Clinical Chemistry
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Clinical Chemistry 46: 715-718, 2000;
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(Clinical Chemistry. 2000;46:715-718.)
© 2000 American Association for Clinical Chemistry, Inc.

Technical Briefs

Semiautomated PCR-Single-Strand Conformation Polymorphism Method for Detection of a Novel Sequence Polymorphism (Ile1000Val) in Human {alpha}2-Macroglobulin

Yan-Yun Wu1, Rosario M. Delgado1, Trey Sunderland2 and Gyorgy Csako1,a

1 Clinical Pathology Department, Warren G. Magnuson Clinical Center

2 Geriatric Psychiatry Branch, National Institute of Mental Health, NIH, Bethesda, MD 20892
a address correspondence to this author at: Clinical Pathology Department, NIH, Bldg. 10, Rm. 2C-407, Bethesda, MD 20892-1508

{alpha}2-Macroglobulin ({alpha}2M) biochemically is a glycoprotein, structurally is a tetramer of identical subunits (182 000 Da each), and functionally is a major human plasma pan-proteinase inhibitor (1). Its gene (A2M) has been mapped to chromosome 12p12-13 (2). Investigations into the pathogenesis of Alzheimer disease (AD) revealed that {alpha}2M is associated with senile plaques (3), binds to Aß peptide, the major component of ß-amyloid (4); attenuates fibrillogenesis and neurotoxicity of Aß (4)(5); and mediates Aß degradation (6)(7). Activated {alpha}2M, apolipoprotein E-enriched lipoproteins, and amyloid precursor protein share the same neuronal cell surface receptor, LDL receptor-related protein ({alpha}2M-r/LRP) (3)(7). Consequently, there has been a growing interest in exploring possible associations between alterations of the A2M gene and AD risk. Two A2M polymorphisms, both involving functional domains, have been suggested to be related to AD risk (8)(9)(10)(11). One involves a pentanucleotide deletion adjacent to a consensus splice site in intron 17 (5' to exon 18) of A2M, and it may cause exon skipping (12). The other involves an A-to-G transition in exon 24, at position 1000 based on the cDNA sequence (13)(14) or at position 976 based on the mature protein (9), and it changes Ile (ATC) to Val (GTC) near the thiol ester site of {alpha}2M (9). The resulting polymorphism is of high frequency, with an allele distribution of 65–70% A and 30–35% G in Caucasian populations (13)(14)(15).

Current evidence for an A2M gene-AD risk connection is, however, conflicting. Several studies have failed to establish an association between AD and the pentanucleotide deletion near exon 18 (11)(16)(17)(18)(19)(20)(21) and/or the G allele of exon 24 (15)(21) of the A2M gene, and one study found that the A allele instead of the initially proposed G allele was a risk factor (11). On the other hand, because {alpha}2M is a major pan-proteinase inhibitor (1), it has been suggested that, similar to {alpha}1-antitrypsin deficiency, defects in the A2M gene might also be involved in the pathogenesis of pulmonary disease (14). For disease associations of A2M gene alterations, large populations need to be studied with practicable genotyping methods. With its robustness and effectiveness in detecting mutation, single-strand conformation polymorphism (SSCP) is such a method. For SSCP, the target DNA sequence is amplified by PCR, and then the resulting double-stranded product is rendered single-stranded by heating in a denaturing buffer. Rapid cooling prevents the DNA strands from reannealing, and the single strands fold back on themselves into a conformation determined by the primary DNA sequence. The different conformers are separated on a nondenaturing polyacrylamide gel and detected either radioactively or with silver staining. Although the concept of SSCP is simple, because of the complexity of thermodynamics of single-stranded folding, it currently is impossible to predict which nucleotide change will alter the conformation. Thus, development of these methods remains largely empirical. In a previous work, we described a rapid semiautomated non-radioactive PCR-SSCP method for genotyping the above-described pentanucleotide deletion in A2M (22). Here we report a similar method that is capable of screening large numbers of samples for the A and G coding variants in exon 24 of the A2M gene.

The study was approved by the review board of the National Institute of Mental Health as part of a larger study following people at risk of developing AD. All subjects gave informed consent. Genomic DNA was extracted from EDTA-anticoagulated whole blood with the QIAamp blood kit (Qiagen). For PCR-SSCP, the A2M target DNA sequence was amplified using a forward primer (5'-GAGACATATTAGGCTCTGCC-3') and the reverse primer (5'-GTAACTGAAACCTACTGGAA-3'). The PCR reaction mixture of 100 µL contained 1 µg of human genomic DNA, 20 pmol of each primer, 200 µmol/L dNTPs, 2.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 0.1 g/L gelatin, and 3 U of AmpliTaq Gold polymerase (PE Applied Biosystem). In addition, 5 µmol/L dUTP and 10 kU/L uracil glycosylase (GeneAmp Carryover Prevention Kit; PE Applied Biosystem) were added to the PCR mixtures to prevent contamination. PCR conditions were as follows: 37 °C for 10 min; 95 °C for 9 min; 35 cycles of 94 °C for 1 min, 58 °C for 40 s, and 72 °C for 40 s; and then final extension at 72 °C for 5 min. The PCR products were checked for purity by electrophoresis in 1.5% agarose gel (ethidium bromide staining), and the DNA content of the 245-bp band was quantified with reference to a DNA Gel Marker (Research Genetics) (22).

SSCP analysis of the PCR fragments was carried out essentially as described previously (22). Briefly, PCR products were mixed with an equal volume of denaturing/loading solution containing 980 mL/L formamide (Amresco), incubated at 95 °C for 3 min, and then immediately chilled on ice. Precast mini gels with 20% homogenous polyacrylamide and native buffer strips (Pharmacia Biotechnology) were used for electrophoresis. Both gel electrophoresis and band development was carried out with the semiautomated PhastSystemTM (Pharmacia). The gels were prerun at 400 V, 10 mA, 1.0 W for 100 V · h at 4 °C. Sample (1 µL) was applied at 25 V, 10 mA, 1.0 W for 2 V · h at 4 °C, and then electrophoresed at 200 V, 5 mA, 1.0 W for 700 V · h at 4 °C. Gels were silver-stained according to the manufacturer’s protocol.

Representative PCR-SSCP patterns for the three different A2M genotypes that were classified according to the nomenclature of Wavrant-DeVrieze et al. (15) are shown in Fig. 1 A. There were two distinct groups of bands: four bands for the A allele, and three bands for the G allele. The presence of multiple bands for both alleles is likely caused by alternative conformations of single-stranded DNA. The non-denatured, double-stranded PCR product migrated much faster than the single-stranded forms and therefore is not seen in Fig. 1 . After dilution of PCR products from a heterozygote (Fig. 1D ), the two fastest moving bands of both alleles were reproducible over a wide range of DNA loads (0.3–19.0 ng/lane). In turn, the two slower moving bands of the A allele and the single slow moving band of the G allele only were detectable at higher DNA loads (>5 ng/lane). Although these slower moving bands confirm a genotype, they are not necessary for genotyping (Fig. 1A ). Nevertheless, under our usual experimental conditions, they were appropriately present in the SSCP patterns of the 57 patients studied.



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  		Figure 1. PCR-SSCP, PCR-RFLP, and DNA sequence patterns of the three different genotypes for a novel sequence polymorphism (Ile1000Val) of the human A2M gene.

Unless otherwise specified, each lane contained 17–20 ng and 200–400 ng of PCR products for SSCP and RFLP analysis, respectively. (A and D), lane M, linear double-stranded DNA marker (GelmarkerTM; Research Genetics); (B), lanes M1 and M2, different DNA size markers [Gelmarker and 10-bp DNA ladder (Life Technologies), respectively]; (C), DNA sequencing. A, adenosine; C, cytidine; G, guanosine; R, A or G; T, thymidine. (D), serial dilution of a heterozygous specimen showing dependence of band detection with SSCP on the DNA load.

Results of the PCR-SSCP analysis were confirmed in two ways. The first method involved PCR-restriction fragment length polymorphism (RFLP) analysis by minor modification of the method of Wavrant-DeVrieze et al. (15). Briefly, the A2M gene target sequence was amplified by PCR using 1 µg of extracted genomic DNA as a template. The forward primer was 5'-GAGACATATTAGGCTCTGCC-3', and the reverse primer was 5'-CAGTGTTGAGATAGCCAATG-3'. Apart from a smaller number of cycles (30) and no final extension for 5 min at 72 °C, the PCR was carried out as described above, and the DNA content of the resulting 180-bp PCR band was quantified with reference to a DNA Gel Marker (Research Genetics) (22). PCR products (200–400 ng) were subjected to restriction enzyme digestion with 10 U of DpnII (New England BioLabs) for 16 h. After electrophoresis in a 6% polyacrylamide gel, the 140- and 40-bp restriction fragments for the A allele (type 1) and the uncut 180-bp fragment for the G allele (type 2) were visualized with SYBR Green staining (Molecular Probes). Images were captured with a Storm 840 system (Molecular Dynamics). For the 57 human blood specimens that were analyzed, a complete match was found between the PCR-SSCP and PCR-RFLP results that included 22 cases of type 1/1, 28 cases of heterozygous type 1/2, and 7 cases of type 2/2 (Fig. 1BUp ). The distribution of these genotypes is similar to those reported for much larger populations: 39% type 1/1, 49% type 1/2, and 12% type 2/2 in our study subjects vs 42%, 46%, and 12%, respectively, in controls (n = 2925) and 44%, 46%, and 10%, respectively, in AD patients (n = 2537) of an earlier study (15). The allele distribution in our study was also similar to those published earlier: 63% A and 37% G in our 57 subjects vs 65% A and 35% G in 2925 controls (15) and 67% A and 33% G in 2537 AD patients (15).

For additional confirmation of the PCR-SSCP and PCR-RFLP results, a subset of genomic DNA specimens was sequenced with the primer 5'-GAGACATATTAGGCTCTGCC-3', using an ABI Prism 310 genetic analyzer (PE Applied Biosystem) and Big Dye Terminator cycle sequencing kit (PE Applied Biosystem). Before sequencing, PCR products were purified with a PCR Product Purification kit (Roche). All 27 different specimens that were analyzed by DNA sequencing (10 cases each of type 1/1 and type 1/2, and 7 cases of type 2/2) revealed the expected alleles (A/A for type 1/1, A/G for type 1/2, and G/G for type 2/2; Fig. 1CUp ).

The semiautomated nonradioactive PCR-SSCP method described here for detection of a novel single nucleotide polymorphism (A-to-G transition, which produces Ile1000Val) in the human A2M gene is reliable and is simpler to perform and less labor-intensive than previously reported methods such as DNA sequencing (13)(14) and PCR-RFLP (9)(11)(15)(21). From the time a whole blood specimen is received, our PCR-SSCP would take ~10 h to complete (~2 h of labor), our DNA sequencing would take ~16 h to complete (~3 h of labor), and our PCR-RFLP would take ~24 h to complete (~3 h of labor). PCR-SSCP reduces the likelihood of misinterpretation that may occur because of partial digestion in the PCR-RFLP method, and it is easier to use than DNA sequencing for interpreting heterozygotes. Furthermore, PCR-SSCP requires less expensive instrumentation than sequencing (PhastSystem vs DNA sequencer). These advantages favor the use of PCR-SSCP in routine clinical laboratory testing and for large-scale screening.


Footnotes

fax 301-402-1885, e-mail gcsako{at}nih.gov


References

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