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Rapid detection of a pentanucleotide deletion polymorphism in the human [[alpha].sub.2]-macroglobulin gene.

To the Editor:

Alpha-2 macroglobulin ([[alpha].sub.2]M) is a serum glycoprotein and a panproteinase inhibitor found in various tissues, including plasma and cerebrospinal fluid. [[alpha].sub.2]M is thought to inactivate proteinases by a specific trapping mechanism in the so-called "bait" region of the protein (1). [[alpha].sub.2]M is also a ligand for the LDL receptor-related protein, and both are upregulated after brain injury and in regions of the brain affected by Alzheimer disease. Additionally, [[alpha].sub.2]M binds to the amyloid [beta] peptide (2, 3), which leads to attenuation of both fibrillogenesis and neurotoxicity (4) and which is cleared by the LDL receptor-related protein. Recently, a pentanucleotide deletion in the 5' splice site of exon 18, which encodes a portion of the [[alpha].sub.2]M bait region, has been suggested to be genetically associated with an increased risk for developing Alzheimer disease (5). [[alpha].sub.2]M and the 4 [euro] allele of the apolipoprotein E gene seem to confer a similar degree of risk for developing late-onset Alzheimer disease. The conventional methods for measuring [[alpha].sub.2]M are ELISA, immunoblotting, or enzymatic assays, but these methods can not be applied to the detection of [[alpha].sub.2]M pentanucleotide polymorphism. The DNA-based method for detecting this polymorphism deletion is not amenable to large-scale screening (5, 6).


The method described below is based on the observation that the a2M pentanucleotide deletion polymorphism (6) leads to the loss of the HphI restriction site at the intronic sequence in the 5' splice site of exon 18 (Fig. 1A). The primers amplify a 196-bp region in individuals without the pentanucleotide deletion (Fig. 1B). Genomic DNA was extracted from leukocytes, using HQIAamp (Qiagen), and was amplified by PCR using oligonucleotide primers [[alpha].sub.2]MF (5'-GGT GGC AAC TAT TAC ATT CTC TCA-3') and [[alpha].sub.2]MR (5-ACT TAC TTT ACC ACC ACC AAA TCC-3'). In addition to the buffer and nucleotide components, each amplification reaction contained ~200 ng of genomic DNA, 20 [mu]mol of each primer, and 2 U of Taq polymerase (Life Technologies) in a final volume of 50 [micro]L. The reaction mixture was first denatured at 94 [degrees]C for 2 min and then subjected to 35 cycles of PCR (94 [degrees]C for 1 min, 59 [degrees]C for 40 s, 72 [degrees]C for 40 s), after which it was incubated at 72 [degrees]C for 10 min. A 20-[micro]L aliquot of the amplification product was then digested in the presence of 2.2 [micro]L of 1O X buffer and 20 U of HphI for at least 2 h at 37 [degrees]C. Restriction digest products were size fractionated by electrophoresis on a 2% agarose gel with 1 mg/L ethidium bromide for 20 min at 200 V and detected directly under ultraviolet light. (Incomplete digestion may sometimes occur, which can be avoided by a purification step of the PCR product before enzymatic digestion. However, this does not interfere with the scoring of the alleles.) The PCR-amplified digestion products are shown in Fig. 1B along with representative a2M genotypes. In a preliminary study of 367 individuals genotyped by this method, the allele frequency of one or two a2M alleles was 19.1% in patients with sporadic late-onset Alzheimer disease compared with 13.8% in age-matched unaffected individuals. We have encountered no difficulties in the samples tested, and we found this method to be well-suited to high-throughput routine clinical screening.


(1.) Borth W. Alpha 2-macroglobulin, a multifunctional binding protein with targeting characteristics. FASEB J 1992;6:3345-53.

(2.) Du Y, Ni B, Glinn M, Dodel RC, Bales KR, Zhang Z, et al. Alpha2-macroglobulin as a beta-amyloid peptide-binding plasma protein. J Neurochem 1997;69:299-305.

(3.) Hughes SR, Khorkova 0, Goyal S, Knaeblein J, Heroux J, Riedel NG, et al. Alpha2-macroglobulin associates with beta-amyloid peptide and prevents fibril formation. Proc Natl Acad Sci U S A 1998;95:3275-80.

(4.) Du Y, Bales KR, Dodel RC, Liu X, Glinn MA, Horn JW, et al. Alpha2-macroglobulin attenuates beta-amyloid peptidel_QO fibril formation and associated neurotoxicity of cultured fetal rat cortical neurons. J Neurochem 1998;70: 1182-8.

(5.) Blacker D, Wilcox MA, Laird NM, Rodes L, Horvath SM, Go RCP, et al. Alpha-2 macroglobulin is genetically associated with Alzheimer disease. Nat Genet 1998;19:357-60.

(6.) Matthijs G, Marynen P. A deletion polymorphism in the human alpha-2-macroglobulin (A2 M) gene. Nucleic Acids Res 1991; 19:5102.

Richard C. Dodel [1] Kelly R. Bales [2] Martin R. Farlow [3] Thomas Gasser [4] Steven M. Paul [2] Yansheng Du [1] *

Departments of [1] Pharmacology and Toxicology and [3] Neurology Indiana University School of Medicine Indianapolis, IN 46285
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Title Annotation:Letters
Author:Dodel, Richard C.; Bales, Kelly R.; Farlow, Martin R.; Gasser, Thomas; Paul, Steven M.; Du, Yansheng
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Feb 1, 1999
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