Browsing by Author "Laranjeira, Francisco"
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- Data in support of a functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS IIPublication . Matos, Liliana; Gonçalves, Vânia; Pinto, Eugénia; Laranjeira, Francisco; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R.; Pérez, Belén; Alves, SandraThis data article contains insights into the methodology used for the analysis of three exonic mutations altering the splicing of the IDS gene: c.241C>T, c.257C>T and c.1122C>T. We have performed splicing assays for the wild-type and mutant minigenes corresponding to these substitutions. In addition, bioinformatic predictions of splicing regulatory sequence elements as well as RNA interference and overexpression experiments were conducted. The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in "Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II" Matos et al. (2015) [1].
- Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS IIPublication . Matos, Liliana; Gonçalves, Vânia; Pinto, Eugénia; Laranjeira, Francisco; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R.; Perez, Belén; Alves, SandraMucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS, i.e., c.241C>T and c.257C>T, whose presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C>T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3' splice site of exon 3 respectively. For the c.1122C>T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n=3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotides transfection in control fibroblasts led to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis-acting element for 5' splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation.
- Mucopolysaccharidosis type III in PortugalPublication . Caseiro, Carla; Rocha, Sónia; Ferreira, Célia; Ribeiro, Helena; Pinto, Eugénia; Pinto, Fernanda; Sousa, Domingos; Pinto, Eugénia; Ribeiro, Isaura; Laranjeira, Francisco; Coutinho, Maria Francisca; Alves, Sandra; Lacerda, LúciaIntroduction: Mucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) is a rare autosomal recessively inherited disorder composed at least by four different subtypes: type A (OMIM # 252900), type B (OMIM # 252920), type C (OMIM # 252930) and type D (OMIM # 252940). Each subtype is caused by a deficiency in a different enzyme involved in the catabolic pathway for heparan sulfate. From the clinical point of view, type III patients can be classified within the MPS neurodegenerative phenotype, given visceromegaly, coarse facies, joint disease and bone dysplasia are relatively mild and progresses steadily. Behavioural disturbances and hyperactivity are often reported in the early stages of the disease. Objectives: The aim of this study is to draw the attention to the several subtypes which may be diagnosed within type III MPS, pointing out for mucopolysacharidosis-like patients with unclear etiology. Methods: MPS type III patients are diagnosed by the quantification of GAG heparan sulfate in urine, identification of the enzymatic deficiency in peripheral blood and gene sequencing for causal mutations. Results: From 34 MPS type III families, 4 patients were found to be IIIA, 24 IIIB and 6 IIIC. Type IIIB is the most common, accounting for 70% of all Portuguese cases with MPS III. Conclusions: Biochemical and molecular characterization allows carrier identification and informed family planning decisions. A negative diagnosis for subtype A to D does not rule out the disease and those patients with a high clinical suspicion are currently further tested for a recently proposed subtype, MPS IIIE.
- SCARB2 mutations as modifiers in Gaucher disease: the wrong enzyme at the wrong place?Publication . Coutinho, Maria Francisca; Lacerda, Lúcia; Gaspar, Ana; Pinto, Eugénia; Ribeiro, Isaura; Laranjeira, Francisco; Ribeiro, Helena; Silva, Elizabete; Ferreira, Célia; Prata, Maria João; Alves, SandraUnlike most lysosomal proteins, -glucocerebrosidase (GCase) – the hydrolase defective in Gaucher disease (GD) – is specifically delivered to the lysosome through interaction with the lysosomal integral membrane protein type 2 (LIMP-2). Recently, mutations in the LIMP-2 coding gene, SCARB2, were reported to affect the severity of Gaucher phenotype. To understand the role of variations in SCARB2 in the broad phenotype spectrum observed for patients carrying similar GBA mutations, we have screened the gene in the Portuguese GD patients. After analyzing a total of 91 individuals, that constitutes the whole population of affected individuals referenced in our country, we identified 3 different SCARB2 coding variants. Of those, 2 were known polymorphic variations, with high prevalence in the normal population (p.M159V and p.V396I) and the third was a novel coding variant, p.T398M, present in heterozygozity in one Gaucher patient, a severely affected child, born from healthy unrelated parents of Cape Verdean origin. Initial investigations showed low GCase levels, and the child was referenced for enzyme replacement therapy (ERT), having started treatment. Nevertheless, subsequent therapeutic follow-up with assessment of chitotriosidase levels showed enzyme levels which were disparate from the ones expected for a GD patient under ERT treatment, suggesting lower response to treatment. When analyzed both in silico and in vitro, this variation was predicted to be deleterious for protein function. Preliminary results of Western blot assays in COS7 cells transfected with a minigene carrying the mutation show a decrease of LIMP-2 levels when compared to wild-type protein. Recombinant GCase uptake is known to be dependent from the receptor density (mannose 6-phosphate receptors and LIMP-2). That is, indeed, one the major reasons for the variable organ response to ERT. Taking this into account, it would be expectable that any alteration causing either dysfunction or reduction of LIMP-2 lead to a decrease in the efficacy of ERT in GD patients, as observed in our case. To the best of our knowledge this is the first time that a whole GD population is screened for mutations in this gene. From our results on the Portuguese population it was possible to conclude that SCARB2 mutations neither the only nor the most frequent cause of GD phenotype variability. Nevertheless, with the identification of a novel mutation in one of the patients who present a severe GD phenotype and had a poor response to ERT standard treatment and its subsequent evaluation, our study reinforces previous evidence that SCARB2 mutations do act as modifiers of GD.
- Solving a case of allelic dropout in the GNPTAB gene: implications in the molecular diagnosis of mucolipidosis type III alpha/betaPublication . Coutinho, Maria Francisca; Encarnação, Marisa; Laranjeira, Francisco; Lacerda, Lúcia; Prata, Maria João; Alves, SandraWhile being well known that the diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing, not rarely, however, genetic testing needs much perseverance and cunning strategies to identify the causative mutation(s). Here we present a case of a thorny molecular diagnosis of mucolipidosis type III alpha/beta, which is an autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the α/β-subunits of the GlcNAc-1-phosphotransferase. We used both cDNA and gDNA analyses to characterize a mucolipidosis type III alpha/beta patient whose clinical diagnosis was already confirmed biochemically. In a first stage only one causal mutation was identified in heterozygosity, the already described missense mutation c.1196C>T(p.S399F), both at cDNA and gDNA levels. Only after conducting inhibition of nonsense-mediated mRNA decay (NMD) assays and after the utilization of another pair of primers the second mutation, the c.3503_3504delTC deletion, was identified. Our findings illustrate that allelic dropout due to the presence of polymorphisms and/or of mutations that trigger the NMD pathway can cause difficulties in current molecular diagnosis tests.