Subjects
A total of 180 patients who had suffered an episode of MI were included in the present study. All patients were male, no more than 55 years of age, and had at least one angiographically diseased vessel (a vessel was considered diseased when the angiography showed a narrowing of at least 70% in the lumen diameter). These patients were recruited for a search of genetic factors involved in MI [20].
The control group consisted of 250 healthy males younger than 55 years (blood bank donors and Hospital Universitario Central de Asturias staff). Although control subjects did not have a history of cardiovascular disease, they were not angiographically analysed to exclude the presence of diseased vessels. Patients and controls were white and were all from the same region (Asturias, northern Spain, 1 million total population) and gave their informed consent to participate in the study, which was approved by the Ethical Committee of the Hospital Universitario Central de Asturias.
Identification and genotyping of SNPs within the human p27kip1gene
Genomic DNA was obtained from each individual included in the study. In the search for p27kip1 SNPs, we analysed 50 patients and 50 controls using SSCA. The entire p27kip1 coding sequence and 1500 nucleotides of the promoter region were amplified in overlapping fragments of 300–450 base pairs by PCR (Table 1 shows the sequences of the PCR primers; the sequence of the p27kip1 gene was obtained from http://ncbi.nlm.nih.gov, accession number NT009714). SSCA analysis was performed as previously described [21]. The DNA from samples showing atypical electrophoretic patterns was amplified, purified, and sequenced using an automated ABI310 system.
For genotyping of the three SNPs in patients and controls, genomic DNA was amplified by PCR to generate DNA fragments containing the SNP, and these fragments were digested with restriction enzymes that produced distinct patterns of digestion depending on the allele present (primers and restriction enzymes are listed in Table 2). The digested fragments were electrophoresed on 3% agarose gels, and the pattern corresponding to each individual was visualized after ethidium bromide staining of the gel.
Construction of p27kip1reporter vectors
DNA fragments from the 5' flanking region of the p27kip1gene between nucleotides – 1100 to -27, containing either C or A at -838, were generated through PCR of two CC and AA homozygous individuals with forward (TATGATGGTACCAGACGTTCGCTTTGGCTTC) and reverse (GCACGAAAGCTTCTCTCGCACTCTCAAAAA) primers containing KpnI and HindIII sites, respectively. The PCR products were cloned into the pGL3-Basic plasmid (Promega, Madison, WI, USA) to generate pGL3-p27kip1
C (-838C) and pGL3-p27kip1
A (-838A). For each genotype, two plasmids were generated and confirmed by direct sequencing.
Cell culture and luciferase reporter gene assays
Jurkat cells (American Type Culture Collection) were incubated at 37°C in a humidified 5% CO2–95% O2 atmosphere in RPMI medium supplemented with 100U/ml penicillin, 0.1 mg/ml streptomycin, 2 mmol/l L-glutamine, and 10% fetal calf serum. Transfections were performed with SuperFect Transfection Reagent following the manufacturer's protocol (Quiagen, Valencia, CA, USA). In a blinded manner, the promoter/luciferase reporter gene (2 μg) was cotransfected with 1 μg of pEGFP-N1 control vector. Three independent transfections were done for each of the four reporter genes (2 pGL3-p27kip1
C and 2 pGL3-p27kip1
A, a total of six transfections for each SNP). For luciferase activity assay, cells were collected by centrifugation, washed twice with PBS, and lysed with 100 μl of cell lysis reagent (Promega). Insoluble protein was removed by a 10-minute centrifugation at 15,000 g, and the supernatants were immediately assayed for luciferase activity and green fluorescence. Luciferase assays were performed using a Victor 4120 multilabel counter (Perkin Elmer, Boston, MA, USA) and the Promega luciferase assay kit. Cell extract (50 μl) and 100 μl of luciferin mixture were incubated for 5 s and light output was monitored for 5 s. For each sample, arbitrary light units from luciferase were normalized versus the measure of green fluorescence.
Statistical analysis
Allele and genotype frequencies in patients and controls were compared using a chi-square test. This test was also used to determine if the observed genotype frequencies in cases and controls differed from those expected under the Hardy–Weinberg equilibrium. Multivariate analysis was used to compare the genotype and allele frequencies between the groups according to the presence/absence of classical risk factors. Odds ratios (OR) with 95% confidence intervals (CI) were obtained to calculate the relative risk of MI associated with the genotypes. The results of transfections were analysed by two-tailed unpaired t-test. All statistical analyses were performed with the SPSS statistical package (v.11.0).