Two antisense promoters in the immunoglobulin mu-switch region drive expression of c-myc in the Burkitt's lymphoma cell line BL67.

In the Burkitt's lymphoma (BL) cell line BL67 the first exon of the c-myc gene is fused to the mu-switch region of the immunoglobulin heavy-chain gene (IgH). BL67 cells express IgH/c-myc hybrid RNAs which are initiated in the immunoglobulin locus, transcribed across the chromosomal breakpoint into the first exon of c-myc and spliced using the physiological splice donor and acceptor sites of the c-myc gene. We have isolated cDNAs of these hybrid RNAs and characterized the start points in the Ig heavy-chain gene. Two promoters were identified in the mu-switch region of BL67 cells which give rise to antisense transcription of the mu-gene. These promoters are also active in other BL cell lines, in B cells without Ig translocation and in a T-cell line. Both promoters co-localize with DNAase I-hypersensitive sites, HNF and HSW, in the mu-switch region. The structures of IgH/c-myc hybrid RNAs and of the corresponding promoters are described.

The ribose 5-phosphate isomerase-encoding gene is located immediately downstream from that encoding murine immunoglobulin kappa.

The immunoglobulin kappa locus (Ig kappa) is active only in the B-lymphocyte cell lineage. By exon-trapping we found a gene situated downstream from the murine Ig kappa locus. This gene encodes a protein with 53% sequence identity to the ribose 5-phosphate isomerase A (RPI-A) of Escherichia coli and is therefore likely to be the murine homologue (mRPI) of this enzyme. We confirmed this assumption by showing that a glutathione S-transferase (GST)::mRPI fusion protein has enzymatic activity and that an anti-mRPI antibody detects a protein of the predicted mass of RPI (33 kDa). Cloning and sequencing of the human counterpart show that the RPI gene is evolutionarily conserved. The expression of mRPI is not influenced by the rearrangement status of the Ig kappa locus in B cells and mRPI is expressed in all tissues. We thus show that two genes with very different expression patterns, a housekeeping gene and a gene expressed in a tissue-specific manner, can be located on a chromosome in close proximity to each other.

Incidence and clinical relevance of RET proto-oncogene germline mutations in pheochromocytoma patients.

BACKGROUND: Autosomal dominant cancer syndrome--multiple endocrine neoplasia type 2 (MEN 2), may exist more often than expected in patients with pheochromocytoma. Germline mutations identified recently in MEN 2 can be revealed by genetic screening. OBJECTIVE: To evaluate the frequency of RET (rearranged during transfection) mutations in patients with pheochromocytoma. DESIGN AND METHODS: We genetically screened germline mutations in the RET proto-oncogene and clinically re-evaluated patients with pheochromocytoma. A pentagastrin test and other biochemical studies were performed in all patients. SETTING: Department of Internal Medicine and Hypertension, The Medical University of Warsaw, Warsaw, Poland and the Department of Nephrology and Hypertension, Albert Ludwigs University, Freiburg, Germany. PARTICIPANTS: Seventy seven unselected patients with pheochromocytoma (19 men, 58 women, mean age: 51.55 +/- 1.5 years; pheochromocytoma confirmed histopathologically) out of 162 diagnosed and treated in the years 1957-1998 in the Department of Internal Medicine and Hypertension in Warsaw, Poland. The other 85 patients did not respond to the written invitation. MAIN OUTCOME MEASURES: The finding of RET mutations and diagnosis of MEN 2 in patients with pheochromocytoma. RESULTS: Genetic testing revealed germline mutations in the RET proto-oncogene in six patients (7.8%). All carriers had mutation of exon 11, codon 634: TGC to CGC. In four patients with this mutation, medullary thyroid carcinoma (MIC) was diagnosed and in three cases, surgically treated. Biochemical parameters: parathormone 31.88 +/- 2.87 pg/ml, calcitonin: 0 min 0.23 +/- 0.14 ng/ml; 2 min 0.49 +/- 0.21 ng/ml; 5 min 0.48 +/- 0.21 ng/ml, metoxycatecholamines: 601.62 +/- 42.71 microg/24h, epinephrine: 1.94 +/- 0.17 microg/24h, norepinephrine 13.96 +/- 1.3 microg/24h, carcinoembryonic antigen (CEA) 9.94 +/- 4.3 ng/ml. Ambulatory blood pressure monitoring (ABPM): systolic blood pressure (SBP): 116 +/- 1.9 mmHg, diastolic blood pressure (DBP): 73.7 +/- 0.9 mmHg. Clinical, biochemical and imaging procedures did not reveal any recurrence of pheochromocytoma in the 77 patients studied. CONCLUSIONS: Patients with pheochromocytoma should be genetically screened for mutations of the RET proto-oncogene. These patients should undergo clinical screening for MEN 2. In addition, genetic studies can be useful for the screening of the families of the carriers.

Phaeochromocytoma associated with a de novo VHL mutation as form fruste of von Hippel-Lindau disease.

UNLABELLED: Phaeochromocytomas usually occur sporadically but may be associated with dominant inherited cancer syndromes such as multiple endocrine neoplasia type 2 (MEN 2), von Hippel-Lindau disease (VHL) and type 1 neurofibromatosis. We report on a boy presenting at age 8 years with an isolated benign phaeochromocytoma of the left adrenal. Three years later a second adrenal phaeochromocytoma was diagnosed on the right side and removed. His family history was negative. Genetic analysis did not show a mutation in the MEN 2 susceptible proto-oncogene rearranged during transfection; however, we found a germline missense mutation in the VHL gene (nucleotide 695 G to A transversion) which has been described only twice before in the literature. Both parents had normal (wild type) VHL copies indicating that our patient had a de novo germline VHL mutation. Careful clinical evaluation of the patient at 18 years did not reveal any other manifestations of VHL disease. CONCLUSION: Carriers of von Hippel-Lindau germline mutations can present with a form fruste of von Hippel-Lindau disease presenting initially with unilateral phaeochromocytoma and therefore mutation analysis should be carried out.

Reconsideration of biallelic inactivation of the VHL tumour suppressor gene in hemangioblastomas of the central nervous system.

OBJECTIVES: Cerebellar haemangioblastoma occurs sporadically or as a component tumour of autosomal dominant von Hippel-Lindau disease. Biallelic inactivation of the VHL tumour suppressor gene, which is located on chromosome 3p, has been shown to be involved in the pathogenesis of both tumour entities. Mechanisms of VHL inactivation are intragenic mutations, mitotic recombination events, and hypermethylation of the promoter region. The systematic and complete examination of these genetic and epigenetic phenomena in large series of von Hippel-Lindau disease related and sporadic hemangioblastomas has, thus far, not been performed. METHODS: In the largest series to date, 29 von Hippel-Lindau disease associated and 13 sporadic haemangioblastomas were investigated for all suggested inactivating mechanisms of the VHL gene using single strand conformational polymorphism (SSCP), loss of heterozygosity (LOH), and methylation analyses. Additionally, corresponding blood samples of all patients were screened for VHL germline mutations by SSCP and Southern blotting. RESULTS: Germline mutations were identified in 94% of patients with von Hippel-Lindau disease and their tumours and 62% of these hemangioblastomas showed LOH of chromosome 3p. Of the 13 sporadic tumours, 23% showed a single somatic mutation of the VHL gene that was not present in the germline. 3p LOH was identified in 50% of informative sporadic tumours. No von Hippel-Lindau disease related or sporadic tumour demonstrated VHL promoter hypermethylation. CONCLUSIONS: For most von Hippel-Lindau disease related haemangioblastomas, the inactivation or loss of both alleles of the VHL gene, as predicted by the Knudson two hit theory, is required. However, in a subset of tumours including most sporadic haemangioblastomas, the genetic pathways involved in tumorigenesis have yet to be defined and may represent alterations of a different pathway or pathways.

The impact of molecular genetic analysis of the VHL gene in patients with haemangioblastomas of the central nervous system.

OBJECTIVES: Haemangioblastoma of the CNS occurs as a sporadic entity and as a manifestation of the autosomal dominant von Hippel-Lindau disease with the major additional components retinal angioma, renal cancer, and pheochromocytoma. Genetic testing for germline mutations predisposing to von Hippel-Lindau disease has been available since identification of the VHL tumour suppressor gene. The impact of this testing was evaluated in patients with haemangioblastomas seen in this centre. METHODS: A register and database of patients with symptomatic haemangioblastomas for the last 15 years was evaluated. The VHL gene was analysed by the SSCP method for all exons and Southern blotting for mutations and deletions of the gene. RESULTS: 141 patients with haemangioblastoma of the CNS were registered. In 81 patients (57%) there was a disease predisposing germline mutation including eight novel mutations. Population related calculation of patients from the administrative district of Freiburg disclosed VHL germline mutations in 22% of the patients with haemangioblastoma. Analysis of mutation carriers for clinical information suggestive of the syndrome showed (1) a positive family history of a brain tumour in 50%, (2) a history for the patient of extracranial manifestations in 36% (retinal angioma 30%, pheochromocytoma 6%), and (3) 19% presenting with multiple brain tumours when first admitted. By genetic testing of haemangioblastoma patients without any indications of von Hippel-Lindau disease mutation carriers were identified in 14%. Sensitivity of VHL germline testing was 86%. CONCLUSIONS: DNA analysis for VHL germline mutations is clearly superior to clinical information in the diagnosis of von Hippel-Lindau disease. Although the percentage of von Hippel-Lindau disease associated haemangioblastoma decreases after the fourth decade of life and is infrequent in patients without other symptomatic lesions and a negative family history, it is recommended that every patient with CNS haemangioblastoma should be screened for von Hippel-Lindau disease germline mutations. This provides the key information and enables screening for extraneurological tumours of the patients and investigations of the patient's family to ameliorate management of von Hippel-Lindau disease.