim 30, wherein εaid determining εtep iε carried out by isoelectric focusing. 32. A method according to claim 30, wherein said determining step iε carried out by immunoassay. 33. A method according to claim 30, wherein said determining step is carried out by immunoassay with an antibody that selectively binds to the ApoE4 isoform. 34. A method according to claim 25, wherein said subject has not previously been diagnosed as afflicted with Alzheimer's disease. 35. A method according to claim 25, wherein said detecting step comprises detecting whether said subject is homozygouε for the gene encoding the ApoE type 4 iεoform. 36. An apolipoprotein E iεoform-specific antibody, which antibody is selected from the group consisting of: (i) antibodies which selectively bind to the apolipoprotein E type 2 (ApoE2) isoform; (ii) antibodies which selectively bind to the apolipoprotein E type 3 (ApoE3) isoform; and (Hi) antibodies which εelectively bind to the apolipoprotein E type 4 (ApoE4) iεoform. 37. An antibody according to claim 36, wherein said antibody is a monoclonal antibody. 38. An antibody according to claim 36, wherein εaid antibody εelectively binds to the ApoE4 iεoform. 39. An antibody according to claim 36 conjugated to a εolid εupport. 40. An antibody according to claim 36 conjugated to a detectable group. AMENDED CLAIMS [received by the International Bureau on 21 February 1994 (21.02.94) New claims 41-46 added; other claims unchanged (1 page)] 41. The use of a means of detecting apolipoprotein E type 4 isoform (ApoE4) in determining if a subject is afflicted with or at risk of developing Alzheimer's diεeaεe. 42. The use of Claim 41 when applied to diagnoεing or determining the prognoεiε of Alzheimer's disease in a subject. 43. The use of Claim 41 or Claim 42 when effected by detecting for DNA encoding ApoE4. 44. The use of Claim 41 or 42 when effected by directly detecting for ApoE4. 45. The use of any one of Claims 41 and 44 including detecting if said subject is homozygous for the gene encoding ApoE4. 46. A method of ascertaining if a subject is afflicted with or at risk of developing Alzheimer's disease comprising detecting the presence or absence of ApoE4 in a sample from said subject and correlating the reεult of said detection with the presence of ApoE4 indicating Alzheimer's disease or increased risk thereof; optionally said determination including detecting if said subject is homozygous for the gene encoding ApoE4.

Inventors: ENGHILD JAN, ROSES ALLEN D, SALVESEN GUY S, SCHMECHEL DONALD E, STRITTMATTER WARREN J, ENGHILD, JAN, ROSES, ALLEN, D., SALVESEN, GUY, S., SCHMECHEL, DONALD, E., STRITTMATTER, WARREN, J.
Applicants: ENGHILD JAN, ROSES ALLEN D, SALVESEN GUY S, SCHMECHEL DONALD E, STRITTMATTER WARREN J, UNIV DUKE, DUKE UNIVERSITY
Application year: 1993
Main classification: C12Q1
IPC codes: A61K39, C07K1, C07K14, C07K16, C12N15, C12P21, C12Q1, G01N33