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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 11 5438-5443
Copyright © 2003 by The Endocrine Society

A Novel Germ-Line Point Mutation in RET Exon 8 (Gly533Cys) in a Large Kindred with Familial Medullary Thyroid Carcinoma

Adriana M. Álvares Da Silva, Rui M. B. Maciel, Magnus R. Dias Da Silva, Silvia R. C. Toledo, Marcos B. De Carvalho and Janete M. Cerutti

Laboratory of Molecular Endocrinology, Division of Endocrinology (A.M.A.D.S., R.M.B.M., M.R.D.D.S., J.M.C.), and J. F. Perez Genomic Center (R.M.B.M., M.R.D.D.S.), Department of Medicine; Division of Genetics (J.M.C.), Department of Morphology and Institute of Pediatric Oncology (S.R.C.T.), Department of Pediatrics, Escola Paulista de Medicina, Federal University of Sao Paulo; Division of Genetics and Biotechnology (A.M.A.D.S.), Santo Andre Foundation; and Service of Head and Neck Surgery (M.B.D.C.), Heliopolis Hospital, 04039-032 Sao Paulo, Brazil

Address all correspondence and requests for reprints to: Janete M. Cerutti, Ph.D., Laboratory of Molecular Endocrinology, Division of Endocrinology, Department of Medicine, Universidade Federal de Sao Paulo, Rua Pedro de Toledo 781, 12 andar, 04039-032 Sao Paulo SP, Brazil. E-mail: cerutti-endo{at}pesquisa.epm.br.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Familial medullary thyroid carcinoma is related to germ-line mutations in the RET oncogene, mainly in cysteine codon 10 or 11, whereas noncysteine mutations in codons 13–15 are rare. We now report a new missense point mutation in exon 8 of the RET gene (1597G->T) corresponding to a Gly533Cys substitution in the cystein-rich domain of RET protein in 76 patients from a 6-generation Brazilian family with 229 subjects, with ascendants from Spain. It is likely that the mutation causes familial medullary thyroid carcinoma (FMTC), because no other mutation was found in RET, the mutation cosegregates with medullary thyroid carcinoma (MTC) or C cell hyperplasia (CCH) in patients subjected to surgery, and family members without the mutation are clinically unaffected. The histological analysis of 35 cases submitted to thyroidectomy revealed that 21 patients had MTC after the age of 40 yr and 8 before the age of 40 yr, 4 presented MTC or CCH before the age of 18 yr, 2 died due to MTC at the age of 53 and 60 yr, and CCH was found in a 5-yr-old child, suggesting a clinical heterogeneity. To improve the diagnosis of FMTC, analysis of exon 8 of RET should be considered in families with no identified classical RET mutations.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
MEDULLARY THYROID CARCINOMA (MTC) presents either sporadically or in hereditary forms (1). In hereditary forms, MTC and/or its precursor lesion, C cell hyperplasia (CCH), is the single component in familial MTC (FMTC) or is part of the multiple endocrine neoplasia type 2 syndromes (MEN2A and 2B). In MEN2A, in addition to MTC, 50% of patients have a pheochromocytoma, whereas parathyroid hyperplasia or adenoma is observed in 10–30% of the cases. MEN2B syndrome is recognized by MTC, pheochromocytoma, and developmental abnormalities such as marfanoid habitus, skeletal abnormalities, mucosal neuromas, and diffuse intestinal ganglioneuromatosis; parathyroid disease is not observed (1, 2, 3, 4).

Activating germ line mutations of the RET oncogene predispose to hereditary MTC (1, 2, 3, 4, 5). The RET gene is located on chromosome 10q11.2, comprises 21 exons and encodes a tyrosine kinase receptor. Like other tyrosine kinases receptors, RET protein has extracellular, transmembrane, and cytoplasmic domains. The extracellular domain includes regions with homology to the cadherin family and a large cysteine-rich region, and the intracellular domain functions in the phosphorylation of tyrosine residues involved in the interaction with downstream targets and activation of signaling pathways (1, 2, 3, 6). Under normal conditions, RET receptor is activated by a multicomponent complex involving one of its ligands (glial cell line-derived neurotrophic factor, neurturin, artemin, and persephin) and one of their cell surface-bound coreceptors (respectively, GFR{alpha}-1, GFR{alpha}-2, GFR{alpha}-3, and GFR{alpha}-4) (1, 2, 3, 6).

Almost all mutations in MEN2A involve one of the cysteines in the extracellular domain of RET that are encoded by exons 11 (codon 634) or 10 (codons 609, 611, 618, and 620). In MEN2B a single mutation has been found in 95% of patients, Met918Thr, in the tyrosine kinase domain of RET encoded by RET exon 16. In FMTC, RET gene mutations involve either a cysteine codon in exon 10 or exon 11 or, less often, codons 768, 790, and 791 in exon 13; codon 804 in exon 14; and codon 891 in exon 15 (4, 5, 7, 8). In addition, Pigny et al. (9) described an FMTC with a 9-bp duplication in exon 8, which creates an additional cysteine residue in the extracellular cysteine-rich domain of RET.

In this paper we describe a new missense point mutation in exon 8 of RET gene (1597G->T) that corresponds to a Gly533Cys substitution in the cystein-rich domain of RET protein in 76 patients from a 6-generation Brazilian family with 229 subjects of Caucasian background. In addition, DNA-based analysis of the RET oncogene enables the identification of 2 new polymorphisms in intron 8 and 2 already described polymorphisms in exons 7 and 15.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Family history

The pedigree of the family is depicted in Fig. 1Go. This large family is of Caucasian origin, with ascendants from Barcelona, Spain, who immigrated to Brazil in the 19th century, resides mainly in the southeastern region of Brazil, and consists of 6 generations with a total of 229 individuals. Before undergoing genetic testing, a signed letter of informed consent was obtained from all patients or their legal guardians. The study was approved by the ethics and research committee of Universidade Federal de São Paulo and Hospital Heliópolis and was in agreement with the 1975 Helsinki statement, revised in 1983.



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  		FIG. 1. Pedigree of the six-generation family with FMTC. Circles and squares denote female and male family members, respectively. Solid circles indicate subjects with MTC.

 
The index case (IV.77; Fig. 1Go) is a 49-yr-old man, asymptomatic until the age of 47 yr, when he noticed a nodule in the right lobe of the thyroid gland. Thyroid ultrasound showed an 18-mm nodule in the left lobe and a 35-mm nodule in the right lobe. The basal serum calcitonin (sCT) level was 1080 pg/ml (normal range, <11.5 pg/ml). Total thyroidectomy with selective neck dissection (central compartment, level VI) was performed, and histological examination showed the presence of MTC, CCH, and microscopic metastatic disease in three lymph nodes with extracapsular extension to adjacent tissues. His last sCT determination, after surgery, was 181 pg/ml; work-up performed showed no radiological evidence of metastatic disease.

The proband’s mother, a 78-yr-old woman (III.18) had a thyroid nodule in 1972 at the age of 47 yr, when she underwent total thyroidectomy and postoperative irradiation for undifferentiated thyroid carcinoma. Thirty years later, at the time of her son’s surgery, the paraffin-embedded tissue from the thyroid tumor was reviewed and revealed that it was a MTC. Despite being asymptomatic, she has residual MTC, as her sCT is 2200 pg/ml. She has refused further investigation.

During the genetic screening, the proband’s daughter (V.121), a 22-yr-old woman, was identified as a gene carrier and had a basal sCT of 385 pg/ml; she underwent total thyroidectomy and neck dissection, and histological analysis confirmed the presence of MTC with lymph node metastases. Similarly, the proband’s son (V.122), a 21-yr-old man, was identified as a gene carrier. He exhibited increased sCT in response to pentagastrin (Pg; 0.5 µg/kg during 3 min) stimulation (441 pg/ml) and underwent total thyroidectomy; the pathology was consistent with MTC.

There is consanguinity in one of the branches of the family; as can be observed on the right of Fig. 1Go. Patients II.7 and II.8 were cousins, as were patients IV.70 and IV.71.

All family members were evaluated by their own physicians, who referred to us the medical history, physical examination, and results of sCT, serum calcium, and urinary metanephrines; sCT was determined by a chemiluminescence immunoassay from Nichols Institute (San Juan Capistrano, CA; normal values, <11.5 pg/ml). There was no clinical or biochemical evidence of pheochromocytoma, parathyroid disease, or other associated clinical features attributed to MEN2.

Control group

One hundred DNA samples were obtained from healthy volunteers, aged 20–65 yr, living in the southeastern region of Brazil.

DNA analysis

Genomic DNA was extracted from peripheral blood lymphocytes using a GFX Genomic Blood DNA Purification Kit (Amersham Pharmacia Biotech, Piscataway NJ), and DNA from the tumor was extracted using a phenol/chloroform (Life Technologies, Inc., Grand Island, NY) standard method. Primers specific for exons 7–19 of the RET gene (GenBank accession no. AJ243297) were designed and used to generate PCR products, which were purified and directly sequenced. Briefly, the PCR reaction was performed employing 200 ng genomic DNA in a 50-µl volume containing 10 mmol/liter Tris-HCl (pH 8.3), 50 mmol/liter KCl, 200 µmol/liter of each deoxy-NTP, 1.5 mmol/liter MgCl2, 1 U Taq DNA polymerase (Life Technologies, Inc.), and 25 pmol of each specific primer (Life Technologies, Inc., Sao Paulo, Brazil). Cycling conditions to generate PCR products included an initial phase of 5 min at 94 C, followed by 35 cycles of 45 sec at 94 C, an annealing temperature step of 1 min, and an extension step of 1 min at 72 C. PCR products were resolved by electrophoresis in a 1.5% agarose gel stained with ethidium bromide, purified using the CONCERT Rapid PCR Purification System (Life Technologies, Inc., Gaithersburg, MD), and quantified using a DNA mass ladder (Life Technologies, Inc.). Purified PCR products (150 ng) were submitted to direct sequencing using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit and the ABI 3100 sequencer (PE Applied Biosystems, Foster City, CA). Each sample was sequenced at least twice and in both directions. The PCR specific primers, product sizes, and annealing temperatures are summarized in Table 1Go.


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  		TABLE 1. Primers used to amplify exons 7–19 of the RET gene

 

   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
We first screened the proband’s DNA by looking for RET gene mutations in exons 10, 11, 13, 14, and 15, where most of the RET mutations were found in MEN2. As we have not found a mutation that could be associated with the tumor phenotype, we extended the analysis to exons 7–9, 12, and 16–19. A novel mutation located at nucleotide 1597 in the exon 8 of the RET gene was identified in DNA samples obtained from both peripheral blood and tumor tissue of the index case (IV.77). This missense point mutation arises in heterozygosis and causes a substitution of glycine to cysteine residue at codon 533 (Gly533Cys) in the cysteine-rich extracellular domain of the RET protein; this substitution is a G to T transversion and is displayed as two peaks in Fig. 2Go, differently from the single peak observed in the wild-type alleles.



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  		FIG. 2. Direct sequencing of PCR products from exon 8 of the RET oncogene in a normal control and the index case. In the normal control a GGC was observed, whereas in the index case a GGC/TGC was found due to a G to T transversion. The mutation in heterozygosis is displayed as two peaks, in contrast to the one peak observed in the wild-type alleles. The mutation occurs at nucleotide 1597, which corresponds to codon 533.

 
Direct sequencing of RET exon 8 PCR products from genomic DNA of family members showed that 76 of 229 members were carriers for this mutation, and 153 were unaffected. In addition, 14 members of the family were not tested. Eleven of those 14 cases died many years before the genetic testing; 2 died due to MTC with metastatic disease (II.6, III.20), 1 died due to heart disease, but had MTC (II.7), and 8 died due to other causes. Three of those 14 cases refused to perform the genetic test. However, all 14 cases were definitely carriers, because they or their descendants had MTC (Table 2Go).


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  		TABLE 2. Clinical characteristics of 14 carriers not submitted to genetic screening

 
To date, 35 of the 76 gene carriers have undergone total thyroidectomy. Histological examination showed the presence of MTC in 29 patients and of CCH in 6 patients. CCH was described in only 5 patients from 29 with MTC, probably due to methodological differences in processing the tissues for histology (Table 3Go). The postoperative calcitonin levels were undetectable in 20 patients and elevated in 8. The 7 remaining cases were recently submitted to thyroidectomy, and their own physician requested the postoperative sCT measurement. Of these 8 patients with elevated sCT, 6 presented lymph node metastases (III.17, III.18, IV.65, IV.77, IV.87, and V.121), and 2 had no evidence of lymph node involvement (IV.68 and IV.91). Forty-one gene carriers have not yet undergone surgery; 3 have scheduled surgery; 10 presented low levels of sCT after stimulation with Pg, and surgery was delayed; 24 did not complete the clinical evaluation because they had recent molecular diagnosis; 3 refused further clinical investigation; and 1 refused surgery. All 153 non-carriers of RET Gly533Cys mutation family members had normal levels of sCT (normal range, <11.5 pg/ml) and, consequently, were excluded from further clinical investigation.


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  		TABLE 3. Clinical characteristics of 35 gene carriers submitted to total thyroidectomy

 
The clinical course of MTC has been variable in this family (Tables 2Go and 3Go). Before genetic screening, two patients died due to MTC after protracted illness and metastasis. The first patient was a woman (II.6) who died at 53 yr of age after 14 yr of disease; the second patient was a man (III.20) who died at 60 yr of age after 7 yr of disease. Both patients underwent total thyroidectomy, whose histological examination demonstrated the presence of MTC, and before death both had evidence of distant metastatic disease. On the other hand, several gene carriers died at older ages due to other causes, without clinical manifestations of MTC (II.1, II.10, III.4, III.5, III.15, and III.16). In addition, two patients positive for the Gly533Cys mutation, a 75-yr-old man (III.6) and a 69-yr-old man (III.21), are asymptomatic, notwithstanding that they have refused clinical investigation.

Among all family members identified by genetic screening, the youngest affected patient is a 5-yr-old girl (V.145) who exhibited increased sCT after Pg stimulation (basal sCT, 8 pg/ml; after Pg, 120 pg/ml; normal, 40 pg/ml). She underwent total thyroidectomy, and histological examination showed the presence of CCH. The youngest patient to present MTC and lymph node metastases is the 22-yr-old proband’s daughter (V.121; Table 3Go).

Additionally, 2 new single nucleotide polymorphisms (SNPs) were detected in intron 8 of the RET gene, corresponding to an A to G variation at nucleotide 127810 (127810A->G) and an insertion of a C after nucleotide 127813 (127813insC; GenBank accession no. AJ243297) in 11 of 76 affected family members. Also, 2 previously described SNPs were detected in the proband DNA samples; these polymorphisms are 1296G->A in exon 7 and 2712C->G in exon 15.

We did not find the 1597G->T substitution in the genomic DNA of 100 healthy individuals. However, the 127810A->G substitution and 127813insC insertion were found in 23% of them.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present report we describe a novel RET mutation in exon 8, codon 533, which causes substitution of a glycine by a cysteine in the cysteine-rich domain of the RET receptor in a 6-generation FMTC family composed of 229 members and 76 carriers. According to the last consensus statement on MEN2 (5), only 1 abnormality on RET exon 8 has been previously reported by Pigny et al. (9), who identified a FMTC family with 4 affected members with a germ-line 9-bp duplication in the same locus, creating an additional cysteine residue after codon 531.

Several facts indicate that this mutation is associated with the tumor phenotype. First, in the index case, no other germ-line mutation was found in exons 7–19 of the RET gene. Second, the mutation cosegregates with the MTC or CCH in all 35 patients already submitted to surgery. Third, those family members without the mutation are clinically unaffected. Fourth, the cysteine-rich domain is extremely conserved among different organisms (Table 4Go), and probably, the rupture in the balance of cysteine residues causes constitutive phosphorylation of RET protein. However, to demonstrate that Cys533Gly in the RET receptor causes ligand-independent activation, similar to the other cysteine mutations described in MEN2A and FMTC families, in vitro analysis should be performed. It is interesting to note that the described mutation, in which a glycine is changed by a cysteine in the codon 533, is in the same region of the previously described duplication (9), where the 9-bp duplication leads to the introduction of an extra cysteine residue. We therefore believe that this mutation causes the disease.


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  		TABLE 4. Amino acid alignment of the RET thyrosine kinase receptor of six different organisms

 
According to the consensus statement on MEN2 (5), the reported kindred can be rigorously characterized as FMTC and not MEN2A, as there are more than 10 carriers in the family, multiple carriers or affected members are over 50 yr of age, and an adequate medical history has been obtained.

The results of the molecular studies also revealed the presence of 4 polymorphisms. The follow-up of patients with these polymorphisms will allow us to investigate whether the presence of 1 or any of the SNPs could be associated with a different course of the disease, contributing to earlier onset of MTC, as recently suggested for other RET mutations (10, 11, 12). Interestingly, the SNPs in intron 8 of the RET oncogene was found in 11 of 76 affected family members and in 23 normal controls. As the initial reverse primer was designed in exactly the same spot of the SNPs, one of alleles was not amplified by PCR, suggesting a mutation in homozygosis. A new upstream primer was designed, and the PCR product sequencing showed that the mutation occurred in heterozygosis.

The mutation Cys533Gly has never been reported in other FMTC or MEN2A kindreds. This could be secondary to the fact that exon 8 is not routinely screened or that the mutation is clustered in this family. This is a large family with a Caucasian background, and it is conceivable that relatives living in Europe might also bear the same mutation; hence, it is likely to address a founder effect from Spain, and it is feasible that other relatives also migrated to other countries by the time the ancestor moved to Brazil at the end of the 19th century. Recent studies of genetic screening from Spain and countries of South America with a large number of descendants from Barcelona, such as Brazil, Argentina, and Chile, are not performing molecular tests, including RET exon 8 (12, 13, 14, 15, 16, 17, 18, 19, 20, 21). Actually, part of this Brazilian family has recently emigrated to other countries, with two patients living in Australia (IV.80 and IV.125) and two living in the United States (IV.84 and IV.104), justifying the importance of expanding the genetic search over the hot spot region for a mutation in the RET gene.

A clinical heterogeneity is observed in this family with FMTC. Interestingly, some observations indicate a more benign course of this disease. First, 6 gene carriers died from causes not related to MTC. Second, the proband’s mother is still alive at 78 yr of age after a 30-yr course of indolent metastatic disease (III.18); finally, 20 of 35 surgically treated patients are probably cured. However, 2 affected members died due to MTC at ages 53 and 60 yr (II.6 and III.20), 8 of 35 carriers had MTC or CCH before 40 yr of age, and 4 carriers had CCH before 18 yr of age. Therefore, follow-up of these cases is necessary to better understand the prognosis of patients with this mutation.

We expect that the mutation described in this paper will cover a significant proportion of the families with a hitherto undisclosed mutation in the RET gene. To improve genetic testing sensitivity, the analysis of RET exon 8 should be considered in FMTC families with no identified classical mutations.


   Acknowledgments
 
We thank Nildete Gomes, M.D.; M. Inez C. França, M.D.; José R. V. Podestá, M.D.; Antonio Pinto, M.D.; and Fernando Pretti, M.D., for clinical and histological information about the family. We also thank Mauro S. Figueiredo, M.D.; Cassandra Corvelo, Ph.D.; Omar M. Hauache, M.D.; Ana O. Hoff, M.D.; and Gustavo S. Guimarães for helpful discussions; Ilda S. Kunii and Rosana Tamanaha for technical assistance; and Angela Faria for efficient laboratory administration. We express appreciation to all family members for their prompt collaboration.


   Footnotes
 
This work was supported by grants from the Sao Paulo State Research Foundation [FAPESP Grants 99/03688-4 and 01/00246 (to R.M.B.M. and J.M.C.) and 00/03442-2 (to M.R.D.D.S.)], Brazilian Research Council (R.M.B.M. is investigator under Contract 300879/1998–1999), and CAPES-Brazilian Department of Education (J.M.C. is investigator under Contract 1946/01-3).

Abbreviations: CCH, C Cell hyperplasia; FMTC, familial medullary thyroid carcinoma; MEN, multiple endocrine neoplasia; MTC, medullary thyroid carcinoma; Pg, pentagastrin; sCT, serum calcitonin; SNP, single nucleotide polymorphism.

Received June 9, 2003.

Accepted August 6, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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C. Jimenez, M. A. Habra, S.-C. E. Huang, A. El-Naggar, S. E. Shapiro, D. B. Evans, G. Cote, and R. F. Gagel
Pheochromocytoma and Medullary Thyroid Carcinoma: A New Genotype-Phenotype Correlation of the RET Protooncogene 891 Germline Mutation
J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 4142 - 4145.
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