Browsing by Author "Pinto, Eugénia"
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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.
- 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].
- Distinct Haplotype in Non-Ashkenazi Gaucher Patients with N370S MutationPublication . Amaral, Olga; Marcao, Ana; Pinto, Eugénia; Zimran, Ari; Sá Miranda, M.C.A new polymorphism, in intron 7 of glucocerebrosidase gene, has been identified in Gaucher Disease patients. It seems to appear only in Pv1.1- alleles bearing the N370S mutation. This new sub-haplotype was only identified in Portuguese patients, of origins spanning all of the Portuguese continental territory. This finding indicates that, in the Portuguese, mutation N370S has existed in the context of two slightly different haplotypes and thus must be relatively ancient.
- Estudo molecular de Epilepsia Mioclónica Progressiva de Unverritch -LundborgPublication . Pinto, Eugénia; Amaral, Olga; Santos, ManuelThe Progressive Myoclonus Epilepsies (PME) are associated to an heterogeneous group of rare metabolic diseases. They are clinically marked by myoclonic, tonic-clonic episodes and progressive neurological decline, with ataxia and dementia. One of the major causes of PME is the Unverricht- Lundborg disease (EPM1, MIM 254800), an autosomal recessive disorder, caused by loss of function mutations in the cystatin B gene (CSTB). The onset of the symptoms is around the age of 10 yr. and is marked by convulsions. The EPM1 diagnostic is done through a differential diagnostic, first at a clinical level, and being confirmed at a genetic level. The main objective of this work is the implementation of the genetic study and applied research in EPM1. Since EPM1 is a rare disease without laboratory diagnostic in Portugal its real impact at the level of public health is unknown, the methods developed in this thesis will give the possibility of confirming/excluding clinical suspicion of EPM1, and allow the characterization of the patients. The results obtained have permitted the initiation of the study of CSTB, in instances where there was a suspicion of EPM1. It became possible to confirm a case of a patient suspect of having the EPM1, as well as making the evaluation of some polymorphisms present in our population. The new mutation, found and characterized in this work, is a point mutation, apparently silent (p.Q22Q), which alters the normal splicing pattern resulting in partial retention of the intronic sequence and subsequently leading to loss of function. These results show the need for making a non directional approach, doing a comprehensive study of the CSTB gene and complementing the gDNA study with the cDNA one. The study of this disease at the molecular biology level, may contribute to the evaluation and characterization of this pathology as far as its mutations and, at same time, it may contribute for a better understanding of its pathophysiology.
- 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.
- 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.
- 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.
- New splicing mutation in the cystatin B genePublication . Amaral, Olga; Freitas, Joel; Pinto, Eugénia; Duarte, Ana Joana; Ribeiro, Isaura; Ribeiro, Diogo; Chaves, João
- 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.
- Type 1 Gaucher Disease: Identification of and Prevalence of Glucocerebrosidase Mutations in the PortuguesePublication . Amaral, Olga; Pinto, Eugénia; Fortuna, Margarida; La cerda, Lucia; Sá Miranda, M.C.