Tesis doctoral de Ana Isabel Vega Pajares
Congenital disorders of glycosylation (cdg) are a group of human genetic diseases with a broad spectrum of clinical manifestations affecting multiple organs and tissues which makes difficult the clinical diagnosis. The aim of this work has been the study of the molecular bases of these defects to find new diagnostic tools and new target and therapeutic approaches based on the genotype. using three serum biomarkers analyzed by isoelectric focusing (transferrin, ¿1¿ antitrypsin and apociii) and subsequent enzymatic and/or genetics approaches we have diagnosed 19 patients, 14 cdg ia, 2 cdg ib, 2 cdg ie and 1 cdg ij with mutations in the pmm2, mpi, dpm1 and dpgat1 genes, respectively. the mutational spectrum of the pmm2 gene included 13 different allelic variants, 5 of them are novel (t118s, p184t, d209g, ivs7¿9t>g and ivs3¿1g>c). The most frequent mutations were r141h and t237m, both identified in 15% of the mutant chromosomes. three mutations have been identified in the mpi gene, one in the dpm1 gene and two new allelic variants (r301c and l385r) in the dpgat1 gene. the serum proteomic analysis by 2¿de technology of two cdg ia patients has revealed the differential expression of proteins involved in the immune response, inflammation, coagulation mechanism and tissue protection against oxidative stress, indicating that this technique might be useful for the diagnosis of these defects and for the identification of new therapeutic targets. the functional analysis of pmm2 missense mutations in a prokaryotic expression system has demonstrated that all are disease¿causing and the results of pmm 2 activity have allowed the classification of these mutations in null mutations, intermediate mutations and mutations with high residual activity. Most of the patients are functional hemyzygous of a null mutation and a mutation with intermediate residual activity usually associated with a moderate form of the disease or with a mutation with higher residual activity, usually associated with a milder clinical phenotype. In addition the protein stability assays allowed the identification of at least five changes (v44a, d65y, f157s, p184t y f207s) with decreased amount of immunoreactive pmm 2 protein and decreased half life compared to wild type opening up therapeutic options by pharmacological chaperones in several patients. the function analysis by minigenes of three nucleotide changes, ivs7¿9t>g and ivs3¿1g>c identified in the pmm2 gene and ivs7¿15479c>t previously described, suggest that all are disease¿causing mutations. The ivs3¿1g>c mutation causes skipping of exons 3 and 4 in fibroblast cell line and in the minigene expression system. The ivs7¿9t>g mutation was found to be responsible for the activation of a cryptic intronic splice site in fibroblast cell line and in a hybrid minigene when cotransfected with certain sr proteins. The deep intronic change ivs7¿15479c>t was found to be responsible for the activation of a pseudoexon sequence in intron 7. The use of morpholino oligonucleotides allowed the production of correctly spliced mrna that was efficiently translated into functional and immunoreactive pmm 2 protein. The results suggest a novel mutation¿specific approach for the treatment of this genetic disease for which no effective treatment is yet available, and open up therapeutic possibilities for several genetic disorders in which deep intronic changes are identified.
Datos académicos de la tesis doctoral «Caracterización bioquímica y molecular de los defectos congénitos de glicosilación. aplicación de terapias específicas de mutación.«
- Título de la tesis: Caracterización bioquímica y molecular de los defectos congénitos de glicosilación. aplicación de terapias específicas de mutación.
- Autor: Ana Isabel Vega Pajares
- Universidad: Autónoma de Madrid
- Fecha de lectura de la tesis: 25/02/2009
Dirección y tribunal
- Director de la tesis
- cerda Silvestre Perez
- Tribunal
- Presidente del tribunal: jose Fernandez piqueras
- Mª Luisa Martínez frías (vocal)
- paz Briones godino (vocal)
- Antonio Baldellou vazquez (vocal)