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Laboratoire de Génétique, Hôpital Edouard Herriot, and CNRS UMR 5641, Université Claude Bernard (I.S., G.M.L.), Lyon, France; Service de Médecine Interne (M.V-M., P.J.G.), Hôpital Lariboisière, Paris, France; Unité d’Epidémiologie Génétique (R.B, D.G.), Centre International de Recherche sur le Cancer, Lyon, France; Service d’Endocrinologie (B.C.D), Centre Hospitalier Universitaire de Marseille, France; Service d’Endocrinologie (L.L.), Centre Hospitalier Universitaire de Lille, France; Service d’Endocrinologie (O.C.), Centre Hospitalier Universitaire de Grenoble, France; Service de Médecine Nucléaire (A.B.), Centre Claudius Régaud, Toulouse, France; Service de Médecine Interne (J.C.), Centre Hospitalier Universitaire de Reims, France; Service d’Endocrinologie (C.H.), Centre Hospitalier Universitaire de Rouen, France; Service d’Endocrinologie (E.M.), Hôpital Avicenne, Bobigny, France; Service de Médecine Interne (V.R.), Centre Hospitalier Universitaire d’Angers, France; Service de Médecine Nucléaire (M.S.), Institut Gustave Roussy, Paris, France; and Department of Adult Oncology (C.E.), Human Cancer Genetics Unit, Dana-Farber Institute, Harvard Medical School, Boston, Massachusetts; Service d’Endocrinologie, Centre Hospitalier Universitaire de Marseille, Marseille, France
Address all correspondence and requests for reprints to: Gilbert M. Lenoir, CNRS UMR 5641, Université Cloude Bernard, 8 avenue Rockefeller, 69373, Lyon cedex 08, France.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MEN 2 is caused by allelic germ-line mutations of the RET proto-oncogene, which codes for a transmembrane receptor tyrosine kinase protein whose natural ligand is a neurotrophin called GDNF (Glial cell line-derived neurotrophic factor) (10, 11, 12). The majority of mutations occurs in one of two functional regions of the RET protein. A unique point mutation of codon 918 in the tyrosine kinase domain has been identified, thus far, in more than 95% of MEN 2B patients (13, 14, 15, 16, 17). Missense mutations of one of five cysteine codons (609, 611, 618, 620, or 634) in the extracellular cysteine-rich domain have been identified in 98% of MEN 2A cases and in 85% of patients affected with FMTC (17, 18, 19, 20). Codon 634 mutations are, by far, the most frequent in MEN 2A patients, occurring in 85%. Finally, missense mutations at codon 768 or codon 804 in the tyrosine kinase domain have been shown to be responsible for 5–10% of the FMTC cases (17, 21, 22). Known MEN 2 mutations result in constitutive activation of the tyrosine kinase of the RET protein (23, 24, 25, 26, 27).
Correlations between specific mutations and clinical features in MEN 2 have been established. In particular, M918T is pathognomonic for MEN 2B. Also, the presence of pheochromocytoma and HPT in MEN 2 families (excluding MEN 2B) is associated with any mutation of codon 634. This association, found in two preliminary studies (19, 20), has been recently confirmed in the first pooled analysis of the International RET Mutation Consortium dataset (17). Association between the nature of the codon 634 mutation and the familial risk of HPT remains controversial. An association between the C634R mutation and the development of HPT was found in a study by Mulligan et al. (19) but was not observed in our previous study (20) nor in a study published by Frank-Raue et al. (28). Analysis of the Consortium data found a significant correlation between C634R and the presence of HPT in a given family (17), but this might have been influenced by the dataset of Mulligan et al.
To further investigate the C634R-HPT relationship, we analyzed a population of 188 individuals with codon 634 mutations, belonging to 30 French families for which there was comprehensive genotyping data and clinical and biochemical information regarding the diagnosis and follow-up of HPT. In this register-based study, we examined whether prevalence and penetrance of HPT were influenced by the nature of the mutation at codon 634.
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Materials and Methods |
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All the families and patients originated from the French national register of the Groupe d’Etude des Tumeurs à Calcitonine. About 200 families (from 20 participating centers), diagnosed with MEN 2, have been registered since 1984. Of these, 160 have been genotyped and 146 were found to have a RET mutation. Eighty-eight families were found to have a mutation at codon 634, of which 38 families had a C634R mutation, 32 had a C634Y mutation, and 18 had any other missense mutation. With the aim of studying prevalence and penetrance of HPT in relation to the nature of codon 634 mutation, we analyzed a subset of these 88 families: 10 with a C634R mutation, 11 with a C634Y mutation, and 9 with any other less frequent 634 mutation. These families were chosen because these were the only families with exhaustive genotyping data from individuals belonging to at least 2 generations and with comprehensive clinical and biochemical information regarding the diagnosis and follow-up of MTC, pheochromocytoma, and HPT. For each gene carrier, we sent a questionnaire to collect the following data: presence or absence of MTC, pheochromocytoma, and HPT; and age at positive diagnosis or age at last negative screening and biological and clinical data related to thyroid, adrenal, and parathyroid glands. Reliable data were obtained for 188 individuals.
DNA mutation analysis
In each family, identification of the predisposing mutation was obtained by sequencing the RET proto-oncogene, as described elsewhere (20). Genotyping of the relatives of each proband was done by restriction analysis of PCR products, as described by McMahon et al. (29).
Criteria for HPT diagnosis
In 33 patients (92%), the diagnosis of HPT was based on both documented biochemical and histological data, i.e. the association of a preoperative serum calcium level equal to or greater than 2.55 mmol/L-1 (and elevated PTH1–84 levels, if available) and the presence of adenoma or hyperplasia of 1 or more parathyroid(s). Immunoradiometric assay for PTH1–84 was available only for those patients screened after 1985. In 3 patients, the minimal criteria used for HPT diagnosis was the association of a preoperative calcium equal to or greater than 2.55 mmol/L-1 and either elevated PTH levels or clinical data compatible with HPT disease. We excluded HPT diagnosis when the calcium level was below 2.55 mmol/L-1, even in the presence of hyperplastic gland(s). For each of the patients diagnosed with HPT, clinical data regarding parathyroid disease were collected from the questionnaires. For the remaining 152 patients, HPT diagnosis was excluded on the basis of negative screening data.
Criteria for MTC and pheochromocytoma diagnosis
The diagnosis of MTC and pheochromocytoma was based on documented histological examination. MTC screening was considered negative on the basis of negative calcitonin stimulation tests. Pheochromocytoma screening was considered negative on the basis of normal values of 24-h collections of urinary metanephrines and/or catecholamines.
Statistical analysis
Analysis of prevalence data. To examine interfamilial
variability of the HPT prevalence, we compared the likelihood of the
observed data under a series of four models: 1) each family possesses a
unique family-specific risk of HPT; 2) the HPT risk in each family is
dependent on the specific RET codon 634 mutation segregating
in the family; 3) risk heterogeneity, i.e. there are two
types of families (one with a relatively high risk of HPT, the other
associated with a lower risk); and 4) a single HPT risk is appropriate
for all families. The likelihood of the observed data under each model
was computed, assuming that the number of cases of HPT observed in each
family followed a binomial distribution, with the appropriate
probability or set of probabilities estimated for each model. For model
3, the method of maximum likelihood was used to jointly estimate the
risk associated with the high risk group and the proportion of families
of the high risk type. Models were compared using Akaike’s information
criteria (AIC) and through 
-squared approximations to the likelihood
ratio test.
Penetrance analysis. To examine the cumulative probability of HPT for mutation carriers, we used a life-table approach. Kaplan-Meier curves were based on the age and phenotype of all 188 gene carriers. Individuals were considered to be affected at the age of their first diagnosis (see the criteria for diagnosis below), whether they were diagnosed by biochemical screening or at a clinical stage of MEN 2. Individuals not diagnosed with HPT were censored at the age of their last biochemical screening. From the life table results, age-specific penetrance was calculated as 1-si, where si is the Kaplan-Meier estimated survival fraction at age i. Differences in HPT penetrance between C634R and other mutations were assessed by the log-rank test. The S-plus (Stat-Sci Inc., Seattle, WA) package was used for penetrance estimation and testing.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of 188 patients with a RET 634 mutation, 36 had evidence of HPT disease. Thirty-three patients had both biochemical and histological evidence of the disease; 2 patients had documented hypercalcemia associated with elevated PTH1–84 but have not been operated yet; 1 had a serum calcium of 3.0 mmol/L-1 and a history of recurrent nephrolithiasis, but the histological report was not available.
Mean calcium observed in the 36 patients at HPT diagnosis was 2.92 ± 0.12 mmol/L-1 (range: 2.55–3.67 mmol/L-1); 15 patients had uniglandular involvement (13 patients with a solitary adenoma and 2 with a solitary hyperplasia), and 18 had multiple gland involvement.
Mean age at HPT diagnosis was 33.7 yr (12–70 yr). Thirteen patients
had clinical expression of their disease, of which 12 had a history of
nephrolithiasis (complicated with renal failure in 2 of them) and 1 had
fatal hypercalcemic crisis. Repartition of asymptomatic and symptomatic
HPT patients by age is given in Fig. 1
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HPT was found synchronously with MTC or pheochromocytoma in 31
patients. In 1 17-yr-old patient, biochemical screening for MTC and
pheochromocytoma was negative at the time of HPT diagnosis. In 1
29-yr-old woman, HPT occurred 11 yr after MTC. In the remaining 3
patients, clinical expression of HPT was the first presentation of the
MEN 2 syndrome, but CT levels were unknown and MEN 2A was not suspected
at the time of HPT diagnosis.
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Overall prevalence of HPT was 19.1%. The prevalence of HPT was
23.1% in families with a C634R mutation, 17.5% in families with a
C634Y mutation, and 16.3% in families with other mutations of codon
634 (Table 1). Consistent with our
previous findings (20), the mean prevalence of HPT did not vary
significantly in relation with the nature of the mutation
(X2 = 1.08). Among the four models tested, the model
allowing for heterogeneity of risk among the families proved to be the
most parsimonious (i.e. had the minimum AIC) compared with
the other three. The AIC for this model was 179.6, compared with values
of 199.0, 188.6, and 185.7 for the family-specific risk,
mutation-specific risk, and single-risk models, respectively. Under
this model, it was estimated that 40% of the families had an HPT risk
of 34%, whereas the remaining 60% were associated with a
lower risk of about 9%.
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Age-related penetrance was evident (Fig. 2). The penetrance rose to 14% by age 30
(95% confidence limit: 8–20%), 26% by age 40 (95% confidence
limit: 17–34%), 48% by age 60 (95% confidence limit: 29–61%), and
81% by age 70 (95% confidence limit: 42–95%). HPT penetrance was
compared between C634R carriers and patients with other mutations of
codon 634 (Fig. 3). Under the age of 35,
cumulative risks were similar in the two groups. At age 35, penetrance
of HPT was 28% in C634R carriers and 16% in other codon 634 mutated
individuals. At age 50, penetrance of HPT rose to 55% in C634R
carriers and only 28% in patients with other mutations of codon 634.
By age 60, penetrance of HPT rose to 70% in the former group of
patients and remained stable at 37% in the latter. Overall, the
penetrance of HPT was significantly higher in C634R patients
(P = 0.02).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present register-based study, the overall prevalence of HPT was similar to those already reported (4, 7). We could not find a significant association between a particular mutation and the familial risk for HPT, although the observed prevalence of HPT among families with a C634R mutation was slightly higher than the observed prevalence in families with other mutations of codon 634. Mulligan et al. had previously suggested a highly significant correlation between C634R mutation and HPT in MEN 2A families (23). In a preliminary study, we could not confirm this correlation in families from the French MTC register (20) and neither could Frank-Raue et al. in families from the German MTC register (27). The present data reinforce our first analysis. These data are of clinical importance, because they underline that all codon 634 mutation carriers are at some risk for HPT and should be screened for this disease.
Whatever the nature of the codon 634 mutation, observed HPT prevalence showed a high interfamilial variability. The statistical model that best fitted the observed data was that which favored the heterogeneity of the risk for HPT, with 40% of the families showing an HPT risk of 34% and 60% of families showing an HPT risk of 9%. It seems unlikely that this variability is caused by the effect of modifier genes closely linked to the RET locus, because the HPT cases do not seem to cluster in branches of a family.
Our register-based study provided the first estimate of the age-specific and mutation-specific penetrance of HPT in patients with a codon 634 mutation of the RET proto-oncogene. The probability of a predisposed individual developing HPT below the age of 30 represented a low, but significant, risk. The 18% figure observed at age 35 was in accordance with the overall prevalence figures estimated in this study and in other retrospective studies on MEN 2A-affected families. Estimation of HPT was quite imprecise beyond the age of 60 because of the small number of patients documented. Overall, the HPT penetrance curve was probably shifted to the right, because it was based on a mixed population of patients diagnosed by early biochemical screening and patients diagnosed when clinical symptoms of MEN 2 were evident. It is important also to emphasize that the natural history and, hence, the penetrance of HPT might be influenced by thyroid surgery. The mutation-specific penetrance data suggested a higher penetrance of the C634R mutation after 35 yr of age. Beyond this age, the relative risk for developing HPT was found to be 1.8 times higher in C634R carriers than in patients carrying any other 634 mutation. So, although we did not find a significant C634R-HPT correlation on a family-unit basis, C634R was associated with a higher penetrance of HPT within families, i.e. on an individual-unit basis. The differences initially observed between the Mulligan et al. data (19) and our previous results (20) may then be explained by and attributed to the ages of individuals ascertained for these family-as-unit studies. In our families, mean age at diagnosis was 31.6 yr; whereas in Mulligan’s study, individuals ascertained could have been older and, as a consequence, the C634R association revealed. The higher penetrance of HPT in C634R patients could be related to tissue-specific effects. According to Mulligan’s threshold model (19), each type of endocrine cell responds to a different quantum of mitogenic signal. C cells would be the most sensitive to the mitogenic effects of RET; adrenal chromaffin and parathyroid cells being less responsive. Any mutation of exon 10, 11, 13, or 14 would then result in MTC, whereas only the most transforming mutations (those of codon 634, and especially C634R) would have an effect on parathyroid tissue.
Biological, clinical, and histological characteristics of HPT were similar to those already described (4, 7, 8). However, in our series, 16 patients (44%) developed biological and/or clinical signs of the disease before 30 yr, whereas, it is usually noted that HPT develops after the third decade (2, 3, 4, 5). In addition, we were recently informed that, in one family (F 60) with a C634Y mutation, a 2-yr-old girl had biological stigmata of HPT and MTC. Therefore, we had evidence that the onset of HPT can be very precocious. These data underline the importance of an early screening of the disease, especially because calcium and PTH1–84 measurements are simple.
From a genetic point of view, the present study reaffirms the role of RET mutations in MEN 2A-associated HPT. Specific biological effects of these mutations on parathyroid tissue remain to be elucidated, but it is highly probable that the activating mutations of the codon 634 generate or predispose to hyperplasia of parathyroid tissue. Subsequently, other genetic events may be involved in the successive stages of proliferation and hence remain to be identified.
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Acknowledgments |
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Footnotes |
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2 A recipient of a fellowship of the Hospices Civils de Lyon and the
Centre National de la Recherche Scientifique.
3 The Lawrence and Susan Marx Investigator in Human Cancer Genetics;
supported by a Patterson Fellowship.
4 Supported by EMUL Grant 93061 from Assistance Publique des
Hôpitaux de Paris.
Received November 4, 1996.
Revised June 5, 1997.
Revised September 10, 1997.
Accepted October 9, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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patients with clinical signs of HPT; 
, patients with asymptomatic
HPT.
