RET Proto-Oncogene Mutational Analysis in Patients With Medullary Thyroid Carcinoma

Background: The aim of this study was to identify germline mutation of the RET (rearranged during transfection) gene in patients with Medullary Thyroid Carcinoma (MTC) and their rst-degree relatives to nd pre-symptomatic carriers for possible prophylactic thyroidectomy. Methods and results: We examined all six hot spot exons (exons 10, 11, 13, and 14-16) of the RET gene by PCR and bidirectional sanger sequencing in 45 Iranian patients with MTC (either sporadic or familial form) from 7 unrelated kindreds and 38 apparently sporadic cases. First-degree relatives of RET positive cases were also genotyped for index mutation. Moreover, pre-symptomatic carriers were referred to the endocrinologist for further clinical management and prophylactic thyroidectomy if needed. Overall, the genetic status of all of the participants was determined by RET mutation screening, including 61 affected individuals, 22 pre-symptomatic carriers and 29 genetically healthy subjects. In 37.5% (17 of 45) of the MTC referral index patients, seven distinct RET germline mutations were found, including p.C634R (35.3%), p.M918T (17.6%), p.C634Y (11.8%), p.C634F (5.9%), p.C611Y (5.9%), p.C618R (5.9%), p.C630R (5.9%), p.L790F (5.9%) and an uncertain variant p.V648I (5.9%). Conclusion: RET mutation detection is a promising/golden screening test and provides an accurate pre-symptomatic diagnostic test for at-risk carriers (the siblings and offspring of the patients) to consider prophylactic thyroidectomy. Thus, mutation screening of the RET hot spot exons could be suggested for any MTC patient to nd pre-symptomatic RET carriers in their relatives.


Introduction
Calcitonin-secreting parafollicular C cells of the thyroid gland are the origin of 5-10% of thyroid cancers, so called as Medullary Thyroid Carcinoma (MTC). MTC has a worse prognosis than the most common form of thyroid cancer, papillary thyroid carcinoma (PTC), that accounts for 60-80% of thyroid carcinomas and originates from follicular cells [1]. MTC has 2 forms of sporadic (isolated) and hereditary that compromise about 75% and 25% of the cases, respectively. Hereditary forms are transmitted as autosomal dominant pattern of inheritance and are seen either as isolated familial MTC (FMTC) with a prevalence of 10%, or as a syndromic form of cancer, also known as multiple endocrine neoplasia type 2 (MEN2A, MEN2B, which have a prevalence of 85%, 5%, respectively) [2,3]. In familial medullary thyroid carcinoma, the only lesion present is MTC, but MEN2A is characterized by MTC and pheochromocytoma with or without parathyroid hyperplasia or adenoma [1][2][3].
PTC and MTC have clear clinical and pathological differences but RET proto-oncogene contributes somehow to the carcinogenesis of both types. Activation of the RET gene by rearrangement (inversion or translocation) appears to be only seen in patients suffering from PTC [5] or thyroid adenoma [6], and germline missense mutations of the RET gene have been shown to be the cause of the hereditary form of MTC (FMTC and MEN2) [7]. Interestingly, somatic mutations in RET have been found in 23-70% of sporadic MTCs and 10-15% of sporadic pheochromocytoma patients, as well [8,9]. Gain of function mutations in exon 10,11,13,14, and 15 of the RET gene have mostly been reported in FMTC and MEN2A (e.g. codons 609, 611, 618, 620, and 634) but about 95% of the patients with MEN2B have a mutation in exon 16 (codon 918). Therefore, to obtain a reliable screening genetic test for MTC patients, exons 10, 11, 13, 14, 15 and 16 should be considered as the hot spots for RET gene mutations.
Identi cation of the germline mutation of the RET gene in a patient with MTC provides an accurate pre-symptomatic diagnostic test for the siblings and offspring of the patients. Here we report the results of the mutational analysis of the RET proto-oncogene in 45 Iranian patients with MTC (either sporadic or familial form) from 7 unrelated kindreds and 38 apparently sporadic cases. We examined all six exons (10, 11, 13, 14, 15 and 16) by PCR and direct Sanger sequencing. First-degree relatives of RET positive cases were also genotyped and underwent prophylactic thyroidectomy if they had the causative mutation. According to the results, 37.5% (17 of 45) of the MTC referral index patients were found to have nine distinct RET germline mutations, including p.C634R (35.3%), p.M918T (17.6%), p.C634Y (11.8%), p.C634F (5.9%), p.C611Y (5.9%), p.C618R (5.9%), p.C630R (5.9%), p.L790F (5.9%) and an uncertain variant p.V648I (5.9%). The distribution of the detected RET mutations was as following; 64.7% in exon 11, 17.6% in exon 16,11.8% in exon 10, 5.9% in exon 13, and no mutation in exons 14 and 15 was found. Overall, the genetic status of all participants (61 affected individuals, 22 pre-symptomatic carriers and 29 genetically healthy) was determined by RET mutation screening, regarding all six exons of RET (10, 11, 13, 14, 15 and 16). Moreover, 22 genetically pre-symptomatic carriers were referred to the endocrinologist for further clinical management.

Patients and Specimen Collection
Seven unrelated families and thirty-eight apparently sporadic cases with medullary thyroid carcinoma referred to Endocrinology and Metabolism Research Institute of Tehran University of Medical Sciences, participated in this study.
First, 45 index cases whose MTC was diagnosed by an endocrinologist and con rmed histopathologically and underwent thyroidectomy based on the ATA guideline were genetically consulted. Then, each index case was evaluated for mutations in six hot spot exons of the RET gene and segregation analysis was done for their at-risk relatives if a causative mutation was found [3]. Overall, 112 participants were examined for RET mutations. All procedures were in accordance with the ethical standards of Tehran University of Medical Sciences and the Helsinki Declaration of 1975 revised in 1983. Informed consent was obtained from all participants or their parents.

Molecular Analysis
Genomic DNA was extracted from EDTA peripheral venous blood using the standard salting out/proteinase K method.
Primers were designed to amplify all six exons; 10, 11, 13, 14, 15 and 16, and their exon-intron boundaries using the NCBI Primer-design tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and gene runner software (version 6.0.28). Fifty nanograms of the extracted DNA was used as a PCR template. PCR conditions are available upon request. The PCR products were run on a 2% agarose gel and subsequently, all six exons were bi-directionally sequenced using the ABI3130 automated sequencer and analyzed with the Chromas version 2 (http://chromas.software.informer.com/2.0/). The sequence data were compared with the RET sequence data (Ref Seq NM_020975) from the NCBI BLAST human database (http://www.ncbi.nlm.nih.gov/BLAST/) and Ensembl human database (GRCh37).

Clinical and molecular ndings
Of 45 referred MTC patients, 7 cases had FMTC or MEN syndrome, with more than one affected members. Thirty-eight patients seemed to be apparently sporadic MTC. All six RET exons (10, 11, 13, 14, 15 and 16 exons) were sequenced in 45 index patients. Eight RET known distinct mutations, one unclassi ed variant, and one novel variant were detected (Table 1).
The family IR-F4 was an extended kindred with two affected individuals in the second generation (II 2 and II 3 ), two affected (III 3 and III 10 ) and three carriers in the third generation (III 5 − 7 ), and two carriers in the fourth generation (IV 2 and IV 8 ) ( Fig. 1-a). The 44-year-old proband III 3 and other affected individuals of the pedigree had a known heterozygous mutation (c.2370G > T) in exon 13 (p.L790F). According to the ATA risk classi cation, p.L790F mutation is strati ed as level A, indicating moderate risk of aggressive MTC with ~ 10% incidence of PHEO. Carriers of this mutation are advised to undergo prophylactic thyroidectomy only if it occurs as FMTC or they have a high level of calcitonin. Therefore, these carriers were referred to the endocrinologist for more clinical follow-up.
The proband of the family IR-F15 (II 2 ) also had a known mutation (c.1900T > C) in exon 11 (p.C634R). Her mother (I 2 ) died because of MTC and was not available but two other family members did not have any RET mutations in the six tested exons ( Fig. 1-b).
The family IR-F18 was another family with three affected daughters (II 2 , II 6 and II 7 ), one affected son (II 4 ), three affected grandchildren (III 3 , III 4 and III 8 ), and one carrier granddaughter (III 6 ) ( Fig. 1-c). A known heterozygous mutation in exon 11 [c.1900T > C (p.C634R)] was found in all affected individuals (II 2 , II 4 , II 6 , II 7 , III 3 , III 4 and III 8 ) and one carrier granddaughter (III 6 ). Interestingly, individual III 6 had two genetically normal triplets. According to the ATA risk classi cation, she was advised to undergo prophylactic thyroidectomy before 5 years of age.The family IR-F25 was an extended kindred consisting of a healthy father (II 3 ) and an affected mother (II 4 ) with one affected son (III 1 ), one affected daughter (III 2 ), two carrier daughters (III 4 and III 6 ), one phenotypically healthy son (III 3 ) and one genetically healthy daughter (III 5 ), three carrier grandsons (IV 1 , IV 4 and IV 9 ), one carrier granddaughter (IV 2 ), and three genetically healthy grandchildren (IV 3 , IV 7 and IV 8 ) ( Fig. 1-d). The family IR-F31 was another kindred comprising an affected father (I 1 ), a healthy mother (I 2 ), two affected daughters (II 2 and II 4 ), one affected son (II 5 ), one genetically healthy son (II 7 ), and two carrier grandsons (III 3 and III 4 ) ( Fig. 1-e).
MEN2A was diagnosed in the 45-year-old grandfather as the proband with clinical manifestations of MTC and pheochromocytoma. He underwent thyroidectomy and adrenalectomy. His affected children also had MEN2A, including MTC and pheochromocytoma. We identi ed a heterozygous known mutation in exon 11 (c.1900T > C, p.C634R) in this family which has been classi ed as a pathogenic variant with ATA risk level C. Prophylactic thyroidectomy is recommended for this mutation before 5 years of age because of high risk of aggressive MTC (H). To conduct a followup study, two carrier grandsons (III 3 , III 4 ) were referred to the endocrinologist and underwent prophylactic thyroidectomy at age 5.
The family IR-F33 had a known RET mutation (c.1852T > C) in exon 10 (p.C618R) ( Fig. 1-f). The proband (II 5 ) was rst referred for RET genetic testing because of a high calcitonin level (above 1500) and histopathological results showed MTC. Initially, a heterozygous known mutation (c.1852T > C) was seen in exon 10 (p.C618R) in the proband (II 5 ) at age 27 and subsequently, this variant was found in his affected father I 1 and three carrier siblings (II 1 , II 2 and II 3 ). According to the ATA risk classi cation, p.C618R mutation is strati ed as level B (MOD) and carriers are recommended to undergo prophylactic thyroidectomy before 5 years of age. Follow-up visits showed that the calcitonin level was above the normal range (300 pg/ml) in II 5 even after prophylactic thyroidectomy.
Thirty-eight index patients who seemed to be apparently sporadic MTC were tested for RET mutation at six mentioned exons. The results showed that 10 out of 38 patients had germline RET mutations. In addition, in four families, we found four carriers of a causative mutation in presymptomatic children who had affected parents, including family IR-F14 with c.1832G > A mutation in exon 10 (p.C611Y), families IR-F17 and IR-F29 with c.1900T > C mutation in exon 11 (p.C634R) and family IR-F22 with c.1901G > T mutation in exon 11 (p.C634F).
In the family IR-F17, a 32-year-old affected mother (II 8 ) with a p.C634R mutation had a -year-old daughter with elevated calcitonin who was found to have the same mutation as observed in her mother. The proband in the family IR-F29 (III 16 ) had an asymptomatic 10-year-old boy who carried the same mutation (p.C634R). Moreover, a 15-year-old carrier of p.C611Y in the family IR-F14 with an ATA risk level B (MOD) and a 12-year-old carrier of p.C634F in the family IR-F22 were found. All of the carriers in these four families were referred to the endocrinologist for further clinical follow-up and prophylactic thyroidectomy. Furthermore, three index patients in families IR-11, IR-F23 and IR-F37 had a known RET germline mutation (c.2753T > C) in exon 16 (p.M918T) which is a MEN2B speci c mutation with ATA risk level D (highest risk of aggressive MTC-HST). However, no available persons in their families had this mutation. The apparent sporadic patient IR-F39 had a known RET mutation (c.1901G > A) in exon 11 (p.C634Y). Likewise, another apparently sporadic patient (IR-F38) had a RET germline mutation (p.C634Y) and a novel uncertain variant (c.1973A > G) causing p.H658R, both in exon 11. RET novel variant p.H658R was predicted to be of uncertain signi cance by InterVar, benign by polyphen2, disease causing by mutation taster and tolerated with SIFT score 0.5 by SIFT web server ( Table 2). RET sequencing results of another sporadic patient (IR-F19-II 7 ) showed an uncertain variant, c.1942G > A, in exon 11 (p.V648I). This variant was predicted to be likely benign by InterVar, benign by polyphen2, disease causing by mutation taster and tolerated with SIFT score 0.43 by SIFT web server ( Table 2).  [15][16][17][18][19]. It has been stated that mutations at codon 634 are the most frequent mutation in Caucasians [20]. This mutation comprised 53% of all detected mutations in this study. The present study showed that mutations in exon 11, especially at codon 634, are commonly observed in the Iranian population, which is consistent with other studies [15][16][17][18][19].
Interestingly, follow-up studies in IR-F25 family showed that a 19-year-old carrier, IV-2, did not manifest any MEN2A phenotypes unlike other carriers. The carrier IV-2 might have some other modi er genes which give her an opportunity to be a healthy carrier with a normal calcitonin level until 19 years of age. In addition, a review of the literature showed that p.L790F mutation is reported for the second time in Iranian families with FMTC [21]. Similarly, it is a second report of p.C618R mutation in Iranian MTC families (18). Nevertheless, p.C618R mutation is the most common mutation in MEN2A in Saudi Arabian families (22).
The present study showed ve distinct RET mutations in 10 out of 38 (26.3%) apparently sporadic Iranian MTC cases, including two p.C634R, two p.C634Y, one p.C634F, three p.M918T, and one p.C611Y mutations. Additionally, one RET uncertain variant (p.V648I) was detected in one apparently sporadic MTC Iranian patient for the rst time. Moreover, it is a rst report of an apparently sporadic MTC Iranian patient who had a p.C634R mutation and a novel H658R uncertain variant. Further molecular studies are needed to identify the clinical effect of these two variants.
On the other hand, in this study, p.M918T mutation was another common mutation in sporadic Iranian MTC cases, which is inconsistent with ndings in European populations (16,21). To the best of our knowledge, p.M918T mutation has been reported in two cases and has not been detected in Iranian Azari and other populations [15][16][17][18][19]. Different genetic backgrounds could be the reason of the mentioned con icting results which requires more investigations. On the other hand, it seems that some modi er polymorphisms that were found in some carriers of RET mutation in our study might contribute to MTC manifestations [23]. Additional genetic evidence is required to clarify the additive effect of modi er polymorphisms In conclusion, evaluation of RET mutations in hot spot exons resulted in genetic diagnosis of 37.5% of the Iranian MTC patients. The genotype of all participants in this study was precisely determined and 22 pre-symptomatic carriers were referred for further clinical management and prophylactic thyroidectomy. Thus, RET mutation screening could serve as an essential detection method for any MTC patient to nd pre-symptomatic RET carriers.