- Email: [email protected]
Germline polymorphisms on RET proto-oncogene involved in medullary thyroid carcinoma in a Druze family
European Journal of Cancer 82 (2017) 149e152
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.ejcancer.com
Letter to the Editor
Germline polymorphisms on RET proto-oncogene involved in medullary thyroid carcinoma in a Druze family Naiel Azzam a,b, Rinat Bar-Shalom a,b, Anwar Saab c, Fuad Fares a,b,* a
Department of Molecular Genetics, Carmel Medical Center, Haifa, Israel Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa 3498838, Israel c Clalit Health Maintenance Organization, Israel b
Received 28 April 2017; accepted 1 June 2017
Dear Editor,
In this following letter, we describe a case in which the subject diagnosed to have medullary thyroid carcinoma (MTC). We suppose that germline line mutations are associated with the disease. The patient is a 45-year-old Druze male born in Israel and living in a village in the Northern part of the state. He is married to a Druze woman with second-grade consanguinity to him, and he is a father of 4 sons and 2 daughters. Following complains about general weakness, fatigue, lack of appetite and depressed mood, he was hospitalised. Comprehensive inquiry, including imaging and laboratory tests, revealed no extraordinary findings. Physical test witnessed swollenness in left breast, after which the biopsy was diagnosed to be lipoma. He was released from hospital without any treatment. Eight months later in a second test, enlargements of the thyroid and of cervical lymph glands were detected. Histological analysis of the biopsy taken
* Corresponding author: Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa 3498838, Israel. Fax: þ972 4 8288763. E-mail addresses: [email protected] (N. Azzam), rbar-shal@ univ.haifa.ac.il (R. Bar-Shalom), [email protected] (A. Saab), [email protected] (F. Fares). http://dx.doi.org/10.1016/j.ejca.2017.06.007 0959-8049/ª 2017 Elsevier Ltd. All rights reserved.
from the thyroid, diagnosed MTC of the gland (Figs. 1 and 2). The patient underwent a radical surgery for removal of thyroid and started a treatment with laevothyroxine and an oncological follow-up. Six months following surgery, CT and an MRI imaging detected positive pathological absorption in cervical glands and metastatic scattering in the liver. Liver functioning was still maintained, but the patient developed diarrhoea and weight decrease with deteriorating clinical condition. Irradiation treatment was carried out, after which the imaging showed no positive clinical findings. The patient was released in a stable condition with a Calcitonin surveillance. The values of Calcitonin, the most sensitive and specific tumour marker for MTC which is secreted by parafollicular cells [1], continued to increase and crossed the 2000 pg/ml value. The metastatic region in the liver still exists with no improvement, despite the treatment. However, the patients’ general condition is stable, while he is normally functioning in daily life. The patient referred to the Laboratory Molecular Genetics at Carmel Medical Center, Haifa, Israel, for genetic analysis. Exons 2e20 of the RET gene were screened for occurrence of mutations. Blood sample was obtained from the patient. Genomic DNA was extracted using high pure PCR template preparation kit (Roche, Mannnheim, Germany). For sequencing analysis of RET
150
N. Azzam et al. / European Journal of Cancer 82 (2017) 149e152
Fig. 1. Sequencing chromatograms of exon 7 at the region of A432A polymorphism and of exon 13 at the region of L769L polymorphism of a Druze MTC patient from Israel and of his daughter, compared with control normal sequence. MTC, medullary thyroid carcinoma.
exons, PCR products were prepared from patient genomic DNA, using specific primers for each exon. The PCR products, following electrophoresis on agarose gels, were purified and then sequenced, using dyeterminator chemistry (ABI, Foster city, CA) and the forward primer for each amplified exon. The results indicated that excluding exons 7 and 13, the DNA sequences of all other exons were found to be normal. However, screening of exon 7 and exon 13 revealed that the patient is heterozygous for G > A at codon 432 (GCG / GCA) and for T > G at codon 769 (CTT / CTG), respectively. Interestingly, these are silent polymorphisms harbouring no change of the amino acid at both codons; Alanine at codon 432 and Leucine at codon 769. Having this observation, all patients’ family members, wife, son and daughter also referred to Molecular Genetics Laboratory at Carmel Medical Center for
genetic screening. The same procedures of blood collection, genomic DNA extraction, PCR amplifications of exons 2e20 of the RET oncogene, as well as DNA sequencing, were performed. Interestingly, the daughter was found to have the same genotype of her father, with regard to the normality of all exons, but heterozygosity in A432A and L769L in exons 7 and 13, respectively. However, the wife and son found to have the same but different genotypes, being normal for all exons screened, but homozygous for L769L polymorphism on exon 13. We assume that the combination of these two polymorphisms A432A and L769L in compound heterozygous pattern is associated with MTC and may increase the risk for its incidence. In fact mutations in the RET proto-oncogene were found to be frequent (40e50%) in MTC. More than 90 mutations in the RET gene had an association with
N. Azzam et al. / European Journal of Cancer 82 (2017) 149e152
151
Fig. 2. Sequencing chromatograms of exon 7 at the region of A432A polymorphism and of exon 13 at the region of L769L polymorphism of the wife and the son of a Druze MTC patient from Israel, compared with control normal sequence. MTC, medullary thyroid carcinoma.
inherited MTC in exons 5, 8, 10 and 12e16 [2,3]. Other germline mutations were described on exons 10, 11 and 15 in MTC patients [4]. However, it was reported previously that A432A polymorphism, has no association with the risk of sporadic MTC [5], neither in the heterozygote nor in the homozygote pattern [6,7]. Nevertheless, a previous study indicated that compound heterozygosity of A432A and S838S polymorphisms is strongly associated with a significant increased risk for differentiated thyroid carcinoma which arises from papillary or follicular cells [8]. Regarding L769L polymorphism, a meta-analysis carried out by Zhang et al., in 2014 came to the conclusion that the L769L polymorphism alone was not associated with increased risk or development of MTC, neither the association with other polymorphisms [9]. Our assumption, therefore, is compatible with the claim of Severskaya et al. [10] that the combination of polymorphic alleles, which are individually not
associated with hereditary MTC, may increase the risk of the disease. Therefore, we think that the daughter has higher risk for MTC than the wife and son and she needs to be under closed surveillance, to achieve early diagnosis of unfortunate possible MTC to occur. The Druze population is considered as an isolated population with a high rate of consanguinity, which was estimated to be as about 47% of all the marriages until recent decades [11]. The most frequent marriages occurred between first cousins. Since the Druze community has a closed gene pool for 1000 year, due to the fact that Druze people merely marry among themselves and due to that inter-marriage has been common at least until recent years, this community demonstrates high consanguinity [12]. This fact is critical for inheriting rare mutations including those involved in cancer development [13]. Since this case looks to be interesting, specifically in the Druze community, there is a need to perform wider screening in families reported to have
152
N. Azzam et al. / European Journal of Cancer 82 (2017) 149e152
MTC. The fact that mixed marriage in this community is almost absent, it may contribute for characterising the genetic background of MTC.
[6]
Conflict of interest statement None declared.
References [1] Ismailov SI, Piulatova NR. Postoperative calcitonin study in medullary thyroid carcinoma. Endocr Relat Cancer 2004;11: 357e63. [2] Margraf RL, Crockett DK, Krautscheid PM, Seamons R, Calderon FR, Wittwer CT, et al. Multiple endocrine neoplasia type 2 RET protooncogene database: repository of MEN2associated RET sequence variation and reference for genotype/phenotype correlations. Hum Mutat 2009;30:548e56. [3] Wei S, LiVolsi VA, Montone KT, Morrissette JJ, Baloch ZW. Detection of molecular alterations in medullary thyroid carcinoma using next-generation sequencing: an institutional experience. Endocr Pathol 2016;27:359e62. [4] Elisei R, Cosci B, Romei C, Bottici V, Renzini G, Molinaro E, et al. Prognostic significance of somatic RET oncogene mutations in sporadic medullary thyroid cancer: a 10-year follow-up study. J Clin Endocrinol Metabolism 2008;93:682e7. [5] Gimm O, Neuberg DS, Marsh DJ, Dahia PL, Hoang-Vu C, Raue F, et al. Over-representation of a germline RET sequence variant in patients with sporadic medullary thyroid carcinoma
[7]
[8]
[9]
[10]
[11]
[12]
[13]
and somatic RET codon 918 mutation. Oncogene 1999;18: 1369e73. Cebrian A, Lesueur F, Martin S, Leyland J, Ahmed S, Luccarini C, et al. Polymorphisms in the initiators of RET (rearranged during transfection) signaling pathway and susceptibility to sporadic medullary thyroid carcinoma. J Clin Endocrinol Metab 2005;90:6268e74. Figlioli G, Landi S, Romei C, Elisei R, Gemignani F. Medullary thyroid carcinoma (MTC) and RET proto-oncogene: mutation spectrum in the familial cases and a meta-analysis of studies on the sporadic form. Mutat Res/Rev Mutat Res 2013;752:36e44. Ho T, Li G, Zhao C, Wei Q, Sturgis EM. RET polymorphisms and haplotypes and risk of differentiated thyroid cancer. Laryngoscope 2005;115:1035e41. Zhang Y, Wang S, Chen X, Huang S, Li J. Quantitative assessment of the association between L769L and S836S polymorphisms at RET gene and medullary thyroid carcinoma risk. Tumor Biol 2014;35:6641e7. Severskaya N, Saenko V, Ilyin A, Chebotareva I, Rumyantsev P, Isaev P, et al. Germline polymorphisms of RET and GFRA1 genes in patients with medullary thyroid carcinoma. Mol Biol (NY) 2006;40:375e84. Vardi-Saliternik R, Friedlander Y, Cohen T. Consanguinity in a population sample of Israeli Muslim Arabs, Christian Arabs and Druze. Ann Hum Biol 2002;29:422e31. Falik-Zaccai TC, Kfir N, Frenkel P, Cohen C, Tanus M, Mandel H, et al. Population screening in a Druze community: the challenge and the reward. Genet Med 2008;10:903e9. Kristiansson K, Naukkarinen J, Peltonen L. Isolated populations and complex disease gene identification. Genome Biol 2008;9:1.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.ejcancer.com
Letter to the Editor
Germline polymorphisms on RET proto-oncogene involved in medullary thyroid carcinoma in a Druze family Naiel Azzam a,b, Rinat Bar-Shalom a,b, Anwar Saab c, Fuad Fares a,b,* a
Department of Molecular Genetics, Carmel Medical Center, Haifa, Israel Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa 3498838, Israel c Clalit Health Maintenance Organization, Israel b
Received 28 April 2017; accepted 1 June 2017
Dear Editor,
In this following letter, we describe a case in which the subject diagnosed to have medullary thyroid carcinoma (MTC). We suppose that germline line mutations are associated with the disease. The patient is a 45-year-old Druze male born in Israel and living in a village in the Northern part of the state. He is married to a Druze woman with second-grade consanguinity to him, and he is a father of 4 sons and 2 daughters. Following complains about general weakness, fatigue, lack of appetite and depressed mood, he was hospitalised. Comprehensive inquiry, including imaging and laboratory tests, revealed no extraordinary findings. Physical test witnessed swollenness in left breast, after which the biopsy was diagnosed to be lipoma. He was released from hospital without any treatment. Eight months later in a second test, enlargements of the thyroid and of cervical lymph glands were detected. Histological analysis of the biopsy taken
* Corresponding author: Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa 3498838, Israel. Fax: þ972 4 8288763. E-mail addresses: [email protected] (N. Azzam), rbar-shal@ univ.haifa.ac.il (R. Bar-Shalom), [email protected] (A. Saab), [email protected] (F. Fares). http://dx.doi.org/10.1016/j.ejca.2017.06.007 0959-8049/ª 2017 Elsevier Ltd. All rights reserved.
from the thyroid, diagnosed MTC of the gland (Figs. 1 and 2). The patient underwent a radical surgery for removal of thyroid and started a treatment with laevothyroxine and an oncological follow-up. Six months following surgery, CT and an MRI imaging detected positive pathological absorption in cervical glands and metastatic scattering in the liver. Liver functioning was still maintained, but the patient developed diarrhoea and weight decrease with deteriorating clinical condition. Irradiation treatment was carried out, after which the imaging showed no positive clinical findings. The patient was released in a stable condition with a Calcitonin surveillance. The values of Calcitonin, the most sensitive and specific tumour marker for MTC which is secreted by parafollicular cells [1], continued to increase and crossed the 2000 pg/ml value. The metastatic region in the liver still exists with no improvement, despite the treatment. However, the patients’ general condition is stable, while he is normally functioning in daily life. The patient referred to the Laboratory Molecular Genetics at Carmel Medical Center, Haifa, Israel, for genetic analysis. Exons 2e20 of the RET gene were screened for occurrence of mutations. Blood sample was obtained from the patient. Genomic DNA was extracted using high pure PCR template preparation kit (Roche, Mannnheim, Germany). For sequencing analysis of RET
150
N. Azzam et al. / European Journal of Cancer 82 (2017) 149e152
Fig. 1. Sequencing chromatograms of exon 7 at the region of A432A polymorphism and of exon 13 at the region of L769L polymorphism of a Druze MTC patient from Israel and of his daughter, compared with control normal sequence. MTC, medullary thyroid carcinoma.
exons, PCR products were prepared from patient genomic DNA, using specific primers for each exon. The PCR products, following electrophoresis on agarose gels, were purified and then sequenced, using dyeterminator chemistry (ABI, Foster city, CA) and the forward primer for each amplified exon. The results indicated that excluding exons 7 and 13, the DNA sequences of all other exons were found to be normal. However, screening of exon 7 and exon 13 revealed that the patient is heterozygous for G > A at codon 432 (GCG / GCA) and for T > G at codon 769 (CTT / CTG), respectively. Interestingly, these are silent polymorphisms harbouring no change of the amino acid at both codons; Alanine at codon 432 and Leucine at codon 769. Having this observation, all patients’ family members, wife, son and daughter also referred to Molecular Genetics Laboratory at Carmel Medical Center for
genetic screening. The same procedures of blood collection, genomic DNA extraction, PCR amplifications of exons 2e20 of the RET oncogene, as well as DNA sequencing, were performed. Interestingly, the daughter was found to have the same genotype of her father, with regard to the normality of all exons, but heterozygosity in A432A and L769L in exons 7 and 13, respectively. However, the wife and son found to have the same but different genotypes, being normal for all exons screened, but homozygous for L769L polymorphism on exon 13. We assume that the combination of these two polymorphisms A432A and L769L in compound heterozygous pattern is associated with MTC and may increase the risk for its incidence. In fact mutations in the RET proto-oncogene were found to be frequent (40e50%) in MTC. More than 90 mutations in the RET gene had an association with
N. Azzam et al. / European Journal of Cancer 82 (2017) 149e152
151
Fig. 2. Sequencing chromatograms of exon 7 at the region of A432A polymorphism and of exon 13 at the region of L769L polymorphism of the wife and the son of a Druze MTC patient from Israel, compared with control normal sequence. MTC, medullary thyroid carcinoma.
inherited MTC in exons 5, 8, 10 and 12e16 [2,3]. Other germline mutations were described on exons 10, 11 and 15 in MTC patients [4]. However, it was reported previously that A432A polymorphism, has no association with the risk of sporadic MTC [5], neither in the heterozygote nor in the homozygote pattern [6,7]. Nevertheless, a previous study indicated that compound heterozygosity of A432A and S838S polymorphisms is strongly associated with a significant increased risk for differentiated thyroid carcinoma which arises from papillary or follicular cells [8]. Regarding L769L polymorphism, a meta-analysis carried out by Zhang et al., in 2014 came to the conclusion that the L769L polymorphism alone was not associated with increased risk or development of MTC, neither the association with other polymorphisms [9]. Our assumption, therefore, is compatible with the claim of Severskaya et al. [10] that the combination of polymorphic alleles, which are individually not
associated with hereditary MTC, may increase the risk of the disease. Therefore, we think that the daughter has higher risk for MTC than the wife and son and she needs to be under closed surveillance, to achieve early diagnosis of unfortunate possible MTC to occur. The Druze population is considered as an isolated population with a high rate of consanguinity, which was estimated to be as about 47% of all the marriages until recent decades [11]. The most frequent marriages occurred between first cousins. Since the Druze community has a closed gene pool for 1000 year, due to the fact that Druze people merely marry among themselves and due to that inter-marriage has been common at least until recent years, this community demonstrates high consanguinity [12]. This fact is critical for inheriting rare mutations including those involved in cancer development [13]. Since this case looks to be interesting, specifically in the Druze community, there is a need to perform wider screening in families reported to have
152
N. Azzam et al. / European Journal of Cancer 82 (2017) 149e152
MTC. The fact that mixed marriage in this community is almost absent, it may contribute for characterising the genetic background of MTC.
[6]
Conflict of interest statement None declared.
References [1] Ismailov SI, Piulatova NR. Postoperative calcitonin study in medullary thyroid carcinoma. Endocr Relat Cancer 2004;11: 357e63. [2] Margraf RL, Crockett DK, Krautscheid PM, Seamons R, Calderon FR, Wittwer CT, et al. Multiple endocrine neoplasia type 2 RET protooncogene database: repository of MEN2associated RET sequence variation and reference for genotype/phenotype correlations. Hum Mutat 2009;30:548e56. [3] Wei S, LiVolsi VA, Montone KT, Morrissette JJ, Baloch ZW. Detection of molecular alterations in medullary thyroid carcinoma using next-generation sequencing: an institutional experience. Endocr Pathol 2016;27:359e62. [4] Elisei R, Cosci B, Romei C, Bottici V, Renzini G, Molinaro E, et al. Prognostic significance of somatic RET oncogene mutations in sporadic medullary thyroid cancer: a 10-year follow-up study. J Clin Endocrinol Metabolism 2008;93:682e7. [5] Gimm O, Neuberg DS, Marsh DJ, Dahia PL, Hoang-Vu C, Raue F, et al. Over-representation of a germline RET sequence variant in patients with sporadic medullary thyroid carcinoma
[7]
[8]
[9]
[10]
[11]
[12]
[13]
and somatic RET codon 918 mutation. Oncogene 1999;18: 1369e73. Cebrian A, Lesueur F, Martin S, Leyland J, Ahmed S, Luccarini C, et al. Polymorphisms in the initiators of RET (rearranged during transfection) signaling pathway and susceptibility to sporadic medullary thyroid carcinoma. J Clin Endocrinol Metab 2005;90:6268e74. Figlioli G, Landi S, Romei C, Elisei R, Gemignani F. Medullary thyroid carcinoma (MTC) and RET proto-oncogene: mutation spectrum in the familial cases and a meta-analysis of studies on the sporadic form. Mutat Res/Rev Mutat Res 2013;752:36e44. Ho T, Li G, Zhao C, Wei Q, Sturgis EM. RET polymorphisms and haplotypes and risk of differentiated thyroid cancer. Laryngoscope 2005;115:1035e41. Zhang Y, Wang S, Chen X, Huang S, Li J. Quantitative assessment of the association between L769L and S836S polymorphisms at RET gene and medullary thyroid carcinoma risk. Tumor Biol 2014;35:6641e7. Severskaya N, Saenko V, Ilyin A, Chebotareva I, Rumyantsev P, Isaev P, et al. Germline polymorphisms of RET and GFRA1 genes in patients with medullary thyroid carcinoma. Mol Biol (NY) 2006;40:375e84. Vardi-Saliternik R, Friedlander Y, Cohen T. Consanguinity in a population sample of Israeli Muslim Arabs, Christian Arabs and Druze. Ann Hum Biol 2002;29:422e31. Falik-Zaccai TC, Kfir N, Frenkel P, Cohen C, Tanus M, Mandel H, et al. Population screening in a Druze community: the challenge and the reward. Genet Med 2008;10:903e9. Kristiansson K, Naukkarinen J, Peltonen L. Isolated populations and complex disease gene identification. Genome Biol 2008;9:1.