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Keywords:

  • inflammasome;
  • NLRP1;
  • NLRP3;
  • CIAS1;
  • SNP;
  • melanoma;
  • IL-1β

Summary

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements and financial support
  9. Conflict of interest
  10. References

Genetic variants of NLRP3 and NLRP1 are known to modulate levels of pro-inflammatory cytokine interleukin (IL)-1β. The purpose of this study was to investigate the association of NLRP3/NLRP1 polymorphisms with susceptibility and clinical features of malignant melanoma in a Swedish case–control study. Common variants in NLRP3/NLRP1 were investigated in sporadic malignant melanoma patients and healthy controls followed by analysis using logistic regression. NLRP3 variant (rs35829419) was significantly more common in male patients than in controls (OR, 2.22; CI, 1.27–3.86). Upon stratification, significant association with nodular melanoma was observed (OR, 2.89; CI, 1.33–6.30), which intensified in male patients (OR 4.03, CI 1.40–11.59). The NLRP1 variant (rs12150220) was significantly more common in fair-skinned female patients (OR, 1.85; CI, 1.04–3.33) and showed strong associations with nodular melanoma (OR, 6.03; CI, 1.33–25). Our data suggest that NLRP3/NLRP1 polymorphisms are associated with melanoma susceptibility; these findings warrant validation in other independent populations.


Significance

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements and financial support
  9. Conflict of interest
  10. References

An essential role of the pro-inflammatory cytokine IL-1β in tumor invasiveness and angiogenesis has previously been demonstrated. NLRP3/NLRP1 inflammasomes are IL-1β-producing platforms that regulate inflammation. We report an increased susceptibility to sporadic malignant melanoma in the presence of common polymorphisms in the inflammasome genes. A particularly strong association was observed to nodular melanoma. Genetic alterations in the inflammasome genes through dysregulations in IL-1β production might result in increased susceptibility to melanoma.

Introduction

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements and financial support
  9. Conflict of interest
  10. References

NACHT-LRRs (NLRs) are a group of cytosolic receptors that recognize intracellular microbial products as well as metabolic stress (Schroder and Tschopp, 2010). The NLR family includes 22 members and has a key role in regulation of inflammation and apoptosis (Schroder and Tschopp, 2010). The NLRs, NLRP1 (also termed NALP1), and NLRP3 (NALP3 or cryopyrin) upon activation bind to an adaptor protein ASC to form an inflammatory complex termed the inflammsome, which cleaves pro-interleukin (IL)-1β and pro-IL-18 to mature and biologically active IL-1β and IL-18 (Figure 1) (Agostini et al., 2004; Martinon et al., 2002). Another adaptor comprising a function-to-find (FIIND) and caspase-activation and recruitment domain (CARD), termed CARD-8 (TUCAN or Cardinal) (Pathan et al., 2001), is postulated to be a binding partner of NLRP3, whereas its binding is not required by NLRP1, which already contains a C-terminal FIIND-CARD domain.

image

Figure 1.  A simplified picture of IL-1β and IL-18 processing by the NLRP1 and NLRP3 inflammasomes. Upon sensing a stimuli, NLRP1/NLRP3 recruits the adaptor protein ASC and thereby activates caspase1 (and caspase 5 in case of NLRP1), which cleaves pro-IL-1β and pro-IL-18 to form the mature IL-1β and IL-18. These pro-inflammatory cytokines are then secreted out of the cell and upregulate a number of pro-inflammatory genes through a positive feedback loop. CARD-8 is postulated to be a binding partner of NLRP3, whereas its binding is not required by NLRP1 that already contains a CARD-8 domain.

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Missense mutations in the NLRP3 gene lead to familial periodic fevers, where symptoms arise from excess of IL-1β and accordingly, blockade of IL-1β effectively ameliorates the symptoms (Hoffman and Simon, 2009). More recently, a single nucleotide polymorphism (SNP) in NLRP3 (rs35929419), in combination with CARD-8 polymorphism (rs2043211), has been associated with chronic inflammatory conditions (Kastbom et al., 2008; Roberts et al., 2010, 2011; Schoultz et al., 2009; Verma et al., 2008). The CARD-8 protein is additionally suggested to be a negative regulator of caspase-1-mediated IL-1β regulation and NF-κB activation (Fontalba et al., 2008; Razmara et al., 2002). Another SNP in NLRP3 (rs10733113), located downstream of the NLRP3 gene, has been shown to regulate NLRP3 expression and IL-1β levels and has recently been implicated in Crohn’s disease (Villani et al., 2009). NLRP1 SNPs rs12150220 and rs6502867 have been reported to be associated with susceptibility to vitiligo (Jin et al., 2007b), an autoimmune disease affecting the melanocytes in the skin, where patients are reported to have elevated serum IL-1β levels (Tu et al., 2003).

Several recent reports describe the importance of inflammatory microenvironment in the risk of tumor initiation and progression (Clevers, 2004; Coussens and Werb, 2001, 2002; Mantovani et al., 2008). Tumors secrete pro-inflammatory cytokines into the surroundings (Okamoto et al., 2010), which accelerate tumor development and progression in a number of ways, for example, by inducing proliferation and survival of malignant cells, by promoting angiogenesis and metastasis, and by altering responses to chemotherapeutic drugs (Mantovani et al., 2008). The molecular mechanisms linking inflammation and carcinogenesis, however, still remain to be clarified. Malignant melanoma (MM) originates from the pigment-producing melanocytes in epidermis and is the most severe type of skin cancer. Melanoma often displays an aggressive behavior with early metastases and its incidence increases faster than any other cancer type among fair skin population worldwide (Diepgen and Mahler, 2002; Lens and Dawes, 2004). The survival rate of patients with melanoma is directly related to early detection and treatment, and hence, the search for genetic markers of disease risk is of continuing medical interest.

The present study investigates the significance of five inflammasome-related SNPs; rs35829419 and rs10733113 in NLRP3, rs6502867 and rs12150220 in NLRP1, and rs2043211 in CARD-8, in susceptibility and progression of MM.

Results

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements and financial support
  9. Conflict of interest
  10. References

In the present case–control study, the SNPs rs35829419 and rs10733113 in NLRP3, rs2043211 in CARD-8, and rs6502867 and rs12150220 in NLRP1 were studied in 258 patients diagnosed with sporadic MM and 792 controls. Both cases and controls were from the Southern part of Sweden. The description of the study subjects and stratifications of phenotypic features and melanoma characteristics are detailed in Table 1, and the genotype distributions are presented in Table 2. All the SNP frequencies were found to be in Hardy–Weinberg equilibrium.

Table 1.    (a) Patients with sporadic malignant melanoma and controls, (b) Phenotype features of the patients and melanoma characteristics
  1. aSkin type I + 11, Sun-sensitive skin type, Skin type III + IV, Less sun-sensitive skin type.

(a)
 PatientsControls
Total number258792
Sex
 Males120 (47%)396 (50%)
 Females138 (53%)396 (50%)
Mean age59 ± 1454 ± 17
Mean age at diagnosis55 ± 15
(b)
 Number
Age at diagnosis
 ≤3527 (11%)
 36–60129 (54%)
 >6084 (35%)
Skin typea
 I + II153 (60%)
 III + IV100 (40%)
Eye color
 Blue + green211 (82%)
 Brown + mixed46 (18%)
Hair color
 Blond + red216 (84%)
 Brown + Black40 (16%)
Histopathological type
 Superficial spreading melanoma (SSM)150 (64%)
 Nodular melanoma (NM)53 (22%)
 Lentigo malignant melanoma (LMM)9 (4%)
 Acro-lentiginous melanoma (ALM)2 (1%)
 Melanoma in situ22 (9%)
Breslow thickness (mm)
 <2166 (77%)
 ≥250 (23%)
Anatomic site
 Sun-exposed areas176 (72%)
 Rarely/not sun-exposed areas37 (15%)
 Other areas33 (13%)
Table 2.   Distribution of genotypes in sporadic malignant melanoma (MM)
 Sporadic MMControls
NLRP3 rs 35829419257792
 CC218 (85%)692 (87%)
 CA36 (14%)94 (12%)
 AA3 (1%)6 (1%)
NLRP3 rs 10733113257792
 GG181 (71%)574 (72%)
 GA70 (27%)204 (26%)
 AA6 (2%)14 (2%)
NLRP1 rs 6502867255784
 TT169 (66%)497 (63%)
 TC80 (31%)248 (32%)
 CC6 (3%)39 (5%)
NLRP1 rs 12150220254790
 TT66 (26%)157 (20%)
 TA132 (52%)410 (52%)
 AA56 (22%)223 (28%)
CARD-8 rs 2043211257792
 TT114 (44%)330 (42%)
 TA111 (43%)359 (45%)
 AA32 (13%)103 (13%)

NLRP3 and CARD-8 polymorphisms in patients with sporadic MM

No association was detected between the NLRP3 rs35829419 and the overall risk for developing MM (Table 3). Owing to the small numbers (1% in cases as well as controls), AA was analyzed in combination with the CA genotype group. As we have previously observed gender-based differences in genotype frequencies of rs35829419 (unpublished observation), we stratified the patients with sporadic MM and their controls based on gender and found the presence of at least one A allele to be more frequent among male patients (20%) than in male controls (10%) (P = 0.005; OR, 2.22; CI, 1.27–3.86) (Table 3). No corresponding risk was detected among women.

Table 3.   NLRP3 rs35829419 and NLRP3 rs10733113 genotype distributions in patients with sporadic malignant melanoma (MM). Genotype distributions for the two SNPs are also represented after gender stratification and in combination
GenotypesSporadic MMControlsOR (95% CI)P value
  1. Bold values indicate statistical significance.

NLRP3 rs35829419257792 0.22
 CC218 (85%)692 (87%)1
 CA/AA39 (15%)100 (13%)1.27 (0.86–1.88)
NLRP3 rs35829419
 Males
  CC96 (80%)355 (90%)10.005
  CA/AA24 (20%)40 (10%)2.22 (1.27–3.86)
 Females
  CC121 (88%)337 (85%)10.32
  CA/AA16 (12%)60 (15%)0.74 (0.41–1.34)
NLRP3 rs10733113257792  
 GG181 (70%)574 (72%)10.44
 GA/AA76 (30%)218 (28%)1.13 (0.83 – 1.53)
NLRP3 rs10733113
 Males120395 0.97
  CC87 (73%)287 (73%)1
  CA/AA33 (27%)108 (27%)1.01 (0.64 – 1.59)
 Females137397 0.41
  CC94 (69%)287 (72%)1
  CA/AA43 (31%)110 (28%)1.19 (0.78 – 1.82)
NLRP3 rs 35829419/rs 10733113
 CC/GG169 (92%)496 (87%)10.001
 CA + AA/GA + AA22 (8%)22 (13%)2.93 (1.58 – 5.44)

Stratifying the patients by melanoma type, that is, nodular melanoma (NM) versus superficial spreading melanoma (SSM), revealed a significantly increased risk for developing NM in the presence of least one A allele (P = 0.007; OR, 2.89; CI, 1.33–6.30) (Table 4). Furthermore, upon stratifying the patient group by gender (Table 4), a higher frequency of the A allele was observed among the male patients (20%) as compared to the female patients (12%). This difference, however, did not reach statistical significance (P = 0.069; OR, 1.89; CI, 0.95–3.76). Furthermore, an even higher risk was associated with the male NM patients (P = 0.010; OR, 4.03; CI, 1.40–11.59). The other types of melanoma (ALM, LMM) were not analyzed because of their small numbers.

Table 4.   NLRP3 rs35829419 genotype distribution in patients with sporadic malignant melanoma (MM) upon stratifying for gender and melanoma type
StratificationSporadic MMOR (95% CI)P value
  1. NM, Nodular melamona; SSM, Superficial spreading melanoma. Bold values indicate statistical significance.

Gender257  
 Males/females
  CC96 (80%)/121 (88%)10.069
  CA/AA24 (20%)/16 (12%)1.89 (0.95–3.76)
Melanoma type203  
 NM/SSM
  CC38 (72%)/132 (88%)10.007
  CA/AA15 (28%)/18 (12%)2.89 (1.33–6.28)
Gender/melanoma type203 
 Males  NM/SSM
  CC16 (62%)/58 (87%)10.010
  CA/AA10 (38%)/9 (13%)4.03 (1.40–11.59)
 Females   NM/SSM
  CC22 (81%)/74 (89%)10.30
  CA/AA5 (19%)/9 (11%)1.87 (0.57–6.16)

The additional SNP studied in NLRP3, rs10733113, revealed no associations even after stratifications for the clinical parameters (all P-values >0.05) (Table 3). However, the logistic regression analysis demonstrated that the combined genotypes rs35829419 and rs10733113 (CA,AA/GA,AA) were significantly more frequent in subjects with sporadic MM than in controls (P = 0.001; OR, 2.93; CI, 1.58–5.44) suggesting a synergistic effect.

No overall association was found between the CARD-8 SNP rs2043211 and MM. Moreover, no significant association was found after stratification for gender and melanoma type.

Statistical analyses of the three polymorphisms in relation to skin type (I + II to III + IV), eye color (blue +  green to brown + mixed), Breslow thickness (≥2 to <2 mm), age at diagnosis (≤35, 36–60, >60 yrs), and anatomic tumor site (sun exposed to rarely/not sun exposed) were performed; however, no associations were found.

NLRP1 polymorphisms in patients with sporadic MM

No significant overrepresentation of the NLRP1 SNPs was found in patients with sporadic MM when comparing with the control population (Table 2). An overrepresentation of the A allele (TA/AA genotype) in the rs12150220 was observed in the patient group, which did not reach statistical significance (P = 0.06; OR, 1.37; CI, 0.99–1.89).

When stratifying according to skin type, a 1.85 fold increased risk for MM was seen for the A allele in the rs12150220 among individuals with the fair skin types (type I and II) (P = 0.037; OR, 1.85; CI, 1.04–3.33) (Table 5). This risk further increased to 3.06-fold when analyzed only in the female patients (P = 0.049; CI, 1.01–0.09). No association was observed in the respective group of men.

Table 5.   NLRP1 genotype frequencies in patients with sporadic melanoma and NLRP1 rs 12150220 genotype distribution after stratifying for gender, skin type, and malignant melanoma (MM) type
GenotypesSporadic MMControlsOR (95% CI)P value
  1. NM, Nodular melanoma; SSM, Superficial spreading melanoma.

  2. aSkin type I + II, Sun-sensitive; III + IV, Less sun-sensitive. Bold values indicate statistical significance.

NLRP1 rs 6502867255784  
 TT169 (66%)497 (63%)1 
 TT/CC86 (34%)287 (37%)1.18 (0.78–1.59)0.27
NLRP1 rs 12150220254790 
 TT66 (26%)157 (20%)10.06
 TA/AA188 (74%)633 (80%)1.37 (0.99–1.89)
StratificationSporadic MMOR (95% CI)P value
Skin typea
 I + II/III + IV157/97  
  TT32 (21%)/32 (33%)10.037
  TA/AA120 (79%)/65 (67%)1.85 (1.04–3.33)
Gender/skin type
 Females  I + II/III + IV86/45  
  TT20 (23%)/16 (36%)10.049
  TA/AA66 (77%)/29 (64%)3.06 (1.01–9.09)
 Males  I + II/III + IV66/52  
  TT12 (18%)/16 (31%)10.11
  TA/AA54 (82%)/36 (69%)2.00 (0.85–4.72)
Gender/melanoma type
 Females  NM/SSM27/83  
  TT2 (7%)/27 (33%)1 
  TA17 (63%)/40 (48%)5.74 (1.22.27)0.027
  AA8 (30%)/16 (19%)6.75 (1.27–35.7)0.025
  TA + AA25 (93%)/56 (67%)6.03 (1.33–25)0.025
 Males  NM/SSM26/66  
  TT9 (35%)/13 (20%)1 
  TA10 (38%)/35 (53%)0.41 (0.14–1.24)0.12
  AA7 (27%)/18 (27%)0.56 (0.17–1.90)0.35
  TA + AA17 (65%)/53% (80%)0.46 (0.17–1.27)0.14

Upon stratification by melanoma type, no association of any of the NLPR1 SNPs was found (Table 5). However, when analyzing based on gender, a strongly increased risk for developing NM was found for the rs12150220A allele among females (P = 0.020; OR, 6.03; CI, 1.33–25). This increased risk was not detected in men.

Statistical analyses of both the NLRP1 polymorphisms in relation to eye color, Breslow thickness, age of diagnosis, and anatomic tumor site of the body were performed, without statistically significant differences.

Discussion

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements and financial support
  9. Conflict of interest
  10. References

The current study has investigated the association of the SNP’s rs35829419 and rs10733113 in NLRP3, rs2043211 in CARD-8, and rs6502867 and rs12150220 in NLRP1 in patients with sporadic MM. NLRP3 is a quite recently described member of the NLR family that is an essential regulator of the innate immune responses. NLRP3 forms an inflammatory complex called the inflammasome, which activates caspase-1 that subsequently leads to the formation of the potent pro-inflammatory cytokines IL-1β and IL-18. Missense alterations in NLRP3 are postulated to confer a gain-of-function resulting in uncontrolled production of IL-1β, as seen in the patients with hereditary periodic fevers (Aksentijevich et al., 2002). The rs35829419 (Q705K) in NLRP3, because of its prevalence in the healthy population, was initially described as a low-penetrance polymorphism (Aksentijevich et al., 2007), but in recent years, this SNP has been associated with chronic inflammatory conditions. These associations have especially been seen in combination with rs2043211 in CARD-8 (Kastbom et al., 2008; Roberts et al., 2010, 2011; Schoultz et al., 2009; Verma et al., 2008). Using genetic construct for rs35829419 in an in vitro functional model, we have recently shown this SNP to confer a gain-of-function in terms of increased spontaneous IL-1β production from a human monocyte cell line (Verma et al., 2012).

In this study, we found an increased overall risk for sporadic MM susceptibility among Swedish males carrying rs35829419A in NLRP3. The combination of rs35829419A with rs2043211 displayed no further increase in susceptibility risk for MM. Notably, a strong association was observed between NM and NLRP3 rs35829419. Unlike SSM that initially describes a radial growth pattern, NM arises rapidly and grows in a more vertical direction and might be associated with a worse prognosis (Pollack et al., 2011). We hypothesize that this gain-of-function SNP because of increased IL-1β levels, might upregulate other related pro-inflammatory genes like TNF-α and NF-κB through a positive feedback loop, which might promote melanoma cells to resist elimination by the T lymphocytes. This survival phenomenon has previously been demonstrated in melanoma cell lines treated with cytokines like TNF-α (Englaro et al., 1999) and very recently IL-1β (Kholmanskikh et al., 2010).

The fact that the SNP rs35829419A constituted a susceptibility risk only in the male population has previously been observed by us in Crohn′s disease (Schoultz et al., 2009). It is tempting to speculate that this differential risk pattern might depend upon the differences in estrogen levels, because the estrogen receptor by acting as a cofactor for NF-κB can inhibit its signaling (Biswas et al., 2005). Moreover, estradiol augments IL-1β-mediated pro-inflammatory response by downregulating the IL-1 receptor type-1, probably accounting for an increased tolerance level in women (Schaefer et al., 2005).

NLRP1 polymorphisms are strongly associated with increased predisposition to autoimmune diseases (Jin et al., 2007a,b). We report an increased risk with the rs12150220 variant in the individuals with a fair skin. Intriguingly, unlike our previous observation, this SNP was found to constitute a risk only in the women. NLRP1 inflammasome activates caspase-5 in addition to caspase-1. Caspase-5 is an upstream activator of caspase-1, and hypothetically, these two caspases together would result in a more effective IL-1β processing. Jin and colleagues have reported several NLRP1 variants (including rs6502867 and rs12150220) that confer susceptibility to autoimmune and autoinflammatory diseases that are associated with vitiligo (Jin et al., 2007b). The fact that the SNP rs121150220 (L155H) is highly conserved through primate evolution (human, chimpanzee, rhesus monkey, and bush baby) (Jin et al., 2007b) indicates that this region is critical for the function of the NLRP1 protein. This region has also been implicated in NOD2-NLRP1 interaction (Wagner et al., 2009). Functional studies elucidating the role of rs12150220 are lacking, but it is tempting to speculate that this SNP leads to an overactivation of the inflammasome and hence contributes to melanoma predisposition.

This is the first study, to our knowledge, showing inflammasome polymorphisms to be associated with tumor susceptibility and melanoma type. Many cancers are known to arise from sites with inflammation (Mantovani et al., 2008). In addition, IL-1β is also detected in various tumor types (Culig et al., 1998; Jin et al., 1997; Oelmann et al., 1997; Saijo et al., 2002), where its expression is associated with tumor invasiveness and angiogenesis (Carmi et al., 2009; Voronov et al., 2003), suggesting a possible role for the inflammasome in tumorgenesis. In line with this, late stage human melanoma cells were demonstrated to spontaneously secrete active IL-1β via constitutive activation of the NLRP3 inflammasome in the absence of exogenous stimulation, exhibiting a feature of autoinflammatory diseases (Okamoto et al., 2010). We hence postulated that the gain-of-function SNP because of increased IL-1β production might contribute toward increased melanoma susceptibility. In a recent study using a mouse melanoma model, it was demonstrated that inflammasomes contributed toward inhibition of the endogenous antitumor response and facilitated tumor growth (Van Deventer et al., 2010). Further, in the highly invasive melanoma cells, the expression of matrix metalloproteinase-1 (MMP), which is essential for tumor invasion and metastasis, is seen to be increased in an IL-1α- and FGF2-dependent manner (Loffek et al., 2005). Notably, both IL-1α and FGF2 are regulated by caspase-1 activity (Keller et al., 2008), providing further support for the role of inflammasomes in melanoma. IL-18, another cytokine associated with inflammasome, has been shown to be highly expressed in malignant skin tumors (Park et al., 2001), further emphasizing the role of inflammasomes in tumorgenesis.

Additional support for a critical role of IL-1β in the tumor microenvironment is evident by the success of IL-1 blockade in preventing angiogenesis and metastasis in animal models (Carmi et al., 2009; Chirivi et al., 1993; Vidal-Vanaclocha et al., 1994, 1996) as well as in human melanoma cell lines (Chirivi et al., 1993; Okamoto et al., 2010). A recent paper demonstrated increased IL-1β expression in primary and metastatic human melanomas and a decrease in melanoma cell growth was observed upon blocking the IL-1 signaling pathway (Qin et al., 2011). A potential role of IL-1β and inflammasomes in the pathogenesis of melanoma has recently been postulated (Dunn et al., 2012; Zitvogel et al., 2012). Above reports suggest that targeting IL-1 in tumors might indeed be a promising therapeutic option.

One caveat of the present study is the small number of patients resulting from subgroup analyses. However, we believe that the consistent trends in our results allow for the conclusion that NLRP3/NLRP1 polymorphisms influence susceptibility to MM and the development of NM.

In conclusion, we report inflammasome polymorphisms to be one link between innate immunity and susceptibility to sporadic MM. Further investigations are needed to confirm this finding in additional cohorts, and the importance of NLRP proteins in the pathogenesis of melanoma and other cancer types are warranted.

Methods

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements and financial support
  9. Conflict of interest
  10. References

Study subjects

This study was approved by the Regional Ethics Committee at the Linköping University, Linköping, Sweden. 258 patients (53% women, 47% men, ≥18 yrs) diagnosed with sporadic MM in the skin were included in the study. The patients were recruited from dermatology clinics in Southeastern region of Sweden (Linköping, Norrköping, and Kalmar). The mean age at inclusion was 59 yrs (±14 yrs, range 23–87 yrs). A detailed description of the study subjects is listed in Table 1. The mean age of the patients with melanoma at the time of diagnosis was 55 yrs (±15 yrs, range 18–86 yrs). The skin type was classified according to Fitzpatrick (Fitzpatrick, 1988) and grouped in sun-sensitive (I and II) and less sun-sensitive (III and IV) skin types. The eye color of the patients with melanoma was divided into two groups, blue/green and brown/mixed and hair color into blond/red and brown/black. Histopathological types of melanoma were in the following categories: superficial spreading melanoma (SSM), nodular melanoma (NM), lentigo malignant melanoma (LMM), acro-lentiginous melanoma (ALM), and melanoma in situ, and the Breslow thickness was grouped into <2 and ≥2 mm. Tumor location was registered using a schematic body chart as previously described (Synnerstad et al., 2004) and was classified into sun exposed (D,G,I,K,L,M,N) and rarely/or not sun-exposed (C,H,J) areas.

The control population comprised 792 healthy individuals (50% women, 50% men) with mean age of 54 yrs (±17 yrs, range 20–80 yrs), randomly collected from the population register from the same geographic region as the patients with sporadic MM (Table 1).

Genotyping

Genomic DNA was isolated from blood using the DNA blood maxi kit from Qiagen (Hilden, Germany). The rs35829419 and rs10733113 in NLRP3, rs6502867 and rs12150220 in NLRP1, and rs2043211 in CARD-8 were genotyped using TaqMan® SNP Genotyping assays (C_25648615_10, C_30713847_10, C_11708080_1, C_29222211_20, and C_1600653_10, respectively), according to the manufacturer’s recommendations (Applied Biosystems, Foster City, CA, USA). All analyses were performed in an ABI Prism 7500 Sequence Detection System, using the sds 2.3 software for allelic discrimination (Applied Biosystems).

Statistical analysis

The stata v. 10 (Stata Corp., College Station, TX, USA) statistical package was used to analyze the results. A comparison between cases and controls was performed with logistic regression analysis. Results are expressed as odds ratio (OR) with a corresponding 95% confidence interval (CI). Logistic regression was also used for calculations of the interactions between genes by using linear combinations of the coefficients of the variables. The variables were included one by one, together with the cross-product term. The combination of variables was calculated by adding the coefficients and translated into an OR.

Acknowledgements and financial support

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements and financial support
  9. Conflict of interest
  10. References

We thank Ms. Mona-Lisa Sandh for technical support. Funding for this study was provided by grants from the Medical Research Council of Southeast Sweden (FORSS-8984), the County Council of Östergötland, the Swedish Research Council (PS, K2010-55X-20451-04-03) and the Welander Finsen Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements and financial support
  9. Conflict of interest
  10. References
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