Germline RET proto-oncogene mutations in two Taiwanese families with multiple endocrine neoplasia type 2A

To elucidate the germline RET proto-oncogene mutations in Taiwanese families with multiple endocrine neoplasia type 2A (MEN 2A), we extracted DNA from peripheral blood leukocytes of 28 members of two families with MEN 2A. Oligonucleotide primers for exons 10 and 11 were used to analyze the nucleotide sequence of codons 609, 611, 618, and 620 of exon 10, and codon 634 of exon 11 of the RET proto-oncogene. Two fragments of genomic DNA were amplified by polymerase chain reaction (PCR). The amplified PCR products were separated and purified from primers and free nucleotides in agarose gels, and the expected 187-bp and 234-bp bands were cut from the gels and sequenced. Thirteen family members in the two MEN 2A kindreds had mutations in codon 634 of exon 11. In kindred 1 (15 members available for this study), a heterozygous codon 634 mutation in nine members and a homozygous codon 634 mutation in one member led to the substitution of Phe (TTC) for Cys (TGC). Three members of kindred 2 (13 members available for this study) had a heterozygous base pair change in codon 634, which led to the substitution of Arg (CGC) for Cys (TGC). In this study, we found two mutation events occurring in two MEN 2A kindreds and also discovered a homozygous point mutation in one woman that led to heterozygous mutations in all of her children.     

The physical map of the human RET proto-oncogene

The RET proto-oncogene, a transmembrane tyrosine kinase receptor, is involved in the development of at least five different disease phenotypes. RET is activated through somatic rearrangements in a number of cases of papillary thyroid carcinoma while germ-line point mutations are associated with three inherited cancer syndromes MEN 2A, MEN 2B and FMTC. Moreover, point mutations or heterozygous deletions of RET are found in the dominant form of Hirschsprung disease or congenital colonic aganglionosis. We cloned the entire RET genomic sequence in a contig of cosmids encompassing 150 kb, from the CA repeat sTCL-2 to the region upstream the RET promoter, and established the position of the 20 exons of the RET gene with respect to a detailed restriction map based on eight endonucleases. A new highly polymorphic CA repeat sequence was identified within intron 5 of RET (RET-INT5). Finally the orientation of RET on chromosome 10q11.2 made it possible to orientate three other genes rearranged with RET in papillary thyroid carcinomas, namely H4/D10S170 on 10q21, R1 alpha on 17q23 and RFG2/Ele1 on 10q11.2.     

Oncological implications of RET gene mutations in Hirschsprung's disease

BACKGROUND: Germline mutations of the RET proto-oncogene identical to those found in the tumour predisposition syndrome multiple endocrine neoplasia type 2A (MEN2A), were detected in 2.5-5% of sporadic and familial cases of Hirschsprung's disease. Some patients with Hirschsprung's disease may therefore be exposed to a highly increased risk of tumours. AIMS: To define clinical use of RET gene testing in Hirschsprung's disease and related patient management from an oncological point of view. METHODS: Sixty patients with Hirschsprung's disease were screened for RET mutations. In three, MEN2A type RET mutations were detected. Case reports for these three patients are presented. RESULTS AND CONCLUSIONS: Only 22 families or sporadic patients with Hirschsprung's disease and MEN2A type RET mutations have been reported. Therefore, it is difficult to predict tumour risk for patients with familial or sporadic Hirschsprung's disease, and their relatives, who carry these mutations. For these mutation carriers, periodic screening for tumours as in MEN2A is advised, but prophylactic thyroidectomy is offered hesitantly. RET gene testing in familial or sporadic Hirschsprung's disease is not recommended at present outside a complete clinical research setting. In combined MEN2A/Hirschsprung's disease families RET gene testing, tumour screening, and prophylactic thyroidectomy are indicated as in MEN2A.     

Mutation analysis of glial cell line-derived neurotrophic factor, a ligand for an RET/coreceptor complex, in multiple endocrine neoplasia type 2 and sporadic neuroendocrine tumors

Causative germline missense mutations in the RET proto-oncogene have been associated with over 92% of families with the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). MEN 2A is characterized primarily by medullary thyroid carcinoma (MTC) and pheochromocytoma, both tumors of neural crest origin. Parathyroid hyperplasia or adenoma is also seen in MEN 2A, but rarely in MEN 2B, which has additional stigmata, including a marfanoid habitus, mucosal neuromas, and ganglioneuromatosis of the gastrointestinal tract. In familial MTC, MTC is the only lesion present. Somatic RET mutations have also been identified in a subset of sporadic MTCs, pheochromocytomas, and rarely, small cell lung cancer, but not in sporadic parathyroid hyperplasias/adenomas or other neuroendocrine tumors. Glial cell line-derived neurotrophic factor (GDNF) and its receptor molecule GDNFR-alpha, have recently been identified as members of the RET ligand binding complex. Therefore, the genes encoding both GDNF and GDNFR-alpha are excellent candidates for a role in the pathogenesis of those MEN 2 families and sporadic neuroendocrine tumors without RET mutations. No mutations were found in the coding region of GDNF in DNA samples from 9 RET mutation negative MEN 2 individuals (comprising 6 distinct families), 12 sporadic MTCs, 17 sporadic cases of parathyroid adenoma, and 10 small cell lung cancer cell lines. Therefore, we find no evidence that mutation within the coding regions of GDNF plays a role in the genesis of MEN 2 and sporadic neuroendocrine tumors.     

Familial medullary thyroid carcinoma: not a distinct entity? Genotype-phenotype correlation in a large family

BACKGROUND: Multiple endocrine neoplasia type 2A (MEN 2A) is a hereditary syndrome characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism. Familial MTC (FMTC) is characterized by MTC only. Both MEN 2A and FMTC are caused by germline mutations of the RET proto-oncogene. PURPOSE: To assess genotype/phenotype correlations, large families have to be examined periodically over a long period using an extensive screening program. PATIENTS AND METHODS: Since 1973, we screened a large family with hereditary C cell carcinoma for MTC, pheochromocytoma, and parathyroid disease by clinical tests and imaging methods. A germline codon Cys618 to Ser mutation in the RET proto-oncogene was recently identified in this family. The disease phenotype associated with this mutation was compared with that of Cys634 mutations in some other large MEN 2A families. RESULTS: The distinct course of disease in the family described here is similar to that in other FMTC families and MEN 2A families with a Cys618 mutation of the RET gene, but clearly different from that in families with a Cys634 mutation. The frequency of pheochromocytomas and parathyroid disease is clearly lower, whereas cure rates and life expectancy are higher. However, in families with a Cys618 mutation, pheochromocytoma and parathyroid disease do occur. CONCLUSION: In FMTC families with cysteine codon mutations of the RET proto-oncogene, screening for other endocrinopathies is mandatory, since these may not be MTC-only families. Therefore, we suggest that MEN 2A families should not be subclassified into MEN 2A and FMTC, but rather according to their specific mutation in the RET protein (i.e., for this family MEN 2A RET C618S).     

Full activation of MEN2B mutant RET by an additional MEN2A mutation or by ligand GDNF stimulation

Germline mutations of RET gene, encoding a receptor tyrosine kinase, have been associated with the MEN2A and MEN2B inherited cancer syndromes. In MEN2A mutations affecting cysteine residues in the extracellular domain of the receptor cause constitutive activation of the tyrosine kinase by the formation of disulfide-bonded homodimers. In MEN2B a single mutation in the tyrosine kinase domain (Met918Thr) has been identified. This mutation does not lead to dimer formation, but has been shown (both biologically and biochemically) to cause ligand-independent activation of the Ret protein, but to a lesser extent than MEN2A mutations. Intramolecular activation by cis-autophosphorylation of RetMEN2B monomers has been proposed as a model for activation, although alternative mechanisms can be envisaged. Here we show that the activity of RetMEN2B can be increased by stable dimerization of the receptor. Dimerization was achieved experimentally by constructing a double mutant receptor with a MEN2A mutation (Cys634Arg) in addition to the MEN2B mutation, and by chronic exposure of RetMEN2B-expressing cells to the Ret ligand GDNF. In both cases full activation of RetMEN2B, measured by 'in vitro' transfection assays and biochemical parameters, was seen. These results indicate that the MEN2B phenotype could be influenced by the tissue distribution or concentration of Ret ligand(s).     

Usefulness of the genetic study in the diagnosis of medullary carcinoma of the thyroid

Germ-line mutations in the RET proto-oncogene are associated with multiple endocrine neoplasia type 2A (MEN 2A) and with familial medullary thyroid carcinoma (FMTC). Detection of these mutations allows the identification of the affected kindred members, who will develop medullary thyroid carcinoma (MTC) in 100% of cases. We studied 24 patients of two kindreds (MEN 2A and FMTC). Basal calcitonin levels and pentagastrin-stimulated calcitonin were measured in all patients. The RET mutations were detected by DNA analysis. The RET mutations were identified in 14 patients. Two of them had been operated in the past, 2 refused operation and 4 were living abroad. In the 6 remaining, only one showed a thyroid mass, basal calcitonin was normal in all patients except one, and pentagastrin-stimulated calcitonin was negative in 2 patients. Total thyroidectomy was performed in all cases. Histology showed C-cell hyperplasia in all patients and MTC in 5 of them. In MEN 2A and FMTC DNA analysis allows the identification of RET mutation carriers, in which presymptomatic thyroidectomy allows and improvement in survival.     

RET protooncogene mutational analysis in multiple endocrine neoplasia syndrome type 2B: case report and review of the literature

Multiple endocrine neoplasia, type 2B (MEN 2B), is a phenotypic variant of a group of autosomal-dominant neurocristopathies. MEN 2B is associated with medullary thyroid carcinoma and pheochromocytoma with oral, ocular, and alimentary submucosal ganglioneuromas and Marfanoid body features. Approximately 50% of cases are thought to be spontaneous mutations. The RET protooncogene (RET) is a 21-exon gene encoding a tyrosine kinase receptor. A codon 918 germ line mutation, which converts a highly conserved methionine to a threonine in the intracellular tyrosine kinase portion of this receptor of RET, has been identified in 95% of patients with MEN 2B. This mutation is easily detected by a direct deoxyribonucleic acid sequencing or restriction enzyme (Fok 1) analysis of amplified polymerase chain reaction products. The RET gene is normally expressed in the oral and gastrointestinal submucosal neural ganglia, and the codon 918 mutation is thought to cause neuromas by virtue of its transforming activity in these ganglia. Identifying clinical features of MEN 2B in an 11-year-old boy by an oral pathologist led to confirmation by mutational analysis. Before genetic testing was available, the patient, and at a later date his mother, underwent thyroidectomies based solely on biochemical testing. Results indicated the patient had the codon 918 mutation, whereas his phenotypically normal mother, father, and older brother had normal RET analyses. Studies in families have demonstrated that the mutant allele is derived from the father with possible acquisition during spermatogenesis. We believe the mother of our affected patient to be normal; the absence of phenotypic features of MEN 2B and a normal genotype suggest her calcitonin abnormalities and minimal evidence for C-cell hyperplasia were inconsequential. Molecular analysis for RET abnormalities will likely supplant biochemical methods of diagnosis in patients with MEN 2B.     

Anti-HCV envelope prevalence in blood donors from Baden-Württemberg

It has been slightly less than 3 years since mutations of the RET proto-oncogene were identified in multiple endocrine neoplasia type 2. The period since the initial report has been one of intense activity with a wide spectrum of mutations identified in exons 10, 11, 13, 14, 15 and 16 of this gene. Recent studies have provided experimental evidence for an activating effect of the exon 11 and 16 mutations. Two questions are addressed in this report: the high rate of false positive tests in prospective screening studies and the implications for future interpretation of this test: and the mechanism of transformation in MEN 2.     

The RET gene in thyroid pathology]

The RET proto-oncogene encodes a receptor tyrosine kinase which plays a crucial role during the embryonic development of the enteric nervous system and of the kidney. Cytogenetic analyses of papillary thyroid carcinoma (PTC), a neoplasm which originates from thyrocytes, have revealed that somatic rearrangements of the RET gene are involved in the etiology of a significant proportion of this tumour. Medullary thyroid carcinoma (MTC) which arises from neural-crest derived C-cells is the cardinal disease feature of multiple endocrine neoplasia type 2 (MEN 2), a dominantly inherited cancer syndrome. Recent studies have provided evidence that germline mutations of the RET gene are the underlying genetic events responsible for MEN 2. This review focuses on the role of RET mutations in the pathogenesis of PTC and MTC and summarizes our present knowledge on the consequences of these alterations on the RET tyrosine kinase function. We further describe a transgenic mouse model for hereditary MTC. Mice carrying a MEN 2A allele of RET under the control of the CGRP/calcitonin promoter develop bilateral and multifocal MTC, morphologically and biologically similar to human MTC.     

Analysis of RET proto-oncogene abnormalities in patients with MEN 2A, MEN 2B, familial or sporadic medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) may occur either as a sporadic or familial (FMTC) disease. Multiple endocrine neoplasia (MEN) type 2, inherited as an autosomal dominant disease, is characterized by coexistence of MTC with other endocrine neoplasia. Activating mutations of the RET proto-oncogene, involving the somatic or the germinal cell lineage, are found in both inherited and acquired forms. In this study, RET mutations were screened in 47 individuals either affected by MTC or belonging to families with hereditary MTC. Exons 10, 11, 13, 14, 15 and 16 of the RET gene were amplified by polymerase chain reaction and examined by DNA sequence and/or restriction enzyme analysis to detect mutations in purified amplicons. Six MEN 2A families with a germline mutation at codon 634, one FMTC family carrying a mutation at codon 618 and two MEN 2B families with a mutation at codon 918 were identified. In affected members of a MEN 2A family no known RET mutations were observed. Besides, we identified a germline mutation in a patient with apparently sporadic MTC and in two out of three sons, indicating the presence of a sporadic misclassified familial disease. In all of the families examined we were able to distinguish the affected vs unaffected (not at risk) members. A somatic mutation of codon 918 was detected in three out of ten patients with apparently sporadic MTC.     

Sporadic phaeochromocytomas are rarely associated with germline mutations in the von Hippel-Lindau and RET genes

OBJECTIVE: von Hippel-Lindau (VHL) disease and multiple endocrine neoplasia type 2 (MEN2) are autosomal dominant cancer syndromes. In both conditions, phaeochromocytoma is a prominent feature. It has recently been suggested that phaeochromocytoma can be the presenting and sole clinical manifestation of these multi-organ syndromes. The aim of this study was to ascertain the incidence of VHL and MEN2 among patients with sporadic phaeochromocytoma by mutational analysis. PATIENTS: Twenty-seven unrelated patients with biochemically and/or anatomically proven sporadic phaeochromocytoma were evaluated. DESIGN AND MEASUREMENTS: Constitutional DNA obtained from the patients was analysed by single stranded conformational analysis (SSCP) for mutations within the VHL gene coding sequence and by denaturing gradient gel electrophoresis (DGGE) for predominant mutations in exons 10, 11 and 16 of the RET proto-oncogene. The incidence of patients positive for either VHL or RET germline mutations was assessed. RESULTS: Twenty-six of 27 patients had normal SSCP patterns in all three VHL gene exon segments and only one patient, with an atypical clinical presentation, had an aberrant pattern in exon 3 which upon DNA sequencing was shown to harbor a G to A transversion mutation at nucleotide 695. All patients had normal RET exon 10, 11 and 16 DGGE migration patterns. CONCLUSION: Most, if not all, patients with typical unilateral sporadic phaeochromocytoma do not have von Hippel-Lindau disease or MEN2. Thus, clinical and/or molecular investigation for von Hippel-Lindau disease and MEN2 in this patient population does not appear to be indicated.     

Gene structure of the human MET proto-oncogene

By direct sequencing of cosmids using primers designed from the known cDNA sequence, we identified 19 exons in the human MET proto-oncogene, and sequenced the corresponding 5' and 3' exon-intron junctions. By homology search in the database of the Washington University Genome Sequence Center (GSC), we identified one additional exon. These 20 exons, together with a previously reported exon, bring the total exon number of MET to 21. Oligonucleotide primers were designed to amplify each exon and adjacent intronic sequences to permit examination of each exon for mutations. By restriction mapping, we assembled a 110 kb genomic contig that covered almost the entire MET proto-oncogene. This information is relevant for the screening of recently reported mutations of the MET gene which cause hereditary papillary renal carcinomas and for the search for additional mutations of the same gene which may play a role in the pathogenesis of common human carcinomas including carcinomas of the breast, ovary and pancreas.     

The molecular pathology of RET protooncogene in families with multiple endocrine neoplasia type 2A

BACKGROUND: Multiple endocrine neoplasm type 2A (MEN 2A) is an autosomal dominantly inherited disease characterized by medullary thyroid carcinoma, pheochromocytoma and hyperparathyroidism. Mutations have been identified in the extracellular domain of the RET proto-oncogen product (10q11.2) in MEN 2A patients. In each case a single base pair substitution results in replacement of cysteine with another amino acid. Most MEN 2A patients have mutations of codon 634. PATIENTS AND METHODS: Sixty-five unrelated MEN 2A patients from seven families were studied. Polymerase chain reaction, segregation, sequence analysis and restriction enzyme digestion were performed. RESULTS: Of seven families, four had the TGC to TAC transition, two families the TGC to TGG transversion and one family the TGC to CGC transition in codon 634 of RET. CONCLUSIONS: We found all the mutations in codon 634. The characterization of MEN 2A mutations allows early and presymptomatic diagnosis in this syndrome.     

Characterization of ret oncogenic activation in MEN2 inherited cancer syndromes

Germline mutations of c-ret, encoding a receptor-type tyrosine kinase, were found to be associated with variants of multiple endocrine neoplasia type 2 (MEN2A, MEN2B), and familial medullary thyroid carcinoma. NIH/3T3 stable transfectants expressing RET with a mutation of MEN2A (MEN2A/RET) or MEN2B (MEN2B/RET) gained a transformed morphology, formed colonies in soft agar, and formed tumors in nude mice. These results confirmed that both MEN2A/RET and MEN2B/RET exert dominant transforming activities in NIH/3T3 cells. However, in contrast to their clinical manifestation, transfectants expressing MEN2A/RET exhibited a higher tumorigenicity in nude mice than transfectants expressing MEN2B/RET may depend on the presence of its ligand and/or substrates that are absent in NIH/3T3 cells. No change in the cellular localization of the mutated RET proteins was observed compared to c-RET. Interestingly, ret activation in NIT/3T3 cells appeared to be associated with up-regulation of homologous gap-junctional intercellular communication and increased expression of a gap-junctional protein, connexin43.     

Molecular genetic diagnosis of von Hippel-Lindau disease in familial phaeochromocytoma

Inherited predisposition to phaeochromocytoma is seen in multiple endocrine neoplasia type 2 syndromes, von Hippel-Lindau (VHL) disease, and neuro-fibromatosis type 1. In addition familial phaeochromocytoma alone has been reported. To investigate the genetic basis for familial phaeochromocytoma alone, we screened three affected kindreds for mutations in the RET proto-oncogene and the VHL tumour suppressor gene. We did not detect MEN 2 associated RET mutations in any family, but missense VHL gene mutations (V155L and R238W) were identified in two kindreds with no clinical evidence of VHL disease. Patients with familial, multiple, or early onset phaeochromocytoma should be investigated for germline VHL and RET gene mutations as the molecular diagnosis of multisystem familial cancer syndromes enables appropriate counselling and screening to be provided.     

Mutations in the extracellular domain cause RET loss of function by a dominant negative mechanism

The RET proto-oncogene encodes a tyrosine kinase receptor expressed in neuroectoderm-derived cells. Mutations in specific regions of the gene are responsible for the tumor syndromes multiple endocrine neoplasia types 2A and 2B (MEN 2A and 2B), while mutations along the entire gene are involved in a developmental disorder of the gastrointestinal tract, Hirschsprung's disease (HSCR disease). Two mutants in the extracellular domain of RET, one associated with HSCR disease and one carrying a flag epitope, were analyzed to investigate the impact of the mutations on RET function. Both mutants were impeded in their maturation, resulting in the lack of the 170-kDa mature form and the accumulation of the 150-kDa immature form in the endoplasmic reticulum. Although not exposed on the cell surface, the 150-kDa species formed dimers and aggregates; this was more pronounced in a double mutant bearing a MEN 2A mutation. Tyrosine phosphorylation and the transactivation potential were drastically reduced in single and double mutants. Finally, in cotransfection experiments both mutants exerted a dominant negative effect over protoRET and RET2A through the formation of a heteromeric complex that prevents their maturation and function. These results suggest that HSCR mutations in the extracellular region cause RET loss of function through a dominant negative mechanism.     

A complex nine base pair deletion in RET exon 11 common in sporadic medullary thyroid carcinoma

Genetic alteration of the RET proto-oncogene is associated with multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B), familial medullary thyroid carcinoma (FMTC) and Hirschprung's disease. Oncogenically activated RET has also been demonstrated in sporadic medullary thyroid tumors, which in some cases show somatic missense mutations. We have recently described a complex 9 bp deletion in RET exon 11 in a single case of sporadic MTC. In order to determine the prevalence of this mutation among sporadic MTC tumors, we have now analysed 15 cases and five normal controls by PCR-based nonradioactive single-strand conformational polymorphism analysis (PCR-SSCP) and fragment size analysis of exon 11. DNA was extracted from microdissected tumor tissue or normal cells and subjected to nested PCR prior to analysis. A markedly divergent SSCP pattern and a PCR fragment 9 bp shorter than normal were demonstrated in 14 of the 15 MTC tumors. Sequencing revealed the deletion of nine bases encompassing a key cysteine at codon 634, often altered in MEN 2A. Four lymphocyte controls and normal thyroid tissue from one patient failed to show the deletion. Several factors in the DNA sequence environment immediately surrounding the deletions, including an extended inverted repeat, several direct repeats and a so-called symmetric element suggest that the deletional events may be non-random.     

Ectopic accumulation of 99mTc-HMDP in primary lung cancer in comparison with CT findings

Hypertension attributable to pheochromocytoma is a very attractive model for the elucidation of the pathogenesis of hypertension. Sixteen different point mutations in the RET proto-oncogene and 30 mutations in the Von Hippel-Lindau (VHL) tumor suppressor gene have been identified so far associated with expression of pheochromocytoma. Each of these mutations initiates either the syndrome of multiple endocrine neoplasia type 2 (MEN 2) (MEN 2A and MEN 2B) or the VHL disease. Certain mutations in both genes are associated with the presence of pheochromocytoma. In general, these pheochromocytomas produce catecholamines that result in hypertension. Therefore, analysis for germline mutations in these genes are of practical value, because susceptibility to these diseases can be predicted in as yet clinically unaffected relatives.