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IL-1RN VNTR polymorphism as a susceptibility marker for nasopharyngeal carcinoma in Portugal Hugo Sousa a,b,c,*, Eduardo Breda d, Alexandra M. Santos a, Raquel Catarino a,c, Daniela Pinto a,b, Paulo Canedo e, Jose´ Carlos Machado e,f, Rui Medeiros a,b,c a
Molecular Oncology Group, Portuguese Institute of Oncology of Porto, Porto, Portugal Molecular Virology Laboratory of Virology Service, Portuguese Institute of Oncology of Porto, Porto, Portugal c ICBAS, Abel Salazar Institute for the Biomedical Sciences, University of Porto, Porto, Portugal d Otorhinolaryngology Service, Portuguese Institute of Oncology of Porto, Porto, Portugal e Institute of Molecular Pathology and Immunology of University of Porto (IPATIMUP), Porto, Portugal f Faculty of Medicine of University of Porto (FMUP), Porto, Portugal b
Background: Nasopharyngeal carcinoma (NPC) is a rare malignancy in Western countries
Accepted 10 February 2013
that is widely associated with the infection by Epstein–Barr virus (EBV). Several studies have showed that a common allele (allele 2) of the 86-bp variable number of tandem repeats
(VNTR) polymorphism within intron 2 of the interleukin 1 receptor antagonist (IL-1RN) gene
is associated with several disorders, including viral-associated cancers.
Methods: We have developed a hospital-based case–control study to characterise the role of
the IL-1RN 86-bp VNTR polymorphism in the development of NPC with 112 patients with the
disease and 433 healthy individuals from the northern region of Portugal. IL-1RN genotypes
were combined according to the number of repeats: allele 2 (A2), the short allele that corresponds to two repeats, and L, the long allele that corresponds to three or more repeats. Results: Our study revealed that 31.2% of NPC patients were IL-1RN A2*A2, compared with 9.7% observed in the control group. The statistical analysis revealed that IL-1RN*A2 homozygosity for the A2 allele was associated with a fourfold increased risk for NPC development ( p < 0.001). Additionally, cumulative hazard analysis revealed that estimated median age of onset of NPC is significantly ( p < 0.001) different for A2*A2 homozygous versus non-A2*A2 (57.0 vs. 74.0, respectively). Conclusions: This is the first study to evaluate the role of the IL-1RN VNTR in NPC development in Portugal. Our study indicates IL-1RN*A2 homozygosity as a significant risk marker in our population and that it should be further investigated for the potential role in the definition of a susceptibility profile for NPC onset. # 2013 Elsevier Ltd. All rights reserved.
Nasopharyngeal carcinoma (NPC) is a frequent malignancy in Southeast Asia, particularly in Southern China, while
in Western countries its incidence rates are below 1 per 100,000.1–3 NPC is considered to be a multifactorial disease, where environmental factors such as tobacco smoking, alcohol consumption, exposure to wood dust and consumption of salted fish and foods containing
* Corresponding author at: Grupo de Oncologia Molecular – CI, Laborato´rios 48 Piso, Instituto Portugueˆs de Oncologia do Porto FG, EPE, Rua Dr. Anto´nio Bernardino Almeida, 4200-072 Porto, Portugal. Tel.: +351 22 508 4000x5410; fax: +351 22 508 4001. E-mail addresses: [email protected]
, [email protected]
(H. Sousa). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.02.004
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nitrosamine or its precursors have been associated with its development.3–5 In addition, epidemiological evidence suggests that undifferentiated NPCs (UNPCs) are associated up to 100% with infection by the Epstein–Barr virus (EBV).6–10 Recently it has been discussed that deficiencies in the immune response to viral infection can play a role in the different individual susceptibility to the development of viral-associated neoplasias.11–16 Upon viral infection the host immune system activates a response that is mediated mainly by the interleukin 1 (IL-1) family. This family combines potent pro-inflammatory cytokines, IL-1a and IL-1b, and its negative regulator with antiinflammatory effect, the IL-1 receptor antagonist (IL-1RN).17–22 The last-named competes with IL-1a and IL-1b for the IL-1 cell receptor, preventing the transmission of pro-inflammatory signals.17,23–25 Moreover, IL-1RN avoids cellular damages in the case of a severe and prolonged inflammatory response and can also be used to follow the immune response, as its levels increase during the final steps of inflammatory response.18–27 Genetic polymorphisms have been considered to be important regarding the definition of a susceptibility profile for the development of several cancers.28 Several genetic studies have indicated that polymorphisms of the IL-1RN gene sequence could influence its expression and therefore have an important role in the development of several disorders.27–30 In fact, several authors have been referring that a 86-bp variable number of tandem repeats (VNTR) polymorphism within intron 2 is associated with the development of several cancers: gastric,31–41 oesophageal,42 bladder,43–45 breast,46 colorectal,47,48 lung,49–51 brain,52,53 gallbladder,43 cervical54 and nasopharyngeal.55 A previous study from our group has demonstrated that that IL-1RN VNTR polymorphism is associated with the development of cervical cancer.54 Hence, with this hospitalbased case–control study we aimed to characterise the role of the IL-1RN VNTR polymorphism in the development of NPC within a southern European population from the northern region of Portugal.
Table 1 – Distribution of clinical data in cases and controls included in the study. Gender, no. (%) Female Controls NPC patients WMDNPC (n = 11) UNPC (n = 101)
196 33 2 31
(45.3) (29.0) (18.2) (30.7)
Age, y (mean SD)
Male 237 79 9 70
(54.7) (71.0) (81.8) (69.3)
42.2 15.2 47.2 14.3 49.5 12.7 47.0 14.5
WMDNPC, well or moderated differentiated NPC; UNPC, undifferentiated nasopharyngeal cancer; SD, standard deviation.
age of 42.2 years (standard deviation 15.3) recruited from a community-based Healthy Blood Donors Database at the Institute of Molecular Pathology and Immunology of University of Porto (IPATIMUP). All individuals were submitted to a previous clinical evaluation and have no individual history of cancer.
This study does not include any clinical procedure other than collection of blood samples and it has not interfered with the routine procedures decided upon by clinicians. Clinico-pathological data were collected from individual clinical records and entered into a database with unique codification to assure confidentiality. Informed consent according to the Declaration of Helsinki was obtained from each individual.
Sample collection and DNA extraction
Peripheral blood samples were collected following a standard venipuncture technique in ethylene diamine tetraacetic acid (EDTA)-containing tubes and the DNA was extracted from the white blood cell fraction using a salting-out protocol.
Materials and methods
Type of study and population
The analysis of the IL-1RN VNTR polymorphism located within intron 2 was performed by polymerase chain reaction (PCR) with the primers described previously37 (forward: 50 CCCCTCAGCAACACTCC-30 ; and reverse: 50 -GGTCAGAAGGGCAGAG-30 ). Briefly, the PCR reaction was performed in a total volume of 50 ml including: 1 Taq buffer, 2.0 mM of MgCl2, 0.4 mM of deoxynucleotide triphosphates (dNTPs), 0.30 mM of each primer and 1 U Taq DNA polymerase. Amplification was performed with an initial denaturation step of 4 min at 94 8C, followed by 35 cycles of 30 s at 94 8C, 30 s at 57 8C and 60 s at 72 8C and a final extension step of 5 min at 72 8C. PCR amplification products (allele 1 – 410 bp; allele 2 – 240 bp; allele 3 – 325 bp; allele 4 – 500 bp; and allele 5 – 595 bp) were separated by electrophoresis in a 3% agarose gel (Cambrex Bio Science Rockland Inc., Rockland, ME, USA) stained with 5% ethidium bromide – Fig. 1. Possible genotype combinations include: A1*A1, A1*A2, A1*A3, A1*A4, A1*A5, A2*A2, A2*A3, A2*A4, A2*A5, A3*A3, A3*A4, A3*A5, A4*A4, A4*A5 and A5*A5.
We have performed a hospital-based case–control study considering unrelated individuals from a Caucasian population from the northern region of Portugal (Table 1). The group of patients consisted of 112 individuals with mean age of 47.2 years (standard deviation 14.3) who attended consecutively at the Otorhinolaryngology Department of the Portuguese Institute of Oncology, of Porto. All cases were histologically confirmed by a senior pathologist from our institution according to the World Health Organisation (WHO) classification: 11 type I (keratinising squamous cell carcinoma) or type II (non-keratinising squamous cell carcinoma), combined as well or moderated differentiated (WMDNPC) and 101 type III characterised as undifferentiated carcinoma (UNPC). The control group consisted of 433 (237 males and 196 females) randomly selected healthy individuals with mean
Analysis of IL-1RN VNTR polymorphism
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5% level of significance. The chi-squared (x2) analysis was done to compare the categorical variables and the odds ratio (OR) and its 95% confidence interval (CI) were used as a measure of the association between the IL1-RN genotypes, distributed into a dichotomic variable considering the status on allele A2 homozygosity and the risk of developing NPC. Logistic regression analysis was performed with adjustment for age and gender. Kaplan–Meier methodology with the logrank and Breslow tests were used to calculate the cumulative hazard function plots of the genotype-specific distributions according to age of disease onset. Fig. 1 – PCR analysis of IL-1RN VNTR polymorphism. Agarose gel (3% w/v) stained with ethidium bromide: M – 100 bp DNA ladder; (–) negative control; lanes: (1) A2*A2, (2) A1*A1, (3) A1*A2, (4) A1*A4, and (5) A1*A3.
Genotyping was performed without discrimination of cases and controls and was independently analysed by two authors. Ten percent of all samples were randomly selected and resubmitted to a genotyping to confirm the genotypes, which were concordant in 100% among duplicates.
The Hardy–Weinberg equilibrium (HWE) was calculated by combining IL-1RN alleles according to the number of repeats stratified as A2*A2, A2*L and L*L (A2, the short allele that corresponds to two repeats and L, the long allele that corresponds to three or more repeats) with the public software at http://www.oege.org/software/hwe-mr-calc. available shtml.56,57 Statistical analysis was performed with Statistical Package for Social Sciences – SPSS for Windows (version 16.0) – using a
IL-1RN VNTR genotype distribution
The distribution of the IL-1RN VNTR polymorphism genotypes among controls and patients considering the possible multiple combinations of genotypes is shown in Table 2. In our population it was not possible to find individuals carrying the following genotypes: A2*A5, A3*A3, A3*A4, A3*A5, A4*A4, A4*A5 and A5*A5. The HWE analysis revealed that genotypes were according to the expected ones in the control group ( p = 0.373) while patients with NPC did not respect the HWE ( p < 0.001), which was expected as we observed an increased frequency of allele 2 homozygosity (A2*A2) in patients (Table 2).
3.2. IL-1RN VNTR polymorphism and nasopharyngeal carcinoma The genotype distribution of IL-1RN VNTR polymorphism in the NPC group revealed that 31.2% of all patients were IL-1RN A2*A2, compared with 9.7% observed in the control group (Table 3). This study revealed that IL-1RN A2*A2 homozygosity represents a fourfold increased risk of developing both all
Table 2 – Distribution of IL-1RN VNTR genotypes. A1*A1 Controls (n = 433) NPC (n = 112) WMDNPC (n = 11) UNPC (n = 101)
207 52 2 50
(47.8) (46.4) (18.2) (49.5)
A1*A2 169 17 2 15
(39.0) (15.2) (18.2) (15.2)
10 5 1 4
– 2 (1.8) – 2 (2.0)
1 (0.2) – – –
(2.3) (4.5) (9.1) (4.0)
A2*A2 42 35 6 29
(9.7) (31.2) (54.5) (28.7)
3 (0.7) – – –
1 (0.2) 1 (0.9) – 1 (1.0)
WMDNPC, well or moderated differentiated NPC; UNPC, undifferentiated nasopharyngeal cancer.
Table 3 – Association of IL-1RN VNTR genotypes and nasopharyngeal carcinoma.
Controls (n = 433) NPC (n = 112) UNPC (n = 101)
391 (90.3) 77 (68.8) 72 (71.6)
42 (9.7) 35 (31.2) 29 (28.7)