DGH - Posters/abstracts em congressos internacionais
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- Arylsulfatase B mutations in Portuguese MPS VI patientsPublication . Amaral, Olga; Dias, Aureliano; Pinto, Eugénia; Ribeiro, Isaura; Sá Miranda, M.C.Mucopolysaccharidosis type VI (MPS VI, OMIM 253200) is a rare autosomal recessive disorder characterized by the deficient activity of arylsulfatase B (ARSB, EC 3.1.6.12). In Portugal, the birth prevalence of the rare MPS VI is 0.42/100000. With the emerging availability of promising enzyme replacement therapy for this disease, mutation analysis becomes an important tool not only for the genetic counselling of individuals at risk, but also in the prognosis of the disease and identification of cases which might benefit of an early therapeutic intervention. In this work we present the preliminary results obtained through mutation analysis of 12 Portuguese patients. This study involved PCR amplification and direct automated sequencing analysis of exons and intron boundaries. The identification of seven mutations is reported: two recently described deletions, three missense mutations (two of them new), one novel nonsense mutation and also one new splicing mutation. Additionally, two previously described point mutations were detected in the form of a complex allele. Seven patients were homozygous for various mutations, while the remaining five were compound heterozygotes. Interestingly, three mutations seem to have an increased frequency in the Portuguese sample studied jointly c.1533del23 (Petry et al.,2003), c.427delG (Karageorgos et al.,2004) and R315Q (Villani et al.,1999) represent 58% of the patients alleles. In some of the cases Western blot analysis was also carried out. The impact of the various mutations at the protein level and the resulting phenotypic implications are discussed.
- Molecular Characterization of the three Portuguese patients with Mucopolysaccharidosis IIICPublication . Coutinho, Maria Francisca; Lacerda, Lúcia; Ribeiro, Helena; Ferreira, Célia; Lopes, Lurdes; Prata, Maria João; 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, 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.
- Common origin of the worldwide-spread mutation c.3503_3504delTC causing the lysosomal storage disease mucolipidosis type IIPublication . Coutinho, Maria Francisca; Encarnação, Marisa; Gomes, Rui; Prata, Maria João; Bargal, Ruth; Filocammo, Mirella; RaasRothschild, A.; Tappino, Barbara; Alves, Sandra
- UGT1A1 gene variations in individuals with and without clinical diagnosis of Gilbert SyndromePublication . Rodrigues, Carina; Vieira, Emília; de Carvalho, João; Costa, Elísio; Santos, Rosário; Santos-Silva, Alice; Bronze-da-Rocha, Elsa
- Novel method for picking up large heterozygous deletions with semiquantitative PCR in patients with mucolipidosis III alpha/betaPublication . Coutinho, Maria Francisca; Encarnação, Marisa; Lacerda, Lúcia; Ribeiro, Helena; Prata, Maria João; Alves, SandraMucolipidosis II (ML II alpha/beta) or I-cell disease is a rare genetic disease in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is 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 alpha/beta, there are some patients that were previously reported as possessing only one heterozygous mutation while the second one was missing. Here we support the idea that some of these cases may be attributable to the methodological problem of direct sequencing since large heterozygous deletions are undetectable through this method. Methods: We developed a semiquantitative approach by multiplex PCR and capillary electrophoresis. In order to do so, we co-amplified two exon fragments in each PCR reaction, including the one corresponding to GNPTAB exons and an internal fragment that would work as a control (primer pairs for LIPA exons, the gene that codes for acid lipase, another lysosomal enzyme). Results: After testing this technique in the two Portuguese patients that present heterozygous missense mutations but with the second mutation missing, we found evidences that support the idea that, in both patient, the last exons (20 and 21) of the GNPTAB gene are missing in one of the alleles. Conclusion: Here we present the first molecular evidence of the existence of large deletions in the GNPTAB, underlying Mucolipidosis type III as well as a novel method for detecting these types of alterations when present in heterozygous patients. This semiquantitative PCR approach is a valuable tool that allows the screening of large deletions in the GNPTAB gene. In conclusion, ML III cases with only one heterozygous mutation already detected trhpugh direct sequencing methods should be further screened for the possible presence of a large heterozygous deletion mutation.
- 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
- Study of Mutation Impact on Lysosomal Proteins using Computational AnalysisPublication . Duarte, Ana Joana; Amaral, OlgaReduction in time and cost involved in mutation analyses led to a rapid increase in the number of molecular lesions identified at the major loci involved in several diseases. Laboratories where DNA analyses of patients samples are carried out, are constantly confronted with the need to further investigate the causal nature of the mutations found. In many cases this type of approach may be useful to rapidly confer the genotyping results with a prognosis value. Although the establishment of a genotype/phenotype correlation cannot be fully accomplished through such a simple strategy, the multi-step approach used seems to be useful for predicting the causal potential of new, private or unique mutations, when the 3D structure is available. Thus, in silico analyses combining multiple information seem to be a fast, inexpensive, reliable tool, providing more accurate results, and thus allowing better predictions.
- 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.