Hippokratia 2016, 20(3):187-191

Rovcanin B1, Damjanovic S2, Zivaljevic V1, Diklic A1, Jovanovic M1, Paunovic I1
1Center for Endocrine Surgery , 2Institute of Endocrinology, Diabetes and Metabolic Disease, Clinical Center of Serbia, Faculty of Medicine, University of Belgrade, Belgrade, Serbia.

Background: Medullary thyroid carcinoma (MTC) is a type of thyroid neoplasm which originates from parafollicular cells, and it is commonly diagnosed by calcitonin screening. Besides the sporadic form, the heritable form of MTC is characterized by constitutive activation of the RET (REarranged during Transfection) proto-oncogene caused by different mutations.
Method: We collected data regarding RET genetic screening performed in the Center for Endocrine Surgery in Belgrade during a 20-year-period. The study group included 249 MTC patients who were genetically tested for RET mutations by Sanger’s sequencing method.
Results: Genetic screening of the study population revealed nine different mutations of the RET gene in 42 carriers. The most common mutation was C634F, and it has been detected in 31 % (13/42) of individuals, while C618R, L790F, and S904S were present in only 2 % (1/42) each in the study group. Detected mutations were unequally distributed in different RET gene exons. Among MTC patients, 67 % (28/42) had mutation harbored in exon 11, while the rarest mutation was located in exons 10 and 15, each present in only 2 % (1/42) of patients.
Conclusions: The RET gene mutation profile has a unique distribution in this study population when compared with the other European populations. The mutations in codon 634 are most common; therefore the cost-reducing genetic screening should primarily target this codon, and if the negative outcome appears, then other codons should be examined in the order that depends on their occurrence. Hippokratia 2016, 20(3): 187-191.

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Key words: Genetic testing,medullary thyroid carcinoma, mutations, RET proto-oncogene, retrospective study

Corresponding author: Branislav Rovcanin, MD, PhD, Specialist of genetics, Center for Endocrine Surgery, Clinical Center of Serbia, Faculty of Medicine, University of Belgrade, Koste Todorovica 8, 11000, Belgrade, Serbia, tel: +381113663261, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.



Medullary thyroid carcinoma (MTC) is a type of thyroid neoplasm which originates from the parafollicular C-cells, which produce the hormone calcitonin. MTC accounts for about 5 % to 10 % of all thyroid carcinomas, and it is responsible for 8 % to 15 % of all thyroid cancer-related deaths1. The early diagnosis and the preoperative management of MTC are most frequently established by calcitonin screening2. On the molecular level, the key pathogenetic event of MTC initiation is constitutive activation of RET proto-oncogene. The RET (REarranged during Transfection) gene consists of 21 exons, and it encodes a 170 kDa tyrosine receptor protein. This protein has a transmembrane domain, an extracellular domain with a ligand binding site, and an intracellular tyrosine kinase (TK) domain3. The product of RET gene plays an important role in the cell signaling in the neural tissue, especially at the early stages of embryonic development. Mutations in the RET gene lead to abnormalities in cell proliferation and differentiation in neural crest-derived tissues, such as the C-cells and the adrenal medulla cells4. Medullary thyroid cancer appears in two distinct forms. The rare familial form is genetically determined, while the sporadic form is more common, but its etiology has not been clearly defined so far5. Germline mutations of RET gene lead to the development of heritable forms of MTC, while somatic mutations are discovered in a major quantity of sporadic MTC6. Mutations which activate TK receptor by ligand-independent dimerization and cross-phosphorylation are located in the codons 609, 611, 618, 620, 630, and 634 (exon 11) which encode the extracellular domain of the protein. The intracellular domain is affected when mutations are harbored in codons 768, 790, 791 (exon 13), 804 (exon 14), 883 and 891 (exon 15), and 918 (exon 16). These mutations create abnormalities in protein structure which interfere with the intracellular ATP binding for the TK receptor7. About 25 % of MTC cases represent a familial autosomal dominant form of the disease. The inherited forms appear as multiple endocrine neoplasia II (MENII) syndromes, which are characterized by the co-existence of MTC and pheochromocytoma (PHEO) together with primary hyperparathyroidism (PHP) in the case of MENIIa. In the case of MENIIb syndrome, mucocutaneous neuroma and muscular hypotonia are present8. The genetic screening of the RET gene mutations is a very important diagnostic tool for detection of inherited disease and identification of mutation carriers in families affected with MTC. Different human populations differ in the allelic frequencies of RET mutations, and therefore it is important to determine the mutation frequencies in each considerable population. In this study, we present the results of the RET genetic screening, which was performed at the Center for Endocrine Surgery of Clinical Center of Serbia in the past 20 years on all patients who were diagnosed with MTC based on the postoperative histopathological examination. Presently there are no published articles which describe the MTC’s genetic epidemiology in the Southeast Europe. We believe that these data will bring a greater light on the incidence, genetic structure and clinical characteristics of MTC patients from this region.

Material and Methods


Over a period of 20 years (1995-2015), 3,244 patients underwent surgical treatment for different types of thyroid cancer, and 249 of them were diagnosed with MTC in our referral center for the Republic of Serbia. These patients were submitted to genetic testing for RET proto-oncogene mutations in exons 10, 11, 13, 14, 15, and 16. The genetic screening for RET mutations was routinely established in 2004, and the patients who were previously diagnosed with MTC, based on their pathohistological examination, were then retrospectively tested. They were genetically examined either they were diagnosed with MTC, or because they had a family member with the same disease. Before genetic testing was performed, all involved patients gave the informed consent. The study was approved by the ethical committee of the Clinical Center of Serbia (decision No: 1575/7, 21-5-2015).

DNA extraction and molecular genetic analyses

Peripheral venous blood was collected by cubital venipuncture in 5 ml vacutainers containing Na-citrate. Total genomic DNA was extracted from the peripheral blood lymphocytes using the salting out protocol9. Following DNA extraction, the RET exons 10, 11, 13, 14, and 15 were amplified by polymerase chain reaction (PCR) using the Applied Biosystems Thermal Cycler 2720 (Applied Biosystems, Waltham, MA, USA). The PCR products were purified with Thermo Scientific ExoI-FastAP mixture (Thermo Fisher Scientific, Waltham, MA, USA), and the final volume of sequencing mixture was 20 μl, containing Ready Reaction Mix, Big Dye Sequencing Buffer (Applied Biosystems, Waltham, MA, USA), primers (10 μmol/l) and the PCR product. After the PCR sequencing, the products were precipitated with sodium acetate/ethanol procedure and sequenced by Sanger’s sequencing method in Applied Biosystems Genetic Analyzer 2500 (Applied Biosystems, Waltham, MA, USA)10.


Genetic screening in the study population revealed nine different mutations in the RET gene in 42 carriers. Of these 42 carriers of RET gene mutations, 19 individuals (7 men and 12 women) were not related, while 23 (10 men and 13 women) of them were related (cousins). The demographic data, molecular genetics and clinical characteristics of the 42 carriers of RET gene mutations, are presented in Table 1. Of the 42 MTC patients, 33 % (14/42) had MEN IIa syndrome, 29 % (12/42) had familial MTC, while the rest (38 %) of patients (16/42) had either sporadic MTC or C-cell hyperplasia. The proportion of familial MTCs in the total number of MTC patients was 4.8 % (12/249). The pedigrees of the eight families with FMTC are displayed in Figure 1. In the study population, the proportion of hereditary MTC was 17% (42/249). The most common prevalent mutation in the 42 carriers of RET gene mutations, was C634F detected in 31 % (13/42) of them, while C618R, L790F, and S904S were present in only 2 % (1/42) each in the study group. The clinical manifestation in the first family where the mutation Tyr791Phe was detected, was C-cell hyperplasia, as well as in the single sporadic case of the same mutation, where the C-cell hyperplasia was accompanied with PHP. The C-cell hyperplasia was also the clinical manifestation of a single sporadic case who carried Leu790Phe mutation. Detected mutations were not equally distributed in different exons of the RET gene. Among these MTC patients, 67 % (28/42) had mutation harbored in exon 11, while the rarest mutations were located in exons 10 and 15, each present in only 2 % (1/42) of patients. Figure 2 shows the percentage of mutation distribution in the different RET gene exons.

Figure 1: The pedigrees of the eight families with heritable form of medullary thyroid carcinoma treated in the Serbian Referral Center for endocrine surgery. The black color stands for the affected individual, while the arrows represent probands. The familial mutation is shown at the bottom of each box.

Figure 2: The percentage of the different RET (REarranged during Transfection) gene mutations presented by their localization in different RET gene exons.


Genetic screening for germline mutations of RET proto-oncogene has been widely used as a mean for diagnosing, preventing and treating cancer, having the greatest impact on patients with MTC. In families with identified germline mutations in the RET gene, testing of the family members is usually trouble-free, because a targeted approach focuses the specific codon which harbors the mutation11. In new families with familial MTC, the usual strategy for genetic testing is to sequence the most frequently affected exons. If no RET mutation is found, it is necessary to sequence the entire coding region of the RET gene, especially in kindred members of families where the heritable form of MTC has not been diagnosed based on the most often tested codons. The vast majority of laboratories that perform genetic testing for RET mutation sequence selected exons (most commonly 10, 11, 13, 14, 15, and 16, and in some laboratories, exon 8) or the entire coding region12. According to previous studies, 3-7 % of patients with presumed sporadic MTC, are confirmed as carriers of familial MTC, and therefore it is important to test new MTC patients for RET mutations in order to detect the increased risk at family members13,14. Genetic counselors and physicians require a considerable effort when they have to provide information to patients with MTC, regarding clinical expression, risks, and disease outcome. This results from the differences encountered in disease’s time of initiation, symptoms, prognosis, and response to therapy. Different human populations and ethnic groups display a considerable variability in the frequencies of RET mutations. Table 2 shows the comparison of RET mutation frequencies obtained in the Serbian population with other European cohorts15-19.

The characteristic of the Serbian ethnic group is the Cys634Phe mutation, which is prevalent in 31 % of genetically confirmed cases of MTC. Like in other cohort studies, the mutations in codon 634 predominate in all European populations. When mutations in codon 634 are considered, there is no pattern in the profile of clinical manifestations and the time of diagnosis, as it can be witnessed from the displayed results. The majority of MENIIa cases were carriers of this mutation. However, the mutations detected in codon 13 (Leu790Phe and Tyr791Phe) have a common clinical characteristic which is C-cell hyperplasia. The consensus document of the 7th international workshop on Multiple Endocrine Neoplasia and National Cancer Centers Network states that patients with mutations found in codons 790 and 791 have the relatively smaller risk for developing the aggressive form of MTC20,21. According to the American Thyroid Association, the highest risk is attributed to the carriers of Val804Met and mutations in codon 63422. Carriers of mutations in codon 618 hold a medium risk for developing aggressive MTC, based on the findings of the North American Neuroendocrine Tumor Society23. In accordance to the other studies, we detected only a single carrier of the Cys618Arg mutation, which is also only occasionally present in the other European cohort groups. Although the Ser904Ser mutation has a silent phenotype effect on protein structure, its role in MTC development remains controversial. In the current study, a single case of Ser904Ser carrier was found to have C-cell hyperplasia after pathohistological examination of the excised thyroid tissue. Other studies also describe this mutation among patients with MTC, but with inconclusive evidence regarding any genetic association, expressing the need for further studies to demonstrate the link between Ser904Ser mutation and MTC development24,25. The American Thyroid Association and European Thyroid Association recommend the genetic screening as a very useful diagnostic tool for all MTC patients22,26. Since the mutations in codon 634 are most common in Serbian and in other European populations, a cost-efficient genetic screening should primarily target this codon, and in case of negative findings, the other codons should be examined for mutations according the order of their prevalence. The mutations in the cysteine-rich codon 634 are most prevalent since they cause the early onset of disease, especially MENIIa syndrome, where they are present in about 85 % of cases7,27,28. There is a possibility that one could be a carrier of multiple RET gene mutations either in sporadic or familial cases, which was previously published in the paper of Jindrichiva et al, and therefore the testing of multiple exons is necessary for adequate genetic diagnosis29. Thyroid carcinoma is the most common endocrine malignancy worldwide with its diagnostic approach comprising of relevant imaging, clinical examination, and fine-needle aspiration biopsy. In approximately 25 % of patients with thyroid nodules, the diagnosis cannot be established by cytological examination30. The influence of molecular genetic and epigenetic biomarkers is expected to play a great role in the future diagnostic strategy for thyroid carcinoma31. The current whole-gene sequencing of the RET gene will imminently be replaced by the next-generation sequencing in the future, which will provide us with more conclusive evidence about the genetic features of the MTC and prediction of its evolution.        

Conflict of Interest

The authors declare that there is no conflict of interest.


1. Pusztaszeri MP, Bongiovanni M, Faquin WC. Update on the cytologic and molecular features of medullary thyroid carcinoma. Adv Anat Pathol. 2014; 21: 26-35.
2. Tiedje V, Ting S, Dralle H, Schmid KW, Führer D. [Medullary thyroid carcinoma]. Internist (Berl). 2015; 56: 1019-1031.
3. Ceccherini I, Bocciardi R, Luo Y, Pasini B, Hofstra R, Takahashi M, et al. Exon structure and flanking intronic sequences of the human RET proto-oncogene. Biochem Biophys Res Commun. 1993; 196: 1288-1295.
4. Ponder BA. The phenotypes associated with ret mutations in the multiple endocrine neoplasia type 2 syndrome. Cancer Res. 1999; 59: 1736s-1742s; discussion 1742s.
5. Kalezic NK, Zivaljevic VR, Slijepcevic NA, Paunovic IR, Diklic AD, Sipetic SB. Risk factors for sporadic medullary thyroid carcinoma. Eur J Cancer Prev. 2013; 22: 262-267.
6. Nikiforov YE, Rowland JM, Bove KE, Monforte-Munoz H, Fagin JA. Distinct pattern of ret oncogene rearrangements in morphological variants of radiation-induced and sporadic thyroid papillary carcinomas in children. Cancer Res. 1997; 57: 1690-1694.
7. Eng C, Clayton D, Schuffenecker I, Lenoir G, Cote G, Gagel RF, et al. The relationship between specific RET proto-oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2. International RET mutation consortium analysis. JAMA. 1996; 279: 1575-1579.
8. Moline J, Eng C. Multiple endocrine neoplasia type 2: an overview. Genet Med. 2011; 9: 755-764.
9. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988; 16: 1215.
10. Zeugin JA, Hartley JL. Ethanol Precipitation of DNA. Focus. 1985; 7: 1-2.
11.    Wells SA Jr, Asa SL, Dralle H, Elisei R, Evans DB, Gagel RF, et al; American Thyroid Association Guidelines Task Force on Medullary Thyroid Carcinoma.. Revised American Thyroid Association guidelines for the management of medullary thyroid carcinoma. Thyroid. 2015; 25: 567-610.
12. GTR: Genetic Testing Registry. National Center for Biotechnology Information, U.S. National Library of Medicine. Available at: http://, last accessed on: 19/2/2017.
13. Elisei R, Romei C, Cosci B, Agate L, Bottici V, Molinaro E, et al. RET genetic screening in patients with medullary thyroid cancer and their relatives: experience with 807 individuals at one center. J Clin Endocrinol Metab. 2007; 92: 4725- 4729.
14. Eng C, Mulligan LM, Smith DP, Healey CS, Frilling A, Raue F, et al. Low frequency of germline mutations in the RET proto-oncogene in patients with apparently sporadic medullary thyroid carcinoma. Clin Endocrinol (Oxf). 1995; 43: 123-127.  
15. Jindrichová S, Vcelák J, Vlcek P, Neradilová M, Nemec J, Bendlová B. Screening of six risk exons of the RET proto-oncogene in families with medullary thyroid carcinoma in the Czech Republic. J Endocrinol. 2004; 183: 257-265.
16. Machens A, Lorenz K, Sekulla C, Höppner W, Frank-Raue K, Raue F, et al. Molecular epidemiology of multiple endocrine neoplasia 2: implications for RET screening in the new millenium. Eur J Endocrinol. 2013; 168: 307-314.
17. Fernández RM, Navarro E, Antiñolo G, Ruiz-Ferrer M, Borrego S. Evaluation of the role of RET polymorphisms/haplotypes as modifier loci for MEN 2, and analysis of the correlation with the type of RET mutation in a series of Spanish patients. Int J Mol Med. 2006; 17: 575-581.
18. Pinna G, Orgiana G, Riola A, Ghiani M, Lai ML, Carcassi C, et al. RET proto-oncogene in Sardinia: V804M is the most frequent mutation and may be associated with FMTC/MEN-2A phenotype. Thyroid. 2007; 17: 101-104.
19. Sarika HL, Papathoma A, Garofalaki M, Saltiki K, Pappa T, Pazaitou-Panayiotou K, et al. Genetic screening of patients with medullary thyroid cancer in a referral center in Greece during the past two decades. Eur J Endocrinol. 2015; 172: 501-509.
20. Brandi ML, Gagel RF, Angeli A, Bilezikian JP, Beck-Peccoz P, Bordi C, et al. Guidelines for diagnosis and therapy of MEN type 1 and type 2. J Clin Endocrinol Metab. 2001; 86: 5658-5671.
21. Tuttle RM, Ball DW, Byrd D, Daniels GH, Dilawari RA, Doherty GM, et al; National Comprehensive Cancer Network. Medullary carcinoma. J Natl Compr Canc Netw. 2010; 8: 512-530.
22. American Thyroid Association Guidelines Task Force, Kloos RT, Eng C, Evans DB, Francis GL, Gagel RF, et al. Medullary thyroid cancer: management guidelines of the American Thyroid Association. Thyroid. 2009; 19: 565-612.
23. Chen H, Sippel RS, O’Dorisio MS, Vinik AI, Lloyd RV, Pacak K; North American Neuroendocrine Tumor Society (NANETS). The North American Neuroendocrine Tumor Society consensus guideline for the diagnosis and management of neuroendocrine tumors: pheochromocytoma, paraganglioma, and medullary thyroid cancer. Pancreas. 2010; 39: 775-783.
24. Sheikholeslami S, Zarif Yeganeh M, Hoghooghi Rad L, Golab Ghadaksaz H, Hedayati M. Haplotype Frequency of G691S/S904S in the RET Proto-Onco-gene in Patients with Medullary Thyroid Carcinoma. Iran J Public Health. 2014; 43: 235-240.
25. Machens A, Frank-Raue K, Lorenz K, Rondot S, Raue F, Dralle H. Clinical relevance of RET variants G691S, L769L, S836S and S904S to sporadic medullary thyroid cancer. Clin Endocrinol (Oxf). 2012; 76: 691-697.
26. Elisei R, Alevizaki M, Conte-Devolx B, Frank-Raue K, Leite V, Williams GR. 2012 European thyroid association guidelines for genetic testing and its clinical consequences in medullary thyroid cancer. Eur Thyroid J. 2013; 1: 216-231.
27. Niccoli-Sire P, Murat A, Rohmer V, Franc S, Chabrier G, Baldet L, et al; French Calcitonin Tumors Group (GETC). Familial medullary thyroid carcinoma with noncysteine ret mutations: phenotype-genotype relationship in a large series of patients. J Clin Endocrinol Metab. 2001; 86: 3746-3753.
28. Machens A, Gimm O, Hinze R, Höppner W, Boehm BO, Dralle H. Genotype-phenotype correlations in hereditary medullary thyroid carcinoma: oncological features and biochemical properties. J Clin Endocrinol Metab. 2001; 86: 1104-1109.
29. Jindrichova S, Vlcek P, Duskova J, Ryska A, Bendlova B. Multiple mutations in the ret proto-oncogene detected in medullary thyroid carcinoma. Ann Endocrinol. 2005; 33: 310.
30. Patel HH, Goyal N, Goldenberg D. Imaging, genetic testing, and biomarker assessment of follicular cell-derived thyroid cancer. Ann Med. 2014; 46: 409-416.
31. Emes RD, Farrell WE. Make way for the ‘next generation’: application and prospects for genome-wide, epigenome-specific technologies in endocrine research. J Mol Endocrinol. 2012; 49: R19-R27.