Hiltrud Brauch

Germline mutations in the Von Hippel-Lindau disease (VHL) gene in families from North America, Europe, and Japan

Germline mutation analysis was performed in 469 VHL families from North America, Europe, and Japan. Germline mutations were identified in 300/469 (63%) of the families tested; 137 distinct intragenic germline mutations were detected. Most of the germline VHL mutations (124/137) occurred in 1–2 families; a few occured in four or more families. The common germline VHL mutations were: delPhe76, Asn78Ser, Arg161Stop, Arg167Gln, Arg167Trp, and Leu178Pro. In this large series, it was possible to compare the effects of identical germline mutations in different populations. Germline VHL mutations produced similar cancer phenotypes in Caucasian and Japanese VHL families. Germline VHL mutations were identified that produced three distinct cancer phenotypes: (1) renal carcinoma without pheochromocytoma, (2) renal carcinoma with pheochromocytoma, and (3) pheochromocytoma alone. The catalog of VHL germline mutations with phenotype information should be useful for diagnostic and prognostic studies of VHL and for studies of genotype‐phenotype correlations in VHL.

Up-regulation of hypoxia-inducible factors HIF-1α and HIF-2α under normoxic conditions in renal carcinoma cells by von Hippel-Lindau tumor suppressor gene loss of function

Hypoxia induces transcription of a range of physiologically important genes including erythropoietin and vascular endothelial growth factor. The transcriptional activation is mediated by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric member of the basic helix–loop–helix PAS family, composed of α and β subunits. HIF-1α shares 48 per cent identity with the recently identified HIF-2α protein that is also stimulated by hypoxia. In a previous study of hemangioblastomas, the most frequent manifestation of hereditary von Hippel-Lindau disease (VHL), we found elevated levels of vascular endothelial growth factor and HIF-2α mRNA in stromal cells of the tumors. Mutations of the VHL tumor suppressor gene are associated with a variety of tumors such as renal clear cell carcinomas (RCC). In this study, we analysed the expression of the hypoxia-inducible factors HIF-1α and HIF-2α in a range of VHL wildtype and VHL deficient RCC cell lines. In the presence of functional VHL protein, HIF-1α mRNA levels are elevated, whereas HIF-2α mRNA expression is increased only in cells lacking a functional VHL gene product. On the protein levels, however, in VHL deficient cell lines, both HIF-α subunits are constitutively expressed, whereas re-introduction of a functional VHL gene restores the instability of HIF-1α and HIF-2α proteins under normoxic conditions. Moreover, immunohistochemical analyses of RCCs and hemangioblastomas demonstrate up-regulation of HIF-1α and HIF-2α in the tumor cells. The data presented here provide evidence for a role of the VHL protein in regulation of angiogenesis and erythropoiesis mediated by the HIF-1α and HIF-2α proteins.

Von Hippel-Lindau (VHL) disease with pheochromocytoma in the Black Forest region of Germany: evidence for a founder effect.

We identified a germline missense mutation at nucleotide 505 (T to C) of the VHL tumor suppressor gene in 14, apparently unrelated, VHL type 2A families from the Black Forest region of Germany. This mutation was previously identified in two VHL 2A families living in Pennsylvania (USA). All affected individuals in the 16 families shared the same VHL haplotype indicating a founder effect. This missense mutation at codon 169 (Tyr to His) would probably cause an alteration in the structure of the putative VHL protein. The association of this distinct mutation with the pheochromocytoma phenotype in VHL may help to elucidate the genetic mechanism of carcinogenesis in this multi tumor cancer syndrome.

Mutations in the VHL tumor suppressor gene and associated lesions in families with von Hippel-Lindau disease from central Europe.

Abstract von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome predisposing to retinal, cerebellar and spinal hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic tumors. Clinically two types of the disease can be distinguished: VHL type 1 (without pheochromocytoma) and VHL type 2 (with pheochromocytoma). We report VHL germline mutations and trends in phenotypic variation in families from central Europe. We identified 28 mutations in 53/65 (81.5%) families with 18 (64%) mutations being unique to this population. Whereas types and distribution of mutations as well as a strong correlation of missense mutations with the VHL 2 phenotype were similar to those identified in other populations, these families have provided new insights into the molecular basis for variability in the VHL 2 phenotype. Seven different missense mutations in exons 1 and 3 varied in their biological consequences from a minimal VHL 2 phenotype with pheochromocytoma only to a full VHL 2 phenotype with RCC and pancreatic lesion. These findings contribute to a better understanding of the fundamental mechanisms of VHL disease and its phenotypic variability. Further, we have provided rapid VHL screening for the families in central Europe, which has resulted in improved diagnosis and clinical management.

Fixed archival tissue. Purify DNA and primers for good PCR yield

The analysis of archival formalin-fixed, paraffin-embedded tissue samples becomes increasingly important for molecular biology studies. As fixation and paraffin-embedding cause alterations of proteins and nucleic acids, archival sources of DNA must be handled with care. To address the need for specificity and reproducibility, we developed an improved protocol for semi-automated DNA extraction adapted to fixed, embedded tissue samples, and a PCR approach using HPLC-purified primers.

Detection of a germline mutation and somatic homozygous loss of the von Hippel-Lindau tumor-suppressor gene in a family with a de novo mutation : A combined genetic study, including cytogenetics, PCR/SSCP, FISH, and CGH

Abstractvon Hippel-Lindau (VHL) disease is a pleioropic disorder featuring a variety of malignant and benign tumors of the eye, central nervous system, kidney, and adrenal gland. Recently the VHL gene has been identified in the chromosomal region 3p25-26. Prognosis and successful management of VHL patients and their descendants depend on unambiguous diagnosis. Due to recurrent hemangioblastomas, a 29-year-old patient without familial history of VHL disease was diagnosed to be at risk for the disease. Histopathological examination of a small renal mass identified a clear cell tumor with a G1 grading. Genetic characterization of the germline and of the renal tumor was performed. Polymerase chain reaction/single strand conformation polymorphism (PCR/SSCP) analysis with primers from the VHL gene identified a deletion of a single nucleotide in exon 2 in the patients germline and in the tumor, but not in the DNA of his parents. This deletion therefore must be a de novo mutation. Comparative genome hybridization (CGH) and fluorescence in situ hybridization (FISH) analysis of the G1 tumor with differentially labelled yeast artifical chromosome (YAC) clones showed loss of 3p and of the 3p26 signals, respectively. In conclusion, we identified a de novo germline mutation in the VHL gene of a young patient and a somatic chromosome 3p loss at the homologous chromosome 3 in his renal tumor. Our results suggest a recessive mode of inactivation of the VHL gene, providing solid evidence for its tumor-suppressor gene characteristics. Our data show the diagnostic potential of genetic testing, especially in patients without VHL family history. Furthermore, the findings of homozygous inactivation of the VHL gene in a G1 tumor support the notion that the inactivation of the VHL gene is an early event in tumorigenesis of renal cell carcinoma.