27-bp Deletion in the ret proto-oncogene as a somatic mutation associated with medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) occurs as a sporadic tumor or in connection with the inherited cancer syndromes of multiple endocrine neoplasia (MEN) types 2A and 2B and familial MTC. Missense RET proto-oncogene mutations of one of cysteine codons in exons 10 and 11 are found in the majority of families with MEN 2A and or familial MTC. In MEN 2B, mutations at codon 918, exon 16, have been identified in most of the affected individuals. In a significant amount of sporadic MTC somatic codon 918 mutations appear. In addition to these, a 6-bp deletion including codon 630 and a 24-bp deletion including codon 634 combined with a 6-bp insertion have been observed. We report on a 27-bp deletion in exon 10 as a somatic mutation associated with a sporadic medullary thyroid carcinoma. Received: 15 September 1997 / Accepted: 19 December 1997     

Detection of RET mutations in multiple endocrine neoplasia type 2a and familial medullary thyroid carcinoma by denaturing gradient gel electrophoresis

Germline missense mutations within the coding region of the RET proto-oncogene have recently been described in patients with the dominantly inherited cancer syndromes, multiple endocrine neoplasia type 2a (MEN 2a) and familial medullary thyroid carcinoma (FMTC). To date, the sequence variations occur in RET exons 10 and 11 and alter highly conserved cysteine residues in the proposed extracellular domain at codons 609, 611, 618, 620, and 634. To expedite rapid screening of populations at risk of MEN 2a or FMTC, we developed a PCR-based denaturing gradient gel electrophoresis (DGGE) strategy that detects polymorphisms occurring at all five Cys codons in both RET exons using identical gel conditions. In this report, the screening results from DGGE analysis of 15 distinct MEN 2a and FMTC mutations are shown. Each mutation generated a clearly distinguishable and unique homo- and heteroduplex band pattern. Given the highly efficient, reproducible, and sensitive nature of this approach, DGGE is particularly appropriate for rapid, large-scale screening of patients. Since prior knowledge of the RET mutation is unnecessary for analysis, DGGE is potentially valuable for distinguishing germline from seemingly sporadic medullary thyroid cancer as well as identifying novel sequence changes. © 1996 Wiley-Liss, Inc.     

Mutational analysis of the RET proto-oncogene in 71 Japanese patients with medullary thyroid carcinoma

Multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinomas (FMTC) are caused by germline mutations in the RET proto-oncogene. To investigate the spectrum of RET mutations among Japanese patients, we screened the RET gene in 71 patients with thyroid carcinomas. The panel included representatives of 44 families carrying FMTC or MEN2, 22 sporadic medullary thyroid carcinomas (MTCs), and five MTCs without familial information. Mutations in nucleotide sequences encoding one of three specific cysteine residues in the extracellular domain of the RET protein were found in 33 of the 34 MEN2A patients and in five of the six FMTC patients examined. A mutation at codon 918, causing the substitution of threonine for methionine in the tyrosine kinase domain of the protein, was found in germline DNAs of all four patients with MEN2B and in two of the 22 patients with sporadic MTCs; codon 918 was mutated somatically in tumor DNAs from three other sporadic cases. Germline mutations of codon 768, GAG to GAC (Glu to Asp), were detected in one FMTC, in one patient with sporadic MTC, and in one of the patients without familial information. Two somatic mutations, an Asp to Gly substitution at codon 631 and a Cys to Arg substitution at codon 634, had not been reported previously. Of five germline mutations found among the 22 sporadic cases, four were confirmed as de novo mutations since in each case neither parent carried the mutation. As nearly one-fourth of the patients with sporadic MTCs carried germline mutations and 50% of their children are expected to develop MTC and other endocrine tumors, these results indicated the importance of careful clinical surveillance of family members of any patient with MTC. Received: August 22, 1997 / Accepted: October 22, 1997     

RET Proto-Oncogene and Thyroid Cancer

TheRET proto-oncogene has not only conclusively been identified as responsible for the three subtypes of the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN-2) but also shown to be involved in the molecular evolution of sporadic medullary and papillary thyroid carcinoma as well as Hirschsprung’s disease. A variety of recent studies have elucidated the pathophysiological mechanisms leading to neoplastic disease and we now understand that dominant activating germline mutations lead to MEN-2A, MEN-2B, and familial MTC; somatic mutations to sporadic medullary thyroid carcinoma;RET rearrangements to papillary thyroid carcinoma; and inactivating alterations to Hirschsprung's disease. The clinical significance, however, ofRET alterations especially in sporadic thyroid tumors is still controversial and therapeutic concepts in MEN-2 gene carriers only start to emerge. This article is a short summary of the recent findings on the structure and physiology of theRET proto-oncogene and its role in familial and sporadic thyroid cancer. This article was presented in part at the Endocrine Pathology Society Companion Meeting of the USCAP in Orlando, FL, March 1, 1997.     

Molecular Screening for RET Proto-Oncogene Mutations in a German MEN2A Pedigree

Multiple endocrine neoplasia type 2A (MEN2A), a dominantly inherited cancer syndrome, is defined by the presence of medullary thyroid carcinoma (MTC), pheochromocytoma (pheo), and primary hyperparathyroidism (p-HPT). Along with multiple endocrine neoplasia type 2B (MEN2B) and familial medullary thyroid carcinoma (FMTC), it is associated with germline mutations of theRET proto-oncogene localized in 10q11.2. In FMTC and MEN2A, point mutations result in the substitution of one of five Cys residues in the extracellular domain ofRET. In a larger pedigree from Saarland, several individuals were observed with C-cell thyroid carcinoma. We screened 16 members of this extended family by single-strand conformation polymorphism analysis (SSCP), polymerase chain reaction (PCR), followed by restriction enzyme analysis, and by sequencing the mutated regions. In 7 family members, all of whom had been earlier operated on because of MTC, a DNA transition from T to C was observed, causing an amino acid substitution Cys⁶³⁴Arg. Nine members of the kindred did not carry the mutation and may be excluded from yearly biochemical testing. One of these persons seems to have been unnecessarily operated on owing to a borderline pentagastrin test.     

Mutation of the RET protooncogene in sporadic medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) occurs sporadically or as part of the inherited cancer syndrome multiple endocrine neoplasia (MEN) type 2. In MEN 2A, germline missense mutations are found in one of five cysteine codons within exons 10 and 11 in the extracellular domain of the RET protooncogene. In MEN 2B, germline mutations occur in codon 918 (exon 16) within the catalytic core of the tyrosine kinase domain. To determine if RET mutations similar to those in MEN 2A and 2B play a role in the pathogenesis of sporadic MTC, we analysed 71 sporadic tumours comprising 68 primary tumours and three cell lines, for mutations in RET exons 10, 11, and 16. We found that 23% of sporadic MTC had RET codon 918 mutations, while only 3% had exon 10 mutations, and none had mutations in exon 11. We found no exon 16 mutations in MTC from 14 MEN 2A cases. Thus, exon 10 and 11 mutations, commonly found in familial MTC and MEN 2A, rarely occur in sporadic MTC; somatic mutation of RET codon 918 appears to play a role in the tumourigenesis of a significant minority of sporadic MTC but not MEN 2A tumours. In addition to their biological interest, these findings may have some clinical application in determining whether a patient presenting with isolated MTC is truly sporadic or is part of an inherited cancer syndrome.     

Presymptomatic genetic screening in families with multiple endocrine neoplasia type 2

Medullary thyroid carcinoma occurs sporadically or as a part of the inherited cancer syndrome multiple endocrine neoplasia (MEN) type 2. The MEN 2 gene has been identified as the RET proto-oncogene on chromosome 10. In MEN 2A, RET mutations are detectable in one of five cysteine codons within exons 10 and 11 and in MEN 2B in codon 918 (exon 16). Direct DNA testing for RET proto-oncogene mutations is the method of first choice in presymptomatic screening of MEN 2 families. Gene carriers should be offered prophylactic thyroidectomy. The process of DNA analysis for RET proto-oncogene mutations is demonstrated in one family with hereditary medullary thyroid carcinoma. RET mutations were detectable in five of the nine family members at risk.Abbreviations MEN Multiple endocrine neoplasia - MTC Medullary thyroid carcinoma - FMTC Familial medullary thyroid carcinoma - PCR Polymerase chain reaction - C-cells Calcitonin-producing parafollicular cells     

Penetrance of inherited medullary thyroid carcinoma and genotype-phenotype correlation in a large multiple endocrine neoplasia type 2A family with C634Y RET mutatiom

Multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC) are characterized by development of medullary thyroid carcinoma (MTC) and caused by germline RET mutations. Patients with MEN 2A also develop pheochromocytoma and/or hyperparathyroidism (HPT). However, MEN 2A-affected individuals could display the FMTC phenotype at first clinical manifestation. To establish the correct phenotype and improve clinical management of patients affected by hereditary MTC, clinical screening, RET mutational analysis, penetrance of MTC, and genotype-phenotype correlation were performed in a large, suspected FMTC kindred of 86 individuals. Germline C634Y RET mutation was confirmed in 22 individuals, 15 of whom were thyroidectomized when high serum calcitonin levels were detected. MTC was confirmed in 12 individuals and C-cell hyperplasia in 3. HPT was detected in two patients. High penetrance of MTC at young age (79% at 30 yr of age) was found. This family was considered to be affected by FMTC for several years because MTC was the sole clinical manifestation. However, our results allowed reclassifying the family as MEN 2A, thereby improving clinical management of family members. Our findings regarding penetrance and genotype-phenotype correlation suggest that patients considered to have FMTC may in fact have MEN 2A in some kindreds.     

RET proto-oncogene mutations in French MEN 2A and FMTC families

Constitutional mutations of the RET proto-oncogene have beenidentified in multiple endocrine neoplasia type 2A (MEN 2A),type 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC)families. We sequenced RET exons 10 and 11 in 86 unrelated patientswith an inherited predisposition to MTC (excluding MEN 2B).Germ-line mutations were Identified in 93% of the MEN 2A familiesand 67% of the FMTC families tested. All were missense mutationsaffecting one of three cysteines in the extracellular domainof the RET tyrosine kinase receptor. The prevalence of phaeochromocytomaand hyperparathyroidism was significantly higher in familieswith a mutation of cysteine 634. These data confirm the preferentiallocalisation of MEN 2A and FMTC associated mutations and thestrong correlation between clinical manifestations and the positionof RET mutation. Although direst sequencing of RET exons 10and 11 allows the identification of a constitutional mutationin a large proportion of MEN 2A and FMTC families, our datasustain the existence of other MTC predisposing mutations elsewherein RET coding or regulating region.     

Integrated DNA-based/biochemical screening for early diagnosis of multiple endocrine neoplasia type 2A (MEN2A)

Multiple endocrine neoplasia type 2A (MEN2A), a subtype of MEN2, is characterized by medullary thyroid cancer, pheochromocytoma, and primary hyperparathyroidism. A Han Chinese pedigree with MEN2A was investigated following confirmation of the proband''s diagnosis by pathological findings and DNA/biochemical screening. DNA samples from 4 other family members were collected and exon 5, 8, 10, 11, 13, 16 and 18 of the RET proto-oncogene were sequenced and then analyzed. A missense mutation of TGG (Trp) to TGC (Cys) at codon 634 (the classic MEN2A mutation) in exon 11 of the RET gene was detected in 3 family members, including the proband. Sequencing data were compared with the human gene mutation database. Elevated serum calcitonin level was detected initially; medullary thyroid carcinoma was revealed in the 3 cases and adrenal pheochromocytoma was also found in the proband. Elective operations were successfully performed on the adrenal and thyroid glands because of pheochromocytoma and medullary thyroid carcinoma. Our case study confirms that integrated DNA-based/biochemical screening is crucial for early diagnosis of MEN2A and is helpful in the screening of their relatives. In addition, DNA-based screening may occasionally uncover a previously unknown RET sequence.     

Molecular Diagnosis of Multiple Endocrine Neoplasia (MEN) in Paraffin-Embedded Specimens

In this article, we summarize our recent findings on rearranged during transfection (RET) mutations in a series of 46 sporadic as well as multiple endocrine neoplasia (MEN) type 2-associated tumors and present results of our family screening efforts to identify MEN 2 and MEN 1 gene carriers. A nonisotopic polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP) analysis and heteroduplex gel electrophoresis method was used to screen DNA extracted from archival specimens of 22 patients with MEN 2-associated and 24 patients with sporadic tumors for mutations inRET exons 10, 11, 13, and 16. Point mutations were identified by nonisotopic cycle sequencing of PCR products using an automated DNA sequencer. We found six different missense germline mutations at cysteine residues encoded by exons 10 and 11 in all patients with MEN 2A or familial medullary thyroid carcinoma (FMTC). The frequency of mutations at codon 634 was higher in patients with MEN 2A than with FMTC and a₆₃₄ Cys→Arg mutation was associated with parathyroid disease. A germline Met→Thr point mutation at codon 918 of theRET tyrosine kinase domain encoded by exon 16 was identified in all MEN 2B patients. Nonpredicted inheritable medullary thyroid carcinomas (MTCs) were detected in two patients and a mosaic postzygotic mutation was found in one additional patient. Tumor-specific (somatic) Met→Thr point mutations at codon 918 were identified in 5 of 13 sporadic MTCs and 2 of 8 sporadic pheochromocytomas (PCCs). The remaining sporadic tumors lacked mutations in all fourRET exons tested. In exon 13, a nucleic acid polymorphism (CTT/CTG; Leu) at codon 769 was identified, which is present in approx 40% of the examined population. Our study demonstrates that the molecular methods used are not only suitable to identify asymptomatic individuals at risk for MEN 2A, FMTC, and MEN 2B, but also to distinguish sporadic from inherited tumors using archival tissue specimens; and that more tumors than clinically expected are inheritable, indicating the need for genetic analysis of all MTC and PCC patients.     

Multiple endokrine Neoplasien Typ 2

Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant inherited cancer syndrome with the major components medullary thyroid carcinoma, pheochromocytoma and hyperparathyroidism. Due to the clinical course three distinct subtypes are distinguished, MEN 2A, MEN 2B and familial medullary thyroid carcinoma. The disease is caused by germ-line mutations of the RET proto-oncogene and the localization of these mutations correlates with the onset of the development of medullary thyroid carcinoma, which is crucial for the clinical course and outcome of the disease. It therefore has a substantial influence on the clinical management of the affected patients and their relatives. This review summarizes the morphology and clinic of MEN 2-associated tumors and their respective precursor lesions.     

Clinical spectrum of MEN2A in a large family caused by the infrequent RET mutation Cys609Phe

Mutations in RET proto‐oncogene cause multiple endocrine neoplasia 2A (MEN2A). Mutations in codons 609 and 611 are not frequent. We identified two MEN2A families with the Cys609Phe RET mutation, which turned out to be the same family. This mutation has been described a couple of times with no clinical details. We have characterized the clinical phenotype of this large kindred. A 54‐year‐old woman, with a medullary thyroid carcinoma (MTC), and a 33‐year‐old woman, who was operated on for an adrenal pheochromocytoma, were the index cases. 35 relatives were studied. Sixteen turned out to be carriers and 12 of them have been operated on. This family showed eight patients with C‐cell hyperplasia, six patients affected by MTC and two showing pheochromocytoma. A papillary thyroid carcinoma was also found, together with the MTC, in one of the carriers. The phenotype in this large kindred is clearly of MEN2A. In carriers presenting the Cys609Phe mutation, the timing of the presentation of the syndrome is highly unpredictable. Therefore, a strict follow up of MTC must be carried out because of risk, and pheochromocytoma should not be ignored. These results reinforce the scarce data observed on this particular mutation.     

Novel point mutation in exon 10 of the RET proto-oncogene in a family with medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) may occur sporadically or as part of the autosomal dominant multiple endocrine neoplasia type 2 (MEN 2). Three hereditary forms of MEN 2 have been identified: MEN 2A, MEN 2B, and familial MTC (FMTC). Missense germ-line mutations in the RET proto-oncogene have been identified as cause of these endocrine diseases. Mutations are found in exons 10 and 11 in MEN 2A and FMTC families and in a small number of families in exons 13, 14, and 15. Although a strong correlation between codon mutations and phenotypes has been described, not all the expected cystein codon mutations have been found. Therefore, the more mutations are found, the better it is possible to establish phenotype-genotype correlations. We report on a novel RET mutation at codon 611 in a family with MTC without other clinical manifestations and of rather benign course. Am. J. Med. Genet. 78:271–273, 1998. © 1998 Wiley-Liss, Inc.     

Mutation of RET codon 768 is associated with the FMTC phenotype

Multiple endocrine neoplasia type 2A (MEN 2A), type 2B (MEN 2B), and familial medullary thyroid carcinoma (FMTC) are inherited cancer syndromes resulting from mutations in the RET proto-oncogene. Missense mutations of five codons in exons 10 and 11 are found in both MEN 2A and FMTC families, while mutations at codon 768 in exon 13 have been identified in three FMTC families. We report here the results of mutation analysis on a large multi-generation family with multiple cases of medullary thyroid carcinoma (MTC) or C-cell hyperplasia and two individuals with isolated adrenal medullary hyperplasia. A mutation in exon 13, which alters codon 768 from a GAG (Glu) to a GAC (Asp), was found to segregate with the FMTC phenotype in this family but not with the adrenal medullary hyperplasia. These findings suggest that the codon 768 mutation does not predispose to adrenal medullary hyperplasia, but is an accurate predictor of the MTC phenotype in this family.     

Long-term follow up of a “sporadic” unilateral pheochromocytoma revealing multiple endocrine neoplasia MEN2A-2 in an elderly woman

A unilateral, apparently sporadic pheochromocytoma was removed from the right adrenal of a 73-yr-old Caucasian woman. At the time of surgery, germline DNA from the patient was not available. However, a continuous cell line (KNA) established from the tumor showed a heterozygous sequence variant TGC (cysteine) to TGG (tryptophan) in exon 10, codon 611 of the RET proto-oncogene. Subsequent genetic testing of the patient and her offspring revealed the same base-change in herself, one daughter, one son, and the only grandson, confirming hereditary disease classified as MEN2A-2. Clinical follow up of the patient revealed elevated serum calcitonin after 6 yr. Thyroidectomy was performed and revealed a small medullary thyroid carcinoma. The patient’s children thus far show no evidence of MEN2, but C-cell hyperplasia has been diagnosed in the grandson. Our serendipitous finding of a MEN2A-2 mutation in a patient with initial diagnosis of late onset, unilateral, “sporadic” pheochromocytoma would argue for routine mutation screening of even elderly patients presenting with a pheochromocytoma.     

Novel germline mutation in the transmembrane region of RET gene close to Cys634Ser mutation associated with MEN 2A syndrome

Two mutations on the same allele of RET gene were revealed in a family with predisposition to multiple endocrine neoplasia (MEN) type 2A. The first mutation changes codon 634 from cysteine to serine. The second, a novel mutation in codon 641, changes alanine to serine in the transmembrane domain of the RET protein. Two mutations were present in close proximity in both the patients germline and tumor DNA and were absent in DNA isolated from healthy family members and control blood donors. All MEN 2A affected family members suffered from medullary thyroid carcinoma and two of ten patients for pheochromocytoma. No parathyroid gland alterations were observed in patients with two RET gene mutations. Analysis of four genetic polymorphisms in the RET gene showed higher incidence of polymorphisms of exons 11 and 15. The observed allelic imbalance in favor of mutated allele in pheochromocytoma corresponded to higher expression of the RET gene. These observations confirm the multifactorial process leading to development of MEN 2A syndrome.     

Diagnosis of multiple endocrine neoplasia [MEN] 2A, 2B and familial medullary thyroid cancer [FMTC] by multiplex PCR and heteroduplex analyses of RET proto-oncogene mutations

Multiple endocrine neoplasia type 2 [MEN 2] is an autosomal dominant cancer syndrome with two subtypes, 2A and 2B. MEN 2A and medullary thyroid cancer [MTC] are caused by >25 different point mutations in exons 10, 11, and 13 of the RET proto-oncogene, whereas MEN 2B is caused by a single exon 16-point mutation. Various molecular methods have been used to identify the different mutations, including DNA sequencing, restriction enzymatic analyses, chemical cleavage mismatch, Single Stranded Conformational Polymorphism [SSCP], and Denaturing Gradient Gel Electrophoresis [DGGE]. These techniques, although useful and accurate, are labor intensive and some involve the use of radioactivity. We have developed a multiplex PCR assay simultaneously to amplify exons 10, 11, and 13 of the RET proto-oncogene. The multiplex PCR product is then analyzed on a modified Mutation Detection Enhancement [MDE] matrix for heteroduplex identification and visualized with ethidium bromide. Distinct heteroduplexes were detected for each known RET proto-oncogene mutation available in our laboratory (nine in exon 10, five in exon 11, one in exon 13, and the single exon 16 mutation). Presymptomatic DNA diagnosis of MEN 2 is essential since pentagastrin-stimulated calcitonin studies can occasionally produce false positive results and lead to unnecessary thyroidectomies. Prophylactic thyroidectomy is recommended by age 5 or 6 once a mutation is identified in a patient, since penetrance is very high. MDE heteroduplex detection provides a quick, efficient, and inexpensive method of screening for RET mutations in MTC patients with unknown mutations, or for presymptomatic diagnosis in individuals at risk for inheriting a known RET mutation. Confirmation of the specific mutation can be achieved by restriction enzymatic digestion (if feasible) or by DNA sequencing. © 1996 Wiley-Liss, Inc.     

The pathology of preclinical medullary thyroid carcinoma

Medullary carcinoma of the thyroid (MTC) occurs sporadically, or in familial forms in familial medullary thyroid carcinoma and multiple endocrine neoplasia types 2A and 2B. In the familial forms it is associated with well-characterized, germline mutations in the RET protooncogene. The mutation sites differ in MEN2A and MEN2B, and MTC develops at an earlier age and is more aggressive in MEN2B. Screening of relatives of affected individuals for such mutations can identify those at risk of developing MTC and total thyroidectomy can be carried out in the first decade of life before the development of clinical disease. Analysis of such removed thyroid glands shows abnormalities of the parafollcular C-cells in almost all cases. The abnormalities range from C-cell hyperplasia, either diffuse or nodular, to microcarcinoma and occasionally frank MTC. The abnormalities are bilateral and affects the upper two thirds of the thyroid lobes. Microcarcinomas may be visible with the naked eye, but often they are identified only on microscopy. Histopathological examination of the entire gland is essential.     

Novel intronic polymorphisms in the RET proto-oncogene and their association with Hirschsprung disease

Germline mutations of the RET proto-oncogene have been found in familial and sporadic forms of Hirschsprung disease (HSCR), but also in the autosomal dominantly inherited multiple endocrine neoplasia type 2 (MEN2) syndromes, which comprise the medullary thyroid carcinoma (MTC) as an obligatory feature. Besides mutations various polymorphisms of the RET proto-oncogene are associated with the HSCR. In this study, we have characterized seven intronic RET polymorphisms (IVS2+9G>A, IVS4+48A>G, IVS12+47C>T, IVS14-24G>A, IVS19+47T>C, IVS20+96C>T, 3'UTR+124A>G) and investigated these variants by DNA sequencing in populations of 76 HSCR patients and 40 sporadic MTC patients as well as in a control population. Variants of four of these seven polymorphisms have a strong association with the HSCR phenotype. In contrast, none of the investigated polymorphisms show a significant difference in the genotype distribution and the allele frequencies in patients with sporadic MTC when compared to controls. These findings support the hypothesis that specific RET haplotypes cause or modify the HSCR phenotype.