Browsing by Author "Silva, Iris Alexandra Lopes da"
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- Genetic and functional analysis of the PDB6 locus in Paget's disease of bonePublication . Silva, Iris Alexandra Lopes da; Cancela, LeonorPaget’s disease of bone (PDB) is the most frequent metabolic bone disease after osteoporosis, and despite having a strong genetic component, Sequestosome 1 is the only gene directly linked, so far, to this disease. Genome wide linkage and association studies have suggested an association of PDB6 locus with PDB, however no functional studies were performed to try to explain that association. The main goal of this work was to assess the relative contribution of each significant variant identified in the PDB6 locus to the functionality of the candidate genes and their involvement in PDB pathophysiology. In this regard we analyzed the entire PDB6 locus to search for the best candidate genes likely to be involved in bone metabolism. OPTN, CCDC3, UCMA/GRP, CAMK1D, PHYH and SEPHS1 were selected and screened for genetic variants. Together with the variants found in a previous work from our group (Michou et al 2012) we studied 82 genetic variants, from which we selected rs2234968 and rs3829923 for an association study. The results showed that only rs2234968 was significantly associated with PDB (p-value = 6 x 10-3). In addition, we assessed the effect of a rare variant (RV) found in one PDB patient, RV -9906, and rs1561570, a SNP in OPTN strongly associated with PDB. Our functional studies showed that (i) rs3829923 was responsible for an increase of OPTN promoter activity due to the appearance of new binding sites for activator nuclear factors E47 and E2F1, while (ii) RV -9906 was responsible for an increase of OPTN promoter activity but due to the loss of SP1 binding and consequent loss of its inhibitory effect in gene transcription. Rs2234968 was found to be in linkage disequilibrium with two SNPs that changed OPTN splicing, thus creating a premature stop codon, and probably resulting in a nonfunctional protein. In this work we showed that rs1561570, the SNP most associated to PDB, was responsible for the loss of a methylation site that subsequent increase in OPTN expression, which in turn increases NF-κB translocation into the nucleus and activation of NF-κB target genes. This will explain the increase in number and activity of the osteoclasts of PDB patients carrying of the rs1561570. In this work we provide new insights about OPTN regulation and its role in bone metabolism, with emphasis in osteoclastogenesis, and more importantly the contribution of OPTN variants to PDB pathophysiology.
- Molecular cloning and analysis of Xeroderma pigmentosum group-D (xpd) in zebrafishPublication . Silva, Iris Alexandra Lopes da; Cancela, Leonor; Conceição, N.