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RET Exon 11 (G691S) Polymorphism Is Significantly More Frequent in Sporadic Medullary Thyroid Carcinoma Than in the General Population

Departments of Endocrinology and Metabolism (R.E., B.C., C.R., V.B., M.S., A.P.), and Human and Environmental Sciences (R.B.), University of Pisa, 56100 Pisa, Italy; Section of Endocrinology (R.L.), Ospedale F. Lotti, 56025 Pontedera, Pisa, Italy; Department of Internal Medicine, Endocrinology, and Metabolism and Biochemistry (F.P.), University of Siena, 53100 Siena, Italy; and AMBISEN Center (R.B., A.P.), High Technology Center for the Study of the Environmental Damage of the Endocrine and Nervous Systems, University of Pisa, 56124 Pisa, Italy

Address all correspondence and requests for reprints to: R. Elisei, M.D., Department of Endocrinology, University of Pisa, Via Paradisa 2, 56124 Pisa, Italy. E-mail: relisei{at}endoc.med.unipi.it.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
The RET protooncogene is constitutively activated by point mutations in hereditary medullary thyroid carcinomas (MTCs). RET somatic point mutations have also been reported in 40–50% of sporadic MTCs. Several single nucleotide polymorphisms of the RET gene have been described in the general population as well as in patients with MTC. These allelic variants do not seem to confer any transforming activity to the tyrosine kinase domain of the RET gene. Because the exon 11 RET polymorphism determines an important aminoacidic variation (G691S), we studied its frequency in 212 subjects, 106 sporadic MTC patients and 106 normal age-, sex-, race-, and geographic origin-matched controls. In 46 cases of sporadic MTCs, we also studied the cosegregation of somatic RET gene mutation and G691S polymorphism as well as the linkage of the polymorphism with RET germline mutation in 60 members of eight multiple endocrine neoplasia type 2 families. The influence of this polymorphism on the RET gene transcription has also been studied. In parallel we analyzed the frequencies of another three neutral polymorphisms (L769L, S836S, S904S). We found a statistically significant (P = 0.029) higher allelic frequency of G691S polymorphism in MTCs (27.83%) than that found in normal controls (18.86%), at variance with the three neutral polymorphisms whose frequencies were not different in patients and controls. With this study we excluded the influence of the G691S polymorphism on RET mRNA expression, the development of the somatic RET mutation, the linkage with the germline RET mutation, the younger onset of the MTCs, and the clinical outcome of the disease. A putative role of the G691S polymorphism as genetic modifier in the normal subjects remains to be established.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
MEDULLARY THYROID CARCINOMA (MTC) derives from the parafollicular C cells and may develop in either sporadic or hereditary form (75–80% and 20–25% of cases, respectively) (1). The hereditary form may be isolated familial MTC or part of multiple endocrine neoplasia type 2. In the latter syndrome, MTC is associated with pheochromocytoma and/or parathyroid adenoma and other phenotypic abnormalities such as mucosal neurinomas and cutaneous lichen amyloidosis (2).

In 1993 RET protooncogene was shown to be involved in the pathogenesis of the hereditary form of MTC (3, 4). In the following years, several germline activating point mutations of this gene, affecting exons 10, 11, 13, 14, 15, and 16, were found to be associated with multiple endocrine neoplasia type 2 and familial MTC (5, 6). RET somatic point mutations and gene deletions were also described in 40–50% of sporadic MTCs (7, 8, 9, 10, 11, 12).

RET gene is also involved in the pathogenesis of Hirschsprung syndrome, a congenital disease characterized by the absence of the intramural ganglia in the hindgut resulting in functional intestinal obstruction (13). Loss of function germline and somatic RET mutations, mainly affecting the extracellular domain of the RET gene, are present in up to 50% and less than 30% of Hirschsprung familial and sporadic cases, respectively (14).

Several single nucleotide polymorphisms of the RET gene have been described in the general population (15, 16) as well as in patients with MTC (17, 18) and with Hirschsprung syndrome (19, 20). By definition, polymorphisms are allelic variants of a gene that do not alter the functional activity of the encoded protein, regardless of changes in the sequence of the protein. The gene will then code for a protein different to the wild-type but apparently without any phenotypic consequence. Among the seven known polymorphisms of the RET gene, only one (exon 11) determines an amino acidic substitution (G691S). Aims of this study were to analyze the frequency of G691S RET gene polymorphism in sporadic MTC patients and normal matched controls, verify its correlation with somatic and germline RET gene mutations and/or with the clinical features of MTC patients, study the influence of the G691S polymorphism on the RET gene mRNA transcription, and compare the frequency of the other three neutral RET gene polymorphisms localized in the portion of RET gene involved in the pathogenesis of MTC with that of G691S polymorphism.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

We studied 106 subjects, 48 males and 58 females, affected by sporadic MTC and 106 normal subjects, 44 males and 62 females, as controls. The 106 MTC cases selected for this study had no familial history of MTC and no germline RET point mutations in exons 10 or 11 or 13–16. The diagnosis of MTC was based on histopathology of thyroid tumors after thyroidectomy.

One hundred six healthy volunteers were selected as controls, and it was established that there were no relatives among the group or any relationship to the MTC patients. Control cases were matched with patients for age, sex, and geographic area of birth (Fig. 1). Informed consent to this study was given by both MTC patients and controls.

View larger version (23K): [in this window] [in a new window]   FIG. 1. Distribution of MTC patients and matched controls by age (decades) and sex.

We also analyzed 60 members of eight families, with the index case carrying a germline-activating RET mutation and G691S polymorphism to study the linkage of these two gene alterations in these pedigrees.

Genomic DNA extraction and PCR amplification

Two hundred microliters of blood were used for genomic DNA extraction using a method based on the proteinase K digestion (QIA amp kit, Quiagen, Valencia, CA). Genomic DNA was also extracted from 46 tissues with a homemade method based on an overnight proteinase K digestion at 55 C, followed by a phenol/chloroform extraction and an ethanol precipitation. DNA was kept in Tris EDTA at –20 C.

The genomic DNA of both patients and controls was amplified twice for exons 11, 13, 14, and 15 of the RET gene by PCR using a PTC-100 instrument (MJ Research Inc., Watertown, MA). In patients with MTC, exons 10 and 16 were also studied by sequencing analysis searching for somatic RET mutations. Because polymorphisms of these two exons have never been described and were not found in our MTC cases, we did not search for them in our controls. The PCR amplifications were performed following the method previously described (10) and using primers reported by Ceccherini et al. (15).

Sequencing and restriction analysis

Ninety-five microliters of one of the two PCR products were purified with a commercial kit (Concert rapid PCR purification system, Life Technologies, Inc., Gaithersburg, MD) and sequenced using an automated system employing dye fluorescent terminators (ABI Prism 310, Perkin-Elmer, Foster City, CA).

The presence or absence of each polymorphism within each amplicon was also assessed by restriction digestion of 20–30 µl of the second PCR product using the appropriate endonuclease able to recognize restriction sites created or deleted by the presence of a different nucleotide typical of the polymorphic allele (18). The digested products were electrophoresed on a 3% agarose gel to resolve the different digested products.

Real time RT-PCR

RNA was extracted from 46 MTC fresh-frozen tissues, and cDNA was then prepared and tested for quality as previously reported (21). Quantitative RT-PCR for RET mRNA was performed using the real-time sequence detection system 7700 (PE Applied Biosystems, Foster City, CA). A standard curve with serial dilution of cDNA (100–100,000 pg) obtained from total RNA extracted from TT human MTC cell line was constructed. The PCR primers and probe sequences were: sense, 5'-GCCCTCGCTGGACTCCAT-3'; antisense, 5'-CAAGTTCTTCCGAGGGAATTCC-3'; probe, 5'-FAM-AACCAGGTCTCCGTGGATGCCTTCAA-TAMRA-3'. Each sample was assayed in triplicate according to conditions recommended by Applied Biosystems. Samples omitting reverse transcriptase and cDNA were included in each run as negative controls of potential laboratory and/or assay contamination. cDNAs derived from nonthyroid cell line [Chinese hamster ovary (CHO)] and human poorly differentiated (NPA) and anaplastic (ARO and FRO) human thyroid carcinoma cell lines, which were expected not to express RET mRNA, were used as negative controls.

To normalize for differences in the amount of total RNA added to the reaction, amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was performed as endogenous control. Primers and probe for GAPDH were purchased from PE Applied Biosystems.

Statistical analysis

The statistical analysis of the frequencies of the polymorphic alleles was performed by ² test. P < 0.05 was considered as significant.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Frequency of RET G691S polymorphism and neutral variants

The analysis of G691S RET gene polymorphism showed that 49 of 106 (46.2%) MTC patients and 35 of 106 (33%) normal matched controls harbored a heterozygous and/or homozygous polymorphism. In particular, 39 MTCs and 30 controls were heterozygous and 10 MTCs and 5 controls were homozygous for the G691S RET polymorphism. These differences in the frequency of G691S polymorphism became statistically significant when comparing the allelic distribution (Table 1). In fact, the allelic frequency of RET G691S polymorphism in MTC patients was 59 of 212 (27.83%) and the allelic frequency of RET G691S polymorphism in controls was 40 of 212 (18.86%). By ² statistical analysis, RET exon 11 G691S polymorphism allelic frequency was significantly higher in MTC patients with respect to normal controls (27.83% vs. 18.86%, P = 0.029). On the contrary, the allelic frequency of the other neutral polymorphisms in exons 13–15 was not different in MTC cases and controls (Table 1). With the exception of the higher frequency of G691S polymorphism in the MTC patients, all the other polymorphism frequencies found in our controls and MTCs were similar to those previously reported in the general population (18). We also analyzed the distribution of the G691S polymorphic alleles (GG, GS, SS) in both MTC and normal subjects. We found that in the homozygous group (SS), the ratio of MTC/normals was 2:1 and in the heterozygous group (GS) the ratio was 1.3:1, whereas in nonmutated subjects (GG), the ratio was inversed 0.8:1, thus suggesting a greater influence of the biallelic presence of the polymorphism on the development of MTC. However, these differences did not show any statistical significance.

We also analyzed the cosegregation of RET G691S polymorphism with one or more of the other neutral polymorphisms in both MTC and normal subjects. G691S polymorphism showed a positive significant cosegregation with S904S (exon 15) polymorphism when compared with the association of any other polymorphism, both in MTCs and controls (P < 0.005). The frequency of S836S exon 14 polymorphism alone is quite rare (0.47% both in MTCs and controls). We also observed that exon 13 L769L polymorphism was rarely associated with other RET gene polymorphisms, with the exception of a cosegregation with S836S exon 14 polymorphism. When we compared the frequency of the cosegregation of exon 13 L769L polymorphism with S836S exon 14 polymorphism or with any other polymorphism, the difference was statistically significant (P = 0.0001) both in MTCs and controls (Table 2).

We compared the presence of the exon 11 polymorphism with the presence of a somatic RET gene mutation. Eighteen of 46 tumoral tissues (39.0%) showed a somatic RET gene mutation in exons 16, 15, 11, or 10. As shown in Table 3, 11 of 18 (61.1%) patients with RET somatic mutation also harbored one or more polymorphisms similarly to 20 of 28 (71.4%) patients with no somatic RET mutation. In particular, no cosegregation between RET mutation and G691S and/or any other neutral polymorphism was found (Table 3).

To measure RET mRNA, we used real-time quantitative RT-PCR with TaqMan assay. RET mRNA was detected in all samples tested with the exception of four cell lines not deriving C cells (CHO, NPA, ARO, and FRO), used as negative controls. The expression level was measured comparing RET mRNA absolute quantity with that of the housekeeping gene GAPDH. The relative mean ratio value of RET mRNA level in 46 MTC patients was 14.2 ± 31.63. There was no difference in RET mRNA expression in the tumors with or without RET G691S polymorphism (18.99 ± 46.2 vs. 14.23 ± 31.6) or any other polymorphism.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
In a small series of MTCs previously analyzed (22), we already demonstrated that the frequency of RET G691S polymorphism was significantly higher than that found in normal subjects (23.3% vs. 12.5%). In the present study, we confirmed the significantly higher frequency of this polymorphism in a larger series of 106 patients affected by MTCs with respect to that found in 106 normal race-, age-, sex-, and geographic origin-matched normal subjects. We found that the allelic frequency of exon 11 G691S polymorphism, which determines an amino acid substitution, is significantly more frequent (27.83%) when compared with the population matched controls (18.86%). Although the frequency of G691S exon 11 polymorphism is also well represented in normal subjects, the higher frequency of this polymorphism in the MTC patients might suggest a role of this genetic variant in the pathogenesis or predisposition to develop sporadic MTCs.

Recently it has been reported that G691S exon 11 polymorphism was associated with the C cell hyperplasia in the peritumoral tissue of differentiated radiation induced thyroid tumors (23). Because C cell hyperplasia is considered by many authors as a preneoplastic lesion (24), it is conceivable to hypothesize that the G691S polymorphisms can predispose to C cell hyperplasia. However, we were not able to find an association between the presence of C cell hyperplasia and G691S exon 11 polymorphism in our series of sporadic MTCs. Because about 60% of normal healthy subjects have detectable basal levels of calcitonin (25) and 33% of adult normal subjects showed C cell hyperplasia at autopsy (26), a genetic predisposition to develop C cell hyperplasia, based on the RET gene polymorphisms distribution, might also be present in normal subjects. Unfortunately at the moment it is difficult to demonstrate that normal subjects harboring the G691S polymorphism are at risk of developing MTCs. Only a long-term follow-up could give an answer to the question of whether the presence of G691S polymorphism is a true predisposition to the development of MTC. As a matter of fact, the two amino acids, glycine in the wild-type RET protein and serine in the polymorphic RET variant, confer different electrochemical and conformational structures to the RET protein, thus suggesting a different functional activity. A similar situation has been reported in diabetic patients for the Gln192Arg polymorphism of paroxonase gene, which was recently found to be strongly associated with a higher risk of developing cardiovascular disease (27). To support the hypothesis of a predisposing role of G691S polymorphism to MTC is the observation that, at variance with the higher frequency of G691S exon 11 polymorphism, we did not find any difference in the frequency of exons 13, 14 and 15 neutral polymorphisms between MTC and normal controls, in keeping with the idea that neutral polymorphisms should not have any functional role.

Because it has been reported that polymorphic sequence variants can lead to production of different amounts of mRNA (28), another hypothesis to explain the theoretical role of this polymorphism in the pathogenesis of MTCs is that the G691S allelic variant might influence RET mRNA expression. In this study we did not find any significant difference in the levels of RET mRNA, as assessed by real-time quantitative PCR, when comparing cases with or without G691S polymorphism. Similarly, the other neutral polymorphisms did not show any evident influence on the RET mRNA expression, thus suggesting that the polymorphic allelic variants do not modify transcriptional levels of the RET gene.

In a previous study (18), a significant association between S836S neutral polymorphism and the somatic M918T RET mutation has been found. In our series of 46 sporadic MTC tissues, we did not find any difference of polymorphism distribution between the group of mutated (n = 18) and nonmutated (n = 28) cases. The analysis of the cosegregation of G691S polymorphism with the RET mutation was further performed in a group of eight families, whose index case carried both RET alterations. We did not find any cosegregation of the germline RET mutation, with either G691S or the other neutral polymorphisms. It could be interesting to analyze the segregation of the G691S polymorphism with MTCs in families without any established RET mutation. However, we had only one family in this condition in our series of hereditary MTCs (47 families), but the affected members of the family were not found to carry the G691S polymorphism.

A similarly higher frequency of G691S polymorphism in MTC patients has been recently reported by Robledo et al. (29), who mainly focused their study on the cosegregation of G691S and S904S polymorphisms. We also observed a significant association (P = 0.001) of G691S and S904S polymorphisms in MTC patients. However, this frequency was not different from that found in normal controls, thus suggesting the absence of a predisposing role of the cosegregation to the development of MTC. Moreover, similar to Robledo et al. but at variance with Wiench et al. (30), we did not find any difference in the frequency of single or cosegregated polymorphisms and the age of diagnosis of MTC patients.

At variance with our data, no significant difference in the frequency of the RET G691S polymorphism was observed between MTC patients and controls in a study performed on a German-American population (18). The same authors reported that the frequency of the S836S (exon 14) neutral polymorphism was significantly overrepresented in their MTC patients with respect to the general population. Similar findings have been reported in two other studies (31, 32). On the contrary, in our series, the allelic variant frequency of the exon 14 S836S polymorphism was found to be lower (6.1%) in the group of affected patients with respect to that found in the group of normal controls (8.49%), although this difference was not statistically significant. These differences may reflect a random variability in the genetic background (Italian vs. German vs. Spanish) or may be due to a different sensitivity of the techniques used to detect RET gene polymorphisms. It is worth noting that we used two independent techniques, sequencing and restriction analysis, and that only cases with polymorphisms detected by both techniques were considered positive.

In conclusion, we found a significantly higher frequency of the RET G691S polymorphism in MTC patients with respect to normal controls. Because this polymorphism determines an aminoacidic variation, a predisposing role to develop MTCs can be postulated. However, we excluded the influence of the G691S polymorphism on RET mRNA expression, the development of the somatic RET mutation, the linkage with the germline RET mutation, the younger onset of the MTC, and the clinical outcome of the disease. A putative role of the G691S polymorphism as genetic modifier in the normal subjects remains to be established.

	Footnotes

 
This work was supported in part by grants from Ministero dell’Istruzione dell’Università e della Ricerca (ex 40%) 2002 and Associazione Italiana per la Ricerca sul Cancro.

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; MTC, medullary thyroid carcinoma.

Received November 3, 2003.

Accepted March 29, 2004.

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Top Abstract Introduction Patients and Methods Results Discussion References

 

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