Browsing by Author "Macedo-Ribeiro, Sandra"
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- Lysosomal multienzymatic complex-related diseases: a genetic study among Portuguese patientsPublication . Coutinho, Maria Francisca; Lacerda, Lúcia; Macedo-Ribeiro, Sandra; Baptista, Estela; Ribeiro, Helena; Prata, Maria João; Alves, SandraThe functional activity of lysosomal enzymes sialidase, β-galactosidase and N-acetylaminogalacto-6-sulfate-sulfatase in the cell depends on their association in a multienzyme complex with cathepsin A. Mutations in any of the components of this complex result in functional deficiency thereby causing severe lysosomal storage disorders. Here, we report the molecular defects underlying sialidosis (mutations in sialidase; gene NEU1), galactosialidosis (mutations in cathepsin A; gene PPGB) and GM1 gangliosidosis (mutations in β-galactosidase; gene GLB1) in Portuguese patients. We performed molecular studies of the PPGB, NEU1 and GLB1 genes in biochemically diagnosed Portuguese patients. Gene expression was determined and the effect of each mutation predicted at protein levels. In the NEU1 gene, we found three novel missense mutations (p.P200L, p.D234N and p.Q282H) and one nonsense mutation (p.R341X). In the PPGB gene, we identified two missense mutations, one novel (p.G86V) and one already described (p.V104M), as well as two new deletions (c.230delC and c.991-992delT) that give rise to non-functional proteins. We also present the first molecular evidence of a causal missense mutation localized to the cathepsin A active site. Finally, in the GLB1 gene, we found six different mutations, all of them previously described (p.R59H, p.R201H, p.H281Y, p.W527X, c.1572-1577InsG and c.845-846delC). Seven novel mutations are reported here, contributing to our knowledge of the mutational spectrum of these diseases and to a better understanding of the genetics of the lysosomal multienzymatic complex. The results of this study will allow carrier detection in affected families and prenatal molecular diagnosis, leading to the improvement of genetic counseling.
- Molecular Characterization of Portuguese Patients with Pathologies Related to the Lysosomal Multienzymatic Complex: Sialidosis, Galactosialidosis and GM1 Gangliosidosis.Publication . Coutinho, Maria Francisca; Macedo-Ribeiro, Sandra; Lacerda, Lúcia; Prata, Maria João; Ribeiro, Helena; Baptista, Estela; Rodrigues, M.C.; Alves, SandraThe functional activity of lysosomal enzymes sialidase, beta-galactosidase and N-acetylaminogalacto-6-sulfate in the cell depends on their association in a multienzyme complex with the lysosomal carboxipeptidase, cathepsin A. Mutations in any of these complex components results in their functional deficiency causing severe lysosomal storage disorders. Here we report the molecular defects underlying sialidosis (mutations in sialidase; gene NEU1), galactosialidosis (mutations in cathepsin A; gene PPGB) and GM1 gangliosidosis (mutations in beta-galactosidase; gene GLB1) in the Portuguese population. Methods: Using gDNA extracted from patient’s fibroblasts, we performed a molecular study of the PPGB, NEU1 and GLB1 genes in the biochemically diagnosed Portuguese patients with galactosialidosis, sialidosis and GM1 gangliosidosis, respectively. The expression of these genes was determined by qRT-PCR. The effect of each mutation was evaluated at protein levels using bioinformatic tools. Results: In the PPGB gene, we identified two missense mutations, one novel (p.G85V) and one previously reported (p.V132M) as well as two new deletions (c.228-229delC and c.1075-1076delT) both giving origin to transcripts that lead to the synthesis of truncated non-functional proteins. In the NEU1 gene, we found two novel missense mutations (p.P200L and p.D234N). At protein levels, these mutations result in the substitution of two aminoacids located in a surface region of the molecule, already proposed to be involved in the interface sialidase/cathepsin A. Finally, in the GLB1 gene, we found four different mutations, all of them previously described: one missense mutation (R59H), one nonsense (W527X), one insertion (1572-1577InsG) and one deletion (845-846delC). Interestingly, in the Portuguese population the missense mutation R59H has a higher prevalence among the other ones. This is totally in accordance with which has been described for Brazilian, Iberian and Italian populations. Conclusion: Seven novel mutations are here reported for the first time, which contributes to enrich the knowledge on the mutational spectrum of these diseases and, by extension, to understand better the genetics of the lysosomal multienzymatic complex (LMC). The knowledge of the sialidosis, galactosialidosis and GM1 gangliosidosis mutational spectrum is also an important contribute to a better diagnosis, as well as to allow carrier detection in affected families and prenatal molecular diagnosis, leading to the improvement of genetic counseling with great benefits for the affected families. The existence of a molecular approach to the diagnosis is also of particular important strategy since it helps to overcome the difficulties associated to the neuraminidase enzymatic assay.
- Sortilin/Neurotensin Receptor 3: Three-dimensional Insights on its Coding VariantsPublication . Coutinho, Maria Francisca; da Silva Santos, Liliana; Lacerda, Lúcia; Macedo-Ribeiro, Sandra; Prata, Maria João; Alves, SandraIntroduction: Sortilin is a multifunctional sorting receptor required for Golgi-to-lysosome protein transport by a M-6-P independent pathway. It presents a unique structural organization with a 10-bladed β-propeller domain forming a tunnel where several ligands bind and is able to mediate the lysosomal targeting of sphingolipid activator proteins (SAPs), acid sphingomyelinase (AS) and cathepsins D and H. For some of these proteins no alternative receptor is known. The inner rim of the tunnel, along with several patches which function as additional binding sites have particularly high sequence conservation. Such structural organization is a key element for regulation of sortilin’s function and any sequence alterations changing it may potentially result in impaired lysosomal transport. As a result from the “1000Genomes Project”, several coding variants were identified for this protein with no known individuals carrying two of those variant alleles ever identified. Methods: In silico mutagenesis of sortilin was performed with SwissModelServer and the effect of each variant on its 3-dimensional structure was evaluated. The results were cross-linked with the potential pathogenicity predictions of PolyPhen and SIFT. Results/Discussion: Here we present the complete results of this in silico analysis, highlighting the potential of each one to disrupt sortilin’s structure and, consequently, impair its biological function and, ultimately, cause lysosomal storage and disease.