Alpha-2-macroglobulin isotype diagnostic test for Alzheimer's disease

Abstract

The disclosed invention relates to a diagnostic method for Alzheimer's disease based on genotyping the Alpha-2-Macroglobulin locus. A statistically significant correlation was found between inheritance of particular alleles of the Alpha-2-Macroglobulin gene and the occurrence of Alzheimer's disease. The diagnostic method involves the isolation of nucleic acid from an individual and subsequent genotyping by means such as sequencing or restriction fragment length polymorphism analysis. The invention also provides a means of genotype analysis through protein isotyping Alpha-2-Macroglobulin variant proteins. Finally, kits for nucleic acid analysis or protein analysis are taught.

What is claimed is:  
     
         1 . A method of diagnosing Alzheimer's disease comprising the step of genotyping the Alpha-2-Macroglobulin locus of an individual.  
     
     
         2 . The method of  claim 1 , wherein said genotyping step comprises the steps of: 
 (a) isolating nucleic acid from an individual;    (b) amplifying the nucleic acid to generate an A2M fragment; and    (c) analyzing the fragment thereby correlating A2M genotype with the occurrence of Alzheimer's disease.    
     
     
         3 . The method of  claim 2 , wherein the nucleic acid is DNA.  
     
     
         4 . The method of  claim 2 , wherein the nucleic acid is RNA.  
     
     
         5 . The method of  claim 2 , wherein said step (b) utilizes polynucleotide primers X and Y, wherein said primers X and Y anneal to nucleotides flanking the site of the mutation present in the A2M-2 allele.  
     
     
         6 . The method of  claim 2 , wherein said step (b) utilizes polynucleotide primers X and Y, wherein said primers X and Y anneal to nucleotides flanking the site of the mutation present in the A2M-G allele.  
     
     
         7 . The method of  claim 2 , wherein said step (c) comprises sequencing the fragment to determine A2M genotype.  
     
     
         8 . The method of  claim 2 , wherein said step (c) comprises RFLP analysis of the fragment to determine A2M genotype.  
     
     
         9 . The method of  claim 2 , wherein said step (c) comprises size fractionation of the fragment to determine A2M genotype.  
     
     
         10 . The method of  claim 5 , wherein said step (c) comprises sequencing the fragment to determine A2M genotype.  
     
     
         11 . The method of  claim 5 , wherein said step (c) comprises RFLP analysis of the fragment to determine A2M genotype.  
     
     
         12 . The method of  claim 5 , wherein said step (c) comprises SSCP analysis of the fragment to determine A2M genotype.  
     
     
         13 . The method of  claim 6 , wherein said step (c) comprises sequencing the fragment to determine A2M genotype.  
     
     
         14 . The method of  claim 6 , wherein said step (c) comprises RFLP analysis of the fragment to determine A 2M genotype.  
     
     
         15 . The method of  claim 6 , wherein said step (c) comprises SSCP analysis of the fragment to determine A2M genotype.  
     
     
         16 . The method of  claim 1 , wherein said genotyping step comprises the steps of: 
 (a) isolating DNA from an individual    (b) subjecting said DNA to RFLP analysis thereby correlating A2M genotype with the occurrence of Alzheimer's disease.    
     
     
         17 . The method of  claim 16 , wherein said RFLP analysis utilizes a restriction endonuclease specific for a restriction site created or deleted due to the pentanucleotide deletion found in A2M-2.  
     
     
         18 . The method of  claim 16 , wherein said RFLP analysis utilizes a restriction endonuclease specific for a restriction site created or deleted due to the substitution mutation found in A2M-G.  
     
     
         19 . A method for diagnosing Alzheimer's disease comprising: isotyping the Alpha-2-Macroglobulin protein of an individual.  
     
     
         20 . The method of  claim 19  comprising the steps of: 
 (a) isolating protein from said individual  
 (b) analyzing the protein thereby correlating Alpha-2-Macroglobulin isotype with the occurrence of Alzheimer's disease.  
 
     
     
         21 . The method of  claim 20 , wherein said step (b) comprises western blot analysis of the protein to determine A2M genotype.  
     
     
         22 . The method of  claim 20 , wherein said step (b) comprises ELISA analysis of the protein to determine A2M genotype.  
     
     
         23 . The method of  claim 20 , wherein said step (b) comprises α2M electrophoretic mobility assay analysis of the protein to determine A2M genotype.  
     
     
         24 . The method of  claim 21 , wherein said western blot analysis utilizes an antibody specific for the α 2 M-2 variant.  
     
     
         25 . The method of  claim 21 , wherein said western blot analysis utilizes an antibody specific for the α 2 M Val 1000  variant.  
     
     
         26 . The method of  claim 22 , wherein said ELISA analysis utilizes an antibody specific for the α 2 M-2 variant.  
     
     
         27 . The method of  claim 22  wherein said ELISA analysis utilizes an antibody specific for the α 2 M Val 1000  variant.  
     
     
         28 . A diagnostic kit for diagnosing Alzheimer's disease according to the method of  claim 2 , comprising: (i) a carrier means compartmentalized in close confinement therein to receive one or more container means; (ii) a container means containing polynucleotide primers X and Y, wherein said primers X and Y anneal to nucleotides flanking the site of the mutation present in the A2M-2 allele; (iii) a container means containing α 2 M-1 DNA, or fragment thereof; and (iv) a container means containing α 2 M-2 DNA, or a fragment thereof.  
     
     
         29 . A diagnostic kit for diagnosing Alzheimer's disease according to the method of  claim 2 , comprising: (i) a carrier means compartmentalized in close confinement therein to receive one or more container means; (ii) a container means containing the polynucleotide primers X and Y, wherein said primers X and Y anneal to nucleotides flanking the site of the mutation present in the A2M-G allele; (iii) a container means containing α 2 M Ile 1000  DNA, or fragment thereof; and (iv) a container means containing α 2 M Val 1000  mutant DNA, or a fragment thereof.  
     
     
         30 . A diagnostic kit for diagnosing Alzheimer's disease according to the method of  claim 20 , comprising: (i) a carrier means compartmentalized in close confinement therein to receive one or more container means ; (ii) a container means containing an antibody specific for the α 2 M-2 variant; (iii) a container means containing an antibody specific for α 2 M-1; (iv) a container means containing the α 2 M-2 variant, or fragment thereof; and (v) a container means containing α2M-1, or fragment thereof.  
     
     
         31 . A diagnostic kit for diagnosing Alzheimer's disease according to the method of  claim 20 , comprising: (i) a carrier means compartmentalized in close confinement therein to receive one or more container means; (ii) a container means containing an antibody specific for the α2M Val 1000  variant; (iii) a container means containing an antibody specific for the α 2 M Ile 1000  protein; (iv) a container means containing the α 2 M Val 1000  variant, or fragment thereof; and (v) a container means containing the α 2 M Ile 1000  protein, or fragment thereof.  
     
     
         32 . A diagnostic kit for diagnosing Alzheimer's disease according to the method of  claim 23 , comprising: (i) a carrier means compartmentalized in close confinement therein to receive one or more container means; (ii) a container means containing a protease; (iii) a container means containing a substantially purified sample of the α2M-2 variant; and (iv) a container means containing a substantially purified sample of the fast form of α 2 M-1.