Characterization of Polymorphic Structure of Cathepsin G Gene

The ECTIM Study

ECTIM is a study of patients with MI from regions covered by the World Health Organization’s Monitoring Trends and Determinants in Cardiovascular Disease (MONICA) registers and of control subjects representative of each geographic area. The ECTIM Study design was originally described in 1992.^9,10 Recently, the study population has been extended by the recruitment of population samples from the United Kingdom in Belfast and Glasgow.¹¹ The results reported of the present study are based on samples of subjects selected from populations covered by MONICA registers of Strasbourg (France), Belfast (Northern Ireland), and Glasgow (Scotland). The 2 centers in the United Kingdom recruited men and women, whereas only men were recruited in France. Cases (n=990, mean±SD age 55.8±8.1 years, 26.2% women), aged 25 to 64 years for men and 25 to 69 years for women, were recruited between 3 and 9 months (2 years for women in Belfast) after the index MI. Controls of comparable sex and age (n=900 controls, free of coronary heart disease, mean±SD age 56.4±8.3, 27.5% women) were recruited from the lists of general practitioners in the same areas in the United Kingdom and from the electoral rolls in France. Genetic informed consent was obtained from all study subjects. Plasma fibrinogen was assayed by using the thrombin time method described by Clauss.¹²

The GENIC Study

The GENIC Study, of case-control design, examined genetic susceptibility for BI and involved 12 French neurological departments. Details of the protocol have been reported elsewhere.^13,14 Briefly, cases (aged 18 to 85 years) were consecutively recruited in the week after the onset of clinical symptoms if they fulfilled these criteria: clinical symptom suggestive of stroke, no brain hemorrhage on CT scan, infarct proven by MRI, and white parents. Cases (n=510) were classified into etiological subtypes of BI (ie, atherothrombotic, cardioembolic, lacunar, arterial dissection, rare etiologies, and BI of unknown or undetermined cause) according to prespecified criteria by 2 neurologists (on the basis of clinical symptoms and results of investigations). Controls without a history of stroke were recruited among individuals hospitalized at the same institutions for any reason other than neurological diseases. One control was matched by sex, age (±5 years), and center to each case. Also, both parents had to be white. Information about a previous history of cardiovascular disease (MI, angioplasty, coronary bypass surgery, or lower-limb arteritis) or stroke was obtained. In total, our results for the identified polymorphisms are reported for 444 patients with BI (mean±SD age 65.9±13.5 years, 37.4% women) and 466 controls (mean±SD age 65.8±13.2 years, 38.6% women) from the GENIC Study for whom DNA was available.

Screening of CTSG Gene for Polymorphisms and Genotyping

Genomic DNA was prepared from white blood cells by using either phenol-chloroform extraction or a salting out method.¹⁵ From the published sequences of the CTSG gene,⁸ 16 overlapping fragments <320 bp in length from 96 high-risk MI patients (192 alleles) from the ECTIM Study were enzymatically amplified to cover 973 bp of the upstream region and the entire coding region. Each amplification was performed by using 20 ng DNA in a total volume of 25 μL containing 10 mmol/L Tris-HCl (pH 9), 50 mmol/L KCl, 1.5 to 3.0 mmol/L MgCl₂, 0.1% Triton X-100, 0.2 mg/mL BSA, 200 μmol/L dNTPs, 25 pmol of each primer, and 0.2 U Taq polymerase. Specific amplification protocols for each primer pair and genotyping conditions for all polymorphisms can be obtained online (http://genecanvas.idf.inserm.fr).

For SSCP, 4 μL of the PCR product was mixed with 6 μL of formamide containing 0.0125% bromophenol blue and 0.75% Ficoll 400 in 1× Tris-borate-EDTA buffer and denatured for 5 minutes at 95°C. Samples were subsequently chilled on ice and then loaded on a 10% polyacrylamide gel (acrylamide-to-bisacrylamide ratio 49:1; 110 mm×120 mm×1.0 mm, Multigel-Long/Biometra) containing 0.5× Tris-borate-EDTA buffer. Gels were allowed to run for 14 to 20 hours at 6 V/cm at room temperature and also at 4°C. Bands were then visualized by silver staining.¹⁶ DNA from patients presenting different migration patterns on the polyacrylamide gels were then sequenced twice (both DNA strands with sense and antisense primers) with the use of an automated sequencing device (ABI PRISM 377, Perkin-Elmer). Genotyping of both study populations was performed by using allele-specific oligonucleotides, as previously described.¹⁷

Statistical Analysis

Amplification and Subcloning of Human CTSG Promoter Fragments

On the basis of the sequence of the human CTSG gene (GI 179951 and 179914), we performed a genomic PCR by using the primers CTSG forward (5′-AGAAGCTTTTGCTCCTGGGA-3′ [sense]) and CTSG reverse (5′-CTTTCCTGAAAGGCTGCCC-3′ [antisense]) and different genomic templates, including the following allelic promoter constructs: (1) C (−160), C (−179), G (−317), and G (−618); (2) T (−160), C (−179), G (−317), and G (−618); (3) C (−160), T (−179), A (−317), and G (−618); and (4) C (−160), C (−179), G (−317), and C (−618), with positions relative to the putative translation initiation start codon. The resulting PCR products (976 bp of the 5′-regulatory region directly upstream from the putative translation initiation start codon) were subcloned into the luciferase reporter vector pGL3basic (Promega Corp). The authenticity of the inserts was confirmed by sequencing.

Cell Culture

The human histiocytic lymphoma cell line U937 SLC-1 (a generous gift from Prof H. Stein, Berlin, Germany) was cultured in RPMI 1640 medium (Biochrom) supplemented with 100 U/mL penicillin plus 100 μg/mL streptomycin (Bio-Whittacker), 10% FCS (Biochrom), 2 mmol/L l-glutamine (GIBCO-BRL, Life Technologies), 1 mmol/L sodium pyruvate (Biochrom), and 10 mmol/L HEPES (Biochrom).

Transient Transfection Experiments

Cells were plated into 6-well plates (6×10⁵ cells per well) and transfected 48 hours later with the promoter-luciferase constructs and the promoterless control plasmid pGL3-basic (Promega), respectively, by using Fugene-6 (Boehringer-Mannheim, 0.5 μg plasmid and 3 μL Fugene per well). Cotransfection with pSV-β-galactosidase control vector (0.5 μg per well, Promega) was carried out to standardize for transfection efficiency. Cells were harvested 72 hours after the beginning of the transfection procedure by using reporter lysis buffer (Promega). Luciferase and galactosidase activities were measured in a Lumat LB 9501 (Prof Berthold, Laboratorium, Bad Wildbad, Germany) by using Luciferase Assay Substrate (Promega) and a Galacton/Light Emission Accelerator (Tropix), respectively. A construct-specific transfection represents the mean value of at least 6 single transfections.

Identification of Polymorphisms in the CTSG Gene and Distribution of Genotypes and Alleles in Patients and Controls in the ECTIM and GENIC Studies

Five genetic polymorphisms were identified, 4 in the 5′-flanking region (G-618C, G-315A, C-179T, and C-160T) and 1 already described variant in the coding region (Asn125Ser)²⁰ of the gene.

There was no significant deviation from Hardy-Weinberg equilibrium. The allele frequencies and pairwise linkage disequilibrium coefficients in the whole control sample of the ECTIM Study can be obtained at our site online (http://genecanvas.idf.inserm.fr). The −618C allele was relatively rare (<1%); the polymorphisms G-315A and C-179T were in nearly complete concordance (1% recombinants). There was a complete negative linkage disequilibrium between Asn125Ser and G-315A, whereas an incomplete negative linkage disequilibrium was observed between C-160T and G-315A.

The genotype and allele frequencies of the different polymorphisms were not significantly different in patients and controls from the ECTIM Study (Table 1). None of the polymorphisms was related to coronary artery stenosis, previous MI, blood pressure, body mass index, factor VII levels, plasminogen activator inhibitor-1 levels, or any lipid parameter measured in the ECTIM Study.

However, in cases, the Ser125 allele was significantly associated with elevated plasma fibrinogen levels (385 mg/dL in Ser125 allele carriers versus 344 mg/dL in noncarriers, P=0.006; Table 2). This effect was not observed in controls (case-control heterogeneity, P=0.04). Among cases, there was a significant interaction between CTSG Asn125Ser and the β-fibrinogen (FIBB) gene polymorphism G-455A (also known as the βHaeIII polymorphism) on plasma fibrinogen levels (P=0.04, Table 3), with the difference observed with Asn125Ser being confined to FIBB-455A carriers.

In the GENIC Study, there was no overall significant difference in the distribution of genotypes between cases and controls (OR 1.33, 95% CI 0.89 to 1.99; P=0.17; Table 4). According to specific BI subtypes, there were suggestive trends of association between the CTSG Asn125Ser polymorphism and BI for lacunar and cardioembolic stroke, although they were not significant. Among cases, a positive cardiovascular or cerebrovascular history was less frequent among carriers of the Ser125 allele (38.5%) than among noncarriers (18.6%, P=0.003), whereas no relationship between a positive cardiovascular history and the Ser125 allele was present among controls (P=0.6). Thus, when analyses were restricted to subjects free of cardiovascular or cerebrovascular history, the frequency of carriers of the Ser125 allele was significantly higher in cases (17.1%) than in controls (10.2%; OR 1.82, 95% CI 1.16 to 2.84; P=0.008; Table 4). This association was present in males (OR 1.71, 95% CI 0.97 to 3.03; P=0.06) and females (OR 2.02, 95% CI 1.00 to 4.10; P=0.05), and it remained significant after adjusting for traditional vascular risk factors (OR 1.88, 95% CI 1.19 to 2.97). As reported in Table 4, a significant association was found between the Ser125 allele and lacunar strokes or strokes of unknown cause; a similar trend was observed for cardioembolic strokes; however, this association did not reach significance. On the other hand, our results show no association between CTSG Asn125Ser and atherothrombotic strokes.

CTSG Promoter Activity in U937 Cells

The putative functional significance of the 3 downstream promoter polymorphisms at −160 (C/T), −179 (C/T), and −317 (G/A) relative to the putative translation initiation start codon was examined by subcloning 3 different allelic promoter fragments into a luciferase reporter vector: (1) C (−160)/C (−179)/G (−317), (2) T (−160)/C (−179)/G (−317), and (3) C (−160)/T (−179)/A (−317); all of them contained a G at position −618. When transfected into U937 cells, these promoter-luciferase constructs displayed a relative luciferase activity (RLA) of ≈5, indicating that the subcloned CTSG promoter was transcriptionally active. However, there were no differences in RLA between the different allelic promoter constructs. The putative functional significance of the polymorphism at position −618 was tested (Figure) by comparing only the haplotypes CCGG and CCGC, because all other existing haplotypes in our population sample exhibited a G at position −618. Again, in our in vitro experiments, these 2 promoter sequences displayed a similar promoter activity, indicating that the G/C substitution at position −618 does not exert a major effect, at least in this experimental context.

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CTSG promoter activity in U937 cells according to different allelic constructs. a, The promoter constructs with the nucleotide sequences (5′ to 3′ orientation) CCGG, TCGG, and CTAG represent the different alleles at positions −160, −179, −317, and −618, respectively. RLA is the ratio of the luciferase/galactosidase mean values of each construct related to the promoterless reporter plasmid pGL3 basic [Luc (construct)/Gal(SV40-βGal)/Luc(pGL3 basic)/Gal(SV40-βGal)]. Standard deviations related to Luc (pGL3 basic)/Gal(SV40-βGal) are indicated. Differences between the constructs were not significant. b, The luciferase activity of the wild-type construct (CCGG) was set to 100; standard deviations are indicated. The C/G substitutive polymorphism at position −618 relative to the translational start codon does not affect CTSG promoter activity in U937 cells.