Germline sequence variant S836S in the RET proto-oncogene is associated with low level predisposition to sporadic medullary thyroid carcinoma in the Spanish population

OBJECTIVE: The molecular basis of sporadic medullary thyroid carcinoma (MTC) remains elusive. While germline gain-of-function mutations in the RET proto-oncogene cause hereditary MTC, somatic activating RET mutations and loss of heterozygosity of markers in various chromosomal regions representing deletions of tumour suppressor genes, have been described in a variable number of sporadic MTC. A previous report suggested that the presence of a germline variant at RET codon 836 (S836S) was associated with the development of sporadic MTC and, furthermore, that the presence of S836S was highly correlated with somatic RET M918T mutation in the MTC. Thus, we sought to determine if the S836S variant would be associated with sporadic MTC from a completely different population base, that of Andalucia. DESIGN: This is a case-control study to determine whether the presence of RET germline S836S is correlated with sporadic MTC in Andalucia. PATIENTS: Thirty-two patients with sporadic MTC from the Andalucia region of Spain, serviced by our University Hospital, were ascertained throughout the period 1995-99. Sporadic MTC was defined as a lack of personal or family history suggestive of multiple endocrine neoplasia type 2 (MEN 2) and lack of germline RET mutations which define any MEN 2 subtype. A region and race matched cohort of 250 controls was also obtained. MEASUREMENTS: The frequency of the S836S allele was determined in cases and controls and compared using the standard chi-squared statistic and Fisher's exact test. RESULTS: The polymorphic allele frequency at codon 836 in the control population (18/500 chromosomes, 3.6%) differed significantly from the MTC case cohort, 9.3% of case chromosomes (six of 64 alleles, Fisher's exact test, two-tailed, P = 0.043). CONCLUSIONS: Germline RET S836S variant is associated with a two- to three-fold risk of sporadic MTC in the Spanish population, in accordance with a previous study based on German cases. Our observations suggest that this phenomenon might be universal and not limited to Germany.     

RET polymorphisms and sporadic medullary thyroid carcinoma in a Portuguese population

The genetic basis of the sporadic form of medullary thyroid carcinoma, derived from "C" cells, is still poorly understood. Somatic mutations of RET proto-oncogene have been reported at a variable frequency ranging from 23% to 69%. The hypothesis that low penetrance factors, such as polymorphisms, might contribute to the phenotype of this neoplasm has been addressed in a few studies conducting to conflicting results. Herein, we studied 100 individuals (50 patients and 50 controls) aiming to compare the frequencies of G691S, L769L, S836S, and S904S RET polymorphisms observed in patients with respect to controls. Furthermore, meta-analysis of published studies including the present results was conducted. To test the contributory role of the above polymorphisms for the development of "C"-cell hyperplasia, we studied a group of 10 individuals selected for having a positive pentagastrin test despite the absence of a RET germline mutation. An over-representation of the G691S polymorphism, particularly in females, was observed in patients with respect to controls, although not reaching the level of significance. Allelic frequencies of the other three polymorphisms were not different in patients and controls. Results obtained in the admittedly small group of individuals with a positive pentagastrin test are unlikely to support a major influence of any polymorphism in the development of "C"-cell hyperplasia. The meta-analysis provided evidence for a significant association of the S691 allele with MTC (odds ratio 1.54, 95% confidence interval 1.12-2.12, p=0.008) and found no significant associations for the other polymorphisms.     

RET genotypes in sporadic medullary thyroid cancer: studies in a large Italian series

Background  Highly discrepant data about the different distribution of RET germline single nucleotide polymorphisms (SNPs) among patients with sporadic medullary thyroid cancer (sMTC) and controls are available.
Design and patients  In the present case-control study, a wide panel of seven RET SNPs has been tested in the largest sMTC series and in a matched control group.
Results  None of the investigated polymorphisms show a significantly different distribution in patients with sMTC when compared to controls. Twenty haplotypes and 57 genotypes were generated, and their association with the disease and with the clinical features were statistically evaluated. Interestingly, 14 genotypes were found to be unique to sMTC patients and 25 to controls. Two haplotypes and three genotypes, all including the intronic variants IVS1-126 and IVS14-24, were significantly associated with sMTC patients and with a higher tumour aggression. The functional activity of the only nonsynonymous RET variant (c.2071C > A, G691S) was tested for the first time. Interestingly, Western blot analyses showed that the fraction of Ret9-G691S protein located at the plasma membrane level was overrepresented when compared to Ret9-WT, suggesting facilitated targeting at the cell membrane for this variant. However, no transforming activity was shown in a focus formation assay on cells carrying the Ret9-G691S, against a possible oncogenic role of G691S variant.
Conclusions  RET genotypes including two intronic RET variants were associated with the risk of developing sMTC and to more aggressive behaviour. Further studies are warranted to elucidate whether these RET genotypes are in linkage disequilibrium with another susceptibility gene or whether these variants could play a role in the genesis of sMTC per se .     

Significance of the RET proto-oncogene polymorphisms in Turkish sporadic medullary thyroid carcinoma patients

Several single nucleotide polymorphisms (SNP) of the RET gene have been identified in medullary thyroid carcinoma (MTC) patients as well as in the general population. However, the relevance of SNP for MTC patients is still controversial, whether these allelic variants play other interacting, predisposing or modifying roles in clinical behavior of MTC. The aim of this work is to elaborate allelic frequencies of the RET proto-oncogene polymorphisms in Turkish sporadic MTC patients and to demonstrate if there is an association between SNP and the clinical disease features, specifically the age at onset of MTC and lymph node involvement at diagnosis. We analyzed the allelic frequencies of SNP of the exon 11, 13, 14 and 15 of the RET proto-oncogene in blood samples from 50 sporadic MTC patients, using the polymerase chain reaction methodology followed by DNA sequencing. The observed allelic frequencies were 24% for G691S polymorphism in exon 11, 29% for L769L polymorphism in exon 13, 5% for S836S polymorphism in exon 14, and 26% for S904S polymorphism in exon 15. These frequencies are similar to those reported in other countries. We did not observe any significant association of all four SNP with the age at onset of MTC. Our results indicate a possible association between the presence of lymph node involvement at the time of diagnosis (extent of disease) and L769L or S836S polymorphism. However, it is not possible to draw definitive conclusions that these two polymorphisms play a significant role in clinical behavior of MTC. Further studies are needed to evaluate the role of this polymorphism in the clinical behavior of MTC.     

Analysis of RET polymorphisms and haplotypes in the context of sporadic medullary thyroid carcinoma.

CONTEXT: Little is known about the etiology of sporadic medullary thyroid carcinoma (sMTC). While germline gain-of-function mutations in the RET proto-oncogene cause hereditary MTC, the molecular mechanisms leading to the sporadic forms remain obscure. Our group had evidence about the existence of a low-penetrance susceptibility locus for sMTC in linkage disequilibrium with RET variants S836S/IVS1-126G>T, and probably in 5' with respect to both variants. In this study we sought to identify such locus. On the other hand, because an overrepresentation of G691S/S904S variants in patients with sMTC had been previously reported, we sought to determine if such association was present in our series. DESIGN: We performed a case-control study analysing a wide spectrum of RET variants in the 5' region of the gene, as well as the variants G691S/S904S. Haplotype distribution were also analyzed. A total of 58 patients with sMTC were included in the study. In addition, 100 unselected, unrelated race-, age-, and gender-matched normal controls were also evaluated. MAIN OUTCOME: Although the overrepresentation of IVS1-126G>T remains present in our current sMTC series, thus supporting our previous hypothesis, no differences were obtained among cases and controls in the distribution of the variants tested upstream this position. On the other hand, the frequency and distribution of G691S/S904S variants were similar in both groups of study, leading to exclude their role in sMTC in our series. CONCLUSIONS: These findings would suggest that the major genetic events contributing to the appearance of sMTC may reside in several different RET loci. In this way, we could hypothesize about the existence of at least two sMTC loci, linked to S836S-IVS1-126G>T, or to G691S-S904S, respectively.     

Prognostic significance of somatic RET oncogene mutations in sporadic medullary thyroid cancer: a 10-year follow-up study

BACKGROUND: Medullary thyroid carcinoma (MTC) is a well-differentiated thyroid tumor that maintains the typical features of C cells. An advanced stage and the presence of lymph node metastases at diagnosis have been demonstrated to be the most important bad prognostic factors. Somatic RET mutations have been found in 40-50% of MTCs. Although a relationship between somatic mutations and bad prognosis has been described, data are controversial and have been performed in small series with short-term follow ups. The aim of this study was to verify the prognostic value of somatic RET mutations in a large series of MTCs with a long follow up. METHODS: We studied 100 sporadic MTC patients with a 10.2 yr mean follow-up. RET gene exons 10-11 and 13-16 were analyzed. The correlation between the presence/absence of a somatic RET mutation, clinical/pathological features, and outcome of MTC patients was evaluated. RESULTS: A somatic RET mutation was found in 43 of 100 (43%) sporadic MTCs. The most frequent mutation (34 of 43, 79%) was M918T. RET mutation occurrence was more frequent in larger tumors (P=0.03), and in MTC with node and distant metastases (P<0.0001 and P=0.02, respectively), thus, a significant correlation was found with a more advanced stage at diagnosis (P=0.004). A worse outcome was also significantly correlated with the presence of a somatic RET mutation (P=0.002). Among all prognostic factors found to be correlated with a worse outcome, at multivariate analysis only the advanced stage at diagnosis and the presence of a RET mutation showed an independent correlation (P<0.0001 and P=0.01, respectively). Finally, the survival curves of MTC patients showed a significantly lower percentage of surviving patients in the group with RET mutations (P=0.006). CONCLUSIONS: We demonstrated that the presence of a somatic RET mutation correlates with a worse outcome of MTC patients, not only for the highest probability to have persistence of the disease, but also for a lower survival rate in a long-term follow up. More interestingly, the presence of a somatic RET mutation correlates with the presence of lymph node metastases at diagnosis, which is a known bad prognostic factor for the definitive cure of MTC patients.     

In silico and in vitro analysis of rare germline allelic variants of RET oncogene associated with medullary thyroid cancer

Germline and somatic RET oncogene mutations are found in 98% hereditary and 40% sporadic medullary thyroid carcinomas. Our aim was to analyse by in silico and in vitro assays the transforming activity of six rare RET mutations (T338I, V648I, M918V, A883T, S904F and M848T). Six known RET mutations were used as controls. The in silico analysis showed the highest score value (i.e. 65) for S904F, M848T, M918T and C634R, whereas L790F, G691S, T338I and V648I had 0 score. Intermediate score values were obtained by A883T (score=55), M918V, V804M and Y791F (score=15). The in vitro focus formation assay showed that cells transfected with S904F, M918T, M848T or C634R generated the largest number of focus formation units (FFU). Intermediate numbers of FFU were observed in cells transfected with M918V, V804M, Y791F or A883T, while cells transfected with L790F, G691S, T338I or V648I showed a number of FFU similar to control cells. A positive correlation between the in silico score and in vitro FFU was found (P=0.0005). Only cells transfected with M918T or C634R grew faster and generated higher number of colonies in soft agar than control cells. However, the cells that were transfected with V804M produced an intermediate number of colonies. In conclusion, two of the six rare RET mutations, S904F and M848T possessed a relatively high transforming activity but a low aggressiveness; the other four mutations T338I, V648I, M918V and A883T were low or non-transforming, and their ability to induce tumoural transformation might be related to particular genetic conditions.     

Polymorphisms in the initiators of RET (rearranged during transfection) signaling pathway and susceptibility to sporadic medullary thyroid carcinoma

CONTEXT: Medullary thyroid carcinoma (MTC) is a characteristic tumor occurring in individuals with multiple endocrine neoplasia type 2 who carry germ-line mutations in RET (rearranged during transfection). However, most MTC occur in individuals without a family history. OBJECTIVES: The objective of this study was to explore the possibility that susceptibility in these cases results from low penetrance alleles of RET, its coreceptors, and ligands. DESIGN: We carried out an association study in 135 sporadic MTC (sMTC) patients and 533 controls from the United Kingdom population. RESULTS AND CONCLUSIONS: We analyzed 33 polymorphisms in all nine genes involved in the glial cell line-derived neurotropic factor receptor-alpha (GFRalpha)-RET complex. This is the first association study in which all genes involved in this complex have been investigated for susceptibility to sMTC. We did not find any association between single nucleotide polymorphisms in the exonic regions of the GFRalpha2, GFRalpha3, GFRalpha4, glial cell line-derived neurotropic factor, neurturin, or persephin genes and risk of developing sMTC. We found a strong association between the disease and specific haplotypes of RET. We not only confirmed the previously described association with G691S and S904S (for heterozygotes: odds ratio, 1.85; range, 1.22-2.82; P = 0.004), but we found a novel protective effect associated with a specific haplotype (odds ratio, 0.39; range, 0.21-0.72; P = 0.005) revealing the existence of different genetic variants in the RET oncogene that either increase or decrease risk of sMTC.     

Clinical utility of genetic diagnosis for sporadic and hereditary medullary thyroid carcinoma

Medullary thyroid cancer (MTC) is a rare thyroid tumor whose prevalence is 3–5% among all thyroid tumors. The pathogenesis of MTC is mainly related to germline or somatic RET activating point mutations that are causative of hereditary and sporadic cases, respectively. Hereditary MTC can occur as multiple endocrine neoplasia type 2A (MEN 2A), type 2B (MEN 2B) and familial MTC (FMTC) that differ for the association with other endocrine neoplasia. Germline RET point mutations are prevalently localized in exons 5, 8, 10–11, 13–16 and a significant genotype-phenotype correlation has been observed. RET genetic screening is mandatory in all patients with a diagnosis of MTC regardless from their apparent sporadic origin. The identification of RET germline mutation in an apparently sporadic case is of great clinical utility because it allows the identification of those subjects who will develop the tumor. RET positive relatives must undergo clinical and biochemical tests to verify if the MTC is already present and, according to the type of RET mutation, they have to be screened for the presence of pheochromocytoma and/or hyperparathyroidism. If a MTC is already present patients must be surgically treated. If MTC is not yet present subjects will be followed up with basal and stimulated calcitonin serum measurement, which is the serum marker of MTC. Indeed, RET negative subjects can be reassured that they do not run any risk to develop the disease as well as their children. In conclusions RET genetic screening allows the identification of the hereditary/sporadic nature of MTC and of a relevant percentage of hidden familial MTC. Furthermore, it favors the early diagnosis of MTC in RET positive subjects. RET positive patients and no clinical evidence of MTC can be followed and surgical treatment can be delayed. Finally RET negative relatives do not need to be further monitored.     

Low frequency of germline mutations in the RET proto-oncogene in patients with apparently sporadic medullary thyroid carcinoma

BACKGROUND AND OBJECTIVES Medullary thyroid carcinoma (MTC) occurs both sporadically and In the autosomal domlnantly inherited multiple endocrine neoplasia (MEN) type 2 syndromes. The distinction between true sporadic MTC and a new mutation familial case is important for future clinical managment of both the patient and family. The susceptibility gene for MEN 2 is theRET proto-oncogene. Systematic analysis for germ-line mutations of theRET proto-oncogene was performed in a series of 67 patients with apparently sporadic MTC to determine whether they were true sporadic cases or unsuspected de novo MEN 2 cases. DESIGN AND PATIENTS Sixty-seven unselected patients with sporadic MTC were randomly ascertained from clinic patients from four centres. The diagnosis of MTC was confirmed by hlstopathology. Germline DNA was extracted from peripheral blood leucocytes or from paraffin-embedded tissue and subsequently used for polymerase chain reaction amplification. MEASUREMENTS Polymerase chain reaction basedRET mutation analysis was performed by direct double-stranded cycle sequencing of axons 10, 11, 13 and 16, within which the majority ofMEN2 mutations have been shown to occur. RESULTS In this series, there was one proven case of germline mutation InRET codon 620, which previously has been shown to be responsible for MEN 2, thus Indicating heritable disease. No germline mutation in codon 918, typical of MEN 2B, was found. CONCLUSIONS A figure of 1.5% germline mutations In 67 apparently sporadic MTC Is lower than the incidence of familial disease reported in previous series Involving clinical and biochemical screening. The presence of a germline mutation in theRET proto-oncogene in a patient with MTC indicates heritable disease. The absence of germlineRET exon 10,11,13 or 16 mutation appears to rule out MEN 2A to a high probability, although the presence of a familial form of MTC other than classical MEN 2A cannot be excluded conclusively.     

Somatic trinucleotide change encompassing codons 882 and 883 of the RET proto-oncogene in a patient with sporadic medullary thyroid carcinoma

OBJECTIVE: Restriction analysis is a straightforward procedure for mutational analysis. It is commonly used for screening RET mutations. Incomplete digestion is a well-known cause of false results. Herein, we report another limitation of the method. DESIGN AND METHODS: Screening for somatic mutations in RET exons 16, 13 and 15 was performed in a patient with a sporadic medullary thyroid carcinoma. Genetic study was carried out by both restriction analysis and direct sequencing. RESULTS: A somatic trinucleotide change encompassing codons 882 and 883 of the RET proto-oncogene (GTA GCT to GTT TTT) was documented. Particular to this case is the silent mutation (GTA-->GTT) at codon 882. Independently, both the novel silent mutation and the missense mutation at codon 883 may disrupt the same AluI restriction site. Based on the restriction pattern we were able to say that both mutations occurred in the same allele. CONCLUSIONS: Restriction analysis is an easy approach for screening RET mutations; however, it is not enough to assign a final diagnosis.     

Diagnosis and therapy of sporadic and familial medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) is a rare thyroid malignancy. About 75% are sporadic (sMTC) while the remaining 25% are hereditary (hMTC). The treatment of choice for both sMTC and hMTC is surgery. An adequate initial operation provides the best chance of cure. Hence, the diagnosis of MTC should be made preoperatively. In sMTC, ultrasound, ultrasound-guided fine-needle aspiration cytology and measurement of calcitonin levels (basal and after injection of calcitonin-stimulating reagents, e.g., pentagastrin) are sensitive diagnostic tools. In hMTC, identification of a germline mutation in the proto-oncogene RET is sufficient for making the diagnosis. Total thyroidectomy is recommended in all patients, sporadic and hereditary. In addition, lymphadenectomy of the cervicocentral and both cervicolateral compartments should be performed. The only indication to perform a less extensive operation may be given in young patients with hMTC. Sufficient treatment of MTC beyond local disease is still non-existent. Future research should concentrate on this issue. Received: 16 May 2000 / Accepted: 16 June 2000