Distal and proximal interleukin (IL)-10 promoter polymorphisms associated with risk of cutaneous melanoma development: a case–control study

Abstract

Inherited promoter polymorphisms of the interleukin (IL)-10 gene resulting in altered IL-10 production may contribute to a genetic susceptibility for melanoma. We investigated the role of a haplotype from distal as well as proximal polymorphic sites [−7400InDel, −6752AT (rs6676671), −3538AT (rs1800890), −1087AG (rs1800896), −597AC (rs1800872)] of the IL-10 5′-flanking region in a hospital-based case–control study of 165 Caucasian patients with cutaneous melanoma from Germany in comparison with 162 healthy cancer-free Caucasian control participants from the same area matched by age. Using multivariate analysis for the number of nevi and skin type, the IL-10 ‘higher producing’ haplotype ITAGC was found to be significantly associated with a reduced risk of developing melanoma (adjusted P=0.02). Although our findings need to be confirmed by independent and larger multicenter studies, we have described for the first time the association of distal gene variants of the IL-10 gene as an independent risk factor for melanoma.

Introduction

Cutaneous melanoma is the most serious form of skin cancer and has been increasing in frequency among Caucasian populations faster than that of any other cancer.1 Between 1940 and 2000 there was a progressive worldwide rise in the incidence of cutaneous melanoma.2 The mortality rate from melanoma has increased from 2 per 100 000 in 1969 to 3 per 100 000 in 1999, and this was mainly because of a high mortality among men aged 65 years.3 In the United States, approximately 64 939 new melanoma cases are predicted for 2008. The lifetime probability of developing melanoma is 1 in 41 for men and 1 in 61 for women.4 Phenotypic melanoma risk factors include age,5 sunlight exposure6 (particularly intense intermittent exposure),5 family history of melanoma, dysplastic nevi or atypical nevi, number of nevi, skin sensitivity to sun, freckling, fair hair, eye and skin colour.2

The role of molecular factors that could mediate susceptibility to and prognosis of sporadic melanoma is a subject of ongoing investigation. Polymorphisms associated with melanoma susceptibility or the course of disease have been described in genes involved in DNA repair,7 in the regulation of skin pigmentation, such as the melanocortin-1 receptor gene,8 in the production of proteins of the steroid and thyroid hormone superfamily, including vitamin D and the peroxisome proliferator-activated receptor genes,9 or the detoxification of oxidative stress metabolites, such as the glutathione S-transferase genes.10

Interleukin (IL)-10 is an important immunoregulatory cytokine. It is part of a balanced network of cytokines and can be cancer-promoting (immunosuppressive, stimulation of cell proliferation) or cancer-inhibiting (anti-angiogenic).11, 12, 13 Elevated IL-10 serum levels have been found in many cancers, including melanoma.14 The effects of IL-10 expression in melanoma are not fully understood and is a subject of controversy. IL-10 has been implicated in inhibition of the T-cell immune response and also found to exhibit anti-tumour activities in melanoma on the one hand.14, 15 But on the other hand, IL-10 can act as an autocrine growth factor on melanoma cells and downregulates the expression of HLA molecules on melanoma cells, indicating a immunosuppressive effect of IL-10.16

Polymorphisms in the IL-10 5′-flanking region genetically affect inter-individual differences in IL-10 production.17 Variable associations between the capability of IL-10 production and IL-10 microsatellite alleles, single-nucleotide polymorphisms (SNP) or SNP haplotypes in a 7-kbp IL-10 5′-flanking region have been reported.17 In most studies the major proximal IL-10 promoter haplotypes GCC, ACC and ATA formed by the SNPs IL-10−1087AG, IL-10−824CT and IL-10−597AC were found to be related to the in vitro capability of IL-10 production. GCC/GCC genotypes can be defined as ‘high producer’, GCC/ACC and GCC/ATA as ‘medium producer’, and ACC/ACC, ACC/ATA and ATA/ATA as ‘low producer’.18

More distal IL-10 promoter SNPs have been shown to be associated with high and low IL-10 production, sustaining the hypothesis of a cooperation of elements within the IL-10 promoter.19 The three base pairs’ GGA insertion/deletion polymorphism IL-10−7400InDel exhibited a high capability of IL-10 production for individuals homozygotic for the deletion (IL-10−7400Del). The allelic and genotypic comparison of IL-10 production by lipopolysaccharide (LPS)-stimulated leukocytes with the distal IL-10−6752AT and IL-10−6208CG polymorphisms revealed that the IL-10−6752T and IL-10−6208C genotypes can be considered as ‘high IL-10-producing’ genotypes.20 Unfortunately, the IL-10−3538AT polymorphism has not been assessed separately with regard to IL-10 production so far. This indicates complex and interactive relations between IL-10 polymorphisms and the capability of IL-10 production, rendering it difficult to ascribe a certain production capacity to a single IL-10 SNP.20

Both low and high IL-10-producing genotypes have been linked to susceptibility of cancer or unfavourable outcome.21 However, conflicting data are presented, when comparing different studies with respect to melanoma. Only seven studies investigated the role of proximal IL-10 promoter polymorphisms in melanoma susceptibility and in the course of metastasized melanoma.22, 23, 24, 25, 26, 27, 28 Martinez–Escribano et al.22 found that the above-mentioned proximal IL-10 promoter genotypes (SNPs IL-10−1087AG, IL-10−824CT and IL-10−597AC) were not associated with melanoma susceptibility. However, IL-10 low-producing haplotypes like ACC/ATA were associated with a shorter survival time and greater tumour thickness. Similarly, Nikolova et al.23 reported that the low-producing haplotypes like ATA were significantly increased in melanoma cell lines compared with controls, suggesting an association with melanoma susceptibility. Finally, Howell et al.24 found that the IL-10−1087AA low-expression genotype was significantly increased among melanoma patients compared with controls. In addition, this genotype was a risk factor for poor disease outcome. In contrast, Liu et al.25 reported that the IL-10−1087AA low-expression genotype was associated with a longer survival in metastasized melanoma patients, whereas von Euw et al.28 found an accelerated tumour progression in AA genotypes. Alonso et al.26 reported no association of the IL-10−1087AG, IL-10−824CT and IL-10−597AC polymorphisms with melanoma susceptibility; however, once again, decreased survival was observed in patients with the IL-10−1087AA genotype. According to a study of Vuoristo the ATA haplotype confers melanoma susceptibility as assessed in 108 melanoma patients and 393 healthy subjects.27 In most studies an increased risk for poorer outcome of melanoma could be demonstrated in association with low IL-10-producing genotypes as defined by proximal IL-10 gene variations. However, respective case–control studies are still missing taking into account not only age and gender but also nevus count, skin type, as well as hair and eye colour. This underlines the necessity of independent studies to verify these pilot findings.

Therefore, the aim of our study was to analyse haplotypes from distal together with proximal gene variations within the 5′-flanking regions of the IL-10 gene in a case–control study to estimate their effect on susceptibility to cutaneous melanoma.

Results and discussion

Study design

To estimate the effect of gene variations within a 7400 bp fragment of the 5′-flanking region of the IL-10 gene on melanoma susceptibility, a case–control study of 165 melanoma patients in comparison with 162 age-matched healthy volunteers was carried out. Healthy volunteers were recruited from donors at blood transfusion services located in Munich and Goettingen or from local health-care personnel. It was ensured that the controls were healthy and cancer-free. Blood samples were obtained from each patient and control subject after completion of personal interviews, and DNA was extracted and stored as described.7 The following parameters were registered by standardized procedures in the patients and the controls (Table 1): sex, age, hair colour, eye colour, skin type, number of nevi (on both forearms, diameter >2 mm), family history of melanoma, type and localization of melanoma, primary tumour thickness, month to metastasis, and localization of metastases, multiple primary melanomas, and the presence of dysplastic nevi. The recruitment period was between 2001 and 2003. The studies are in accordance with national protocols approved by the Institutional Review Boards of the Universities of Munich and Goettingen. Informed consent was obtained from all study participants.7All patients had histopathologically confirmed cutaneous melanoma.

Table 1 Phenotypic melanoma risk factors in controls and melanoma patients

For identification of the phenotypic risk factors relevant in the study, logistic regression analyses were carried out to predict melanoma from nevus count and skin type as continuous covariates, as well as from gender, hair colour (red vs other) and eye colour (green or blue vs grey or brown) as binary covariates. A backward selection eliminating covariates with P0.05 was conducted. From the resulting model, odds ratios and 95% confidence intervals were calculated for each remaining factor. To explore the effect of single polymorphisms, these were added to the logistic regression model. Odds ratios and 95% confidence intervals were calculated for each polymorphism. To determine whether the genotype frequencies conformed with the Hardy–Weinberg equilibrium, the equivalence test proposed by Wellek was used (5% test level) with ɛ=0.1.29 Frequencies of haplotypes were estimated with the expectation–maximization algorithm. The effect of the estimated haplotype frequencies was analysed using generalized linear models allowing for ambiguous haplotypes, including the covariates identified in the logistic regression model.30 As the main objective was to test for association between haplotypes and melanoma risk, analyses for single polymorphisms were only exploratory. For the haplotype analysis, all frequent haplotypes were tested for association adjusted for multiple testing using a Bonferroni–Holm correction (Table 4).

Table 4 The extended ITAGC haplotype of the IL-10 gene proved to be a novel independent molecular factor for predicting a reduced melanoma risk (indicated in bold)

Genotype distribution of IL-10 promoter polymorphisms and melanoma susceptibility

The IL-10 5′-flanking gene variations at IL-10−7400InDel, IL-10−6752AT, IL-10−3538AT, IL-10−1087AG and IL-10−597AC were genotyped using multiplex PCR and TaqMan real-time PCR, as described recently.17 Allele frequencies, genotypes and haplotypes were defined and compared with corresponding healthy controls. In Table 2 genotypic data are summarized for the IL-10 gene variations. All genotype frequencies conformed with the Hardy–Weinberg equilibrium.

Table 2 Genotype distributions of the IL-10 gene variations in healthy controls and melanoma patients

Logistic regression analyses showed that a lower number of nevi (OR 0.98, 95% CI: 0.96–0.99) protected from melanoma development. Skin type I (vs II–IV) was associated with an increased melanoma risk (OR 2.28, 95% CI: 1.67–3.12) (Table 1). This indicates that our study population is representative of the German population, as described earlier.7, 31

Exploratory multivariate logistic regression analyses accounting for the number of nevi and skin type indicated that the SNPs at IL-10−6752AT, IL-10−3538AT and IL-10−597AC were independent risk factors for the development of cutaneous melanoma in our study population (Table 3). Carriage of the IL-10−6752T, IL-10−3538A and IL-10−597C alleles was associated with a reduced risk of melanoma (OR 0.56, 95% CI: 0.34–0.92, exploratory P=0.02; OR 0.52, 95% CI: 0.32–0.86, exploratory P=0.01; OR 0.34, 95% CI: 0.13–0.88, exploratory P=0.03, respectively), whereas the genotypes IL-10−6752AA, IL-10−3538TT and IL-10−597AA were associated with a higher risk of melanoma development in our study group. Thereby, individuals carrying the IL-10−597AA seem to have the highest risk of melanoma. Carriage of the IL-10−6752T and IL-10−597C alleles was associated with an increased IL-10 production, implying that a high capability of IL-10 production protects from melanoma development.20, 32, 33 Indeed, this is in line with most other publications regarding IL-10 polymorphisms and melanoma.22, 23, 24, 26, 27, 28 In contrast, Nagano et al.34 reported that ‘low-expression’ IL-10 polymorphisms significantly protected from non-melanoma skin cancer. This may indicate that the effects of IL-10 differ in different skin cancer entities.

Table 3 Distal and proximal regulatory gene variations of the IL-10 promoter with respect to melanoma susceptibility

Haplotype analysis

The analysis of respective haplotypes showed a strong linkage of the loci and a representative distribution of all haplotypes, as described recently (Table 4).17 The four major haplotypes, IATAA, IATAC, ITAGC and DTAGC, are present for the respective IL-10 gene variations, IL-10−7400InDel, IL-10−6752AT, IL-10−3538AT, IL-10−1087AG and IL-10−597AC. Further analysis of haplotypes revealed that the ITAGC haplotype of the IL-10 gene was an independent predictor for a reduced risk to develop cutaneous melanoma, if corrected for the phenotypic risk factors skin type and number of nevi in our study population (adjusted P=0.02).

Compared with recent analyses on the association of IL-10 gene variations limited to proximal polymorphisms, our study indicates that the combination of distal and proximal genetic elements of the 5′-flanking region of the IL-10 gene may serve as a novel molecular predictor for the risk of developing cutaneous melanoma. This may be associated with a higher capability of IL-10 production, but it is also clear that our results have to be validated in independent samples in order to confirm the presented findings. Furthermore, the possibility of extending the haplotypic description to neighbouring genes, such as IL-19 should be considered, because skin tissues seem to represent a major target for IL-10-related cytokines, such as IL-19, IL-20, IL-22 and IL-24.35, 36, 37

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Acknowledgements

We are grateful to the Deutsche Forschungsgemeinschaft DFG (Graduate College 1034, www.gcpg.de) for supporting this work and the colleagues of the GRK1034 for helpful discussions.

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Correspondence to D Kube.

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Schoof, N., von Bonin, F., König, I. et al. Distal and proximal interleukin (IL)-10 promoter polymorphisms associated with risk of cutaneous melanoma development: a case–control study. Genes Immun 10, 586–590 (2009). https://doi.org/10.1038/gene.2009.40

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Keywords

  • cutaneous melanoma
  • interleukin-10
  • cytokines
  • gene polymorphisms
  • risk factor

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