Test and model for Alzheimer's disease

Abstract

Model systems of Alzheimer's disease comprise a DNA sequence encoding an amyloid precursor protein (APP) isoform or fragment that has an amino acid substitution. The substituted amino acid may be other than valine at the amino acid position corresponding to amino acid residue position 717 of APP770. Methods of determining genetic predisposition to Alzheimer's disease are also disclosed.

What is claimed is:  
     
         1 . An isolated polynucleotide comprising a nucleic acid sequence encoding a codon 717 mutant of human amyloid precursor protein 770 (APP770), or an isoform or fragment of APP770 having a mutant amino acid residue at the position encoded by codon 717.  
     
     
         2 . An isolated polynucleotide of  claim 1 , wherein the amino acid at the position encoded by codon 717 is an isoleucine, glycine, or phenylalanine.  
     
     
         3 . An isolated polynucleotide of  claim 1 , wherein the nucleic acid sequence is a cDNA.  
     
     
         4 . A composition comprising a polynucleotide probe capable of specifically hybridizing to an amyloid precursor protein 770 (APP770) allele exhibiting a mutation at codon 717.  
     
     
         5 . A composition of  claim 4 , wherein codon 717 of the mutant allele encodes an isoleucine, phenylalanine, or glycine.  
     
     
         6 . A composition of  claim 4 , wherein the probe is labeled.  
     
     
         7 . A composition of  claim 4 , wherein the probe comprises at least about 10 nucleotides spanning amino acid 717 of the APP770 allele.  
     
     
         8 . A transgenic host comprising a nucleic acid segment encoding a position 717 mutant of human amyloid precursor protein 770 (APP770), or an APP770 isoform or fragment of APP770 having the mutation.  
     
     
         9 . A host of  claim 8 , which is a primary or immortalized eukaryotic cell line.  
     
     
         10 . A host of  claim 8 , which is a bacterium.  
     
     
         11 . A host of  claim 8 , wherein the segment is integrated into the host genome.  
     
     
         12 . A host of  claim 8 , which is a non-human animal having the DNA segment incorporated into its germline and which is capable of expressing the mutant APP770 protein.  
     
     
         13 . A host of  claim 12 , wherein the mutant APP770 protein is the sole APP770 protein produced by the animal.  
     
     
         14 . A transgenic non-human animal with germ cells or somatic cells comprising a heterologous gene encoding a position 717 mutant amyloid precursor protein 770 (APP770), which gene upon expression promotes neuropathological characteristics of Alzheimer's disease in the animal.  
     
     
         15 . A cultured human primary or immortalized cell, comprising a nucleic acid segment encoding a position 717 mutant of human amyloid precursor protein 770 (APP770), or an APP770 isoform or fragment of APP770 having the mutation.  
     
     
         16 . A method of screening for an agent capable of treating Alzheimer's disease, comprising: 
 contacting a host of  claim 8  with the agent; and monitoring expression or processing of proteins encoded by the mutant APP770 gene.    
     
     
         17 . A diagnostic method for determining an inherited predisposition to Alzheimer's disease in a subject, comprising detecting in the subject the presence of an allele of amyloid precursor protein (APP), an isoform or fragment thereof, wherein said allele has a sequence polymorphism at a position 5 corresponding to codon 717 of APP770.  
     
     
         18 . A method of  claim 17 , wherein said sequence polymorphism is a nucleotide substitution, whereby an isoleucine or glycine is substituted at codon 717 of APP770.  
     
     
         19 . A method of  claim 17 , wherein said sequence polymorphism is a single nucleotide substitution.  
     
     
         20 . A method according to  claim 17 , wherein the detecting step comprises sequencing a genomic DNA segment from chromosome 21 of the subject.  
     
     
         21 . A method according to  claim 17 , wherein the detecting step comprises (i) mixing a nucleic acid sample from the subject with one or more polynucleotide probes capable of hybridizing selectively to an APP gene allele in a reaction and (ii) monitoring the reaction to determine the presence of the gene allele in the sample, thereby indicating whether the subject is at risk for Alzheimer's disease.  
     
     
         22 . A method according to  claim 20 , wherein one probe is a polynucleotide comprising a sequence of at least about 10 nucleotides spanning codon 717 of an APP770.  
     
     
         23 . A method according to  claim 22 , wherein the probes are oligonucleotides capable of priming polynucleotide synthesis in a polymerase chain reaction, wherein a reaction product comprises a sequence of at least 25 contiguous nucleotides from exon 17 of the APP gene.  
     
     
         24 . A method according to  claim 21 , wherein at least one oligonucleotide specifically hybridizes to a sequence present in an intron or flanking region of an APP770 gene.  
     
     
         25 . A method according to  claim 21 , wherein the monitoring step comprises analyzing sequencing gel reaction products from the PCR reaction.  
     
     
         26 . A method according to  claim 21 , wherein the monitoring step comprises analyzing an autoradiograph of a BclI digest of reaction products from the PCR reaction.  
     
     
         27 . A method according to  claim 17 , wherein the detecting step comprises (i) mixing in an immunological assay an APP770 or isoform protein sample from the subject with an antibody reagent specific for the allele and (ii) monitoring the assay to determine specific binding between the antibody reagent and the protein sample, thereby indicating whether the subject is at risk for Alzheimer's disease.  
     
     
         28 . A method according to  claim 27 , wherein the antibody reagent is a monoclonal antibody specifically reactive with an antigenic determinant specific for an allele.  
     
     
         29 . A method for genetic analysis of a human subject which comprises detecting the presence or absence of at least one polymorphism at codon 717 of an APP770 gene of an amyloid precursor protein (APP) gene in the subject.  
     
     
         30 . A method according to  claim 29 , wherein the polymorphism is detected by digesting genomic DNA from the subject with at least one restriction endonuclease and hybridizing resulting fragments with a detecting probe  
     
     
         31 . A composition comprising a polypeptide free from human proteins, comprising a core sequence: 
 Ile-Ala-Thr-Val-Ile-X-Ile-Thr-Leu-[SEQ ID NO:6]   wherein X is any of the twenty conventional amino acids except valine.    
     
     
         32 . A transgenic nonhuman animal containing a polypeptide of  claim 31 .  
     
     
         33 . A transgenic nonhuman animal of  claim 32 , wherein the polypeptide is present in the brain.  
     
     
         34 . An isolated polynucleotide, comprising a nucleic acid sequence encoding a mutant human APP allele that cosegregates with a genetic predisposition to Alzheimer's disease.  
     
     
         35 . An isolated polypeptide of  claim 34 , wherein said mutant human APP allele comprises a codon 717 mutant.  
     
     
         36 . A method of determining a genetic predisposition of a subject to Alzheimer's disease, the method comprising detecting in the subject's DNA the presence of an allele of a gene encoding amyloid precursor protein (APP).  
     
     
         37 . A method as claimed in  claim 36 , wherein the step of detection is carried out on material removed from, and not returned to, the subject's body.  
     
     
         38 . A method as claimed in  claim 36  or  37 , wherein the allele of the gene encodes a substitution mutant of APP.  
     
     
         39 . A method as claimed in  claim 38 , wherein a single amino acid is substituted for another.