Analysis of mitochondrial DNA in discordant monozygotic twins with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is the most frequent neurocutaneous disorder with autosomal dominant inheritance. Phenotype variability is high ranging from merely several café-au-lait spots to malignant peripheral nerve sheath tumors or severe disfigurement through plexiform neurofibromas. Identification of genetic factors that modify the NF1 phenotype would contribute to the understanding of NF1 pathophysiology and improve patient counselling. As even monozygotic (MZ) twins with NF1 may differ phenotypically, we wondered whether these variations might be inherited in a non-Mendelian fashion. Mitochondrial DNA (mtDNA) is inherited extrachromosomally through the cytoplasm of the oocyte and often harbours heteroplasmic sequence variations. At the time of blastomere separation, these variants may be skewedly distributed and effect phenotypic differences. Because of their co-localization with the tumor suppressor protein neurofibromin, which is mutated in NF1, mitochondria were particular attractive candidates for investigation. MtDNA was extracted from nucleated blood cells of four pairs of discordant MZ twins with NF1 and from cutaneous neurofibromas of one twin pair. We sequenced the entire mitochondrial genome and determined the state of heteroplasmy by investigating a microsatellite region of the mitochondrial D-loop (D310-tract). The clinical diagnosis was confirmed in all patients by detection of pathogenic mutations in the NF1 gene. Monozygosity was verified by genotyping. However, we did not detect evidence for mtDNA sequence differences or for different degrees of heteroplasmy between individuals of the same twin pair. The phenotypic discordance of MZ twins with NF1 cannot be explained by skewed distribution of mtDNA mutations or polymorphisms.

Expression of prothrombin, thrombin and its receptors in human scars.

The coagulation system is thought to play a pivotal role during the initial phase of wound healing, but mechanisms and cells involved are only partly understood. We have therefore examined human scars for the expression of thrombin, its precursor prothrombin and the thrombin receptors, thrombomodulin (TM) and protease-activated receptor-1 (PAR-1), compared with normal skin. Biopsies of scars were obtained from primary excision sites of melanoma patients (n = 20) and were compared with normal skin distant from the scar (n = 10), using immunohistochemistry. In addition, polymerase chain reaction analyses were performed on scar versus normal tissue and on cultured keratinocytes, fibroblasts and endothelial cells before and after stimulation with selected cytokines known to be active in wound healing. Normal epidermis was stained for prothrombin, thrombin, TM and PAR-1, and dermal tissue was stained only for TM and PAR-1. In scar tissue, thrombin and TM were upregulated in the epidermis and all four molecules in the dermis, independent of the age of the scars. In tissue extracts, mRNA expression of PAR-1 and prothrombin expression were, however, unchanged and TM even slightly decreased in scars, compared with normal skin. On analysis of cultured cells, keratinocytes expressed mRNA for PAR-1, TM and prothrombin, endothelial cells for PAR-1 and TM, and fibroblasts for PAR-1. An upregulation of PAR-1 mRNA was induced in fibroblasts on exposure to tumor necrosis factor-alpha (TNF-alpha), while it remained unchanged in endothelial cells in response to TNF-alpha. A downregulation of TM was induced in endothelial cells on exposure to TNF-alpha. These findings, showing a marked modulation of thrombin, PAR-1 and TM even in older human scar tissue, suggest that the coagulation system is not only involved during clotting, but also during the inflammatory and tissue remodelling phases of wound healing.

Subclassification of nerve sheath tumors by gene expression profiling.

Nerve sheath tumors are the most common tumors of Neurofibromatosis type 1 (NF1) patients. Dermal neurofibromas develop in nearly all NF1-patients, whereas plexiform neurofibromas are only observed in one-third of the patients. NF1-patients have about a 10% lifetime risk for developing malignant pheripheral nerve sheath tumors (MPNST). The origin of these tumors is thought to be the Schwann cell lacking functional neurofibromin. However, additional genetic alterations are likely to modulate tumor biology and to contribute to individual nerve sheath tumor entities. To gain insight into the molecular events and to determine whether these tumors can be classified according to gene expression profiles, we performed expression analysis applying cDNA array technology. Nine dermal neurofibromas, 7 plexiform neurofibromas, ten MPNST and two MPNST cell cultures were examined. All tumors but 6 sporadic MPNST were obtained from NF1-patients. We detected significant differences in gene expression patterns between neurofibromas and MPNST and between dermal neurofibromas and plexiform neurofibromas. Tumor class prediction agreed in all but one case with histological and clinical classification. NF1-associated and sporadic MPNST could not be distinguished by their gene expression patterns. We present a panel of discriminating genes that may assist subclassification of nerve sheath tumors.