Cornelis J. M. Lips

Clinical Screening as Compared with DNA Analysis in Families with Multiple Endocrine Neoplasia Type 2A

BACKGROUND Multiple endocrine neoplasia type 2A (MEN-2A) is characterized by medullary thyroid carcinoma in combination with pheochromocytoma and sometimes parathyroid adenoma. Missense mutations in the RET proto-oncogene are associated with MEN-2A. Their detection by DNA analysis allows the identification of carriers of the gene, in whom the risk of medullary thyroid carcinoma is 100 percent. We compared the reliability of biochemical tests with that of DNA analysis in identifying carriers of the MEN2A gene. METHODS Starting in 1975, we screened 300 subjects in four large families with MEN-2A for expression of the disease, using measurements of plasma calcitonin after stimulation with pentagastrin or calcium and urinary excretion of catecholamines and catecholamine metabolites. We tested for carrier status by DNA analysis, including linkage analysis, and more recently by analysis of mutations in the RET gene. RESULTS Of 80 MEN2A gene carriers (in 61 of whom carrier status was proved by DNA analysis), 66 had abnormal plasma calcitonin values and medullary thyroid carcinoma. Fourteen young carriers had normal results of plasma calcitonin tests. In 8 of these 14, thyroidectomy revealed small foci of medullary thyroid carcinoma; the remaining 6 have not yet been operated on. Of the other 220 family members, 68 were found by DNA analysis not to carry the MEN2A gene. None of these 68 subjects had medullary thyroid carcinoma or pheochromocytoma; 6 had elevated plasma calcitonin concentrations and underwent thyroidectomy but had only C-cell hyperplasia. CONCLUSIONS Unlike biochemical tests, DNA analysis permits the unambiguous identification of MEN2A gene carriers.

A second human calcitonin/CGRP gene

The calcitonin (CT) gene is alternatively expressed in a tissue‐specific fashion producing either the calcium regulatory hormone CT in the thyroid or the neuropeptide calcitonin gene related peptide (CGRP) in the brain. In medullary carcinoma of the thyroid both peptides are produced. We present here evidence for the existence in the human genome of a second CT gene, which is also expressed in human medullary thyroid carcinoma. This gene encodes a second human CGRP, differing from the known human CGRP in 3 of the 37 amino acids.

Menin Links Estrogen Receptor Activation to Histone H3K4 Trimethylation

The product of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene, menin, is an integral component of MLL1/MLL2 histone methyltransferase complexes specific for Lys4 of histone H3 (H3K4). We show that menin is a transcriptional coactivator of the nuclear receptors for estrogen and vitamin D. Activation of the endogenous estrogen-responsive TFF1 (pS2) gene results in promoter recruitment of menin and in elevated trimethylation of H3K4. Knockdown of menin reduces both activated TFF1 (pS2) transcription and H3K4 trimethylation. In addition, menin can directly interact with the estrogen receptor-alpha (ERalpha) in a hormone-dependent manner. The majority of disease-related MEN1 mutations prevent menin-ERalpha interaction. Importantly, ERalpha-interacting mutants are also defective in coactivator function. Our results indicate that menin is a critical link between recruitment of histone methyltransferase complexes and nuclear receptor-mediated transcription.

Von Hippel-Lindau disease: new strategies in early detection and treatment.

UNLABELLED Von Hippel-Lindau disease is an autosomal dominant inherited disorder causing hemangioblastomas of the central nervous system (CNS), retinal hemangiomas, renal cell carcinomas, pheochromocytomas, pancreatic and liver cysts, and epididymal cystadenomas. PURPOSE Since 1976, we have periodically screened for the lesions in a large affected family and were able to evaluate new strategies in detection and treatment. PATIENTS AND METHODS A total of 23 individuals underwent the screening program. A multidisciplinary team of physicians was involved. RESULTS In 13 patients (7 females and 6 males), a total of 31 tumors was detected; hemangioblastoma of the CNS (9), retinal angioma (4), renal involvement (8), pheochromocytoma (4), pancreatic lesions (4), and liver lesions (2) were diagnosed by periodic family screening. On the basis of more than 10 years of experience and current literature, new criteria for diagnosis and treatment have been proposed. CONCLUSION The von Hippel-Lindau disease gene appears to be a tumor suppressor gene, and its absence or a defect in its structure is responsible for the predisposition to the disease. Tumor development depends on a somatic second mutation in the homologous allele. That means, in disease-gene carriers, tumor growth may begin at any age. Most of the lesions can be treated successfully when diagnosed in time. Periodic screening by a multidisciplinary team has to be continued lifelong.

Multiple endocrine neoplasia type 2B mutation in human RET oncogene induces medullary thyroid carcinoma in transgenic mice

Multiple endocrine neoplasia type 2B (MEN 2B) is a familial cancer syndrome, in which the cardinal feature is medullary thyroid carcinoma (MTC), a malignant tumor arising from the calcitonin producing thyroid C-cells. MEN 2B is associated with a germline point mutation in the RET proto-oncogene, leading to a Met→Thr substitution at codon 918 in the kinase domain, which alters the substrate specificity of the protein. We used the human calcitonin gene (CALC-I) promoter to generate transgenic mice expressing either the human RET oncogene with the MEN2B-specific 918 Met→Thr mutation (CALC-MEN2B-RET) or the human non-mutated RET proto-oncogene (CALC-WT-RET) in the C-cells. At 20–22 months of age three out of eight CALC-MEN2B-RET transgenic founders presented with macroscopic bilateral MTC. In two founders nodular C-cell hyperplasia (CCH) was observed. Thyroid abnormalities were never observed in CALC-WT-RET transgenic mice or control non-transgenic mice analysed at this age. In some mice from established CALC-MEN2B-RET transgenic lines nodular CCH was observed from 8 months on whereas MTC was detected in 13% of mice from one CALC-MEN2B-RET line, from the age of 11 months on. These results show for the first time that the MEN2B mutation in the RET oncogene predisposes mice for MTC.

Inhibition of amyloid fibril formation of human amylin by N-alkylated amino acid and alpha-hydroxy acid residue containing peptides

Amyloid deposits are formed as a result of uncontrolled aggregation of (poly)peptides or proteins. Today several diseases are known, for example Alzheimers disease, Creutzfeldt-Jakob disease, mad cow disease, in which amyloid formation is involved. Amyloid fibrils are large aggregates of beta-pleated sheets and here a general method is described to introduce molecular mutations in order to achieve disruption of beta-sheet formation. Eight backbone-modified amylin derivatives, an amyloidogenic peptide involved in maturity onset diabetes, were synthesized. Their beta-sheet forming properties were studied by IR spectroscopy and electron microscopy. Modification of a crucial amide NH by an alkyl chain led to a complete loss of the beta-sheet forming capacity of amylin. The resulting molecular mutated amylin derivative could be used to break the beta-sheet thus retarding beta-sheet formation of unmodified amylin. Moreover, it was found that the replacement of this amide bond by an ester moiety suppressed fibrillogenesis significantly. Introduction of N-alkylated amino acids and/or ester functionalities-leading to depsipeptides-into amyloidogenic peptides opens new avenues towards novel peptidic beta-sheet breakers for inhibition of beta-amyloid aggregation.

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).

The spectrum of carcinoid tumours and carcinoid syndromes.

Carcinoids are neuroendocrine tumours of the gut which may also be found in the bronchus, pancreatic islets and retroperitoneum. They probably arise from gastrointestinal or bronchopulmonary pluripotential stem cells. Carcinoid tumours derived from these cells are potentially malignant; the strength of the tendency for aggressive growth correlates with the site of origin, depth of local penetration and the size of the tumour. Carcinoids occur sporadically or result from specific hereditary tumour syndromes. Mutations and/or aberrant expression of specific genes induce and promote tumour growth. Clinical features include local symptoms due to angulation or obstruction and hepatomegaly due to liver metastases. The carcinoid syndrome commonly involves flushing, diarrhoea, bronchospasm and hypotension. Other distinct syndromes may be caused by tumour release of products that may also be used as biochemical markers in diagnosis and follow-up. Scanning using radiolabelled octreotide, an analogue of somatostatin, sensitively identifies occult primary and metastatic deposits. Localized carcinoid tumours should be resected. Some patients benefit from hepatic resection. Palliation of symptoms is best achieved with octreotide. Hepatic artery chemoembolization may produce long-acting palliation. Further genetic characterization of the different types and stages of carcinoid development as well as assessment of gene expression profiles may improve differential diagnosis, prognosis and treatment.

The Multiple Endocrine Neoplasia Type 1 (MEN1) Tumor Suppressor Regulates Peroxisome Proliferator-Activated Receptor γ-Dependent Adipocyte Differentiation

ABSTRACT Menin, the product of the MEN1 (multiple endocrine neoplasia type 1) tumor suppressor gene, is involved in activation of gene transcription as part of an MLL1 (mixed-lineage leukemia 1)/MLL2 (KMT2A/B)-containing protein complex which harbors methyltransferase activity for lysine 4 of histone H3 (H3K4). As MEN1 patients frequently develop lipomas and peroxisome proliferator-activated receptor γ (PPARγ) is expressed in several MEN1-related tumor types, we investigated regulation of PPARγ activity by menin. We found that menin is required for adipocyte differentiation of murine 3T3-L1 cells and PPARγ-expressing mouse embryonic fibroblasts. Menin augments PPARγ target gene expression through recruitment of H3K4 methyltransferase activity. Menin interacts directly with the activation function 2 transcription activation domain of PPARγ in a ligand-independent fashion. Ligand-dependent coactivation, however, is dependent on the LXXLL motif of menin and the intact helix 12 of PPARγ. We propose that menin is an important factor in PPARγ-mediated adipogenesis and that loss of PPARγ function may contribute to lipoma development in MEN1 patients.

Medullary thyroid carcinoma: role of genetic testing and calcitonin measurement

All patients with a thyroid nodule should have their plasma CT measured. Stimulated CT is generally better than basal, but in the lower ranges false negatives and false positives still occur. In families with hereditary MTC, RET gene mutation analysis has superseded measurement of plasma CT in the detection of asymptomatic disease gene carriers. All individuals with apparently sporadic MTC, but in whom there is some suspicion of familial disease, should also have RET genetic analysis. A negative DNA result practically excludes the possibility of hereditary MTC in families where an index case has been investigated and obviates the need for further biochemical evaluation. Disease gene carriers may be divided into three distinct risk groups depending on the specific RET gene mutation in the family. The age at which presymptomatic surgery has to be performed depends on the risk group to which the patient belongs. Compared with the results of DNA analysis, the results of CT stimulation tests have become less important in the assessment of timing of surgery. During follow-up of patients who underwent surgery, measurement of plasma basal CT is still useful. The high sensitivity of measuring stimulated CT levels does not outweigh the burden of life-long periodic stimulation tests and the limited clinical consequences of slightly elevated levels. Stimulation tests are inevitable for persons at risk who prefer not to have genetic testing.