Atherosclerosis 151 (2000) 587 – 589
Letter to the Editor
www.elsevier.com/locate/atherosclerosis
Rapid genotype analysis of the stromelysin gene 5A/6A polymorphism
Stromelysin (matrix metalloproteinase-3, MMP-3) degrades structural proteins of the blood vessel wall and is implicated in vascular remodelling associated with atherosclerosis and aneurysm [1 – 3]. Previously, we identified a bi-allelic single nucleotide polymorphism in the promoter region of the stromelysin gene, with one allele having a cluster of five adenosines (5A) and the other six adenosines (6A) [4]. We showed that the 5A allelic stromelysin promoter had a greater transcription activity than the 6A allelic promoter [5]. Our data also indicate that the difference in promoter activity be-tween the two alleles is due to preferential binding of a transcription repressor to the 6A allele [5,6]. A recent study by Borghaei et al. supports the importance of the promoter element containing the 5A/6A polymorphic site in the transcriptional regulation of the stromelysin gene [7].
A number of genetic epidemiological studies have suggested that this functional polymorphism in the stromelysin gene may be an important genetic factor contributing to various cardiovascular diseases. The 5A allele was found to be associated with susceptibility of myocardial infarction in a study by Terashirna et al. [8] and with risk of abdominal aortic aneurysm by Yoon et
al. [9]. On the other hand, the 6A allele has been found to be associated with accelerated growth of coronary atheromas [4,10,11] and predisposition to post-angio-plasty restenosis [11]. The above diseases associated with the 5A allele (i.e. myocardial infarction which is often due to plaque rupture, and aneurysms) are, to a large extent, caused by weakening of the connective tissue matrix of the diseased blood vessel wall. Since the 5A allelic stromelysin promoter has a higher promoter activity, it seems plausible that weakening of vascular matrix in the 5A allele carriers is a result of increased stromelysin expression. In contrast, those cardiovascu-lar phenotypes associated with the 6A allele (i.e. atheroma growth and restenosis) are largely at-tributable to accumulation of vascular matrix. Since the amounts of matrix will be determined by the balance between its synthesis and degradation, the accumula-tion of vascular matrix in 6A allele carriers might be explained by insufficient stromelysin expression due to the low stromelysin promoter activity of the 6A allele. Several different methods have been used to type the stromelysin gene 5A/6A polymorphism in the literature. These include allele-specific oligonucleotide hybridisa-tion [4,8,11], electrophoresis of fluorescence labelled PCR products using ABI sequencers [10], and denatur-ing polyacrylamide gel electrophoresis of radioisotope labelled PCR products [9]. All these techniques are
Fig. 1. Schematic presentation of the restriction endonuclease digestion based method.
Letter to the Editor 588
Fig. 2. Results of the restriction endonuclease digestion based method. PCR were carried out using the two primers shown in Fig. 1 and digested withXmn1 (see Section 1). Lane M, a 25 bp DNA ladder; lanes 1 – 19,Xmn1 digested PCR products of DNA templates from 19 different individuals. Inferred genotypes are indicated on the bottom.
from the 5A allele but not those deriving from the 6A allele, allowing discrimination of the two alleles follow-ing gel electrophoresis. In the development of this method, we tested a number of different PCR primers with different mismatched nucleotides and different length of the poly-T track at the 3% end of the primers
that were complementary to the poly-A track of the DNA template. We found that the PCR primer shown in Fig. 1 produced the cleanest bands after XmnI digestion. Genotypes determined using this method were completely consistent with those determined using denaturing polyacrylamide gel electrophoresis of radio-labelled PCR products (Figs. 2 and 3).
1. Methods
1.1. Polymerase chain reaction
The sequences of PCR primers were 5%
-GATTACAGACATGGGTCACA-3% (forward primer)
and 5%-TTTCAATCAGGACAAGACGAAGTTT-3%
(reverse primer). PCR was carried out in a total vol-ume of 25 ml, containing 15 ng genomic DNA, 5 pmol
each primer, 200 mM each dATP, dCTP, dGTP and
dTTP, 20 mM Tris – HCl (pH 8.4), 50 mM KCl, 0.05% (v/v) WI (Gibco BRL), 5 mM MgCl2, and IU Taq
polymerase (Gibco BRL). The solution was overlaid with 25ml of liquid paraffin and incubated for 2 min at
95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 53°C, and 30 s at 72°C, and an additional 2 min extension at 70°C at the end of the 35 cycles.
1.2. Restriction endonuclease digestion
A 10ml aliquot of PCR products was mixed with a 5 ml solution containing 1.5ml 10× NEBuffer 2 (50 mM
NaCl, 10 mM Tris – HCl, 10 mM MgCl2, 1 mM
dithio-threitol, pH 7.9), 1.5 ml bovine serum albumin (1 mg/ ml), 0.3 ml XmnI (20 U/ml) and 1.7 ml sterile deionised
H20. The solution was overlaid with 15 ml of liquid
paraffin and incubated at 37°C for 16 h.
1.3. Gel electrophoresis
A 5ml aliquot of the digests was mixed with 2 ml of
loading buffer and electrophoresed on a 10% horizatol non-denaturing polyacrylamide gel at 130 volts for 1 h and 30 min. The gel was then stained with Vistra Green (Amersham) and scanned with a fluodmager.
Acknowledgements
This work was supported by the British Heart Foun-dation (PG/98183 and PG/98182).
Fig. 3. Autoradiograph of denaturing polyacrylamide gel elec-trophoresis of radiolabelled PCR products. PCR were carried out using two primers (5%-TGGTTCTCCATTCCTTTGATG and 5%-TG-GCCAAATATTTTCCCWTGT) that flanked the stromelysin 5A/6A polymorphic site. The amplicons (73 bp for the 5A allele and 74 bp for the 6A allele) were subjected to denaturing polyacrylamide gel (7%) electrophoresis, followed by autoradiography. Sample numbers (corresponding to those in Fig. 2) are labelled above the autoradio-graph. Inferred genotypes (consistent with those in Fig. 2) are indi-cated below the autoradiograph.
expensive, time-consuming and low throughput. Re-ported here is a simple, rapid method for typing this polymorphism, based on restriction endonuclease di-gestion. The method will be very useful in further studies, particularly large molecular genetic epidemio-logical studies, of this promising candidate for genetic susceptibility of cardiovascular disorders.
As shown in Fig. 1, three mismatched nucleotides are introduced in a PCR primer annealed to the prox-imity of the 5A/6A polymorphism, creating a recogni-tion sequence (5%-GAANNNNTTC-3%) for restriction
endonuclease XmnI when the DNA template contains 5As (but not 6As) at the polymorphic site. Thus, an
Letter to the Editor 589
References
[1] Henney AM, Wakeley PR, Davies MJ, et al. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci USA 1991;88:8154 – 8. [2] Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression
of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994;94:2493 – 503.
[3] Newman KM, Ogata Y, Malon AM. et al. Identification of matrix metalloproteinases 3 (stromelysin-1) and 9 (gelatinase B) in abdom-inal aortic aneurysm. Arterioscler Thromb 1994;14:1315 – 20. [4] Ye S, Watts GF, Mandalia S, Humphries SE, Henney AM. Genetic
variation in the human stromelysin promoter is associated with progression of coronary atherosclerosis. Br Heart J 1995;73:209 – 15.
[5] Ye S, Eriksson P, Hamsten A, et al. Progression of coronary atherosclerosis is associated with a common genetic variant of the human stromelysin-1 promoter which results in reduced gene expression: preliminary report: genetic variation in the human stromelysin promoter is associated with progression of coronary atherosclerosis. J Biol Chem 1996;271:13055 – 60.
[6] Ye S, Whatling C, Watkins H, Henney A. Human stromelysin gene promoter activity is modulated by transcription factor ZBP-89. FEBS Lett 1999;450:268 – 72.
[7] Borghaei RC, Sullivan C, Mochan E. Identification of a cytokine-induced repressor of interleukin-1 stimulated expression of stromelysin 1 (MMP-3). J Biol Chem 1999;274:2126 – 31.
[8] Terashima M, Akita H, Kanazawa K, et al. Stromelysin pro-moter 5A/6A polymorphism is associated with acute myocar-dial infarction. Circulation 1999;99:2717 – 9.
[9] Yoon S, Tromp G, Vongpunsawad S, et al. Genetic analysis of MMP3, MMP9, and PAI-1 in Finnish patients with abdominal aortic or intracranial aneurysms. Biochem Biophys Res Com-mun 1999;265:563 – 8.
[10] Humphries SE, Luong LA, Talmud PJ, et al. The 5A/6A polymorphism in the promoter of the stromelysin-1 (MMP-3) gene predicts progression of angiographically determined coro-nary artery disease in men in the LOCAT gernfibrozil study lopid coronary angiography trial. Atherosclerosis 1998;139:49 – 56.
[11] deMaat MP, Jukerna JW, Ye S, et al. Effect of the stromelysin-1 promoter on efficacy of pravastatin in coronary atherosclero-sis and restenoatherosclero-sis. Am J Cardiol 1999;83:852 – 6.
Louise Dunleavey, Seyyare Beyzade, Shu Ye
Department of Human Genetics,
Uni6ersity of Southampton,
Southampton SO16 6YD,
UK
E-mail: [email protected]
