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A Novel Val⁶⁴⁸Ile Substitution in RET Protooncogene Observed in a Cys⁶³⁴Arg Multiple Endocrine Neoplasia Type 2A Kindred Presenting with an Adrenocorticotropin-Producing Pheochromocytoma

Endocrine Genetics Unit (A.B.N., M.C.L.E., S.P.A.T.), Department of Endocrinology, and Laboratory of Genetics and Molecular Cardiology (A.C.P., J.E.K.), University of Sao Paulo School of Medicine, 01246-903 Sao Paulo, Brazil

Address all correspondence and requests for reprints to: Dr. Adriana B. Nunes, University of Sao Paulo School of Medicine, Endocrine Genetics Unit, Avenue Dr. Arnaldo 455, 5th Floor, 02146-903 Sao Paulo, Brazil. E-mail: adr001{at}uol.com.br.

Abstract

Multiple endocrine neoplasia type 2 (MEN 2) comprises a heterogeneous group of neoplasic disorders that most commonly have a single missense substitution of the RET protooncogene (RET) involving exons 10 and 11. It was previously reported a MEN 2A kindred in which the father presented with a rare phenotype consisting of bilateral ACTH-producing pheochromocytoma and medullary thyroid carcinoma. We recently performed mutational analysis of the father and his 4 children using a denaturing gradient gel electrophoresis approach and PCR-amplified genomic DNA, followed by direct sequencing or restriction fragment length polymorphism testing. All 4 children showed a RET sequence variation. The common exon 11 Cys⁶³⁴Arg RET mutation was present in 2 of the 4 children who had undergone thyroidectomy for C cell disease. The remaining 2 children, who did not harbor the Cys⁶³⁴Arg mutation and are negative for C cell and adrenal disease, carry a previously unreported Val⁶⁴⁸Ile missense change in RET exon 11. This novel substitution was not found in the unaffected mother or in 200 control alleles. Both RET variants were present in the father affected with MEN 2A and the unusual ACTH-producing pheochromocytoma. We speculate that the double RET mutation may have modified and contributed to the rare MEN 2A phenotype in the father.

MULTIPLE ENDOCRINE NEOPLASIA type 2 (MEN 2) is a syndrome of familial cancers with autosomal dominant inheritance basically characterized by medullary thyroid carcinoma (MTC) and/or its presumed precursor lesion, C cell hyperplasia. There are three distinct phenotypes of this syndrome, distinguished by the presence or absence of additional associated pathologies in the family. Thus, in addition to MTC, about 50% of patients with MEN 2A will develop pheochromocytoma (PHEO), and 30–40% of them will present with hyperparathyroidism due to parathyroid hyperplasia or adenomas. In MEN 2B the parathyroid involvement is rare, but, besides PHEO and MTC, affected patients have developmental anomalies, including mar-fanoid habitus, skeletal abnormalities, mucosal neuromas, and sometimes diffuse intestinal ganglioneuromatosis. Familial MTC (FMTC) is the third MEN2 subtype, with MTC as its only feature (1, 2, 3)

Germline activating mutations in RET protooncogene, located on 10q11 were identified as the disease-causing mutation gene for these inherited syndromes (4, 5). The RET gene encodes a tyrosine kinase receptor, and it is mainly expressed during development in subsets of neural crest derivatives and in the embryonic kidney (6).

RET is likely to promote proliferation and differentiation of neural crest-derived cells in addition to promoting cell survival. At least four different ligand systems are involved in RET signaling. Glial cell line-derived neurotrophic factor, neurturin, artemin, persephin, and their membrane-bound coreceptors (respectively, GFR-1, GFR-2, GFR-3, and GFR-4) form complexes with RET (7, 8).

In more than 96% of MEN 2A and FMTC families point mutations in exons 10 and 11 determine the replacement of one of six cysteine residues in the RET extracellular domain (codons 609, 611, 618, 620, 630, and 634) with any of several alternative amino acids (4, 5, 9). Biochemical and biological analysis revealed that these cysteine mutations induce disulfide-linked dimerization of the RET protein, leading to constitutive activation of its intrinsic tyrosine kinase activity (10). Rare FMTC families present mutations in exons 13 and 14 involving noncysteine codons (11, 12). Recently, double mutations in the RET gene have been reported in cases with an extremely late-onset form of FMTC (13) as well as in one MEN 2A patient harboring a calcitonin-producing PHEO associated with a mild MTC phenotype (14).

We describe here a MEN 2A kindred with siblings presenting two different germline RET sequence alterations in exon 11, a C to T transition at codon 634 and a novel G to A transversion at codon 648, both inherited from the affected father who harbored an ACTH-producing PHEO.

Case Report

The index case’s clinical history was first reported in 1988. This patient (Fig. 1, I-1), 34 yr old, presented with Cushing’s syndrome and adrenal nodules in both glands as described previously (15). In brief, he was submitted to bilateral adrenalectomy, and the presence of bilateral PHEO associated with adrenal cortex hyperplasia was documented. This MEN 2A case presented a rare phenotype: MTC associated with an ACTH-producing PHEO. A cold thyroid nodule had been detected, and serum calcitonin levels were high (4660 pg/ml; normal, <50 pg/ml; Diagnostic Products, Los Angeles, CA). Postsurgical basal and stimulated calcitonin levels dropped to the normal range and remained at low levels thereafter. Routine total thyroidectomy revealed multicentric MTC, but lymph node involvement was not reported (15). The classification was T3bN0M0. The patient died due to myocardial ischemia 9 yr later, free of MTC disease.

View larger version (15K): [in this window] [in a new window]   Figure 1. Genealogy of the present family. Confirmed MTC affected members are colored black. An oblique line denotes deceased family members. The arrow denotes the index case. Roman numerals represent generation member. Arabic numerals are the individual members.

Case II-2 was a 9-yr-old girl initially presenting with a normal thyroid gland at clinical examination. Basal and stimulated calcitonin levels were within normal ranges, and no thyroid nodule was seen at ultrasonography. When she was 10 yr old, a 0.5-cm nodule in the right thyroid lobe was detected by ultrasound, and calcitonin levels after pentagastrin administration rose from 4 to 800 ng/liter (normal peak, <350 ng/liter). Serum calcium, phosphate, and PTH were normal, as were urinary catecholamines. The patient underwent total thyroidectomy with central lymph node resection at 12 yr of age. Calcitonin values early after surgery rose from 19 to 4600 ng/liter. Multiple foci MTC and C cell hyperplasia were documented when a detailed pathological study using a highly sensitive immunohistochemical method for calcitonin was performed, as reported previously (16). The classification was T1bN0M0. Parathyroid hyperplasia was documented by pathological studies. Stimulated calcitonin levels after surgery decreased to 14–26 ng/liter and remained normal thereafter.

Case II-3, a 6-yr-old girl, presented with a normal thyroid at physical examination and ultrasonography, whereas calcitonin levels after a pentagastrin stimulation test went from 4 to 790 ng/liter. Calcium, phosphate, PTH, and serum/urinary catecholamines were within normal ranges. She underwent a total thyroidectomy with central lymph node resection soon after the diagnosis was performed. Pathological findings revealed multifocal areas of C cell hyperplasia spread throughout the gland, with higher occurrence in the middle third of the thyroid (16). The classification was T1N0M0.

Replacement therapy with T₄ was instituted in both cases immediately after surgery. Follow-up for 13 yr after thyroidectomy performed in both affected siblings showed no biochemical evidence of persistent MTC disease, and there has been no evidence of PHEO to date in either of the siblings.

Individuals II-1 and II-4 were initially seen at 15 and 3 yr of age, respectively, and have been followed annually for 13 yr. They have had normal basal and stimulated calcitonin levels as well as normal thyroid ultrasonography. Hyperparathyroidism and PHEO have been excluded to date in these individuals by screening with serum calcium, intact PTH molecule, and serum and urinary catecholamine measurements.

We have recently expanded the study of this family by offering DNA screening for RET mutation to those individuals at risk. The genealogy of the family is shown in Fig 1. A total of eight individuals from three generations were screened for RET mutations. Written informed consent was obtained from each family member or his/her parents and from 100 healthy adults before genetic analysis was performed. This study was approved by the local ethic committees.

Materials and Methods

Genomic DNA was isolated from peripheral leukocytes from at risk individuals and from 100 healthy adult volunteers according to established procedures (17). High molecular weight DNA was extracted from 10-µm sections of formalin-fixed, paraffin-embedded, surgical thyroid and adrenal specimens of the deceased father by the Chellex method (18).

RET exon 11 was amplified by PCR using oligonucleotides flanking exon-intron boundaries as primers: 11F, 5'-CCTCACACCACCCCCCACCCA-3'; and 11R, 5'-GCGCCCCCCGCCCCCGCCCCGCCCGCCGACTGGTTCTCCATGGAGTC-3'.

Amplification was performed using 1 µg genomic DNA templates, 2.2 µmol/liter of each primer, 200 µM of each dNTP, 1.25 U TaqDNA polymerase (Life Technologies, Inc., Gaithersburg, MD), 10 mM Tris (pH 8.4), and 50 mM KCl, 2.0 mM MgCl₂ in a total volume of 50 µl. Thermal cycling conditions (MJ Research, Inc., Watertown, MA) were 40 cycles at 95 C for 30 sec and 65 and 72 C for 1 min, followed by a final 10-min denaturing step to facilitate heteroduplex formation.

PCR products were screened for mutation by denaturing gradient gel electrophoresis (DGGE) analysis. Electrophoresis was carried out in a vertical gel electrophoresis apparatus at 65 C and 17 mA/gel for 20 h (DCode System, Bio-Rad Laboratories, Inc., Hercules, CA) through 9% acrylamide/bis-acrylamide (20:1) with a 50–80% linear gradient of denaturants [100% = 7 M urea and 40% (vol/vol) formamide] in 1x Tris-acetate-ethylenediamine tetraacetic acid buffer (pH 7.8). Gels were stained with ethidium bromide for 10 min before UV visualization.

PCR products were purified with the QIAquick PCR purification kit (QIAGEN, Hilden, Germany) before sequencing. PCR products were then subjected to 25 cycles (96 C for 20 sec, 50 C for 10 sec, and 60 C for 4 min) with sense or antisense primer using fluorescence-based dideoxyterminator cycle sequencing (BigDye; Applied Biosystem, Foster City, CA). The products were eluted through a Centri-Sep spin column and subjected to gel electrophoresis. Data collection and analysis were performed on an automated DNA sequencer (ABI PRISM 373, Perkin-Elmer, Foster City, CA).

For restriction analysis, PCR products of exon 11 were digested with the enzyme HhaI (Life Technologies, Inc.) and BsmAI (New England Biolabs, Inc., Beverly, MA), as recommended by the manufacturer.

Results

When the DGGE analysis was performed, an anomalous band pattern was detected in the exon 11 PCR products in all four siblings of generation II. However, a slight difference was observed when the migratory patterns of cases II-2 and II-3 were compared with those of their siblings, II-1 and II-4 (Fig. 2).

View larger version (65K): [in this window] [in a new window]   Figure 2. DGGE assay for RET exon 11. Lanes are numbered from left to the right and represent, respectively, individuals II-1, II-2, III-2, II-3, and II-4. Lanes 2 and 4 show the Cys634Arg mutation, whereas lanes 1 and 5 document the Val648Ile substitution.

Restriction analysis confirmed the presence of the Cys⁶³⁴Arg mutation in patients II-2 and II-3, but not in cases II-1 and II-4. In these two latter individuals, the 275-bp band corresponds to the nondigested PCR product.

Exon 11 PCR product sequencing analysis of individuals II-1 and II-4 showed a germline heterozygous missense GTCATC substitution in codon 648. This transversion leads to the replacement of a valine with an isoleucine. This mutation alters a recognition site for BsmAI; normal sequence shows a 60- and 215-bp band pattern, whereas the mutant presents a 275-, 215-, and 60-bp band pattern (Fig. 3).

View larger version (62K): [in this window] [in a new window]   Figure 3. Novel missense substitution (GTCATC) at codon 648, presented in individuals II-1 and II-4, destroys a BsmAI restriction site.

Next we investigated whether the Val⁶⁴⁸Ile substitution was a DNA sequence polymorphism. This change was not detected in 200 chromosomes from 100 healthy adult individuals.

Considering these findings, DNA purified from thyroid and adrenal tissues from the deceased index case (I-1) was submitted to restriction and sequencing analysis, and both nucleotide changes, Val⁶⁴⁸Ile and Cys⁶³⁴Arg, were observed.

Discussion

This is the first report of a RET analysis in an MEN 2A patient with Cushing’s syndrome due to an ACTH-producing PHEO. This case presented a novel germline sequence, Val⁶⁴⁸Ile substitution, in the RET protooncogene, occurring simultaneously with a Cys⁶³⁴Arg mutation. This case probably presented with a less aggressive form of MTC, as no local lymph node metastases were detected when the patient was 34 yr old, as confirmed by pathological studies. Further, total thyroidectomy was sufficient to cure the thyroid tumor in this case. Few cases of ACTH-producing PHEO were reported to date (19), and only one was a MEN 2 patient. As mutation analyses in non-MEN 2 hormone-producing PHEO cases are lacking to date, it is unknown whether double mutations might occur in such patients.

In two recent reports, a double RET mutation was described in MEN 2A and FMTC cases presenting mild, late phenotypes. Thus, Tessitore et al. (14) reported an MEN 2A patient presenting two germline de novo mutations (Cys⁶³⁴Arg and Ala⁶⁴⁰Gly) in exon 11 in the same allele, harboring a calcitonin-producing PHEO associated with a mild, late form of MTC. Further, Bartsch et al. (13) reported a double RET mutation in the same allele (Val⁸⁰⁴Met and Arg⁸⁴⁴Leu) in a family exhibiting an extremely mild form of FMTC. These findings taken together with our present data raised the question of whether the interaction between both mutations may have played any causative or modifying role in the appearance of these rare phenotypes. Possibly, interactions between two germline events in the same gene, as seen here, should be carefully assessed in similar cases.

The Val⁶⁴⁸Ile substitution described here has not been previously reported to cosegregate either with MEN 2 or in normal phenotypes. Codon 648 lies near the 609–660 cysteine-rich region located at exon 11 of the RET protooncogene. This codon 648 valine residue is not conserved among mammals. However, it lies in a subdomain of the transmembrane domain in the middle of 21-amino acid, highly conserved residues in both mice and humans (20, 21). The absence of this mutation in 200 alleles of 100 healthy adult volunteers analyzed suggest that this is unlikely to be a DNA polymorphism, although a polymorphism at a very low population frequency (<0.5%) cannot be completely excluded. Several polymorphisms in the coding region of the RET protooncogene have been described. A panel of the most frequent polymorphisms has been reported, encompassing those in codons 45, 125, 432, 691, 769, 836, and 904 (4, 22, 23, 24). All of the investigated polymorphisms are silent mutations, except for codon 691, which results in a change in the amino acid residue from glycine to serine.

The first mutation observed in this kindred was a TGCCGC change at codon 634 (Cys⁶³⁴Arg) in RET exon 11. According to the RET Mutation Consortium, mutations in cysteine 634 are by far the most frequently mutations found in MEN 2A and FMTC, and a missense mutation at codon 634 occurs in approximately 87% of all MEN 2A kindreds (1, 3). It would be expected that the six cysteine residues should be affected with equal frequency. Thus, the preponderance of MEN 2A mutations observed at codon 634 may reflect greater disease penetrance and expression. Interestingly, the double RET mutation in the present case was probably located in different alleles, in contrast to the other two reported cases (13, 14). This fact allowed us to observe the occurrence of three different anomalous RET genotypes in this family.

The impact of the Val⁶⁴⁸Ile transition per se in the pathogenesis of MEN 2 is presently unknown. This novel sequence variant may not have a potential effect in tumorigenesis or even act upon the wild-type RET allele, and thus individuals carrying only this substitution would have a normal phenotype. Conversely, its action might occur through a dominant negative mechanism only in presence of its abnormal Cys⁶³⁴Arg allele, modulating the gene action. Further expression and functional studies may help to clarify this issue. The two Val⁶⁴⁸Ile carriers here reported are presently 17 and 32 yr old and free of disease, considering clinical, hormonal, and ultrasonographic findings. Whether in the future these siblings may develop a mild, late-onset form of MEN 2 is unknown. Thus, longer-term annual follow-up of these cases is needed.

Finally, the present report also illustrates the importance of the sensitivity of genetic testing methods offered to MEN 2A and FMTC patients. DGGE with its nonradioactive protocol is an attractive method to screen RET mutations (25). Further, it has an enhanced sensitivity, reproducibility, and ease of performance and analysis compared with SSCP (22). We consider it particularly well suited to MEN 2 because of the dominant genetics and low prevalence of neutral polymorphisms. Otherwise, polymorphisms are the major limitation to DGGE screening, because it will yield false positive results. Furthermore, if individuals II-1and II-4 and their affected father were screened using only restriction enzyme assays searching for Cys⁶³⁴Arg, the gene variant in codon 648 would not be noticed.

Acknowledgments

We are indebted to Dr. Robert Gagel for helpful discussion, to Dr. Homero Vallada Filho for providing control DNA samples, to Drs. Cesar Hayashida and Patricia M. L. Dahia for manuscript review, and to Ms. Marcilene Floriano and Ms. Denise Frediani for technical assistance.

Footnotes

This work was supported by grants from the Brazilian National Research Council (463387/00-9) and Centro de Pesquisa e Desenvolvimento de Informática e Automação (CPDIA/NEC) do Brazil.

A.B.N. is a recipient of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/PICDT) support.

S.P.A.T. is a Brazilian National Research Council researcher (Grant 300346-82-04).

Abbreviations: DGGE, Denaturing gradient gel electrophoresis; FMTC, familial medullary thyroid carcinoma; MEN 2, multiple endocrine neoplasia type 2; MTC, medullary thyroid carcinoma; PHEO, pheochromocytoma.

Received March 5, 2002.

Accepted September 7, 2002.

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