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Polymorphisms in RET and Its Coreceptors and Ligands as Genetic Modifiers of Multiple Endocrine Neoplasia Type 2A

¹ Strangeways Research Laboratory, Cancer Research UK Department of Oncology, University of Cambridge; ² EAMGS Molecular Genetics Laboratory, Addenbrooke's Hospital, Cambridge, United Kingdom; ³ Human Cancer Genetics Programme, Centro Nacional de Investigaciones Oncologicas, Madrid, Spain; ⁴ Service d'Endocrinologie, CHU Timone, Marseilles, France; ⁵ Department of Internal Medicine, Ljungby Hospital, Ljungby, Sweden; and ⁶ Laboratoire de Génétique Humaine, Hopital Edouard Herriot, Lyon, France

Requests for reprints: Arancha Cebrian, Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 8RN, United Kingdom. Phone: 44-1223-740684; Fax: 44-1223-740147; E-mail: arancha{at}srl.cam.ac.uk.

	Abstract

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Germ line missense mutations in the RET proto-oncogene are responsible for the inherited cancer syndrome multiple endocrine neoplasia type 2A (MEN2A). The clinical presentation of the disease and the age at onset varies even within families, where patients carry the same mutation. These variations in phenotypes suggest a role for genetic modifiers, and recently, it has been reported that polymorphisms within RET (G691S/S904S) may have such a modifier effect on the age at onset. Here, we investigate whether this observed association could be confirmed in a larger set of 384 individuals from MEN2 families from four different European populations. In addition, we tested as modifiers four other single nucleotide polymorphisms (SNPs), which we have found in a previous association study of RET, its coreceptors, and ligands to be associated with the risk of developing sporadic medullary thyroid carcinoma. We could not replicate the association between G691S/S904S and modifier effects in MEN2A families in any of the four European families analyzed. Of the other SNPs tested, only RET A432A showed a positive weak effect on tumor spectrum within MEN2A, which requires replication in a larger series. (Cancer Res 2006; 66(2): 1177-80)

	Introduction

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The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2) can be divided in three clinically distinct forms: MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC; ref. 1). All forms are transmitted as an autosomal dominant trait with a high degree of penetrance and variable clinical expression and have in common MTC. In patients with FMTC, only the thyroid gland is affected, whereas patients with MEN2A may also develop phaeochromocytoma and primary hyperparathyroidism. Between 93% and 98% of MEN2A families have mutations of one of the five conserved cysteine residues in exon 10 (codons 609, 611, 618, and 620) or exon 11 (codon 634) in the extracellular domain of the RET proto-oncogene (2). Mutations in Cys⁶³⁴ are the most commonly found in MEN2A families.

The different specific activating mutations in RET are associated with different clinical types of MEN2A. The explanations for the genotype-phenotype correlations are not yet certain. Several lines of evidence suggest that they may result either from different levels of RET activation induced by different mutations or, in one particular class of mutation, from altered substrate specificity of the RET tyrosine kinase.

The range of phenotypic expression seen between families with different mutations and the variation within families with the same mutation provide a potentially interesting and tractable system for analysing both the relationship between phenotype and RET genotype and the effects of modifier genes. We have found six single nucleotide polymorphisms (SNPs) of the RET pathway to be associated with the risk of developing sporadic MTC (sMTC): GFR1 STOP+946bp (rs1061413), RET A45A in exon 2 (rs1800858), RET A432A in exon 7 (rs1800860), RET G691S in exon 11 (rs1799939), RET S904S (rs1800863), and ARTN START-797bp (rs3762422; ref. 3). In a recent report, Robledo et al. have shown that two of these RET SNPs (G691S and S904S) may modify the age at onset of MTC tumor in family members (4). We have analyzed all six of the variants in four sets of Caucasian MEN2A families of different geographic origin to assess their influence on age at onset and on MEN2 phenotype. Because the modifier effect of a variant could depend on the type of mutation, the analyses were done in two different groups; the first considering all RET mutations and the second including only mutations located at codon 634, which were the commonest in all four populations.

	Materials and Methods

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Subjects. Details of the study subjects are shown in Table 1. The U.K. patients were identified through the register of MEN2 families established by the Cancer Research Campaign (now Cancer Research UK) since 1982. The Spanish patients were those used by Robledo et al. (4) with the addition of 10 new MEN2 families. The French patients were recruited through the GETC database (5). One large Swedish family, referred to the U.K. register of MEN2, was also included in the study.

Genotyping. We genotyped all samples for selected polymorphisms using the ABI PRISM 7900 sequence detection system or Taqman (Applied Biosystems, Foster City, CA). We carried out PCR on DNA (20 ng) using 1x Taqman universal PCR master mix, forward and reverse primers (900 nmol/L), and FAM- and VIC-labeled probes (200 nmol/L) in a 5-µL reaction. Amplification conditions on an MJ Tetrad thermal cycler (Genetic Research Instrumentation, MJ Research, Cambridge, MA) were as follows: one cycle of 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. We read the completed PCRs on an ABI PRISM 7900 Sequence Detector and analyzed them using the Allelic Discrimination Sequence Detector Software (Applied Biosystems). For the software to recognize the genotypes, we included four nontemplate controls in each 384-well plate. We designed Taqman primers and probes using the Primer Express Oligo Design Software v2.0 (Applied Biosystems). All sequences are available upon request.

Statistical methods. Unconditional logistic regression was used to test for association between the phenotypes of interest and genotype, while controlling for study center. We have previously observed that each of the SNPs that was significantly associated with predisposition to sporadic MTC had a dominant mode of action (3). We therefore compared individuals who were homozygous for the common allele with those who were carriers of the rare allele (heterozygotes and rare homozygotes).

Permutation testing was used to allow for the effect of multiple hypothesis testing. Case-control status was randomly permuted within study strata and the number of times a P was obtained that was as small as that observed provides an indication of its true significance.

	Results and Discussion

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Genotype frequencies by age at onset of clinically apparent disease and tumor subtype are shown in Tables 2 and 3. Because G691S and S904S are in perfect linkage disequilibrium (6, 7), we analyzed only the nonsynonymous variant G691S, likely to affect the structure or the function of the tyrosine kinase receptor (3, 4). We could not confirm in our combined set of four different populations the association between G691S and age at onset previously reported by Robledo et al. (P = 0.29). This highlights the importance of replication in different populations of any significant association. Apparently, significant results in a small sample set may not be replicated when larger sets are analyzed.

We compared putative modifier genotype frequencies in patients who developed MTC alone to patients who additionally developed clinical symptoms of other characteristic tumors of MEN2A. We found a weak positive association between the SNP A432A (c.1296 g>a) in RET and phenotypic expression (Table 3). The rare allele for this variant (frequency 31% in the normal population) was overrepresented in patients carrying a mutation at codon 634 and who developed MTC alone compared with those with MTC and other MEN2 syndrome components (pheochromocytoma and/or hyperparathyroidism). This suggested a protective effect of c.1296-G/A with a 50% decreased risk of developing other tumors (P = 0.03). This result is consistent with our previous study in sMTC (3) in which the c.1296-A allele seems to protect the general population from developing sMTC, independently of the age at onset. However, this result should be treated with some caution because it may be a chance finding in the context of multiple testing. To examine this, we carried out permutation testing, in which a P 0.036 was achieved 141 times out of 10,000, indicating that a larger series of cases will be needed to assess whether the association is real or not. Subgroup analysis would in principle be useful; however, there are many different codon 634 mutations, and the most common comprises just 42%. Therefore, we do not have the power to show differences in the effect of different mutations.

Some support for a real association would come from a clear functional effect of the c.1296 g>a substitution. Codon 432 is located in cadherin-like domain 4 (8). Although this is not a conserved residue, an effect of synonymous codon usage on gene expression has been supported by the detection of epistatic interactions between nucleotides that are important in maintaining pre-mRNA/RNA secondary structures (9). The presence of the rare allele of SNP A432A might modify the mRNA folding, stability, and translation, leading to decreased amount of mutant 634 protein and consequently leading to less oncogenic activity. Despite this being a weak association, the fact that a similar association has been observed in the four different populations warrants the investigation of the effect of the A432A polymorphism on phenotypic expression in RET mutation carriers at residue 634 in other populations to determine if it is a common real association.

In conclusion, we have used 384 gene carriers from four different European populations; this is a large study for a rare disease, and we could not confirm the previously reported association between G691S and age at onset of the MEN2A syndrome. Our analysis of four other SNPs in RET and its coreceptors and ligands, which we had found to be associated with susceptibility to MTC in a large study of apparently sporadic cases, showed an inconclusive weak effect of the variant c1296 g>a in RET on the probability of multiple organs involvement in RET codon 634 mutation carriers, consistent with the effect of this variant on susceptibility to sporadic MTC.

Each of these results illustrates the necessity, when association studies are used to search for small effects in rare diseases, for very large data sets and for replication of positive results in independent samples.

	Acknowledgments

 
Grant support: Cancer Research UK.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank all the subjects who participated in this study.

	Footnotes

 
Note: F. Lesueur and A. Cebrian contributed equally to this work.

B.A.J. Ponder is a Gibb fellow of Cancer Research UK.

Received 8/22/05. Revised 10/20/05. Accepted 11/ 8/05.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 

1. Mulligan LM, Ponder BA. Genetic basis of endocrine disease: multiple endocrine neoplasia type 2. J Clin Endocrinol Metab 1995;80:1989–95.[CrossRef][Medline]
2. Eng C, Clayton D, Schuffenecker I, 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;276:1575–9.[Abstract/Free Full Text]
3. Cebrian A, Lesueur F, Martin S, et al. Polymorphisms in the initiators of RET signalling pathway and susceptibility to sporadic medullary thyroid carcinoma. JCEM 2005;90:6268–74.[Abstract/Free Full Text]
4. Robledo M, Gil L, Pollan M, et al. Polymorphisms G691S/S904S of RET as genetic modifiers of MEN 2A. Cancer Res 2003;63:1814–7.[Abstract/Free Full Text]
5. Niccoli-Sire P, Murat A, Rohmer V, et al. Familial medullary thyroid carcinoma with noncysteine ret mutations: phenotype-genotype relationship in a large series of patients. J Clin Endocrinol Metab 2001;86:3746–53.[Abstract/Free Full Text]
6. Gil L, Azanedo M, Pollan M, et al. Genetic analysis of RET, GFR alpha 1 and GDNF genes in Spanish families with multiple endocrine neoplasia type 2A. Int J Cancer 2002;99:299–304.[CrossRef][Medline]
7. Rossella E, Cosci B, Romei C, et al. RET exon 11 (G691S) polymorphism is significantly more frequent in sporadic medullary thyroid carcinoma than in the general population. J Clin Endocrinol Metab 2004;89:3579–84.[Abstract/Free Full Text]
8. Anders J, Kjar S, Ibanez CF. Molecular modeling of the extracellular domain of the RET receptor tyrosine kinase reveals multiple cadherin-like domains and a calcium-binding site. J Biol Chem 2001;276:35808–17.[Abstract/Free Full Text]
9. Duan J, Wainwright MS, Comeron JM, et al. Synonymous mutations in the human dopamine receptor D2 (DRD2) affect mRNA stability and synthesis of the receptor. Hum Mol Genet 2003;12:205–16.[Abstract/Free Full Text]

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