From gene mutation to gene expression : studies on multiple endocrine neoplasia type 1 and vascular endothelial growth factors
Multiple Endocrine Neoplasia type 1, MEN1, is an inherited cancer syndrome whose gene was localised to chromosome 11q13 in 1988. A number of !candidate genes were characterised before the MEN1 gene was cloned in 1997. DNA sequencing of MEN1 to search for mutations in patients is used as a complement to clinical diagnosis. Since 1997, a total of 202 index cases were referred to the Department of Clinical Genetics for mutation screening, but no systematic review of their mutations or clinical characteristics has been performed.
By analysing the results of DNA sequencing and deletion detection (using multiplex-ligationdependent probe amplification, MLPA) on blood samples and correlating mutations to clinical data from the referring physicians, 37 unique mutations were found, of which 19 have not been previously reported. Heredity for MEN1 or hyperparathyroidism, an early age of onset and the presence of multiple tumours greatly enhanced the risk of carrying a MEN1 mutation. Mutations were spread all over the gene and there was no genotype-phenotype correlation. The results from this study have led to the addition of MLPA as a standard method of mutation detection in MEN1 patients and have identified patient categories which should be tested for MEN1 mutations. In addition, the compilation of missense mutations and polymorphisms found in the Swedish population will facilitate interpretation of single base pair substitutions in the future.
One of the genes isolated as a MEN1 candidate gene was a novel gene related to vascular endothelial growth factor A (VEGF-A). This gene was called VEGF Related Factor (VRF) and later renamed VEGF-B. It was expressed in all normal tissues examined and consisted of two splice forms: VEGF-B167 and VEGF-B186. They had completely different carboxyl -terminal ends due to different reading frames. VEGF-A was known as a potent inducer of blood vessel growth (angiogenesis) and can also cause inflammation. To further study the role of VEGF-B, two different strategies were used. The first was to produce recombinant VEGF-B protein and to test its function. VEGF-B167 was successfully produced and purified from retrovirally infected HEK293 cells. However, no detectable effect of VEGF-B was found in cell proliferation or monocyte migration assays.
The second strategy was to study the expression of VEGF-B in concert with other angiogenic factors in models of disease that affected organs with high expression of VEGF-B the heart (dilated cardiomyopathy, DCM) and central nervous system (multiple sclerosis, MS). VEGF-A (but not VEGF-B) was significantly increased in a mouse model of DCM due to mitochondrial dysfunction, but there was no parallel increase in capillary density. The expression of VEGF-A (but not VEGF-B) was decreased in the spinal cord resident cells in a rat model of MS, but the invading inflammatory cells did express VEGF-A. It was the heparin-binding splice forms that tended to decrease while the soluble VEGF-A120 isoform remained unaltered. These results were corroborated by a decrease in VEGF-A mRNA in mononuclear cells from cerebrospinal fluid (CSF) from MS patients compared to controls.
Thus, the role of VEGF-B remains largely unknown and our data support the idea that VEGF-A functions in concert with other factors and an increase in VEGF-A as a single factor does not always lead to angiogenesis. VEGF-A may function as a neuroprotective and proinflammatory factor simultaneously in different cell types in the same tissue, which further complicates the picture. Studies in larger clinical materials are warranted to be able to correlate the net effect of VEGF-A with clinical outcome in MS.
List of scientific papers
I. Grimmond S, Lagercrantz J, Drinkwater C, Silins G, Townson S, Pollock P, Gotley D, Carson E, Rakar S, Nordenskjold M, Ward L, Hayward N, Weber G (1996). Cloning and characterization of a novel human gene related to vascular endothelial growth factor. Genome Res. 6(2): 124-31.
https://pubmed.ncbi.nlm.nih.gov/8919691
II. Tham E, Grandell U, Lindgren E, Toss G, Skogseid B, Nordenskjöld M (2006). Clinical testings for mutations in the multiple endocrine neoplasia type I gene in Sweden, a report on 202 unrelated cases. [Manuscript]
III. Tham E, Wang J, Piehl F, Weber G (2002). Upregulation of VEGF-A without angiogenesis in a mouse model of dilated cardiomyopathy caused by mitochondrial dysfunction. J Histochem Cytochem. 50(7): 935-44.
https://pubmed.ncbi.nlm.nih.gov/12070272
IV. Tham E, Gielen AW, Khademi M, Martin C, Piehl F (2006). Decreased Expression of VEGF-A in Rat Experimental Autoimmune Encephalomyelitis and in Cerebrospinal Fluid Mononuclear Cells from Patients with Multiple Sclerosis. Scand J Immunol. 64(6): 609-22.
https://pubmed.ncbi.nlm.nih.gov/17083617
History
Defence date
2006-11-17Department
- Department of Molecular Medicine and Surgery
Publication year
2006Thesis type
- Doctoral thesis
ISBN-10
91-7140-899-1Number of supporting papers
4Language
- eng