- Email: [email protected]
A novel variant in the 3' untranslated region of the CDK4 gene: interference with microRNA target sites and role in increased risk of cutaneous melanoma
Cancer Genetics
-
(2014)
-
LETTER TO THE EDITOR
A novel variant in the 3’ untranslated region of the CDK4 gene: interference with microRNA target sites and role in increased risk of cutaneous melanoma Recent studies confirm the important role of variants within microRNA (miRNA) coding genes and miRNA target binding sites as potential genetic risk factors for the development of cancer (1). Many researchers have focused on the study of germline mutations in miRNA sequences and 3’ untranslated region (UTR) variants (2e5). In a recent study published by our group (6), the presence of germline variants in the miR-34a gene in a cohort of familial melanoma (FAM) patients was investigated to understand the role of the miR-34a gene in cutaneous melanoma (CM) susceptibility. CM (Online Mendelian Inheritance in Man database [OMIM] 155600) is among the most notoriously aggressive and treatment-resistant human cancers, and 5e10% of CM cases show familial clustering (7). To date, two high penetrance genes (CDKN2A and CDK4) have been associated with increased risk of CM, and germline mutations in CDKN2A gene account for susceptibility in 25e40% of FAM patients (8). Among different miRNAs with an important role in pathogenesis and progression of CM (9,10), mir-34a is an oncosuppressor gene, a mediator of p53 (11) with CDK4 and CDK6 as important direct targets (12,13). Our preliminary results led us to hypothesize that miR-34a, although it has a role in late tumorigenesis, does not contribute to inherited susceptibility to CM. In this study, we investigated the presence of variants within the miR-34a binding site located in the 3’-UTRs of the CDK4 and CDK6 genes, with the aim to understand the contribution of mir-34a in CM risk in a cohort of 120 Italian FAM patients. Patients were recruited from the Department of Oncological Dermatology at the Istituti Fisioterapici OspitalierieIstituto di Ricovero e Cura a Carattere Scientifico San Gallicano Institute (Rome, Italy). Most of this study is retrospective and refers to data collected from January 1990 to December 2012. Written informed consent was obtained from each individual. FAM patients were enrolled when at least one first-degree relative had pathologically demonstrated CM or when two or more relatives were affected by CM, regardless of the degree of kinship. Only index cases were considered for this study. Previous molecular analyses of the CDKN2A and CDK4 genes excluded the presence of mutations in coding regions in all patients (14). A total of 200 1 Both authors contributed equally to this work and both are considered first author.
2210-7762/$ - see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cancergen.2014.03.005
unrelated, ethnically matched, healthy individuals were used as controls. Genomic DNA was extracted from blood samples using a Promega Maxwell 16 Blood DNA purification system (Madison, WI). For the molecular study, the 3’UTRs of the CDK4 and CDK6 genes were amplified (primers and conditions available on request) and PCR products were sequenced using the 3500xl Genetic Analyzer (Applied Biosystems, Foster City, CA). Gene nucleotide numbering was based on the reference sequence NM_000075.3 for CDK4 and NM_001145306.1 for CDK6, with the A of the ATG start codon as position þ1. A bioinformatics tool, R/Bioconductor (15) (http://.Rproject.org/), was used to perform a 3’UTR seed-pairing analysis for 2,042 human miRNAs (miRBase 19) (16). The 3’UTR sequences were annotated for all occurrences of short sites complementary to the seed region of a human miRNA. Assayed seed regions were as follows: nucleotides 2e7 in the mature miRNA sequence (6mer-seed); nucleotides 2e8 in the mature miRNA sequence (7mer-seed); and nucleotides 1e8 in the mature miRNA sequence (8merseed). We then selected those seed-pairing site occurrences differing between wild-type and mutated 3’UTRs of CDK4 and CDK6 due to sequence variation introduced by the single nucleotide polymorphism (SNP). Molecular analysis discovered no variant in the 3’UTR of CDK6 and one variant within the 3’UTR of CDK4 (c.*209G>A) in one 47-year-old patient. The patient’s affected sister (one CM at age 39) was not available for mutational screening. The nucleotide substitution was novel and was found in 1 of 200 healthy participants (0.5%). Bioinformatic analysis revealed that the CDK4 variant creates binding sites for three novel microRNAs (hsa-miR-1292-5p, hsa-miR-4471, hsa-miR4803), whereas biding sites for three other microRNAs are deleted (hsa-miR-3144-3p, hsa-miR-1277-5p, hsa-miR-67165p). All miRNAs are marked by low evolutionary conservation and largely unknown function. In conclusion, we found a novel variant (c.*209G>A) in the 3’UTR of the CDK4 gene. Segregation analysis within the family was not possible; however, its presence in healthy control participants and bioinformatic results suggest the absence of a strict correlation between the SNPs and onset of CM in our patient. Although our results support previous evidence defining variants able to destroy or create new miRNA binding sites as rare and unlikely to be functionally important (17,18), further studies are needed to evaluate the role of 3’UTR variants and their effect on mRNA expression and translation. Moreover, these data complete our previous work in suggesting that miR-34a does not contribute to the inherited
2 susceptibility to CM. Finally, we hypothesize that germline mutations within the 3’UTR of the CDK4 and CDK6 genes do not have a pivotal role in heritable predisposition to melanoma in Italian patients.
Acknowledgments The authors thank the patients and their families for collaboration in this study. This work was financially supported by grants from Banca d’Italia.
References 1. Skeeles LE, Fleming JL, Mahler KL, et al. The impact of 3’UTR variants on differential expression of candidate cancer susceptibility genes. PLoS One 2013;8:e58609. 2. Xiong F, Wu C, Chang J, et al. Genetic variation in an miRNA1827 binding site in MYCL1 alters susceptibility to small-cell lung cancer. Cancer Res 2011;71:5175e5181. 3. Jacobs G, Hellmig S, Huse K, et al. Polymorphisms in the 3’untranslated region of the CDH1 gene are a risk factor for primary gastric diffuse large B-cell lymphoma. Haematologica 2011;96:987e995. 4. Wang C, Guo Z, Wu C, et al. A polymorphism at the miR-502 binding site in the 3’ untranslated region of the SET8 gene is associated with the risk of epithelial ovarian cancer. Cancer Genet 2012;205:373e376. 5. Chin LJ, Ratner E, Leng S, et al. A SNP in a let-7 microRNA complementary site in the KRAS 3’ untranslated region increases non-small cell lung cancer risk. Cancer Res 2008;68: 8535e8540. 6. Cozzolino AM, Pedace L, Castori M, et al. Analysis of the miR34a locus in 62 patients with familial cutaneous melanoma negative for CDKN2A/CDK4 screening. Fam Cancer 2012;11: 201e208. 7. Gandini S, Sera F, Cattaruzza MS, et al. Meta-analysis of risk factors for cutaneous melanoma: III. Family history, actinic damage and phenotypic factors. Eur J Cancer 2005;41:2040e2059. 8. Ward KA, Lazovich D, Hordinsky MK. Germline melanoma susceptibility and prognostic genes: a review of the literature. J Am Acad Dermatol 2012;67:1055e1067. 9. Yamazaki H, Chijiwa T, Inoue Y, et al. Overexpression of the miR34 family suppresses invasive growth of malignant melanoma with the wild-type p53 gene. Exp Ther Med 2012;3:793e796. 10. Molnar V, Tamasi V, Bakos B, et al. Changes in miRNA expression in solid tumors: an miRNA profiling in melanomas. Semin Cancer Biol 2008;18:111e122. 11. Hermeking H. p53 enters the microRNA world. Cancer Cell 2007;12:414e418. 12. Sun F, Fu H, Liu Q, et al. Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest. FEBS Lett 2008;582: 1564e1568.
Letter to the Editor 13. He L, He X, Lim LP, et al. A microRNA component of the p53 tumour suppressor network. Nature 2007;447:1130e1134. 14. Pedace L, De Simone P, Castori M, et al. Clinical features predicting identification of CDKN2A mutations in Italian patients with familial cutaneous melanoma. Cancer Epidemiol 2011;35: e116ee120. 15. Gentleman RC, Carey VJ, Bates DM, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004;5:R80. 16. Griffiths-Jones S, Saini HK, van Dongen S, et al. miRBase: tools for microRNA genomics. Nucleic Acids Res 2008;36: D154eD158. 17. Iuliano R, Vismara MF, Dattilo V, et al. The role of microRNAs in cancer susceptibility. Biomed Res Int 2013;2013:591931. 18. Saunders MA, Liang H, Li WH. Human polymorphism at microRNAs and microRNA target sites. Proc Natl Acad Sci USA 2007;104:3300e3305.
Lucia Pedace*,1 Angela M. Cozzolino1 Luana Barboni Carmelilia De Bernardo Paola Grammatico Medical Genetics Department of Molecular Medicine Sapienza University San Camillo-Forlanini Hospital Rome, Italy *Corresponding author. E-mail address: [email protected] (L. Pedace) Paola De Simone Pierluigi Buccini Angela Ferrari Caterina Catricala Department of Dermatologic Oncology San Gallicano Dermatologic InstituteeIstituto di Ricovero e Cura a Carattere Scientifico Rome, Italy Teresa Colombo Institute for Computing Applications Mauro Picone National Research Council Rome, Italy Pietro Donati Dermatopathology Unit San Gallicano IRCCS Rome, Italy Aldo Morrone Healthcare Administration Dermatology San Camillo-Forlanini Hospital Rome, Italy
-
(2014)
-
LETTER TO THE EDITOR
A novel variant in the 3’ untranslated region of the CDK4 gene: interference with microRNA target sites and role in increased risk of cutaneous melanoma Recent studies confirm the important role of variants within microRNA (miRNA) coding genes and miRNA target binding sites as potential genetic risk factors for the development of cancer (1). Many researchers have focused on the study of germline mutations in miRNA sequences and 3’ untranslated region (UTR) variants (2e5). In a recent study published by our group (6), the presence of germline variants in the miR-34a gene in a cohort of familial melanoma (FAM) patients was investigated to understand the role of the miR-34a gene in cutaneous melanoma (CM) susceptibility. CM (Online Mendelian Inheritance in Man database [OMIM] 155600) is among the most notoriously aggressive and treatment-resistant human cancers, and 5e10% of CM cases show familial clustering (7). To date, two high penetrance genes (CDKN2A and CDK4) have been associated with increased risk of CM, and germline mutations in CDKN2A gene account for susceptibility in 25e40% of FAM patients (8). Among different miRNAs with an important role in pathogenesis and progression of CM (9,10), mir-34a is an oncosuppressor gene, a mediator of p53 (11) with CDK4 and CDK6 as important direct targets (12,13). Our preliminary results led us to hypothesize that miR-34a, although it has a role in late tumorigenesis, does not contribute to inherited susceptibility to CM. In this study, we investigated the presence of variants within the miR-34a binding site located in the 3’-UTRs of the CDK4 and CDK6 genes, with the aim to understand the contribution of mir-34a in CM risk in a cohort of 120 Italian FAM patients. Patients were recruited from the Department of Oncological Dermatology at the Istituti Fisioterapici OspitalierieIstituto di Ricovero e Cura a Carattere Scientifico San Gallicano Institute (Rome, Italy). Most of this study is retrospective and refers to data collected from January 1990 to December 2012. Written informed consent was obtained from each individual. FAM patients were enrolled when at least one first-degree relative had pathologically demonstrated CM or when two or more relatives were affected by CM, regardless of the degree of kinship. Only index cases were considered for this study. Previous molecular analyses of the CDKN2A and CDK4 genes excluded the presence of mutations in coding regions in all patients (14). A total of 200 1 Both authors contributed equally to this work and both are considered first author.
2210-7762/$ - see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cancergen.2014.03.005
unrelated, ethnically matched, healthy individuals were used as controls. Genomic DNA was extracted from blood samples using a Promega Maxwell 16 Blood DNA purification system (Madison, WI). For the molecular study, the 3’UTRs of the CDK4 and CDK6 genes were amplified (primers and conditions available on request) and PCR products were sequenced using the 3500xl Genetic Analyzer (Applied Biosystems, Foster City, CA). Gene nucleotide numbering was based on the reference sequence NM_000075.3 for CDK4 and NM_001145306.1 for CDK6, with the A of the ATG start codon as position þ1. A bioinformatics tool, R/Bioconductor (15) (http://.Rproject.org/), was used to perform a 3’UTR seed-pairing analysis for 2,042 human miRNAs (miRBase 19) (16). The 3’UTR sequences were annotated for all occurrences of short sites complementary to the seed region of a human miRNA. Assayed seed regions were as follows: nucleotides 2e7 in the mature miRNA sequence (6mer-seed); nucleotides 2e8 in the mature miRNA sequence (7mer-seed); and nucleotides 1e8 in the mature miRNA sequence (8merseed). We then selected those seed-pairing site occurrences differing between wild-type and mutated 3’UTRs of CDK4 and CDK6 due to sequence variation introduced by the single nucleotide polymorphism (SNP). Molecular analysis discovered no variant in the 3’UTR of CDK6 and one variant within the 3’UTR of CDK4 (c.*209G>A) in one 47-year-old patient. The patient’s affected sister (one CM at age 39) was not available for mutational screening. The nucleotide substitution was novel and was found in 1 of 200 healthy participants (0.5%). Bioinformatic analysis revealed that the CDK4 variant creates binding sites for three novel microRNAs (hsa-miR-1292-5p, hsa-miR-4471, hsa-miR4803), whereas biding sites for three other microRNAs are deleted (hsa-miR-3144-3p, hsa-miR-1277-5p, hsa-miR-67165p). All miRNAs are marked by low evolutionary conservation and largely unknown function. In conclusion, we found a novel variant (c.*209G>A) in the 3’UTR of the CDK4 gene. Segregation analysis within the family was not possible; however, its presence in healthy control participants and bioinformatic results suggest the absence of a strict correlation between the SNPs and onset of CM in our patient. Although our results support previous evidence defining variants able to destroy or create new miRNA binding sites as rare and unlikely to be functionally important (17,18), further studies are needed to evaluate the role of 3’UTR variants and their effect on mRNA expression and translation. Moreover, these data complete our previous work in suggesting that miR-34a does not contribute to the inherited
2 susceptibility to CM. Finally, we hypothesize that germline mutations within the 3’UTR of the CDK4 and CDK6 genes do not have a pivotal role in heritable predisposition to melanoma in Italian patients.
Acknowledgments The authors thank the patients and their families for collaboration in this study. This work was financially supported by grants from Banca d’Italia.
References 1. Skeeles LE, Fleming JL, Mahler KL, et al. The impact of 3’UTR variants on differential expression of candidate cancer susceptibility genes. PLoS One 2013;8:e58609. 2. Xiong F, Wu C, Chang J, et al. Genetic variation in an miRNA1827 binding site in MYCL1 alters susceptibility to small-cell lung cancer. Cancer Res 2011;71:5175e5181. 3. Jacobs G, Hellmig S, Huse K, et al. Polymorphisms in the 3’untranslated region of the CDH1 gene are a risk factor for primary gastric diffuse large B-cell lymphoma. Haematologica 2011;96:987e995. 4. Wang C, Guo Z, Wu C, et al. A polymorphism at the miR-502 binding site in the 3’ untranslated region of the SET8 gene is associated with the risk of epithelial ovarian cancer. Cancer Genet 2012;205:373e376. 5. Chin LJ, Ratner E, Leng S, et al. A SNP in a let-7 microRNA complementary site in the KRAS 3’ untranslated region increases non-small cell lung cancer risk. Cancer Res 2008;68: 8535e8540. 6. Cozzolino AM, Pedace L, Castori M, et al. Analysis of the miR34a locus in 62 patients with familial cutaneous melanoma negative for CDKN2A/CDK4 screening. Fam Cancer 2012;11: 201e208. 7. Gandini S, Sera F, Cattaruzza MS, et al. Meta-analysis of risk factors for cutaneous melanoma: III. Family history, actinic damage and phenotypic factors. Eur J Cancer 2005;41:2040e2059. 8. Ward KA, Lazovich D, Hordinsky MK. Germline melanoma susceptibility and prognostic genes: a review of the literature. J Am Acad Dermatol 2012;67:1055e1067. 9. Yamazaki H, Chijiwa T, Inoue Y, et al. Overexpression of the miR34 family suppresses invasive growth of malignant melanoma with the wild-type p53 gene. Exp Ther Med 2012;3:793e796. 10. Molnar V, Tamasi V, Bakos B, et al. Changes in miRNA expression in solid tumors: an miRNA profiling in melanomas. Semin Cancer Biol 2008;18:111e122. 11. Hermeking H. p53 enters the microRNA world. Cancer Cell 2007;12:414e418. 12. Sun F, Fu H, Liu Q, et al. Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest. FEBS Lett 2008;582: 1564e1568.
Letter to the Editor 13. He L, He X, Lim LP, et al. A microRNA component of the p53 tumour suppressor network. Nature 2007;447:1130e1134. 14. Pedace L, De Simone P, Castori M, et al. Clinical features predicting identification of CDKN2A mutations in Italian patients with familial cutaneous melanoma. Cancer Epidemiol 2011;35: e116ee120. 15. Gentleman RC, Carey VJ, Bates DM, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004;5:R80. 16. Griffiths-Jones S, Saini HK, van Dongen S, et al. miRBase: tools for microRNA genomics. Nucleic Acids Res 2008;36: D154eD158. 17. Iuliano R, Vismara MF, Dattilo V, et al. The role of microRNAs in cancer susceptibility. Biomed Res Int 2013;2013:591931. 18. Saunders MA, Liang H, Li WH. Human polymorphism at microRNAs and microRNA target sites. Proc Natl Acad Sci USA 2007;104:3300e3305.
Lucia Pedace*,1 Angela M. Cozzolino1 Luana Barboni Carmelilia De Bernardo Paola Grammatico Medical Genetics Department of Molecular Medicine Sapienza University San Camillo-Forlanini Hospital Rome, Italy *Corresponding author. E-mail address: [email protected] (L. Pedace) Paola De Simone Pierluigi Buccini Angela Ferrari Caterina Catricala Department of Dermatologic Oncology San Gallicano Dermatologic InstituteeIstituto di Ricovero e Cura a Carattere Scientifico Rome, Italy Teresa Colombo Institute for Computing Applications Mauro Picone National Research Council Rome, Italy Pietro Donati Dermatopathology Unit San Gallicano IRCCS Rome, Italy Aldo Morrone Healthcare Administration Dermatology San Camillo-Forlanini Hospital Rome, Italy