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Detection of a deletion polymorphism of the human [[alpha].sub.2]-macroglobulin gene (A2M-2) by a semi-automated PCR-single-stranded conformational polymorphism method.

Several genes such as the genes that code for apolipoprotein E4, amyloid [beta]-precursor protein, and presenilin 1 and 2 have been linked to the development of Alzheimer disease (AD) (1). Recently, a polymorphism of the human [[alpha].sub.2]-macroglobulin gene (A2M) was also identified as a risk factor for AD (2). The A2M gene is located on chromosome 12p13.3-p12.3 (3), and its product (% macroglobulin) is a serum pan-proteinase inhibitor occurring in many organs and tissues (4). Several studies have suggested that [[alpha].sub.2]-macroglobulin may have protective effect against the deposition of [beta]-amyloids (5,6) and may stimulate their degradation (7, 8). A 5-nucleotide deletion mutation at the 5' splice site of "exon II" of the bait region (exon 18; A2M-2) may lead to loss of this protection because of its location at the functional domain, and this loss confers increased risk for AD (2). Currently available methods based on DNA sequencing (2, 9), heteroduplex formation in nondenaturing gel (9), and PCR-restriction fragment length polymorphism (10) for detection of this deletion mutation are all relatively cumbersome for routine use in clinical laboratories. Here, we describe a semi-automated PCR-single-stranded conformational polymorphism (PCR SSCP) detection method that is amenable to use for large scale screening.

Genomic DNA was extracted from EDTA-anticoagulated whole blood using the QIAamp blood kit (Qiagen). The A2M target DNA sequence was PCR amplified by modifying a previously described technique (9). The 5' upstream primer was 5'-CTT TCC TTG ATG ACC CAA GCG CC-3', and the 3' downstream primer was 5'-GTT GAA AAT AGT CAG CGA CCT C-3'. The PCR conditions were as follows: 37[degrees]C for 10 min; 95[degrees]C for 9 min; 30 cycles of 94[degrees]C for 1 min, 59[degrees]C for 40 s, and 72[degrees]C for 40 s. The PCR reaction mixture of 100 [micro]L contained 1 [micro]g of human genomic DNA, 20 pmol of each primer, 200 [micro]mol/L dNTPs, 2.5 mmol/L Mg[Cl.sub.2], 50 mmol/L KCI, 10 mmol/L Tris-HCI (pH 8.3), 0.1 g/L gelatin, and 3 U of Gold AmpliTaq polymerase (Perkin-Elmer). In addition, 5 [micro]mol/L dUTP (Perkin-Elmer) and 10 kU/L uracil glycosylase (Perkin-Elmer) were added to the PCR mixtures to prevent contamination.

The PCR products were checked for purity by electro phoresis in 1% agarose gel followed by ethidium bromide staining. Images of the patterns were captured with a CCD camera (COHU) coupled with NIH image 1.62 software (NIH). The 326-bp PCR band was quantified by comparison to a DNA gel marker (Research Genetics).

SSCP analysis was performed essentially as described previously (11). In short, the PCR products were mixed with an equal volume of denaturing/ loading solution containing 980 mL/L formamide (Amresco), incubated at 95[degrees]C for 3 min, then immediately chilled on ice. Precast minigels with 20% homogeneous polyacrylamide and native buffer strips (Pharmacia Biotechnology) were used for electrophoresis. Gel electrophoresis and band devel opment were carried out in the semi-automated PhastSys tem~ (Pharmacia). The gels were prerun at 400 V,10 mA, 1.0 W for 100 V*h at 4[degrees]C. Sample (1[micro]L) was applied at 25 V, 10 mA, 1.0 W for 2 V*h at 4[degrees]C, and then electrophoresed at 200 V, 5 mA, 1.0 W for 800 V*h at 4[degrees]C. The gels were silver stained according to the manufacturer's protocol.

To confirm the PCR-SSCP results, all PCR products were also analyzed by sequencing with the primer 5'-GTT GAA AAT AGT CAG CGA CCT C-3', using an ABI Prism 310 genetic analyzer (PE Applied Biosystems) and Big Dye Terminator cycle sequencing kit (PE Applied Biosystems). Before sequencing, PCR products were purified either with a Gel Extraction kit (Qiagen) or a PCR Product Purification kit (Roche/Boehringer Mannheim).

[FIGURE 1 OMITTED]

Representative PCR-SSCP patterns for the three different A2M genotypes are shown in Fig. 1. The same patterns were reproducible over a wide range of PCR products (2.5-30.0 ng), as quantified by the above-described image analysis method. When 30 human blood specimens were analyzed, a complete match was found between the PCR-SSCP and DNA sequencing results that included 16 cases of homozygous wild type (A2M/A2M), 11 cases of heterozygous type (A2M/A2M-2), and 3 cases of homozygous deletion type (A2M-2/A2M-2).

In summary, we have developed a semi-automated PCR-SSCP method that is simpler to perform and less labor-intensive than previous methods for the detection of A2M polymorphisms. In addition, PCR-SSCP avoids misinterpretation that may occur because of partial digestion in the PCR-restriction fragment length polymorphism method.

References

(1.) Blacker D, Tanzi RE. The genetics of Alzheimer disease: current status and future prospects [Review]. Arch Neurol 1998;55:294-6.

(2.) Blacker D, Wilcox MA, Laird NM, Rodes L, Horvath SM, Go RC, et al. a-2 Macroglobulin is genetically associated with Alzheimer disease [Letter]. Nat Genet 1998;19:357-60.

(3.) Devriendt K, Zhang J, van Leuven F, van den Berghe H, Cassiman JJ, Marynen P. A cluster of a2-macroglobulin-related genes ([alpha] 20M) on human chromosome 12p: cloning of the pregnancy-zone protein gene and an [alpha] 2M pseudogene. Gene 1989;81:325-34.

(4.) Borth W. [alpha]2-Macroglobulin, a multifunctional binding protein with targeting characteristics [Review]. FASEB J 1992;6:3345-53.

(5.) Du Y, Bales KR, Dodel RC, Liu X, Glinn MA, Horn JW, et al. [alpha]2-Macroglobulin attenuates [beta]-amyloid peptide 1-40 fibril formation and associated neurotoxicity of cultured fetal rat cortical neurons. J Neurochem 1998;70: 1182-8.

(6.) Hughes SR, Khorkova 0, Goyal S, Knaeblein J, Heroux J, Riedel NG. [alpha]2-Macroglobulin associates with [beta]-amyloid peptide and prevents fibril formation. Proc Natl Acad Sci U S A 1998;95:3275-80.

(7.) Qiu WQ, Borth W, Ye Z, Haass C, Teplow DB, Selkoe DJ. Degradation of amyloid [beta]-protein by a serine protease-a2-macroglobulin complex. J Biol Chem 1996;271:8443-51.

(8.) Narita M, Holtzman DM, Schwartz AL, Bu G. a2-Macroglobulin complexes with and mediates the endocytosis of R-amyloid peptide via cell surface low-density lipoprotein receptor-related protein. J Neurochem 1997;69: 1904-11.

(9.) Matthijs G, Marynen P. A deletion polymorphism in the human [alpha]-2-macroglobulin (A2M) gene. Nucleic Acids Res 1991;19:5102.

(10.) Dodel RC, Bales KR, Farlow MR, Gasser T, Paul SM, Du Y. Rapid detection of a pentanuclectide deletion polymorphism in the human a2-macroglobulin gene [Letter]. Clin Chem 1999;45:307-17.

(11.) Hruszkewycz AM, Delgado RM, Khan MA, Bennett WP. Semiautomated sequence-specific mutation detection of the human K-ras oncogene using "cold" SSCP analysis [Technical Brief]. Clin Chem 1996;42:1717-9.

Yan-Yun Wu, [1] Rosario M. Delgado, [1] Trey Sunderland, [2] and Gyorgy Csako [1] * ([1]Clinical Pathology Department, Warren G. Magnuson Clinical Center, and [2] Geriatric Psychiatry Branch, National Institute of Mental Health, NIH, Bethesda, MD 20892; * address correspondence to this author at: Clinical Pathology Department, NIH, Bldg. 10, Rm. 2C-407, Bethesda, MD 20892-1508; fax 301-402 1885, e-mail gcsako@nih.gov)
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Title Annotation:Technical Briefs
Author:Wu, Yan-Yun; Delgado, Rosario M.; Sunderland, Trey; Csako, Gyorgy
Publication:Clinical Chemistry
Date:Sep 1, 1999
Words:1167
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