Association of interleukin-1B and interleukin-1RN polymorphisms with gastric cancer

Clin Exp Med (2005) 5:169–176 DOI 10.1007/s10238-005-0082-3 ORIGINAL

W. Alpízar-Alpízar • G.I. Pérez-Pérez • C. Une • P. Cuenca • R. Sierra

Association of interleukin-1B and interleukin-1RN polymorphisms with gastric cancer in a high-risk population of Costa Rica

Received: 30 May 2005/ Accepted: 13 September 2005

Abstract Several risk factors have been associated with gastric cancer, among them Helicobacter pylori infection.

This bacterium yields inflammation, the degree of which depends on the bacterial strain and the severity of the host response. The inflammatory response involves a complex cytokine network. Recently, polymorphisms of the genes coding for interleukin-1β (IL-1B), interleukin-1Ra (ILRN) and interleukin-10 have been associated with an increased risk of gastric cancer. In order to determine the association of the IL-1B, IL-1RN and IL-10 polymorphisms with gastric cancer in a high-risk Costa Rican population, we analysed purified DNA of 58 gastric cancer patients, 99 controls and 41 patients classified as group I or II, according to the Japanese classification. Genotyping was carried out by PCR, PCR-RFLP and pyrosequencing analysis. We did not find any association of the IL-1B-31, IL-1B-511 and IL-10 polymorphisms with the risk for

developing gastric cancer in the studied population.

Carriers of the IL-1B+3954T/– had an increased risk for developing gastric cancer (OR 3.7; 95%CI: 1.34–10.2).

Also we found an increased risk for developing gastric cancer for allele 2 heterozygotes of the IL-1RN (OR 2.94; 95%CI: 1.09–7.93). This is the first time that IL-1B+3954 has been associated with gastric cancer. This is one of the first studies trying to describe the role played by IL-1B, IL-1RN and IL-10 genetic polymorphisms in gastric cancer in one of the highest risk American countries. Further investigation on American countries is needed.

Key words Interleukin-1B polymorphisms • Interleukin1RN polymorphisms • Interleukin-10 polymorphisms • Helicobacter pylori • Gastric cancer • Pro-inflammatory response

Introduction

W. Alpízar-Alpízar ( ) • C. Une • P. Cuenca • R. Sierra Instituto de Investigaciones en Salud (INISA), Universidad de Costa Rica, San Pedro, San José, Costa Rica e-mail: awarnercr@yahoo.com / warnera@cariari.ucr.ac.cr Tel.: +506-207-3294 Fax: +506-207-5130 G.I. Pérez-Pérez Division of Infectious Diseases, New York University School of Medicine, New York, USA

Gastric cancer is the second most common cancer causing death worldwide after lung cancer [1]. Several factors have been associated with an increased risk of developing this malignancy, among them genetic predisposition and Helicobacter pylori infection [2–4]. The presence of this bacterium causes chronic inflammation, the magnitude of which depends on the strain of bacterium and the severity of the host response [5, 6]. The immune response to H. pylori involves a complex network of cytokines such as IL-8, IL-1, IL-1Ra, IL-6, TNF-α and IL-10 [7, 8].

IL-1 is a pro-inflammatory cytokine, involving three different proteins, IL-1α, IL-1β and an antagonist receptor of IL-1 (IL-1Ra), which are coded by different genes (IL1B, IL-1A and IL-1RN respectively) [9, 10]. Recently, some polymorphisms of the genes IL-1B and IL-1RN have been associated with an increased risk for developing gastric cancer [11–14]. IL-1β plays an important role in the

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inflammation induced by H. pylori and also alters the gastric physiology because it is one of the more powerful inhibitors of gastric acid secretion known [8, 15]. Polymorphisms related to increased expression of IL-1β and decreased expression of IL-1Ra might be associated with higher risk for developing gastric cancer. However, some studies have failed to demonstrate such association [16, 17]. In addition, there are other functional polymorphisms in the genes coding for other pro-inflammatory and antiinflammatory cytokines. Some of these may act synergistically with IL-1B and IL-1RN polymorphisms [18].

IL-10 is a powerful immunoregulatory cytokine, which modulates expression of pro-inflammatory cytokines such as IL-1α, IL-1β and TNFα. One of the effects of IL-10 on the immune system is to downregulate the inflammatory response [19, 20]. Recently, an association of some IL-10 polymorphisms with increased risk of developing gastric cancer has been reported [21, 22]. The risk for this malignancy might be higher in carriers of genotypes associated with decreased IL-10 expression. Therefore, polymorphisms that are associated with decreased IL-10 expression and polymorphisms associated with increased IL-1β expression, acting together, might result in an increased inflammatory response against H. pylori and consequently in higher gastric cancer risk [21, 23].

Costa Rica has one of the highest incidence and mortality rates for gastric cancer worldwide [1]. The incidence rates are significantly different among regions of the country [24]. However, the prevalence of H. pylori infection is high from childhood in regions at both high and low risk for gastric cancer [25]. Therefore, the difference in incidence among high- and low-risk gastric cancer regions might be associated with differences in the host. The aim of this study was to determine the association of IL-1B, IL-1RN and IL-10 genetic polymorphisms with gastric cancer in a high-risk population of Costa Rica.

Materials and methods

classified as group I or group II, according to the Japanese classification (this group was named dyspeptics); (D) 41 individuals, age- and sex-matched to dyspeptics without suspected gastric cancer according to X-ray examination. The study was approved by the Ethic-Scientific Committee of the University of Costa Rica. All individuals gave and signed an informed consent form. Blood samples were taken from all individuals on the original study and they were stored at –70°C until the date of this study. Genomic DNA was extracted from these blood samples by proteinase K digestion and the phenol/chloroform method.

IL-1B polymorphisms genotyping Polymorphism analysis was performed by PCR-RFLP from 100 ng of genomic DNA. The primers and PCR conditions were the same as previously reported [11]. The PCR products for IL-1B511 and IL-1B+3954 were digested with restriction enzymes AvaI and TaqI (Fermentas, Hanover, MD, USA) respectively and the allele designations for those polymorphisms were the same as previously reported [27]. For IL-1B-31, the PCR product was digested using AluI restriction enzyme (Fermentas, Hanover, MD, USA). Allele T was designated if two bands of 103 and 136 bp were obtained and allele C was designated if a single band of the undigested 239-bp PCR product was obtained. The genotypes were designated as follows: T/T, two bands of 136 and 103 bp; T/C, three bands of 239, 136 and 103 bp; and CC, a single band of 239 bp. Also, we carried out the genotyping of IL-1B polymorphisms using pyrosequencing analysis as a quality control for PCR-RFLP. Primers and PCR conditions used in the pyrosequencing analysis are described in Table 1 and were the same as previously reported [28].

IL-1RN genotyping Genomic DNA (100 ng) was amplified using the same primers and PCR conditions as previously reported [11]. For statistical analysis purposes and because of the low frequency of alleles 3, 4 and 5, this polymorphism was treated as biallelic by dividing alleles into short and long categories, in which the short allele has two repeats (allele 2) and long allele has more than two repeats (allele L).

Study population This study is part of a larger study (n=1300) named “Markers for identifying subjects with high gastric cancer risk”, which was carried out between 1999 and 2000 in the Gastric Cancer Early Detection Center of Costa Rica. From this study we used epidemiologic and clinical information from all the gastric cancer patients and a group of patients with gastric lesions classified as group I or group II according to the Japanese classification [26]. As the control group we selected individuals without evidence of gastric cancer according to an X-ray exam (double contrast gastroduodenal series). We made and compared the following groups: (A) 58 gastric cancer patients according to histopathological diagnosis (28 intestinal type, 18 diffuse type and 12 unknown); (B) 58 individuals age- and sex-matched to cases without suspected gastric cancer according to X-ray examination; (C) 41 individuals histologically

IL-10 polymorphism genotyping The determination of IL-10 polymorphisms (IL-10-1082, IL-10819 and IL-10-592) was performed by pyrosequencing analysis.

Primer sequences and PCR conditions are described in Table 1 and were similar to those previously described [21].

H. pylori serology Serum antibodies to establish the H. pylori status were determined from all individuals of the original study and the results were used

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W. Alpízar-Alpízar et al.: Interleukin polymorphisms and gastric cancer in Costa Rica Table 1 Primers and PCR conditions for pyrosequencing analysis of the IL-1B and IL-10 polymorphisms Polymorphisms

Primers

Pyrosequencing primer

PCR conditions

IL-10-1082 G/A

F: 5’-CACACACACACAAATCCAAGACAA-3’ R: 5’-GCTGGATAGGAGGTCCCTTACTTT-3’

F 5’-CTTTGGGAAGGGGA-3’

95°C by 5’; 30 cycles of 95°C by 30’’, 60°C by 30’’, 72°C by 45’’; 72°C by 5’

IL-10-819 C/T

F: 5’-GGGTGAGGAAACCAAATTCTCA-3’ R: 5’-CATGACCCCTACCGTCTCTATTTTA-3’

R:5’-CAAACTGAGGCACAGAG-3’

95°C by 5’; 30 cycles of 95°C by 30’’, 59°C by 30’’, 72°C by 45’’; 72°C by 5’

IL-10-592 C/A

F: 5’-GGTAAAGGAGCCTGGAACACATC-3’ R: 5’-CCAAGCAGCCCTTCCATTT-3’

R: 5’-CTGGCTTCCTACAG-3’

95°C by 5’; 30 cycles of 95°C by 30’’, 60°C by 30’’, 72°C by 45’’; 72°C by 5’

IL-1B-511 C/T

F: 5’-TCCTCAGAGGCTCCTGCAAT-3’ R: 5’-GGAATCTTCCCACTTACAGATGG-3’

R: 5’-GGTGCTGTTCTCTGCCT-3’

95°C by 5’; 50 cycles of 95°C by 30’’, 58°C by 30’’, 72°C by 45’’; 72°C by 5’

IL-10-31 T/C

F: 5’-CCACCAATACTCTTTTCCCCTTTCC-3’ R: 5’-TCCCTCGCTGTTTTTA-3’ R: 5’-GATTGGCTGAAGAGAATCCCAGAGC-3’

in this study. The determination was measured by an Enzyme Linked Immunosorbent Assay (ELISA) developed in our laboratory based on a modification of a previously described ELISA [29].

Antigen was prepared from five strains of bacteria isolated from patients in Costa Rica. ELISA plates (Immulon 2 plates, Dynel, Charlottesville, VA, USA) were coated with 100 µl of the antigen (10 µg/ml of carbonate buffer)/well and incubated overnight at room temperature. Unspecific binding was blocked with 250 µl of 0.1% gelatine in PBS for 3 h. Between every step the plates were washed 3 times in PBS+0.01% thimerosal and 0.05% Tween 20.

Serum samples, negative and positive controls were diluted 1:800 (7500 for positive controls) in PBS containing 0.1% gelatine and 0.5% bovine gamma globulin (Sigma, Saint Louis, MO, USA) and 100 µl was added to the wells and incubated at 37°C for 60 min.

After washing, 100 µl of anti-human IgG conjugated with alkaline phosphatase (Biosource International) was added. The plates were incubated for 60 min at 37°C and washed 5 times. Thereafter, 100 µl of substrate (Fast p-Nitrophenyl Phosphate Tablet Sets, Sigma, Saint Louis, MO, USA) was added and the plates were incubated at 37°C until the net absorbance at 405 nm exceeded OD 0.4 for positive controls. The absorbance was determined in a Multiscan spectrophotometer (Thermo Labsystems, Finland). The cut-off point was determined for every plate using the mean OD values from the positive controls in a formula obtained from a series of experiments with negative sera to estimate the mean plus three standard deviations. The positive and negative predictive values were 89% and 82% respectively.

95°C by 5’; 50 cycles of 95°C by 30’’, 64°C by 30’’, 72°C by 30’’; 72°C by 5’

Statistical analysis χ2 statistics was used for comparing genotype frequencies and to assess Hardy-Weinberg equilibrium for each one of the studied loci. Haplotype frequencies were assessed using Estimating Haplotype Frequencies (EH) (available from ftp://linkage.rockefeller.edu/software/eh/). Linkage Disequilibrium coefficients D’ and D were calculated using LINKDOS (available from: ftp://ftp.cefe.cnrs-mop.fr/pub/PC/MSDOS/GENEPOP). Odds ratios to assess possible associations of the studied polymorphisms and gastric cancer were calculated using STATA (version8.1, Stata Corporation, TX, USA). P values ≤0.05 were considered statistically significant.

Results Table 2 shows general characteristics of the study population. A high proportion of our population was seropositive for H. pylori (87.4%). No significant differences in H. pylori seropositivity were found comparing either gastric cancer patients and controls or dyspeptic patients and controls. Because of the high prevalence of H. pylori infection, this variable was not considered in the statistical

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analysis. All the studied polymorphisms of IL-1B and IL10 were in Hardy-Weinberg equilibrium in both control groups: the cancer control and the dyspeptic control groups. The IL-1RN locus was not in Hardy-Weinberg equilibrium for the two control groups although the devia-

tion from it was not pronounced (P=0.06 and P=0.05 respectively).

Table 3 shows genotypic and allele frequencies of the studied population. When genotypic frequencies of the gastric cancer patients and their controls were compared, no

Table 2 Characteristics of the subjects in the study Diagnostic

Age (years), mean±SD

Male, n (%)

Female, n (%)

H. pylori+, n (%)

Cancer (n= 58) Controls (n=58) Dyspeptics (n=41) Controls (n=41) Total

59.8±13.8 61.4±9.2 64.3±8.0 65.7±5.6 62.3

46 (79.3) 46 (79.3) 33 (80.5) 33 (80.5) 158 (79.8)

12 (20.7) 12 (20.7) 8 (19.5) 8 (19.5) 40 (20.2)

48 (82.8) 53 (91.4) 37 (90.2) 35 (85.4) 173 (87.4)

Table 3 Frequency of genotypes and allelic presentation for the IL-1B, IL-1RN and IL-10 polymorphisms Genotype

Total

Gastric cancer

Controls

Dyspeptic

Controls

IL-1B-31 C/C C/T T/T Allelic frequency of C IL-B-511 C/C C/T T/T Allelic frequency of T IL-1B+3954 C/C C/T T/T Allelic frequency of T IL-1RN 1/1 1/2 1/3,4 2/2 2/3,4 Allelic frequency of 1 Allelic frequency of 2 IL-10-592 C/C C/A A/A Allelic frequency of A IL-10-819 C/C C/T T/T Allelic frequency of T IL-10-1082 G/G G/A A/A Allelic frequency of A

n=182 49 (0.27) 91 (0.50) 42 (0.23) 0.52 n=182 45 (0.25) 91 (0.50) 46 (0.25) 0.50 n=172 123 (0.72) 49 (0.28) 0 (0.0) 0.14 n=171 85 (0.50) 51 (0.30) 15 (0.09) 17 (0.10) 3 (0.01) 0.68 0.27 n=169 67 (0.40) 86 (0.51) 16 (0.09) 0.34 n=171 75 (0.44) 80 (0.47) 16 (0.09) 0.32 n=169 0 (0.0) 2 (0.01) 167 (0.99) 0.99

n=50 14 (0.28) 22 (0.44) 14 (0.28) 0.50 n=50 14 (0.28) 24 (0.48) 12 (0.24) 0.48 n=45 25 (0.56) 20 (0.44) 0 (0.0) 0.22 n=44 21 (0.48) 17 (0.38) 2 (0.05) 2 (0.05) 2 (0.04) 0.69 0.26 n=44 21 (0.48) 20 (0.45) 3 (0.07) 0.29 n=45 25 (0.56) 16 (0.35) 4 (0.09) 0.26 n=45 0 (0.0) 0 (0.0) 45 (1.0) 1.0

n=50 18 (0.36) 23 (0.46) 9 (0.18) 0.59 n=50 10 (0.20) 23 (0.46) 17 (0.34) 0.57 n=45 37 (0.82) 8 (0.18) 0 (0.0) 0.09 n=45 28 (0.62) 9 (0.20) 4 (0.09) 4 (0.09) 0 (0.0) 0.77 0.19 n=44 18 (0.41) 21 (0.48) 5 (0.11) 0.35 n=45 18 (0.40) 24 (0.53) 3 (0.07) 0.33 n=44 0 (0.0) 1 (0.02) 43 (0.98) 0.99

n=41 6 (0.15) 24 (0.59) 11 (0.27) 0.45 n=41 12 (0.29) 23 (0.56) 6 (0.15) 0.43 n=41 32 (0.78) 9 (0.22) 0 (0.0) 0.11 n=41 15 (0.37) 15 (0.37) 5 (0.12) 5 (0.12) 1 (0.02) 0.61 0.32 n=41 15 (0.37) 21 (0.51) 5 (0.12) 0.37 n=41 18 (0.44) 17 (0.41) 6 (0.15) 0.35 n=41 0 (0.0) 0 (0.0) 41 (1.0) 1.0

n=41 11 (0.27) 22 (0.54) 8 (0.20) 0.54 n=41 9 (0.22) 21 (0.51) 11 (0.27) 0.52 n=41 29 (0.71) 12 (0.29) 0 (0.0) 0.15 n=41 21 (0.51) 10 (0.24) 4 (0.10) 6 (0.15) 0 (0.0) 0.68 0.27 n=40 13 (0.33) 24 (0.60) 3 (0.07) 0.37 n=40 14 (0.35) 23 (0.58) 3 (0.07) 0.36 n=39 0 (0.0) 1 (0.3) 38 (0.97) 0.99

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significant differences for the IL-1B-31, the IL-1B-511 or any of the IL-10 polymorphisms were found. However, there were significant differences when the same comparison was performed for IL-1B+3954 and IL-1RN (χ2=10.3, P=0.006 and χ2=6.4, P=0.04 respectively). Next, a com-

parison between dyspeptic patients and their controls failed to detect differences for any of the polymorphisms of the IL-1B and IL-10 genes. However, there was a significant difference for the polymorphism of the IL-1RN gene (χ2=6.4, P=0.04). No significant differences were observed

Table 4 Estimated haplotype frequencies and linkage disequilibrium coefficients of the study population Group

Cancer (n=58) Controls (n=58) Dyspeptics (n=41) Controls (n=41) Total (n=198)

Loci

IL-1-31/IL-1-511 IL-10-592/IL-10-819 IL-1-31/IL-1-511 IL-10-592/IL-10-819 IL-1-31/IL-1-511 IL-10-592/IL-10-819 IL-1-31/IL-1-511 IL-10-592/IL-10-819 IL-1-31/IL-1-511 IL-10-592/IL-10-819

Haplotypea

Disequilibrium

1–1

1–2

2–1

2–2

D’

χ2

P

0.020 0.056 0.050 0.034 0.012 0.213 0.051 0.000 0.033 0.071

0.480 0.259 0.538 0.270 0.427 0.156 0.486 0.372 0.486 0.264

0.500 0.662 0.378 0.650 0.561 0.430 0.425 0.628 0.464 0.606

0.000 0.023 0.032 0.045 0.000 0.201 0.039 0.000 0.017 0.060

-0.98 -0.81 -0.86 -0.71 -0.99 – -0.92 -0.99 -0.91 -0.62

48.5 26.8 37.3 23.1 40.6 – 35.3 38.0 157.4 66.6

0.0001 0.0001 0.0001 0.0001 0.0001 – 0.0001 0.0001 0.0001 0.0001

aIL-1B-31 and IL-1B-511: allele C is denoted by 1, allele T is denoted by 2; IL-10-592: allele A is denoted by 1, allele C is denoted by 2; IL-10-819: allele C is denoted by 1, allele T is denoted by 2

Table 5 Odds ratios and 95% confidence intervals of IL-1B, IL-1RN and IL-10 polymorphisms for gastric cancer and dyspepsia Genotype

IL-1B-31 T/T C/T C/C C carriers IL-B-511 C/C C/T T/T T carriers IL-1B+3954 C/C C/T IL-1RN L/L L/2 2/2 2 carriers IL-10-592 C/C C/A A/A A carriers IL-10-819 C/C C/T T/T T carriers

Gastric cancer OR (95%CI)

Intestinal type of gastric cancer OR (95%CI)

Diffuse type of gastric cancer OR (95%CI)

Dyspeptics OR (95%CI)

1.0 0.61 (0.22–1.73) 0.50 (0.16–1.53) 0.56 (0.22–1.48)

1.0 0.38 (0.07–1.92) 0.20 (0.03–1.26) 0.29 (0.06–1.34)

1.0 1.2 (0.22–6.99) 1.6 (0.22–10.71) 1.4 (0.28–6.60)

1.0 0.79 (0.27–2.36) 0.40 (0.10–1.61) 0.66 (0.23–1.88)

1.0 0.74 (0.27–2.03) 0.50 (0.16–1.55) 0.64 (0.25–1.64)

1.0 0.29 (0.06–1.49) 0.28 (0.04–1.73) 0.29 (0.06–1.34)

1.0 4.5 (0.61–32.95) 0.64 (0.09–4.34) 1.8 (0.38–8.54)

1.0 0.82 (0.29–2.36) 0.41 (0.10–1.61) 0.68 (0.25–1.86)

1.0 3.7 (1.34–10.2)

1.0 4.5 (1.03–19.56)

1.0 2.0 (0.36–11.53)

1.0 0.68 (0.25–1.86)

1.0 2.94 (1.09–7.93) 0.70 (0.11–4.19) 2.25 (0.96–5.51)

1.0 1.95 (0.49–7.75) 0.27 (0.02–3.05) 1.2 (0.36–4.03)

1.0 5.86 (0.88–38.96) – –

1.0 2.0 (0.73–5.47) 1.04 (0.27–3.96) 1.64 (0.67–3.99)

1.0 0.82 (0.34–1.98) 0.51 (0.10–2.53) 0.76 (0.32–1.77)

1.0 1.24 (0.37–4.54) 1.1 (0.17–7.56) 1.21 (0.35–4.14)

1.0 0.67 (0.13–3.49) Undefined 0.48 (0.1–2.36)

1.0 0.76 (0.29–1.97) 1.44 (0.28–7.44) 0.83 (0.33–2.10)

1.0 0.48 (0.20–1.18) 0.96 (0.19–4.91) 0.53 (0.22–1.25)

1.0 0.54 (0.15–1.97) 2.4 (0.20–30.19) 0.7 (0.20–2.32)

1.0 0.44 (0.08–2.43) 0.33 (0.02–5.19) 0.41 (0.08–2.04)

1.0 0.57 (0.22–1.49) 1.56 (0.32–7.53) 0.69 (0.28–1.70)

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when gastric cancer patients and dyspeptic individuals were compared for any of the studied polymorphisms.

There was linkage disequilibrium between IL-1B-31 and IL-1B-511, with 95% of the inferred haplotypes (IL1B-31/IL-1B-511) consisting of either C-T or T-C (Table 4). Likewise there was linkage disequilibrium between IL10-592 and IL-10-819. In this case 87% of the inferred haplotypes (IL-10-592/IL-10-819) consisting of either A-T or C-C. For dyspeptic individuals there was no linkage disequilibrium between IL-10-592 and IL-10-819 polymorphisms (Table 4).

No association of IL-1B-31, IL-1B-511, IL-10-592, IL10-819 or IL-10-1082 polymorphisms with the risk for developing gastric cancer was found (Table 5). On the contrary, a significant association for IL-1B+3954 and IL1RN polymorphisms with the risk of gastric cancer was observed. Carriers of the genotype C/T on IL-1B+3954 polymorphism showed an increased risk of developing the disease. Also, allele 2 heterozygotes for the polymorphism of IL-1RN gene had an increased risk of gastric cancer.

Carriers of the allele 2 in the polymorphism of the IL-1RN gene showed a tendency towards an increased risk although it was not significant (Table 5). When the histological type of gastric cancer was considered we found that carriers of the genotype C/T of the IL-1B+3954 polymorphism had an increased risk for developing intestinal type gastric cancer (OR 4.5) (Table 5). None of the other polymorphisms for IL-1B, IL-1RN or IL-10 genes was associated with the risk of intestinal or diffuse type of gastric cancer (Table 5). No association was found for any of the studied polymorphisms of IL-1B, IL-1RN or IL-10 genes with the risk of dyspepsia.

Discussion In this study, 87% of the subjects were seropositive for H. pylori. Previous studies carried out with individuals from the same geographic region reported similar H. pylori prevalence [30, 31]. In Costa Rica, a high percentage of people are infected with H. pylori since early in life [32].

The bacterium is considered to be one of the major risk factors for developing gastric cancer [33].

Allele frequencies for IL-1B-31, IL-1B-511 and all three IL-10 polymorphisms in our sample are similar to those reported for studies carried out in East Asian populations [16, 22, 34–37] but are significantly different to those reported for Caucasian populations [11, 14, 38]. The opposite pattern was observed for the IL-1RN polymorphisms where the frequencies were similar to those of Caucasian populations. In the case of IL-1B+3954, the allele frequencies here reported represent an intermediate between those reported in studies carried out in Caucasian and Asiatic countries [11, 27, 36, 38].

The genetic composition of the inhabitants of Costa Rica results from a genetic admixture of several ethnic groups, among them Caucasian, Amerindian and African, which are the more prominent in this admixture. Also, there is an important mongoloid genetic component in the Amerindian population of Costa Rica [39]. The differences and similarities for allele frequencies of the polymorphisms for IL-1B, IL-1RN and IL-10 genes when compared to other ethnic groups must be a product of the genetic admixture process, which has occurred in Costa Rica throughout its history.

We hypothesise that as a result of genetic admixture, selective pressures caused by environmental factors might have resulted in phenotypes with an increased pro-inflammatory response. Tropical regions have an enormous biological diversity because of favourable environmental conditions. The diversity includes pathogens, which results in an increased number and frequency of infectious diseases produced by these microorganisms. Until a few decades ago, infectious diseases were one of the main causes of death in tropical countries like Costa Rica [40, 41]. It is possible that individuals who carried a pro-inflammatory genetic profile were at an advantage when faced with these types of infections. The selective process may have resulted in an increase of the frequencies of pro-inflammatory genotypes, which could partly explain the high frequency of such genotypes in this study.

There are significant differences in gastric cancer incidence between the regions of Costa Rica [24]. The studied population was selected from a region with high risk of gastric cancer. Although H. pylori prevalence is similar in both high- and low-risk areas, the prevalence of atrophic gastritis and intestinal metaplasia are higher in high-risk areas [25]. The factors underlying these differences are unknown. However, we hypothesise that one of the factors involved is the genetic origin. A high frequency of proinflammatory genotypes could partly explain the high incidence of the malignancy in this region.

The IL-1B+3954C/T polymorphism was found to be associated with the risk of gastric cancer. This association is stronger for intestinal gastric cancer. This is the first time that such an association has been reported. Other studies have analysed it but none of them have reported an association [11, 27]. IL-1B+3954T/T has been associated with the risk of duodenal ulcer disease [42]. Nevertheless, the aetiology of duodenal ulcer is different from that of gastric cancer [23]. Further studies will be needed to confirm this finding.

We also found a significant association of IL-1RN L*2 with the risk of developing gastric cancer. Some studies have reported an association of allele 2 of the IL-1RN polymorphism [11, 21, 43]. The IL-1Ra protein (codified

by IL-1RN) plays a role as competitive inhibitor of IL-1β [10]. Carriers of IL-1RN*2/*2 genotype undergo more severe and prolonged immune responses than carriers of

W. Alpízar-Alpízar et al.: Interleukin polymorphisms and gastric cancer in Costa Rica

other genotypes [44]. We suggest that carrying allele 2, by itself, results in increased risk of developing gastric cancer because of a decrease in IL-1Ra expression.

This is one of the first studies trying to describe the role played by IL-1B, IL-1RN and IL-10 genetic polymorphisms in gastric cancer in one of the highest risk American countries. Although the sample size was small, the results raise new questions about the role of the host response against H. pylori in the development of gastric cancer. Further studies in high-risk areas of the American continent will be very important to confirm the role of proinflammatory and anti-inflammatory cytokines in the aetiology of this malignancy.

Acknowledgements This study was supported by the Vicerrectoría de Investigación of the University of Costa Rica (Project No. 742-A2-142), the Consejo Nacional para Investigaciones Científicas y Tecnológicas (CONICIT, Costa Rica) and the Scientific Cooperation Centre of the French Embassy. We wish to thank the German Academic Exchange Service (DAAD) and International Atomic Energy Agency (IAEA) for the donation of laboratory equipment.

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