An intronic mutation in Chd7 creates a cryptic splice site, causing aberrant splicing in a mouse model of CHARGE syndrome.

Alternate splicing is a critical regulator of gene expression in eukaryotes, however genetic mutations can cause erroneous splicing and disease. Most recorded splicing disorders are caused by mutations of splice donor/acceptor sites, however intronic mutations can affect splicing. Clinical exome analyses largely ignore intronic sequence, limiting the detection of mutations to within coding regions. We describe 'Trooper', a novel mouse model of CHARGE syndrome harbouring a pathogenic point mutation in Chd7. The mutation is 18 nucleotides upstream of exon 10 and creates a cryptic acceptor site, causing exon skipping and partial intron retention. This mutation, though detectable in exome sequence, was initially dismissed by computational filtering due to its intronic location. The Trooper strain exhibited many of the previously described CHARGE-like anomalies of CHD7 deficient mouse lines; including hearing impairment, vestibular hypoplasia and growth retardation. However, more common features such as facial asymmetry and circling were rarely observed. Recognition of these characteristic features prompted manual reexamination of Chd7 sequence and subsequent validation of the intronic mutation, highlighting the importance of phenotyping alongside exome analyses. The Trooper mouse serves as a valuable model of atypical CHARGE syndrome and reveals a molecular mechanism that may underpin milder clinical presentation of the syndrome.

Molecular pathways for lymphangiogenesis and their role in human disease.

The lymphatic network functions to return fluid, cells and macromolecules to the circulation. Recent characterization of growth factors that control the growth and development of the lymphatics, and markers which specify lymphatic endothelial cells have enhanced our understanding of this system. Members of the VEGF family of factors are key regulators of these vessels with VEGF-C/VEGF-D and VEGFR-3 being the best validated signalling pathways in lymphangiogenesis. The study of these molecules in various pathologies has shown that they are important in the processes of cancer metastasis and in the formation of lymphoedema. Knowledge of these molecular pathways allows for the generation of modulators of these pathways which could form the basis of novel therapeutic approaches.

Proprotein convertases promote processing of VEGF-D, a critical step for binding the angiogenic receptor VEGFR-2.

Vascular endothelial growth factor (VEGF)-D is a secreted glycoprotein that induces angiogenesis and lymphangiogenesis. It consists of a central domain, containing binding sites for VEGF receptor-2 (VEGFR-2) and VEGFR-3, and N- and C-terminal propeptides. It is secreted from the cell as homodimers of the full-length form that can be proteolytically processed to remove the propeptides. It was recently shown, using adenoviral gene delivery, that fully processed VEGF-D induces angiogenesis in vivo, whereas full-length VEGF-D does not. To better understand these observations, we monitored the effect of VEGF-D processing on receptor binding using a full-length VEGF-D mutant that cannot be processed. This mutant binds VEGFR-2, the receptor signaling for angiogenesis, with approximately 17,000-fold lower affinity than mature VEGF-D, indicating the importance of processing for interaction with this receptor. Further, we show that members of the proprotein convertase (PC) family of proteases promote VEGF-D processing, which facilitates the VEGF-D/VEGFR-2 interaction. The PCs furin and PC5 promote cleavage of both propeptides, whereas PC7 promotes cleavage of the C-terminal propeptide only. The finding that PCs promote activation of VEGF-D and other proteins with roles in cancer such as matrix metalloproteinases, emphasizes the importance of these enzymes as potential regulators of tumor progression and metastasis.

Mechanisms of lymphangiogenesis: targets for blocking the metastatic spread of cancer.

The lymphatic vasculature is an important route of metastatic spread in cancer and recent studies have demonstrated that lymphangiogenesis (the growth of lymphatic vessels) associated with tumors promotes metastasis via the lymphatics. Therefore, the molecular mechanisms that drive lymphangiogenesis are attractive targets for development of novel therapeutics designed to restrict cancer metastasis. Such therapeutics would be of high priority as metastasis is the most lethal aspect of tumor biology. Research over the past seven years has identified protein growth factors and cell surface receptors that signal for lymphangiogenesis during embryonic development, in adult tissues and in cancer. Proteases that process and thereby activate lymphangiogenic growth factors have also been defined. Lymphangiogenic growth factors, the enzymes that activate them and the cell surface receptors signalling for growth of lymphatic vessels are prime targets for anti-lymphangiogenic drugs designed to restrict cancer metastasis. Agents targeting some of these proteins have already shown promise for blocking tumor lymphangiogenesis and lymphatic metastasis in animal models. This article focuses on current and emerging targets for blocking these processes that have been defined in recent studies of the molecular mechanisms controlling lymphangiogenesis. Strategies to block the actions of these proteins in cancer are also explored.

Molecular regulation of the VEGF family -- inducers of angiogenesis and lymphangiogenesis.

The vascular endothelial growth factor (VEGF) family of secreted glycoproteins are critical inducers of angiogenesis (growth of blood vessels) and lymphangiogenesis (growth of lymphatic vessels). These proteins are attractive therapeutic targets for blocking growth of blood vessels and lymphatics in tumors and thereby inhibiting the growth and spread of cancer -- in fact, the first VEGF inhibitor has recently entered the clinic for treatment of cancer. In addition, the VEGFs are being considered for stimulation of angiogenesis in the context of ischemic disease and lymphangiogenesis for treatment of lymphedema. These therapeutic possibilities have focused great interest on the molecular regulation of VEGF family members. Much has been learned in the past five years about the mechanisms controlling the action of the VEGFs, including the importance of hypoxia, proteolysis, transcription factors and RNA splicing. An understanding of these mechanisms offers broader opportunities to manipulate expression and activity of the VEGFs for treatment of disease.

Plasmin activates the lymphangiogenic growth factors VEGF-C and VEGF-D.

Vascular endothelial growth factor (VEGF) C and VEGF-D stimulate lymphangiogenesis and angiogenesis in tissues and tumors by activating the endothelial cell surface receptor tyrosine kinases VEGF receptor (VEGFR) 2 and VEGFR-3. These growth factors are secreted as full-length inactive forms consisting of NH2- and COOH-terminal propeptides and a central VEGF homology domain (VHD) containing receptor binding sites. Proteolytic cleavage removes the propeptides to generate mature forms, consisting of dimers of the VEGF homology domain, that bind receptors with much greater affinity than the full-length forms. Therefore, proteolytic processing activates VEGF-C and VEGF-D, although the proteases involved were unknown. Here, we report that the serine protease plasmin cleaved both propeptides from the VEGF homology domain of human VEGF-D and thereby generated a mature form exhibiting greatly enhanced binding and cross-linking of VEGFR-2 and VEGFR-3 in comparison to full-length material. Plasmin also activated VEGF-C. As lymphangiogenic growth factors promote the metastatic spread of cancer via the lymphatics, the proteolytic activation of these molecules represents a potential target for antimetastatic agents. Identification of an enzyme that activates the lymphangiogenic growth factors will facilitate development of inhibitors of metastasis.