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Búsqueda de nuevas dianas y estrategias terapéuticas en acidura metilmalónica

  • Autores: Ana Jorge Finnigan
  • Directores de la Tesis: María Belén Pérez González (dir. tes.)
  • Lectura: En la Universidad Autónoma de Madrid ( España ) en 2011
  • Idioma: español
  • Tribunal Calificador de la Tesis: José Fernández Piqueras (presid.), Javier Díaz Nido (secret.), Antònia Ribes Rubió (voc.), Carmen Ayuso García (voc.), Maria Isabel Ginestral Tavares de Almeida (voc.), Aurora Martínez Ruiz (voc.), Mercedes Martínez-Pardo Casanova (voc.)
  • UNESCO :
    • 24 Ciencias de la vida
      • 2415 Biología molecular
  • Resumen
    • Vitamin B12, or cobalamin, is an essential nutrient that humans must take in their diet so that it serves as the cofactor of two enzymes: methionine synthase and methylmalonyl-CoA mutase, which need methylcobalamin and adenosylcobalamin as active cofactors, respectively. The study of the inborn intracellular defects of cobalamins has given rise to eight complementation groups, cblA-G and mut. In particular, defects in the synthesis of adenosylcobalamin (groups cblA, B and D) or in the apoenzyme methylmalonyl-CoA mutase give rise to isolated methylmalonic aciduria (MMA).

      Defects in the common pathway of synthesis of methylcobalamin and adenosylcobalamin causes methylcobalamin aciduria combined with homocistinuria (MMAHC; complementation groups cblC, D and F). The objective of this work has been to study the molecular basis of MMAHC and isolated MMA in order to identify molecular targets for the development of new therapeutical approaches to improve the outcome of these severe disorders.

      We have analyzed 48 patients of MMAHC cblC type, identifying the mutational spectrum of the Spanish population for the first time. We have identified eleven nucleotidic changes, including: one small duplication (c.271dupA), two missense (p.M1L and p.R189S), seven nonsense (p.W30X, p.R73X, p.R111X, p.R132X, p.R153X, p.R161X, p.Y205X) and one change that affects the splicing process (IVS1nt+2T>G). Two changes, p.W30X and IVS1nt+2T>G, are novel mutations and the rest have been previously described. The change c.271dupA accounts for 85% of studied alleles, and causes a frameshift and truncation of the protein. All but one of the analyzed patients bear the duplication in at least one allele, so we established a high resolution melting protocol for rapid and cost-effective screening of the mutation in MMACH patients. The specific gene expression analysis of 17 fibroblasts cell lines has indicated that transcripts bearing c.271dupA undergo NMD degradation, while p.R132X and p.R153X bearing mRNA surprisingly escape the NMD system. The transcriptional analysis of eight MMACHC patients and seven controls treated with or without hydroxycobalamin (OHcbl) has not shown any particular group of genes differentially expressed that could account for the B12 responsiveness found in our group of cblC patients.

      To gain insights into the physiopathology of the disease, we have analyzed the differential gene expression of 84 apoptosis-related genes. We have found that MMAHC cblC samples exhibit mostly up-regulated extrinsic pathway apoptotic genes while MMA cblB mostly to over-express the intrinsic pathway genes. The differences could be attributed to homocysteine which is the differential metabolite in cblB and cblC affected patients.

      In this study six MMA allelic variants were analyzed by expression studies in the appropriated expression system. c.733G>A mutation, analyzed by ex vivo splicing expression system using minigenes, affects the splicing process of MMAA gene and causes the skipping of exon 4, although some molecules do not undergo the skipping and show the misssense mutation p.G245S. The p.V153D and p.R629G mutations in MUT gene, drastically affect the enzyme's activity. Since p.V153D responds in vitro to OHcbl, it belongs to the less severe mut- group and since p.R629G does not respond it is classified as the more severe mut0. Finally mutations p.I96T, p.H183L and p.R191W in MMAB gene have been studied using stability/activity assays in prokaryotic expression systems combined with molecular modeling, and residual activity in a cellular disease model in eukaryotic expression systems. Taken together these results suggest that p.I96T, p.H183L and p.R191W are misfolding mutations that destabilize ATR protein, retaining however some biological activity.

      Based on these findings we have tested gene expression modulators specific for lipid metabolism genes, such as statins and bezafibrate, which did not render conclusive results, probably due to the use of a not totally adequate cellular disease model. Pharmacological chaperone stabilization of ATR protein was carried out by screening a commercial library of compounds and selecting six hits by physical-chemical analysis (compound I-VI). Compound V and VI have not inhibited wild-type purified ATR; moreover compound V (currently patent pending) has increased the activity in different degrees in wild-type, p.I96T and p.H183L ATR. Compound V has also increased the activity to the control range in a cblB cell disease model. In summary, the knowledge gained from the comprehensive study of the genetic and functional basis of the disease has enabled us to identify new therapeutical targets and to find a pharmacological chaperone that could be a potential treatment for MMA cblB type patients.


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