| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Letters |
2-Macroglobulin Gene
Departments of,
1
Pharmacology and Toxicology, and
2
Neurology, Indiana University School of Medicine, Indianapolis, IN 46285
To the Editor:
Alpha-2 macroglobulin (
2M) is a serum
glycoprotein and a panproteinase inhibitor found in various tissues,
including plasma and cerebrospinal fluid. 2M is thought
to inactivate proteinases by a specific trapping mechanism in the
so-called "bait" region of the protein (1).
2M is also a ligand for the LDL receptor-related
protein, and both are up-regulated after brain injury and in regions of
the brain affected by Alzheimer disease. Additionally,
2M binds to the amyloid ß 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 2M bait region, has been suggested to be genetically
associated with an increased risk for developing Alzheimer
disease (5). 2M and the 
4 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 2M are ELISA, immunoblotting, or enzymatic
assays, but these methods can not be applied to the detection of
2M 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
2M 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. 1
A). 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 2MF (5'-GGT GGC AAC TAT TAC ATT
CTC TCA-3') and 2MR (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 pmol of
each primer, and 2 U of Taq polymerase (Life Technologies) in a final
volume of 50 µL. The reaction mixture was first denatured at 94 °C
for 2 min and then subjected to 35 cycles of PCR (94 °C for 1 min,
59 °C for 40 s, 72 °C for 40 s), after which it was
incubated at 72 °C for 10 min. A 20-µL aliquot of the
amplification product was then digested in the presence of 2.2 µL of
10x buffer and 20 U of HphI for at least 2 h at
37 °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 2M genotypes. In a preliminary study of
367 individuals genotyped by this method, the allele frequency of one
or two 2M 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.
|
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  R. C. Dodel, Y. Du, K. R. Bales, F. Gao, B. Eastwood, B. Glazier, R. Zimmer, B. Cordell, A. Hake, R. Evans, et al. {alpha}2 Macroglobulin and the risk of Alzheimer's disease Neurology, January 25, 2000; 54(2): 438 - 438. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-Y. Wu, R. M. Delgado, T. Sunderland, and G. Csako Detection of a Deletion Polymorphism of the Human {alpha}2-Macroglobulin Gene (A2M-2) by a Semi-Automated PCR-Single-Stranded Conformational Polymorphism Method Clin. Chem., September 1, 1999; 45(9): 1572 - 1573. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
