Browsing by Author "Ribeiro, Helena"
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- Evidences of large deletions in patients with the Lysosomal Storage Diseases Mucolipidosis type II and III: experimental approaches for picking up both homozygous and heterozygous casesPublication . Coutinho, Maria Francisca; Encarnação, Marisa; Carvalho, Filipa; Lacerda, Lúcia; Willbrand, Flemming; Ribeiro, Helena; Prata, Maria João; Alves, SandraBackgroung/Objectives: Mucolipidosis II and III are rare genetic diseases in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is reduced or absent. GlcNAc-phosphotransferase is a multimeric enzyme encoded by two genes: GNPTAB and GNPTG. Although a wide spectrum of mutations in GNPTAB has recently been reported to cause ML II and III alpha/beta, large deletions have not yet been reported. Furthermore, some previously reported patients present only one heterozygous mutation while the second one is still missing. Here we present evidence that some of these cases may be due to the difficulty to detect large heterozygous deletions through direct sequencing. Methods: We developed a semiquantitative approach by multiplex PCR and capillary electrophoresis, co-amplifying two fragments in each PCR reaction: one corresponding to GNPTAB exons and the other to an internal control fragment. Results: After applying this technique to perform a genomic screen in two Portuguese patients in whom a heterozygous missense was identified while the second mutation was missing, we found that in both patients the last exons (20 and 21) of the GNPTAB gene were missing in one of the alleles. We also found and characterize a gross homozygous deletion involving exon 19 in one Danish patient. Conclusion: Here we present a molecular approach that turned possible to identify for the first time large deletions in the GNPTAB, underlying mucolipidoses in patients who were both homozygous and heterozygous for such alterations. Our semiquantitative PCR based method is a valuable tool that allows the screening of large deletions. Consequently, we propose that cases with only one heterozygous mutation detected through direct sequencing methods, should be further screened for the possible presence of a large heterozygous deletion.
- 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 analyses of genes involved in mannose 6-phosphate independent traffickingPublication . Coutinho, Maria Francisca; Lacerda, Lúcia; Pinto, Eugénia; Ribeiro, Helena; Prata, Maria João; Alves, SandraLysosomes are essential regulators of cell homeostasis, since they harbour a vast repertory of specialized enzymes responsible for degrading endocytosed and intracellular material. The newly-synthesized lysosomal enzymes travel first to the trans-Golgi network (TGN) and then must be driven to the acidic organelle in order to exert their function. Whist the best-known pathway for TGN-to-endosome transport is the delivery of soluble lysosomal hydrolases by the MPRs, additional pathways from the TGN to lysosomes do exist, as recently demonstrated by the identification of two alternative receptors: LIMP-2, shown to be implicated in the delivery of -glucocerebrosidase to the lysosomes, and sortilin, proposed to be involved in the transport of several proteins including the sphingolipid activator proteins prosaposin and GM2 activator protein (GM2AP), acid sphingomyelinase (ASM) and cathepsins D and H. Disruption of the intracellular transport and delivery pathways to the lysosomes may result in lysosomal dysfunction, predictably leading to a range of clinical manifestations suggestive of lysosomal storage diseases (LSDs): impairments of the M6P-dependent pathway are the basis of the rare genetic diseases Mucolipidosis II and III (ML II, OMIM# 252500 and ML III, OMIM# 252600); similarly, impairments on the M6P-independent trafficking processes might also have hazardous effects to the organism, ultimately leading to overt disease as already demonstrated by the existence of a serious autosomal-recessive disorder presently known as action myoclonus-renal failure syndrome (AMRF), which is caused by mutations in the gene that codes for LIMP-2. Having in mind that, for a great percentage of patients presenting LSD manifestations, no condition is successfully diagnosed because their metabolic profile does not fit any known LS, we hypothesized that TGN-to-endosome M6P-independent traffic could be deficient in some of them. For the present study we recruited uncharacterized patients with phenotypes overlapping manifestations that could predictably be due to loss of activities of GCase, saposins, GM2AP and/or ASM, to find out whether those features could be resultant from failures in M6P-independent pathways through which those proteins reach the lysosome. Patients were sorted into five different groups according to their phenotypic picture and screened for SCARB2 and/or SORT1 mutations. No novel mutations were found on the SCARB2 gene and no pathogenic mutations were identified on the SORT1 gene. Other study approaches will be needed to clarify whether sortilin dysfunction may cause disease. The relevance of alternative receptors is demonstrated by their involvement in disease. So, a better understanding on the M6P-independent routes to the lysosome will contribute to a more accurate understanding not only of crucial cellular processes, but also of the pathophysiological bases of severe and disabling diseases. Finally, knowledge of the different trafficking mechanisms responsible for the sorting of lysosomal proteins may be an enormous help for the building of new therapeutic strategies for LSDs.
- Molecular analysis of the GNPTAB and GNPTG genes in 13 patients with mucolipidosis type II or type III - identification of eight novel mutationsPublication . Encarnação, Marisa; Lacerda, Lúcia; Costa, Roberto; Prata, Maria João; Coutinho, Maria Francisca; Ribeiro, Helena; Lopes, Lurdes; Pineda, M.; Ignatius, J.; Galvez, H.; Mustonen, A.; Vieira, P.; Lima, Margarida Reis; Alves, SandraMucolipidosis II (ML II) and mucolipidosis III (ML III) are diseases in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent or reduced, respectively. In the absence of mannose phosphorylation, trafficking of lysosomal hydrolases to the lysosome is impaired. In these diseases, mistargeted lysosomal hydrolases are secreted into the blood, resulting in lysosomal deficiency of many hydrolases and a storage-disease phenotype. GlcNAc-phosphotransferase is a multimeric transmembrane enzyme composed of three subunits (alpha, beta and gamma) encoded by two genes -GNPTAB and GNPTG. Defects in GNPTAB result in ML II and III whereas mutations in GNPTG were only found in ML III patients. We have performed a molecular analysis of the GNPTAB and GNPTG genes in 13 mucolipidosis II and III patients (10 Portuguese, one Finnish, one Spanish of Arab origin and one Indian). Mutations were identified by the study of both cDNA and gDNA. The GNPTAB and GNPTG mRNA expressions were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The study led to the identification of 11 different mutations. Eight of these mutations are novel, six in the GNPTAB gene [c.121delG (V41FfsX42), c.440delC (A147AfsX5), c.2249_50insA (N750KfsX8), c.242G>T (W81L), c.1208T>C (I403T) and c.1999G>T (p.E667X)] and two in the GNPTG gene [c.610-1G>T and c.639delT (F213LfsX7)]. With regard to the mRNA expression studies, the values obtained by qRT-PCR indicate the possible existence of feedback regulation mechanisms between alpha/beta and the gamma subunits.
- Molecular characterization of Portuguese patients with mucopolysaccharidosis IIIC: two novel mutations in the HGSNAT gene.Publication . Coutinho, Maria Francisca; Lacerda, Lúcia; Prata, Maria João; Ribeiro, Helena; Lopes, Lurdes; Ferreira, Célia; Alves, Sandra
- Molecular characterization of Portuguese patients with pathologies related to the lysosomal multienzymatic complex: Sialidosis and GalactosialidosisPublication . Coutinho, Maria Francisca; Lacerda, Lúcia; Prata, Maria João; Ribeiro, Helena; Alves, SandraBackgroung/ Objectives: The functional activity of lysosomal enzymes sialidase, -galactosidase, and N-acetylaminogalacto-6-sulfate in the cell depends on their association in a multienzyme complex with lysosomal carboxipeptidase, cathepsin A. Genetic mutations in any of this complex components results in their functional deficiency causing severe lysosomal storage disorders. Here we study the molecular defects underlying sialidosis (mutaions in sialidase; gene NEU1) and galactosialidosis (mutations in cathepsin A; gene PPGB) in the Portuguese population. Methods: Using gDNA extracted from patient’s fibroblasts, we performed a molecular study of the PPGB and NEU1 genes in the known Portuguese patients with galactosialidosis and sialidosis, respectively. The expression of both genes was determined by qRT-PCR. The effect of each mutation was also analysed at protein levels, through different in silico procedures. Results and Conclusions: On the PPGB gene, we identified two predictably deleterious missense mutations (c.394 G>A -Zhou et al, 1996- and c.254 G>T) and two deletions (c.228-229DelC and c.1075-1076DelT), both of them giving origin to transcripts that lead to the synthesis of truncated non-functional proteins. On the NEU1 gene, we found two novel missense mutations associated to a severe form of the disease (c.700 G>A and c.599 C>T), which, at protein levels, lead to the substitution of two aminoacids localized in a surface region of the molecule, already proposed to be involved in the interface of sialidase binding with cathepsin A (Lukong, 2000). Knowledge about these findings is important to allow carrier detection and molecular diagnostics as well as to understand the genetics of LMC.
- Molecular characterization of Portuguese patients with pathologies related to the lysosomal multienzymatic complex: Sialidosis and Galactosialidosis.Publication . Coutinho, Maria Francisca; Lacerda, Lúcia; Prata, Maria João; Ribeiro, Helena; Alves, Sandra
- 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.
- Molecular characterization of the Portuguese patients with defects in GlcNAc-phosphotransferase: a key enzyme in the M6-P dependent lysosomal traffickingPublication . Coutinho, Maria Francisca; Encarnação, Marisa; Gomes, Rui; Prata, Maria João; Lacerda, Lúcia; Bargal, Ruth; Filocammo, Mirella; Raas-Rothschild; Tappino, Barbara; Laprise, Cathrine; Sirois-Gagnon, D.; Costa, Roberto; Ribeiro, Helena; Lopes, Lurdes; Alves, SandraIntroduction: GlcNAc-phosphotransferase is one of the enzymes responsible for the formation of M6P residues and plays a key role in lysosomal trafficking, since most soluble acid hydrolases reach these organelles through the M6P pathway. It is composed of six subunits (α2β2γ2), products of two genes recently cloned: GNPTAB (mutated in mucolipidosis II/IIIA patients) and GNPTG (mutated in MLIIIC patients). Methods: Using both gDNA and cDNA extracted from patient’s fibroblasts, we performed a molecular study of both genes in 13 MLII/III patients (10Portuguese, 1Finnish, 1Spanish of Arab origin and 1Indian). Expression studies were performed by quantitative real-time PCR. Results: We identified 11 different mutations, 8 of them novel: 6 in the GNPTAB gene (c.121delG;c.440delC;c.2249_50insA;W81L;I403T and E667) and 2 in the GNPTG gene (c.610-1G.T and c.639delT). Interestingly, although the MLII-causing mutations have been mostly found to be private or rare, there is one (c.3503_3504delTC) that shows a broad distribution having been detected among different populations. This same mutation was also the most frequent one in our patients. Such distribution pattern prompted us to perform a haplotypic study. We analysed 37 patients (23Italians, 8Arab Muslims, 1Turkish and 5Portuguese) for 3 intragenic polymorphisms and 2 microsatellite markers flanking the GNPTAB gene, identifying a common haplotype. Regarding the mRNA expression studies, real-time results suggest the existence of feedback regulation mechanisms between α/β and the γ subunits. Discussion/Conclusion: This work enabled the establishment of a strong genotype-phenotype correlation, which is of crucial importance to an improved genetic counselling for ML families. The sharing of an ancestral haplotype by patients carrying the deletion implies a common origin of this mutation, while the higher level of diversity observed at the most distant locus indicates that it is a relatively ancient one. The developed strategies constitute valuable tools that allow carrier detection and prenatal- molecular diagnostics of these diseases.
- Molecular Characterization of the Portuguese Patients with defects in the GLB1 gene: evidences of a strong genotype-phenotype correlation.Publication . Coutinho, Maria Francisca; Lacerda, Lúcia; Ribeiro, Helena; Prata, Maria João; Alves, Sandra