DOI: 10.1111/exd.12778
Letter to the Editor
www.wileyonlinelibrary.com/journal/EXD
Association of microRNA 146a polymorphism rs2910164 and the risk of melanoma in an Italian population Macarena Gomez-Lira1, Silvia Ferronato1, Elisa Orlandi1, Anna Dal Molin1, Giovanni Malerba1, Simona Frigerio2, Monica Rodolfo2,* and Maria Grazia Romanelli1,* 1 Department of Life and Reproduction Sciences, Section of Biology and Genetics, University of Verona, Verona, Italy; 2Unit of Immunotherapy, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Correspondence: Macarena Gomez-Lira, Department of Life and Reproduction Sciences, Section of Biology and Genetics, University of Verona, Strada Le Grazie, 8. 37134 Verona, Italy, Tel.: +39 0458027674, Fax: 0458027180, e-mail: [email protected] *Rodolfo M and Romanelli MG, are both co-senior authors
Key words: melanoma risk – miR-146a
Accepted for publication 24 April 2015
Background and questions addressed
higher in melanoma cases than in controls (8). The limitation of their study lies in the small number of patients (50 patients) examined, and thus, the association of this SNP needs to be confirmed in a larger group of individuals. In this study, we analysed rs2910164G>C in DNA samples from 224 sporadic melanoma patients and 264 controls and observed that allele rs2910164C may represent a factor risk with an additive effect on melanoma predisposition in males.
The genetics of melanoma is complex (1). A large number of genes are being studied for their role in melanoma, including hereditary and acquired genetic alterations (1,2). Mutations in the high-risk genes CDKN2A, CDK4, BAP1, POT1 and in two low-tomoderate risk susceptibility genes (MC1R, MITF) account for familial melanoma cases, which represent 10% of all cases (2,s1,s2, online supporting information). A combination of genetic and environmental modifiers contributes to disease development and progression in sporadic melanoma (3,4,s3,s4, online supporting information). Identification of novel genes predisposing to melanoma will increase our understanding of the genetic pathogenesis of the disease and contribute to the development of novel prevention and treatment of melanoma. Epigenetics have also been recognized to play an important role in melanoma development and progression; changes in methylation patterns of genomic DNA, histone modifications and microRNA expression can alter important cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune evasion (s5, online supporting information). MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules, that function as regulators of gene translation, and can contribute to human cancer predisposition by regulating the expression of proto-oncogenes or tumor suppressor genes (5). Common genetic polymorphisms may alter the processing of miRNA and be associated with the predisposition to different cancers by influencing important cellular pathways (s6, online supporting information). The pre-miR-146a single nucleotide polymorphism (SNP) (G>C rs2910164) alters the expression of mature miR-146a and is associated with an increased risk of several types of cancers. This polymorphism is located in the seed region of pre-miR-146a-3p sequence and leads to the formation of two different isoforms that regulate distinct sets of genes (6). In addition, an enrichment of pre-miR-146a/G during melanoma progression appears to enhance its oncogenic activity (7). Recently, polymorphism rs2910164G>C has been investigated in relation to melanoma predisposition in a Japanese population. The authors showed that CG genotype frequency was significantly
794
Experimental design Patients presenting sporadic melanoma and control individuals were enrolled in the study. Subjects were of Italian or European Caucasians. Patients younger than 20 or older than 66 years, familial or multiple melanoma cases and cases presenting other neoplasias were excluded. Cases were patients hospitalized for surgical treatment of melanoma at the Melanoma and Sarcoma Surgery Unit of the Istituto Nazionale Tumori, Milan, between May 2006 and June 2007. Controls were healthy donors from the Immunohematology and Transfusion Medicine Department, Fondazione IRCCS Istituto Nazionale Tumori. The study was approved by the local Institutional Review Board, and informed consent was obtained from all subjects. The study group is described in Table 1. Genomic DNA was extracted from whole blood using the QIAamp Blood Kit (Qiagen). Polymorphism rs2910164G>C was genotyped as described (s7, online supporting information). The association between genotypes and melanoma was assessed using a logistic regression coding the genotypes according to the count (0,1, or 2) of the risk allele.
Results and discussion The frequency of miR-146a rs2910164G allele in the controls was 0.76, similar to the reported allele frequencies in Caucasians (http://hapmap.ncbi.nlm.nih.gov/) as expected. Genotypes were distributed according to Hardy–Weinberg equilibrium. rs2910164 SNP genotype distribution and allele frequency were different between cases and controls, indicating an association of the polymorphism with the melanoma risk. Logistic regression calculation showed an allelic dosage effect of allele rs2910164C (P = 0.02; OR = 1.09; CI:1.01–1.18, Table 2). These results are reinforced by a recent study showing that miR-146a is regulated
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 790–802
Letter to the Editor
Table 1. Demographic characteristics of the study subjects
Variable Male Female Age, y (mean) Clinical stage 1 I II III IV Clark level 2 I II III IV V 1 2
Table 2. Genotype distribution and allele frequency of polymorphism rs2910164G>C in patients with melanoma and control individuals
Melanoma patients N = 224
Healthy controls N = 264
P
rs2910164G>C
Cases
Controls
OR (CI)
115 109 49 12
127 137 47 15
ns ns 0.05
43 38 96 41
– – – –
– – – –
107 100 17 107/117 207/17 314 134 224
149 105 10 149/115 254/10 403 125 264
1 1.33 2.37 1.42 2.09 1 1.38 1.09
0 6 30 116 14
– – – – –
– – – – –
GG GC CC GG/GC+CC GG+GC/CC Allele G Allele C Log additive 0,1,2 Male GG GC CC GG/GC+CC GG+GC/CC Allele G Allele C Log additive 0,1,2 Female GG GC CC GG/GC+CC GG+GC/CC Allele G Allele C Log additive 0,1,2
44 60 11 44/71 104/11 148 82 115
69 52 6 69/58 121/6 190 64 127
1 1.81 2.88 1.92 2.13 1 1.64 1.14
63 40 6 63/46 103/6 166 52 109
82 53 4 82/57 135/4 217 61 137
1 0.98 1.95 1.05 1.97 1 1.11 1.02
Data for 6 patients are missing. Data for 58 patients are missing.
by BRAF and NRAS and plays an essential role in the initiation and progression of melanoma (7). Stratifying by gender, we observed that the association of the variant with melanoma was restricted to males [P < 0.01; OR = 1.14 (1.04–1.27) (Table 2)]. A gender-specific association of this polymorphism with carcinogenesis has also been observed in several other studies (s8–s10, online supporting information). The way in which gender can influence the effects of a polymorphism is well exemplified in the review written by Bond et al. (s11, online supporting information) where a polymorphism in the MDM2 gene can interfere with sex hormones and alter the p53 response pathway. Ethnicity seems to play an important role in the influence of miR polymorphisms and cancer. For instance, a recent large metaanalysis shows that this SNP is associated with the risk of digestive system cancers in Asians, but not in Caucasians (s12, online supporting information), and with breast cancer risk in the Asians (9), but not in Caucasian population (s13, online supporting information). MiR-146a rs2910164 has been associated with papillary thyroid carcinoma, hepatocellular carcinoma (HCC) in males, prostate cancer, bladder cancer and colorectal cancer in the Chinese population, and with gastric cancer in the Asiatic population. The C allele or CC genotype of miR-146a polymorphism is also associated with increased risk of earlier age of onset of familial breast and ovarian cancers, and gastric cancer (s7, online supporting information). Interestingly, Jazdzewski and colleagues reported that the GC heterozygous genotype is associated with an increased risk of papillary thyroid carcinoma, but both homozygous genotypes are protective (6). The fact that miR-146a rs2910164 has been reported associated only with some types of cancers (s5, online supporting information)
P value
(0.90–1.95) (0.98–5.81) (0.98–2.06) (0.88–5.01
0.06 0.06 0.07
(1.02–1.85) (1.01–1.18)
0.03 0.02
(1.03–3.19) (0.90–9.51) (1.11–3.32) (0.70–6.74)
0.03 0.05 0.14
(1.09–2.48) (1.04–1.27)
0.012 0.008
(0.56–1.72) (0.46–8.67) (0.61–1.80) (0.12–2.10)
0.58 0.85 0.30
(0.72–1.74) (0.92–1.14)
0.61 0.66
Bold numbers correspond to significant p values (
Letter to the Editor
www.wileyonlinelibrary.com/journal/EXD
Association of microRNA 146a polymorphism rs2910164 and the risk of melanoma in an Italian population Macarena Gomez-Lira1, Silvia Ferronato1, Elisa Orlandi1, Anna Dal Molin1, Giovanni Malerba1, Simona Frigerio2, Monica Rodolfo2,* and Maria Grazia Romanelli1,* 1 Department of Life and Reproduction Sciences, Section of Biology and Genetics, University of Verona, Verona, Italy; 2Unit of Immunotherapy, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Correspondence: Macarena Gomez-Lira, Department of Life and Reproduction Sciences, Section of Biology and Genetics, University of Verona, Strada Le Grazie, 8. 37134 Verona, Italy, Tel.: +39 0458027674, Fax: 0458027180, e-mail: [email protected] *Rodolfo M and Romanelli MG, are both co-senior authors
Key words: melanoma risk – miR-146a
Accepted for publication 24 April 2015
Background and questions addressed
higher in melanoma cases than in controls (8). The limitation of their study lies in the small number of patients (50 patients) examined, and thus, the association of this SNP needs to be confirmed in a larger group of individuals. In this study, we analysed rs2910164G>C in DNA samples from 224 sporadic melanoma patients and 264 controls and observed that allele rs2910164C may represent a factor risk with an additive effect on melanoma predisposition in males.
The genetics of melanoma is complex (1). A large number of genes are being studied for their role in melanoma, including hereditary and acquired genetic alterations (1,2). Mutations in the high-risk genes CDKN2A, CDK4, BAP1, POT1 and in two low-tomoderate risk susceptibility genes (MC1R, MITF) account for familial melanoma cases, which represent 10% of all cases (2,s1,s2, online supporting information). A combination of genetic and environmental modifiers contributes to disease development and progression in sporadic melanoma (3,4,s3,s4, online supporting information). Identification of novel genes predisposing to melanoma will increase our understanding of the genetic pathogenesis of the disease and contribute to the development of novel prevention and treatment of melanoma. Epigenetics have also been recognized to play an important role in melanoma development and progression; changes in methylation patterns of genomic DNA, histone modifications and microRNA expression can alter important cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune evasion (s5, online supporting information). MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules, that function as regulators of gene translation, and can contribute to human cancer predisposition by regulating the expression of proto-oncogenes or tumor suppressor genes (5). Common genetic polymorphisms may alter the processing of miRNA and be associated with the predisposition to different cancers by influencing important cellular pathways (s6, online supporting information). The pre-miR-146a single nucleotide polymorphism (SNP) (G>C rs2910164) alters the expression of mature miR-146a and is associated with an increased risk of several types of cancers. This polymorphism is located in the seed region of pre-miR-146a-3p sequence and leads to the formation of two different isoforms that regulate distinct sets of genes (6). In addition, an enrichment of pre-miR-146a/G during melanoma progression appears to enhance its oncogenic activity (7). Recently, polymorphism rs2910164G>C has been investigated in relation to melanoma predisposition in a Japanese population. The authors showed that CG genotype frequency was significantly
794
Experimental design Patients presenting sporadic melanoma and control individuals were enrolled in the study. Subjects were of Italian or European Caucasians. Patients younger than 20 or older than 66 years, familial or multiple melanoma cases and cases presenting other neoplasias were excluded. Cases were patients hospitalized for surgical treatment of melanoma at the Melanoma and Sarcoma Surgery Unit of the Istituto Nazionale Tumori, Milan, between May 2006 and June 2007. Controls were healthy donors from the Immunohematology and Transfusion Medicine Department, Fondazione IRCCS Istituto Nazionale Tumori. The study was approved by the local Institutional Review Board, and informed consent was obtained from all subjects. The study group is described in Table 1. Genomic DNA was extracted from whole blood using the QIAamp Blood Kit (Qiagen). Polymorphism rs2910164G>C was genotyped as described (s7, online supporting information). The association between genotypes and melanoma was assessed using a logistic regression coding the genotypes according to the count (0,1, or 2) of the risk allele.
Results and discussion The frequency of miR-146a rs2910164G allele in the controls was 0.76, similar to the reported allele frequencies in Caucasians (http://hapmap.ncbi.nlm.nih.gov/) as expected. Genotypes were distributed according to Hardy–Weinberg equilibrium. rs2910164 SNP genotype distribution and allele frequency were different between cases and controls, indicating an association of the polymorphism with the melanoma risk. Logistic regression calculation showed an allelic dosage effect of allele rs2910164C (P = 0.02; OR = 1.09; CI:1.01–1.18, Table 2). These results are reinforced by a recent study showing that miR-146a is regulated
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 790–802
Letter to the Editor
Table 1. Demographic characteristics of the study subjects
Variable Male Female Age, y (mean) Clinical stage 1 I II III IV Clark level 2 I II III IV V 1 2
Table 2. Genotype distribution and allele frequency of polymorphism rs2910164G>C in patients with melanoma and control individuals
Melanoma patients N = 224
Healthy controls N = 264
P
rs2910164G>C
Cases
Controls
OR (CI)
115 109 49 12
127 137 47 15
ns ns 0.05
43 38 96 41
– – – –
– – – –
107 100 17 107/117 207/17 314 134 224
149 105 10 149/115 254/10 403 125 264
1 1.33 2.37 1.42 2.09 1 1.38 1.09
0 6 30 116 14
– – – – –
– – – – –
GG GC CC GG/GC+CC GG+GC/CC Allele G Allele C Log additive 0,1,2 Male GG GC CC GG/GC+CC GG+GC/CC Allele G Allele C Log additive 0,1,2 Female GG GC CC GG/GC+CC GG+GC/CC Allele G Allele C Log additive 0,1,2
44 60 11 44/71 104/11 148 82 115
69 52 6 69/58 121/6 190 64 127
1 1.81 2.88 1.92 2.13 1 1.64 1.14
63 40 6 63/46 103/6 166 52 109
82 53 4 82/57 135/4 217 61 137
1 0.98 1.95 1.05 1.97 1 1.11 1.02
Data for 6 patients are missing. Data for 58 patients are missing.
by BRAF and NRAS and plays an essential role in the initiation and progression of melanoma (7). Stratifying by gender, we observed that the association of the variant with melanoma was restricted to males [P < 0.01; OR = 1.14 (1.04–1.27) (Table 2)]. A gender-specific association of this polymorphism with carcinogenesis has also been observed in several other studies (s8–s10, online supporting information). The way in which gender can influence the effects of a polymorphism is well exemplified in the review written by Bond et al. (s11, online supporting information) where a polymorphism in the MDM2 gene can interfere with sex hormones and alter the p53 response pathway. Ethnicity seems to play an important role in the influence of miR polymorphisms and cancer. For instance, a recent large metaanalysis shows that this SNP is associated with the risk of digestive system cancers in Asians, but not in Caucasians (s12, online supporting information), and with breast cancer risk in the Asians (9), but not in Caucasian population (s13, online supporting information). MiR-146a rs2910164 has been associated with papillary thyroid carcinoma, hepatocellular carcinoma (HCC) in males, prostate cancer, bladder cancer and colorectal cancer in the Chinese population, and with gastric cancer in the Asiatic population. The C allele or CC genotype of miR-146a polymorphism is also associated with increased risk of earlier age of onset of familial breast and ovarian cancers, and gastric cancer (s7, online supporting information). Interestingly, Jazdzewski and colleagues reported that the GC heterozygous genotype is associated with an increased risk of papillary thyroid carcinoma, but both homozygous genotypes are protective (6). The fact that miR-146a rs2910164 has been reported associated only with some types of cancers (s5, online supporting information)
P value
(0.90–1.95) (0.98–5.81) (0.98–2.06) (0.88–5.01
0.06 0.06 0.07
(1.02–1.85) (1.01–1.18)
0.03 0.02
(1.03–3.19) (0.90–9.51) (1.11–3.32) (0.70–6.74)
0.03 0.05 0.14
(1.09–2.48) (1.04–1.27)
0.012 0.008
(0.56–1.72) (0.46–8.67) (0.61–1.80) (0.12–2.10)
0.58 0.85 0.30
(0.72–1.74) (0.92–1.14)
0.61 0.66
Bold numbers correspond to significant p values (
