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Cancer Research-UK Department of Oncology, University of Cambridge (F.L., A.Ce., A.Cr. J.L., B.A.J.P.), Strangeways Research Laboratory, Cambridge CB1 8RN, United Kingdom; West Midlands Hospital (T.M.F.), Colman Hill, Halesowen B63 2AH, United Kingdom; Department of Medicine, Watford General Hospital (M.R.C.), Watford, United Kingdom; Department of Human Genetics, Molecular Pathology Program (M.R.), Centro Nacional de Investigaciones Oncologicas, Madrid, Spain; and East Anglian Medical Genetics Service Molecular Genetics Laboratory, Addenbrooke’s Hospital (J.W.), Cambridge, United Kingdom
Address all correspondence and requests for reprints to: A. Cebrian, Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 8RN, United Kingdom. E-mail: arancha{at}srl.cam.ac.uk.
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Abstract |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResults and DiscussionReferences |
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The clinical effect of mutations in codon 804 is disputed. Some studies have suggested that the V804L RET mutation causes low penetrance disease, with late onset and a relatively indolent course (3, 4, 5), whereas other authors have reported that V804L and V804M mutations are more aggressive (6, 7). Clearly there is wide clinical variability (7, 8), and there is not yet agreement as to the therapeutic strategies for mutation carriers. In vitro studies have shown that this mutation is more weakly activating than mutations in exon 10 or 11 (3).
We report three patients with different homozygous mutations at codon 804 (V804M and V804L), and we compare their clinical features and age at diagnosis to six heterozygous cases from the same populations as well as to others previously reported. No clear differences in the age at diagnosis or clinical features were found between the homozygous and heterozygous patients.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResults and DiscussionReferences |
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In the total set of 306 cases, nine were found to have a codon 804 mutation, of which three were homozygous. None of these patients had other affected relatives. Two of the homozygous patients were from the United Kingdom. A woman who had a total thyroidectomy at age 37 yr for MTC and developed pheochromocytoma at age 39 yr was found to be homozygous for V804L (patient M8 in Table 1
). Her asymptomatic 33-yr-old son was heterozygous for the same mutation (Fig. 1A). The second patient, a 54-yr-old woman, was homozygous for V804M (patient M1 in Table 1 and Fig. 1B). She presented with MTC only. In neither case was parental DNA available to study carrier status. However, all parents were clinically without evidence of disease, even at advanced age and despite the obligate carrier status for at least one of each set of parents. The third homozygous patient was from Spain (M6). She developed MTC at the age of 32 and was found to be homozygous for V804M. In this case, we could confirm the heterozygous status for both asymptomatic parents, who were relatives. Patients and asymptomatic carriers were followed up annually with clinical examination, basal calcitonin determination, calcium and PTH determinations, and urinary measurements of catecholamines and derivatives. Signed informed consent for this study was obtained from each patient.
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Results and Discussion |
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TopAbstractIntroductionPatients and MethodsResults and DiscussionReferences |
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A second possibility is the germline loss of one allele of RET. To test this hypothesis, we sequenced four different previously described polymorphisms that flanked codon 804 (A45A in exon2, A432A in exon7, G691S in exon 11, S904S in exon 15) (10). Two different alleles at codon 691 and at codon 904 were present in patient M1 (Table 1
), suggesting that two mutant 804 alleles were present and of separate origin. The parents of patient M6 were related, and each was heterozygous for mutation 804, indicating that M6 was homozygous as the result of consanguinity. Regarding patient M8, all the four polymorphisms tested were homozygous, and neither detection of one allele nor consanguinity could be excluded.
We compared the age at diagnosis, tumor spectrum (MTC and pheochromocytoma), and presence of metastases in our three homozygous patients, six heterozygous cases, and other codon 804 heterozygote subjects with MTC from the literature (Table 1). Considering first V804M, two homozygote subjects were compared with nine heterozygote subjects. There is no systematic difference between homozygote and heterozygote subjects in age at diagnosis; no cases had pheochromocytoma, and none had metastases. Among the V804L cases, one homozygote was compared with seven heterozygotes. The homozygote patient (M8) is one of the two patients to have both MTC and pheochromocytoma, generally regarded as associated with a more strongly activating mutation. In patient M8, the age at diagnosis of MTC at 37 yr was also younger than most of the heterozygous cases; but one heterozygote patient, P5, had extensive metastatic disease, with very early diagnosis at age 12. Two others (P7 and P9) had metastatic disease at older ages (8). There is, therefore, in this small number of cases, no evidence of a more severe phenotype in homozygotes.
Clinical and in vitro evidence suggests a dosage effect of RET activity in relation to clinical phenotype in both gain of function (MEN2) and loss of function (HSCR) mutations (11, 12). It may seem surprising, therefore, that we have seen no decisive effect of homozygous mutation. However, the comparison between homozygote and heterozygote subjects is heavily biased, because we have included only heterozygote patients who presented clinically with MTC. The incomplete penetrance of codon 804 mutations has been well documented. For example, in the family reported by Lecube et al. (5), none of the individuals with heterozygous mutation manifested the disease. Similarly, in the United Kingdom families in our series, the majority of family members shown to carry a single copy of the 804 mutation were clinically normal. Analogous findings were reported in a family with the weakly transforming mutation A883T (13).
Taken together, these observations support the model, previously proposed by others (5, 13), in which individuals heterozygous for weakly transforming mutations of RET require a second germline or somatic mutation in RET or a pathway gene to result in clinical expression of the disease. The occurrence of these second mutations, and their transforming ability, would account for the observed clinical variability in expression of the very low transforming activity germline mutations. In support of this model, patient P5 (Table 1), who presented at age 12 and who had extensive metastatic disease, had a strongly activating somatic mutation (RET M918T) in her tumor (8). Other studies have reported somatic RET mutations in MTC tumors from patients with germline mutations (14, 15). We were unable to investigate this in the present series because of unavailability of pathological material.
Alternatively, there may be a contribution from additional germline mutation. There are several reports of different variants of RET and of its ligands and coreceptors acting as phenotypic modifiers of MEN2 and Hirschsprung disease (HSCR) (Refs. 16, 17, 18, 19, 20 and our unpublished data). There are also three reports of additional different germline mutations (at codons 806, 844, and 904) in patients who have a germline mutation in codon 804 (21, 22, 23). In the patient with the 804/806 mutation, there was in vitro evidence that the compound mutant allele had a much greater transforming activity than was seen with either mutation acting alone. This patient had a severe phenotype resembling MEN2B, which has not been described in association with codon 804 mutation alone (22). However, in each of these cases, the mutations were on the same allele. To date, there have been only two reported cases of biallelic RET germline mutation, both homozygous. Both were associated with clinical FMTC. In one, the family had a V804M mutation (5), and in the other, a novel A883T (13). Both cases showed a history of parental consanguinity, and the mutations were associated with MTC only in the homozygous condition. In the 804 mutation family reported by Lecube et al. only two of four homozygous mutation carriers manifested MTC, but the two without evidence of disease were only 10 and 5 yr of age. The authors speculate that the low transforming activity of the germline mutations at codons 804 and 883 may be augmented in the homozygous state, or by mutation at another site, as in the model we propose here.
To test the model further, we searched for a second germline mutation in the 804 heterozygous cases, by sequencing exons 1–9, 12, and 17–20 in the five patients in our series from the United Kingdom. Of these, we found one (patient M5) with a second mutation (R982C, CGC>TGC). We did not determine whether this was on the same allele as the 804 mutation. No second mutation or sequence variation was found in the other four patients. The significance of the R982C change is not certain. We did not find, in 100 chromosomes from healthy controls, consistent with the previous report, (24) that its frequency in the population was less than 1%. This variant was observed in a family with MEN2A and HSCR (25), and in HSCR patients (26), but the functional consequences of the amino acid change are unknown.
In summary, we describe three additional cases of homozygous germline mutation in RET, apparently unrelated. In one case, analysis of flanking genetic variants suggested that two distinct mutant alleles were involved. Three of the nine 804 mutation cases in our series of apparently sporadic MTC were homozygotes, consistent with previous data that in the heterozygous state weakly transforming mutations of RET have low penetrance for clinical disease, and require a second mutation to become manifest. There are currently insufficient data to estimate in what proportion the presumed second events are germline, in RET or pathway genes; or somatic, in the C-cell population. That four of the five homozygous mutation families that involve codon 804 and have been described so far may reflect the relative frequency of mutation at this codon or the transforming activity of the homozygotes, compared with the other weakly transforming mutations in RET. The lack of compound germline biallelic mutations affecting different codons presumably reflects the rarity of these alleles and so the probability (notwithstanding our patient M1) that most biallelic mutations will result from consanguinity.
Most data on germline mutations in RET in MTC relate to exons 10, 11, and 13–16, where the known mutations have been described. The model for clinical variability in codon 804 and in similar weakly transforming mutations suggests that careful sequencing of the gene should be considered in heterozygous individuals within families, and in tumor from heterozygous individuals when available, to search for other sequence changes. Such data would consolidate the model and, from the clinical standpoint, would indicate the contribution of second germline mutations and thus the possibility of using this information for prediction of the probability of clinically significant disease.
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Acknowledgments |
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Footnotes |
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First Published Online March 1, 2005
1 F.L. and A.Ce. contributed equally to this work. 
Abbreviations: FMTC, Familial MTC; HSCR, Hirschsprung disease; MEN2A, multiple endocrine neoplasia type 2A; MTC, medullary thyroid carcinoma.
Received August 13, 2004.
Accepted February 23, 2005.
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-1 but not GFR-2 or GFR-3 in cases with sporadic medullary thyroid carcinoma. Oncogene 20:2161–2170[CrossRef][Medline]
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