Germline Succinate Dehydrogenase Subunit D Mutation Segregating ...

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multiple endocrine neoplasia (MEN) 2 syndromes (1) in which medullary thyroid carcinoma (MTC) is commonly associated with phaeochromocytoma and less ...
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The Journal of Clinical Endocrinology & Metabolism 88(10):4932– 4937 Copyright © 2003 by The Endocrine Society doi: 10.1210/jc.2002-030008

Germline Succinate Dehydrogenase Subunit D Mutation Segregating with Familial Non-RET C Cell Hyperplasia ´ TEIXEIRA-GOMES, PAULA SOARES, VALDEMAR MA ´ XIMO, JORGE LIMA, JOSE ˜ ES MRINALINI HONAVAR, DILLWYN WILLIAMS, AND MANUEL SOBRINHO-SIMO Institute of Molecular Pathology and Immunology of the University of Porto (J.L., P.S., V.M., M.S.-S.), Porto 4200-465, Portugal; Faculdade de Medicina da Universidade do Porto (J.L., P.S., M.S.-S.), Porto 4200-319, Portugal; Hospital de Pedro Hispano (J.T.-G., M.H.), Matosinhos 4450, Portugal; Strangeways Research Laboratory (D.W.), University of Cambridge, Cambridge CB1 4RN, United Kingdom; and Hospital de S. Joa˜o (M.S.-S.), Porto 4200-319, Portugal C cell hyperplasia is associated with medullary carcinoma of the thyroid in the inherited MEN2 syndromes, in which the great majority of cases have been shown to be due to a mutation in the RET oncogene. We report a study of a family with C cell hyperplasia and hypercalcitoninemia in which no cases of medullary carcinoma have yet occurred and which lacked an identifiable causative RET mutation. Four of the family members showed hypercalcitoninemia, and marked C cell hyperplasia was present in each of the three in whom thyroidectomy has been performed. We investigated the possible involvement of the SDHD gene, because somatic and germline mutations in this gene have been found in a variety of tumors

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CELL HYPERPLASIA IS a consistent finding in the multiple endocrine neoplasia (MEN) 2 syndromes (1) in which medullary thyroid carcinoma (MTC) is commonly associated with phaeochromocytoma and less commonly with parathyroid hyperplasia or adenoma in MEN2A, and with phaeochromocytoma, intestinal ganglioneuromatosis and other lesions in MEN2B. Examples of familial MTC without other tumors (FMTC) also occur (1). The C cell hyperplasia is considered to be a precursor to the development of MTC, and the hypercalcitoninemia it causes has been widely used as a diagnostic test. The vast majority of these inherited syndromes are caused by mutations in the RET (rearranged during transfection) protooncogene (over 95% in MEN2 syndromes, 75% in FMTC). Somatic mutations in RET occur in approximately 50% of sporadic MTCs (1, 2). These mutations lead to a constitutive activation of RET, which then leads to cellular proliferation (C cell hyperplasia) and, ultimately, results in MTC (2). It is surprising that despite the key role played by RET mutations in these settings, there are cases of MEN2/ MTC where no genetic alterations have been detected to date (2), and it is noteworthy that germline RET mutations are most often absent in the least severe form of the inherited disease. Recently, germline and somatic mutations in the gene enAbbreviations: CEA, Carcino-embryonic antigen; FMTC, familial MTC; H&E, hematoxylin and eosin; LOH, loss of heterozygosity; MEN, multiple endocrine neoplasia; MTC, medullary thyroid carcinoma; PGL1, paraganglioma; RET, rearranged during transfection; SDH A–D, succinate dehydrogenase subunits A–D; SSCP, single strand conformation polymorphism.

of neural crest-derived tissue. A germline mutation in exon 2 of the SDHD gene (c149 A-G, His 50 Arg) was found in six members of the family; all the four available members with hypercalcitoninemia possessed the mutation. One of the five available members without hypercalcitoninemia, an 18-yr-old female, also showed the mutation. We conclude that we have identified a new syndrome, characterized by familial nonRET C cell hyperplasia. Our studies suggest that a mutation in SDHD may be causative. These observations have implications for apparently incidental cases of hypercalcitoninemia or C cell hyperplasia. (J Clin Endocrinol Metab 88: 4932– 4937, 2003)

coding for succinate dehydrogenase subunit D (SDHD) have been described in a variety of neuroendocrine tumors, such as paragangliomas, phaeochromocytomas, midgut carcinoids, and Merkel cell carcinomas (3–9). SDHD is part of the succinate-ubiquinone oxidoreductase complex (complex II of the mitochondrial respiratory chain), which is an enzyme complex that catalyzes the oxidation of succinate to fumarate in the tricarboxilic acid cycle, transferring the electrons to ubiquinone, acting therefore as an electron entry site (10). Complex II is composed of four polypeptides, all encoded by the nuclear genome—succinate dehydrogenase subunits A, B, C and D (SDHA, SDHB, SDHC, and SDHD, respectively). SDHA and SDHB constitute the catalytic core of complex II, whereas SDHC and SDHD act as anchor peptides, binding this complex to the inner membrane of mitochondria (10). Germline mutations of SDHD are involved in the development of hereditary head and neck paragangliomas (PGL1) (4, 7, 9). PGL1 is inherited in an autosomal dominant fashion with incomplete penetrance when transmitted through fathers, whereas no disease phenotype appears to occur when transmitted maternally (4). Baysal et al. (4) detected germline mutations in patients with PGL1 whose tumors showed loss of heterozygosity (LOH) exclusively of the maternal allele. Studies of familial and nonfamilial phaeochromocytomas also revealed germline mutations of SDHD with LOH in the tumor tissue (3, 5). Paragangliomas and phaeochromocytomas are tumors that develop from tissues that have their origin in neural crest-derived paraganglia of the autonomous nervous system (5). It well known that C cells of the thyroid have their origin in the neural crest and that MTC and phaeochromo-

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cytomas are tumors of the MEN2A spectrum. Taking all this into account, we hypothesized that paragangliomas, phaeochromocytomas, and MTC might share similar regulatory mechanisms, and that SDHD might also be relevant to C cell hyperplasia. In this paper, we describe the occurrence of hyperparathyroidism and C cell hyperplasia in a 37-yr-old male. This led to the screening of his family and the discovery of three more members with hypercalcitoninemia. We have therefore investigated family members for the occurrence of RET and SDHD mutations. Subjects and Methods Calcitonin assay and pentagastrin stimulation test Calcitonin values were measured by the immunoradiometric assay, using CT-US kit (Medgenix Biosource Diagnostics, Fleurus, Belgium). Pentagastrin test was performed by iv administration of 0.5 ␮g of pentagastrin diluted in 1 ml serum/kg body weight. Values were measured before stimulation and at 0, 2, 6, 10, and 20 min after stimulation. Responses are expressed as the maximum value of peaks of 2 min or 6 min after administration of 0.5 ␮g of pentagastrin. The results were validated by application of the Westgard rules and by the United Kingdom National External Quality Assessment Service.

Patients and lesions We studied a family with a MEN2-like syndrome with one individual affected by C cell hyperplasia and parathyroid hyperplasia (index case; II.6), two individuals affected by C cell hyperplasia alone (II.1, II.2), and another with high level of calcitoninemia (III.3) (Fig. 1). The father (I.2) died from a stroke at the age of 55 yr (he did not have an autopsy) and one of the descendants (II.4) died from ductal adenocarcinoma of the pancreas at the age of 33 yr. This study was approved by the Ethical

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Committee of Hospital de Pedro Hispano (Matosinhos, Portugal), and informed consent was obtained from all individuals studied in the family. The index case, a 37-yr-old male, was admitted in 1996 after several episodes of acute pancreatitis. There was no history of thyroid disease. Elevated serum calcium (2.68 mm; range of normal values: 2.09 –2.42 mm) and calcitonin levels, both basal (16 pg/ml; normal values ⬍10 pg/ml) and pentagastrin-provoked (36 pg/ml; normal values ⬍10 pg/ ml) were recorded. His pancreatitis resolved and subsequent study raised the suspicion of MEN2A: elevated levels of calcium and PTH (79.2 pg/ml; range of normal values: 10 – 65 pg/ml) and high basal and pentagastrin-provoked calcitonin levels. Total thyroidectomy and parathyroidectomy were performed with autotransplantation of one gland to the left forearm. The macroscopic examination revealed hypertrophic parathyroid glands [inferior left parathyroid, 210 mg; inferior right, 130 mg; upper left, 20 mg (the rest was transplanted); upper right, 60 mg]. The patient became hypoparathyroid after surgery (with 3 g/d of calcium he remains, at present, without symptoms). Calcitonin level is, at present, very low (4.7 pg/ml). Family study was then initiated, and two sisters of the index case (II.1 aged 39 yr and II.2 aged 35 yr), showed elevated basal calcitonin levels (11.4 pg/ml; normal values ⬍10 pg/ml and 5.6 pg/ml; normal values ⬍4.6 pg/ml, respectively). Individual II.1 also revealed high pentagastrin-provoked levels (15.6 pg/ml; normal values ⬍10 pg/ml). No hypercalcemia or hyperparathyroidism was noticed in these two sisters or in any other members of the family. Both were subjected to total thyroidectomy. Parathyroid exploration revealed normal glands. Further analyses revealed that, at present, individual III.3, aged 10 yr, presents high basal calcitonin levels (12.9 pg/ml; normal values ⬍10 pg/ml), as well as pentagastrin-provoked levels (31.2 pg/ml; normal values ⬍10 pg/ml). The surgical specimens were studied by routine histological methods, as well as by immunohistochemical and molecular genetics methods. Serial sections were stained with hematoxylin and eosin (H&E) and C cell immunocytochemical markers. Calcitonin and carcino-embryonic antigen (CEA) immunostaining was performed in paraffin-embedded

FIG. 1. Pedigree of the family with C cell hyperplasia and with the SDHD mutation (H50R). At present, individual III.3 has hypercalcitoninemia.

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tissue from individuals II.1, II.2, and II.6 using avidin-biotin-complex method as previously described (11) using calcitonin polyclonal antibody (Dako, Glostrup, Denmark) and CEA polyclonal antibody (Dako) at a dilution of 1:800 and 1:1500, respectively. Positive and negative controls were used in each set of stainings. It is noteworthy that, in generation II, there were three children who died before 1 yr of age and a perinatal death (Fig. 1). This high mortality rate is not uncommon in large, poor families of northern Portugal. Although we have no direct evidence of consanguinity, this family is from a small, remote, and mountainous village.

Lima et al. • Germline SDHD Mutation

random six-oligomer priming using Moloney murine leukemia virus reverse transcriptase at 37 C for 1 h in the presence of 7 mm MgCl2. The recommended conditions were used. RT-PCR of cDNA templates was performed with the described primers (4) and the conditions were: 1 cycle of 5 min at 95 C; 35 cycles of 30 sec at 95 C, 30 sec at 58 C, and 30 sec at 72 C; 1 cycle of 5 min at 72 C. PCR products were subjected to a purifying treatment using Exonuclease I (New England Biolabs, Inc.) and shrimp alkaline phosphatase (Amersham Biosciences). Sequencing analysis was then carried out on purified products using the ABI Prism dGTP BigDye Terminator Ready Reaction Kit (Perkin-Elmer) and an ABI Prism 3100 genetic analyzer (Perkin-Elmer).

DNA extraction Genomic DNA from peripheral blood leukocytes was obtained from individuals I.1, II.1, II.2, II.4, II.5, II.6, III.1, III.2, III.3, and III.5 of the family using NucleoSpin Tissue Kit (Macherey-Nagel, Du¨ ren, Germany). In the available lesions (II.2, II.6) DNA from paraffin-embedded normal thyroid tissue and microdissected hyperplastic C cells was extracted using NucleoSpin Tissue Kit (Macherey-Nagel).

Control population A population of 237 blood donors was used as control. The germline DNA of these individuals was analyzed for alterations in the SDHD gene.

Results Pathological examination

PCR/single strand conformation polymorphism (SSCP) (mutation analysis) PCR amplifications were carried out in 1⫻ PCR buffer (Amersham Biosciences, Piscataway, NJ) that contained 200 ␮m deoxynucleotide triphosphate, 12.5 pmol of each primer, 0.5 U of Taq polymerase (Amersham Biosciences), and 200 ng of DNA template in a 25-␮l volume. PCR conditions were: 1 cycle of 5 min at 95 C; 35 cycles of 30 sec at 95 C, 30 sec at the appropriated annealing temperature, and 30 sec at 72 C; 1 cycle of 5 min at 72 C. The primers for exon amplification (SDHD exons 1– 4 and RET exons 8 –16) were described elsewhere (4, 12). For SSCP analysis, PCR products of SDHD exons 1– 4 were diluted 1:1 with loading buffer (95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol), denatured at 98 C for 10 min and cooled on ice for 5 min. Electrophoresis of the denatured PCR products was carried out in nondenaturing 0.8⫻ mutation detection enhancement gels (Cambrex Bioscience Rockland, Inc., Rockland, ME) at 180 V and 8 C for 15 h. PCR/ SSCP products were visualized by standard DNA silver staining.

Sequencing Aberrant bands were excised from the gel and reamplified using the aforementioned PCR conditions. PCR products were subjected to a purifying treatment using Exonuclease I (New England Biolabs, Inc., Beverly, MA) and shrimp alkaline phosphatase (Amersham Biosciences) and subjected to automatic sequencing, using ABI Prism dGTP BigDye Terminator Ready Reaction Kit (Perkin-Elmer, Foster City, CA) and an ABI prism 3100 genetic analyzer (Perkin-Elmer). Sequencing was performed on both strands using the aforementioned primers. Whenever sequencing revealed a variant, the sample was subjected to repeated mutation analysis, using a separate PCR. RET exons 8 –16 were analyzed by direct sequencing, using the same methodology.

LOH analysis In individuals II.2 and II.6, LOH analysis was performed in DNA from normal thyroid tissue, microdissected hyperplastic foci of C cells and corresponding blood samples using two CA repeats flanking SDHD, D11S5011, and D11S5019, as described elsewhere (4). In individual II.6 LOH analysis was also performed in hyperplastic parathyroid tissue. Radioactive PCR amplification was performed according to the procedures described above for PCR, with the use of [32P]deoxy-CTP. Amplicons were subjected to electrophoresis in a denaturing gel (6% polyacrylamide with 5% cross-linking) and exposed to x-ray film at room temperature. In individual II.1, LOH analysis was not performed for technical reasons: the number of C cells was too small and their distribution too disperse to allow appropriate microdissection.

Gene expression analysis Total RNA was extracted from fresh thyroid and parathyroid tissue (case II.1) and from peripheral blood leukocytes (cases I.1, II.1, and II.5) as previously described (13). cDNA first-strand synthesis was made by

The thyroid glands from the three patients submitted to surgical intervention (II.1, II.2, and II.6) did not reveal any discernible nodules. Despite the nonexistence of clear-cut nodules by light microscopy, foci of neoplastic C cell hyperplasia could be seen in H&E sections of the index case (II.6). The thyroid parenchyma was composed by medium-sized follicules filled with colloid. There were no foci of fibrosis or prominent lymphocytic infiltration. The immunohistochemical study revealed in every case, with both calcitonin and CEA antisera, multiple foci of diffuse, and micronodular C cell hyperplasia involving, to a variable extent, the whole surgical specimens (Fig. 2). The criterion we use for diagnosing C cell hyperplasia was more than 50 C cells per low power field in areas with higher concentration of C cells (14, 15). In every case, there were several foci with more than 100 C cells per low power field. Review of the H&E-stained sections after the demonstration of C cell hyperplasia by immunohistochemistry revealed, in every patient, small solid foci of clear cells that corresponded to C cell micronodules and thus fit with the definition of “neoplastic” C cell hyperplasia advanced by Albores-Saavedra and Krueger (14). In patient II.6, the macroscopical and the histological study confirmed the diagnosis of diffuse hyperplasia of the four parathyroid glands. Molecular genetics study

SDHD analysis. In six individuals of the family, we detected a mutation in exon 2 of SDHD gene (c.149A⬎G) resulting in a substitution of a histidine for an arginine in codon 50 (Figs. 3 and 4). The mutation was present in four of five available individuals of generation II (II.1, II.2, II.4, and II.6), and it was transmitted maternally to two individuals of generation III (III.2 and III.3) (Fig. 1). The mutation segregates with the disease. Because the mother (I.1) had the wild-type allele, the mutation was most probably transmitted by the father (I.2), who had died from stroke at 55 yr of age (no material was available for study). From individuals II.2 and II.6, analysis of DNA from microdissected paraffin-embedded material revealed the presence of the mutated allele as well as the wild-type allele. The expression of SDHD in peripheral blood (I.1, II.1, and II.5), thyroid tissue (II.1), and parathyroid tissue (II.1) was an-

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FIG. 2. Immunohistochemistry for calcitonin showing C cell hyperplasia in thyroid tissue of individual II.1 (⫻100).

FIG. 3. SSCP pattern of wild-type DNA sample (lane 1) and mutated (H50R) DNA sample (lane 2).

alyzed by RT-PCR; in all samples, we detected biallelic expression. Study of SDHD in the control population revealed the presence of the His50Arg mutation in 11 of 474 chromosomes (2.3%). RET analysis. We screened individuals I.1, II.1, II.2, II.4, II.5, and II.6 for mutations in exons 8 –16 of the RET gene, where all the known medullary carcinoma-related mutations are located. Sequencing of these exons revealed a mutation in exon 13 (Tyr791Asn) that results in a substitution of a tyrosine for an asparagine. This variant was present in three of the individuals of the family (I.1, II.1, II.5) and does not segregate with the disease.

FIG. 4. Sequencing of the SDHD gene. A, Pattern of the wild-type SSCP band excised from the gel. B, Pattern of the aberrant SSCP band excised from the gel. Note the substitution of a thymine (arrow) for a cytosine (arrow), resulting in a substitution of a histidine for an arginine.

Discussion

Study of the family of a patient with hyperparathyroidism and hypercalcitoninemia led to the identification of familial C cell hyperplasia but no evidence of any other endocrine tumor. Because of the possibility of MEN2 or FMTC, total thyroidectomy was carried out in the index case and two

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others. All three glands showed marked C cell hyperplasia, but no medullary carcinoma. We first explored the possible involvement of RET in this family because of the role of RET in inherited medullary carcinoma accompanied by C cell hyperplasia. Patients with the MEN2 syndrome show C cell hyperplasia that progresses to medullary carcinoma of the thyroid, usually presenting clinically during adult life in MEN2A and in childhood or adolescence in MEN2B. About 95% of these patients show a germline mutation in RET, close to the transmembrane region (often 634 but between 600 and 650) in 2A and in the tyrosine kinase region (commonly a 918 mutation) in 2B. In FMTC patients, who lack the phaeochromocytomas commonly seen in MEN2A and B, the RET mutations are found in about 75% of cases, in the same region of the gene as in MEN2A. We therefore analyzed exons 8 –16 of the RET gene in six patients in this family. These exons include all the sites that have been found to be mutated in MEN2A, 2B, and FMTC, and those found as somatic mutations in sporadic MTC. A mutation at position 791 (Tyr791Asn) was found in the blood of three of the six family members studied. An identical mutation has not been reported in MEN2A or B, although a mutation at the same position, but with a different amino acid change (Tyr791Phe), has been described by Berndt et al. (16) in two families with MTC only. The mutation found by us did not segregate with the hypercalcitoninemia and C cell hyperplasia in this family, and we therefore conclude that it is a polymorphism of no clinical relevance to this study. We chose to study the SDHD gene because of its link to neuroendocrine tumors, particularly those derived from the neural crest. A mutation at position 149 was found in six of 10 family members studied, including the index case and three of the other cases with hypercalcitoninemia. There were two other mutation carriers: one had died from a ductal adenocarcinoma of the pancreas at the age of 33 yr (II.4), from whom we do not have clinical data on the thyroid, and another one, aged 18 yr, had normal serum calcitonin (III.2). It is known from studies of families with MEN2A syndrome that children with normal calcitonin values can be gene carriers and develop raised calcitonin values in later life, with C cell hyperplasia demonstrated when thyroidectomy is performed, usually for medullary carcinoma. Of the five cases (two adults and three children) with normal serum calcitonin, only one (III.2, aged 18 yr) showed the SDHD mutation. These findings, together with the known role of SDHD mutations in other endocrine lesions lead us to conclude that the SDHD mutation we have observed is the probable cause of the inherited C cell hyperplasia in this family. Mutations in several of the SDH subunits have been described in endocrine tumors, and a variety of mutations in subunit D have been found in familial paragangliomas (4, 5, 8, 17–21). Dannenberg et al. (22) found germline SDHD mutations in all 19 patients with familial parasympathetic paragangliomas, and in 13 of 38 patients with similar tumors but no family history; three different mutations were found but not the H50R mutation found in the present study. The histidine, which is changed to arginine by the mutation is in the region of the gene that codes for the mitochondrial targeting signal peptide (6) and is predicted to lead to a lower

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hydrophobicity (normalized consensus hydrophobicity scale) and higher flexibility of the peptide (us.expasy.org). It has never been observed as a somatic mutation in neuroendocrine tumors, and has been observed as a germline mutation in one case of paraganglioma, one case of Merkel cell carcinoma, and one case of intestinal carcinoid (6, 23). SDH, as an enzyme involved in the mitochondrial respiratory chain, seems an unlikely candidate for oncogene or tumor suppressor gene status. The evidence from a variety of endocrine tumors and particularly from paragangliomas is, however, convincing. In the involved families the germline mutations segregate with the tumors, and the tumors show LOH for the gene (4, 5). Somatic mutation in the SDHD gene also occurs in sporadic tumors. Paragangliomas commonly arise in the carotid bodies, and as these are concerned with oxygen sensing it has been suggested that SDHD mutations could influence the growth of carotid body cells through an effect on oxygen sensing mediated by the role of the mitochondrial respiratory chain in aerobic electron transport (4). Hyperplasia of carotid bodies occurs in individuals living at high altitude, and the incidence of carotid paragangliomas is also increased (24). However, it is difficult to extend this explanation to the other tumors and tissues involved, and our observations provide another example of the apparent specificity of these SDHD mutations for neural crest derived and related endocrine cells. We speculate that the lower hydrophobicity and higher flexibility of the mutant SDHD peptide results in a deficient binding to the mitochondrial inner membrane and in an inappropriate location of enzyme activity, and that increased production of reactive oxygen species could trigger transcription of proliferation inducing genes (4). These do not provide an explanation for the tissue specificity. Our conclusion that the SDHD mutation we have observed is the cause of the familial C cell hyperplasia and hypercalcitoninemia is strengthened by the observation of C cell hyperplasia in one patient with a paratracheal paraganglioma and a germline SDHD mutation (23). Although we have not found any evidence of paragangliomas in the family members we have examined, the death of the father of the index case from a stroke at the age of 55 yr raises the possibility of a catecholamine-secreting tumor. This is necessarily speculative, but it should be noted that the genetic analysis of the family suggests that this patient carried the SDHD mutation but not the RET mutation. One surprising finding in our study was the presence of the H50R mutation in 11 of 237 blood donors, a gene frequency for this mutation of 2.3%. We were careful to confirm each observation and to exclude contamination. Other studies of normal individuals have not found this mutation (6). This raises two interesting questions: first whether this mutation is particularly prevalent in the Portuguese population or this is chance variation, and second, whether the mutation is regularly associated with C cell hyperplasia. For ethical reasons, we could not assess the calcitonin status of the blood donors. Because hypercalcitoninemia is symptomless we cannot exclude its presence, but it seems very unlikely that it would have such a high prevalence. The results may reflect a combination of chance, a high local prevalence, and an unknown penetrance of the mutation.

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These observations raise a number of points of clinical importance. The hypercalcemia and hyperparathyroidism in the index case could represent a low penetrance component of the syndrome. The occurrence of hypercalcemia in some members of the family did not segregate with the hypercalcitoninemia (and C cell hyperplasia) or with SDHD mutation (data not shown). Parathyroid hyperplasia is an occasional finding in MEN2A. We cannot exclude the possibility that the hyperparathyroidism, which was the event that led to the measurement of the calcitonin, is unrelated. Clinically, this syndrome of non-RET C cell hyperplasia can be regarded as the least severe of the range of C cell hyperplasia syndromes. MEN2B is the most severe, with progression from C cell hyperplasia to tumor at an early age. FMTC is the least severe of the RET syndromes with medullary carcinoma often occurring late in adult life. So far, no member of the family we have studied has developed a medullary carcinoma, either as a clinically detectable tumor or as a microcarcinoma in the thyroidectomy specimens. Until more evidence is available, we suggest that family members with non-RET C cell hyperplasia should be followed up by regular ultrasound of the thyroid and by calcitonin assay, with thyroidectomy if any nodularity is detected or if there is a sudden rise in calcitonin values. Unexplained C cell hyperplasia is an occasional finding in thyroidectomy for some other reason, and our findings suggest that SDHD mutations should be sought in these patients, and also in patients with inherited MTC who lack an identifiable RET mutation, particularly FMTC patients. Recognition of this syndrome of non-RET C cell hyperplasia with an H50R mutation in SDHD extends the role of mutations in this gene in endocrine growth and neoplasia, underlines the specificity of SDH mutations for lesions of this range of cells, and suggests that our view of the type of gene that may be involved in hyperplastic and neoplastic growth should be broadened.

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5. 6.

7. 8.

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11. 12.

13. 14. 15. 16.

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Acknowledgments We appreciate the contribution of Dr. Grac¸ a Salcedo in the laboratory studies.

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Received December 30, 2002. Accepted June 14, 2003. Address all correspondence and requests for reprints to: Dr. M. Sobrinho-Simo˜ es, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal. E-mail: [email protected]. This work was supported by a grant from “Fundac¸ a˜ o para a Cieˆ ncia e Tecnologia” (Programa Operacional Cieˆ ncia, Tecnologia, Inovac¸ a˜ o/ Cieˆ ncias Biome´ dicas e Oncolo´ gicas/43944/2001).

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21. 22.

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