Browsing by Author "Coutinho, Maria Francisca"
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- Alu-Alu Recombination Underlying the First Large Genomic Deletion in GlcNAc-Phosphotransferase Alpha/Beta (GNPTAB) Gene in a MLII Alpha/Beta PatientPublication . Coutinho, Maria Francisca; da Silva Santos, Liliana; Lacerda, Lúcia; Quental, Sofia; Wibrand, F.; Lund, A.M.; Johansen, K.B.; Prata, Maria João; Alves, SandraMucolipidosis type II α/β is a severe, autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the α/β subunits of the GlcNAc-phosphotransferase. To date, over 100 different mutations have been identified in MLII α/β patients, but no large deletions have been reported. Here we present the first case of a large homozygous intragenic GNPTAB gene deletion (c.3435-386_3602 + 343del897) encompassing exon 19, identified in a ML II α/β patient. Long-range PCR and sequencing methodologies were used to refine the characterization of this rearrangement, leading to the identification of a 21 bp repetitive motif in introns 18 and 19. Further analysis revealed that both the 5' and 3' breakpoints were located within highly homologous Alu elements (Alu-Sz in intron 18 and Alu-Sq2, in intron 19), suggesting that this deletion has probably resulted from Alu-Alu unequal homologous recombination. RT-PCR methods were used to further evaluate the consequences of the alteration for the processing of the mutant pre mRNA GNPTAB, revealing the production of three abnormal transcripts: one without exon 19 (p.Lys1146_Trp1201del); another with an additional loss of exon 20 (p.Arg1145Serfs*2), and a third in which exon 19 was substituted by a pseudoexon inclusion consisting of a 62 bp fragment from intron 18 (p.Arg1145Serfs*16). Interestingly, this 62 bp fragment corresponds to the Alu-Sz element integrated in intron 18.This represents the first description of a large deletion identified in the GNPTAB gene and contributes to enrich the knowledge on the molecular mechanisms underlying causative mutations in ML II.
- Alu-Alu recombination underlying the first large genomic deletion in GlcNAc-phosphotransferase α/β (GNPTAB) gene in a MLII α/β patientPublication . Coutinho, Maria Francisca; da Silva Santos, Liliana; Lacerda, Lúcia; Quental, Sofia; Flemming, W; Lund, AM; Johansen, KB; Prata, Maria João; Alves, SandraMucolipidosis type II alpha/beta is a severe, autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the alpha/beta subunits of the GlcNAc-phosphotransferase. To date, over 100 different mutations have been identified in MLII alpha/beta patients but no large deletions have been reported. Here we present the first case of a large homozygous intragenic GNPTAB gene deletion (c.3435-386_3602+343del897) encompassing exon 19, identified in a ML II alpha/beta patient. Long range PCR and sequencing methodologies were used to refine the characterization of this rearrangement, leading to the identification of a 21bp repetitive motif in introns 18 and 19. Further analysis revealed that both the 5’ and 3’ breakpoints were located within highly homologous Alu elements (Alu-Sz in intron 18 and Alu-Sq2, in intron 19), suggesting that this deletion has probably resulted from Alu-Alu unequal homologous recombination. RT-PCR methods were used to further evaluate the consequences of the alteration for the processing of the mutant pre mRNA GNPTAB, revealing the production of three abnormal transcripts: one without exon 19 (p.Lys1146_Trp1201del); another with an additional loss of exon 20 (p.Arg1145Serfs*2), and a third in which exon 19 was substituted by a pseudoexon inclusion consisting of a 62 bp fragment from intron 18 (p.Arg1145Serfs*16). Interestingly, this 62 bp fragment corresponds to the Alu-Sz element integrated in intron 18. This represents the first description of a large deletion identified in the GNPTAB gene and contributes to enrich the knowledge on the molecular mechanisms underlying causative mutations in ML II.
- Alu-Alu recombination underlying the first large genomic deletion in GlcNAc-phosphotransferase α/β (GNPTAB) gene in a MLII α/β patient [Poster]Publication . Coutinho, Maria Francisca; da Silva Santos, Liliana; Lacerda, Lúcia; Flemming, Wibrand; Lund, Allan M; Johansen, Klaus B; Prata, Maria João; Alves, SandraMucolipidosis type II α/β is a severe, autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the α/β subunits of the GlcNAc-phosphotransferase. To date, over 100 different mutations have been identified in MLII α/β patients including missense, nonsense, small deletions, small insertions and splice site mutations (Human Gene Mutation Database website [http://www.hgmd.org] and references therein). Large genomic rearrangements were rarely reported (1,6%) with only two large insertions having been described up to now (Tappino et al., 2008; Otomo et al., 2009) but no known large deletions. Results: In this work, through long range PCR and sequencing methodologies we identified a large homozygous intragenic GNPTAB gene deletion, encompassing exon 19, in a ML II α/β patient and refined the characterization of this rearrangement. As a result, it was possible to identify the deletion breakpoints and determine the deletion extension which was 897 bp and included the last 386 nucleotides of intron 18, exon 19, and the first 343 bp of intron 19. A 21bp repetitive motif in introns 18 and 19 was observed at both deletion breakpoints. Further analysis revealed that both the 5’ and 3’ breakpoints were located within highly homologous Alu elements (Alu-Sz in intron 18 and Alu-Sq2, in intron 19), suggesting that this deletion has probably resulted from Alu-Alu unequal homologous recombination. RT-PCR methods were used to further evaluate the consequences of the alteration for the processing of the mutant pre mRNA GNPTAB, revealing the production of three abnormal transcripts: one without exon 19; another with an additional loss of exon 20, and a third in which exon 19 was substituted by a pseudoexon inclusion consisting of a 62 bp fragment from intron 18. Interestingly, this 62 bp fragment corresponds to the Alu-Sz element integrated in intron 18. Conclusion: To the best of our knowledge, this represents the first description of a large deletion identified in the GNPTAB gene. Furthermore, the work adds on the knowledge of the molecular mechanisms underlying causative mutations in ML II and highlights the importance of cDNA analysis on the prediction of the impact of large deletions at protein levels, since a simple gDNA analysis might be misleading.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for a rare diseasePublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana I.; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Omidi, Maryam; Pohl, Sandra; Alves, SandraMucolipidosis II (MLII) is a Lysosomal Storage Disorder caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase, which is responsible for the Mannose- 6-Phosphate marker addition to lysosomal enzymes. Of all MLII mutations, the c.3503_3504delTC in GNPTAB exon 19 is the most frequent, making it a good target for a personalized therapy. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs) for MLII. Previously, on MLII patients’ fibroblasts, ASOs were used to skip exon 19 of the GNPTAB pre-mRNA, successfully resulting in the production of an in-frame mRNA[1]. Now, our aim is to analyze if these results are translated to the enzymatic and cellular phenotype level.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II a/b: in vitro and in vivo studiesPublication . Matos, Liliana; Gonçalves, Mariana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraMucolipidosis type II alpha/beta (ML II alpha/beta) is one of the most severe Lysosomal Storage Disorders and is caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase. This enzyme is responsible for the addition of the mannose 6-phosphate marker to lysosomal enzymes, which allow their targeting to lysosomes. Of the several mutations that occur in ML II alpha/beta, the deletion of 2 nucleotides from GNPTAB exon19 (c.3503_3504del) is the most frequent, making it a good target for a specific mutation therapy as there is no therapy for this disease. In this study, we explored the possibility of an innovative therapeutic strategy based on the use of antisense oligonucleotides (AOs) for ML II alpha/beta. In a previous in vitro study in ML II alpha/beta patient fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, resulting successfully in the production of an in-frame mRNA1. Currently, our objective is to evaluate the therapeutic potential of this approach, both in vitro in C57BL/6 fibroblasts and in vivo in C57BL/6 mice. For this, 18 animals were used, divided into 6 groups: groups 1 and 4 were injected with saline solution, groups 2 and 5 were injected with AO at 25 mg/kg and groups 3 and 6 were injected with AO at 50 mg/kg. All animals were injected by intraperitoneal route and were sacrificed after 4 days (groups 1, 2, 3) or 7 days (groups 4, 5, 6) post-treatment. At the end of the experiment, the organs were collected and frozen at -80ºC, for later RNA extraction, cDNA synthesis and RT-PCR. After results analysis, the exon 19 skipping was not observed using any of the tested doses or incubation periods. So, we can theorize that the doses administered were not sufficient to achieve a response or the AO might have had a high clearance rate. As for the in vitro experience, the C57BL/6 fibroblasts were seeded in 6-well plates and subsequently transfected with concentrations of AO ranging from 10nM to 600nM. After 24h or 48h of incubation, cells were collected and cDNA analysis revealed a full length transcript but also another one of lower molecular weight compatible with exon-skipping. These are preliminary data, so in the near future more experiments will be done. 1. Matos L, Vilela R, Rocha M, et al. Development of an antisense oligonucleotide-mediated exon skipping therapeutic strategy for Mucolipidosis II: validation at RNA level. Hum Gene Ther, 2020, 31(13-14):775-783.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II a/b: in vitro and in vivo studiesPublication . Matos, Liliana; Gonçalves, Mariana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraMucolipidosis type II alpha/beta (ML II) is one of the most severe Lysosomal Storage Disorders and is caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase. This enzyme is responsible for the addition of the mannose 6-phosphate marker to lysosomal enzymes, which allow their targeting to lysosomes. Of the several mutations that occur in ML II, the deletion of 2 nucleotides from GNPTAB exon19 (c.3503_3504del) is the most frequent, making it a good target for a specific mutation therapy as there is no therapy for this disease. In this study, we explored the possibility of an innovative therapeutic strategy based on the use of antisense oligonucleotides (AOs) for ML II. In a previous in vitro study in ML II patient fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, resulting successfully in the production of an in-frame mRNA. Currently, our objective is to evaluate the therapeutic potential of this approach, both in vitro in C57BL/6 fibroblasts and in vivo in C57BL/6 mice. For this, 18 animals were used, divided into 6 groups: groups 1 and 4 were injected with saline solution, groups 2 and 5 with AO at 25 mg/kg and groups 3 and 6 with AO at 50 mg/kg. All animals were injected by intraperitoneal route and were sacrificed after 4 or 7 days post-treatment. At the end of the experiment, the organs were collected and frozen at -80ºC, for later RNA extraction, cDNA synthesis and RT-PCR. After results analysis, the exon 19 skipping was not observed using any of the tested doses or incubation periods. So, we can theorize that the doses administered were not sufficient to achieve a response or the AO might have had a high clearance rate. As for the in vitro experience, the C57BL/6 fibroblasts were seeded in 6-well plates and subsequently transfected with concentrations of AO ranging from 10nM to 600nM. After 24/48h of incubation, cells were collected and cDNA analysis revealed a full length transcript but also another one of lower molecular weight compatible with exon-skipping. These are preliminary data, so in the near future more experiments will be done.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II α/β: in vitro and in vivo studiesPublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraGenetic therapy directed towards the correction of RNA mis-splicing is being investigated at basic research and in late-stage clinical trials. Many mutations that change the normal splicing pattern and lead to aberrant mRNA production have been identified in Lysosomal Storage Disorders (LSDs). Mucopolysaccharidosis IIIC (MPS IIIC) is one of those LSDs caused by mutations in the HGSNAT gene that encodes an enzyme involved in heparan sulphate degradation. Splicing mutations are one of the most frequent (~20%) genetic defects in MPS IIIC. Approximately 55% correspond to 5' splice-site (ss) mutations thus constituting a good target for mutation-specific therapeutic approaches. Recently, we have demonstrated in fibroblast cells that a modified U1 snRNA vector designed to improve the definition of exon 2 5’ss of the HGSNAT can restore splicing impaired by the mutation c.234+1G>A. Currently, our goal is to evaluate in vivo the therapeutic potential of that modified U1 snRNA by testing it in mice expressing the human splicing defect. For this purpose, two full-length constructs were generated by cloning the wild-type (wt) or the mutated HGSNAT splicing-competent cassettes in the pcDNA 3.1 vector. Then, in an in vitro assay, the wt or mutated construct was transfected in Hep3B and COS-7 cells. After molecular analysis it was observed that both minigenes reproduce the healthy control and patient cDNA’s splicing pattern. Therefore, both constructs were used to generate mice of the C57BL/6 strain expressing the human mutation c.234+1G>A in the liver and test its modified U1-mediated rescue in vivo. Wt or mutant minigenes were administrated in mice by hydrodynamic injection following a reported protocol(1). After 48 hours animals were sacrificed, the liver was collected and molecular analysis was performed. Preliminary results showed expression of the HGSNAT cDNA from the mutant construct in the liver of at least one animal. Thus, further tests will be carried out to optimize some limiting points, such as the administration of the minigenes (e.g. increase of injection volume from 7% to 8-9% of mice body weight; inclusion of an in vivo transfection reagent to enhance delivery efficiency) and the use of other mice strain. 1. Balestra D, et al. (2014) J Thromb Haemost 12(2):177–185.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II α/β: in vitro and in vivo studiesPublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraMucolipidosis type II α/β (ML II α/β) is one of the most severe Lysosomal Storage Disorders and is caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase. This enzyme is responsible for the addition of the mannose 6-phosphate marker to lysosomal enzymes, which allow their targeting to lysosomes. Of the several mutations that occur in ML II α/β, the deletion of 2 nucleotides from GNPTAB exon19 (c.3503_3504del) is the most frequent, making it a good target for a specific mutation therapy as there is no therapy for this disease. In this study, we explored the possibility of an innovative therapeutic strategy based on the use of antisense oligonucleotides (AOs) for ML II α/β. In a previous in vitro study in ML II α/β patient fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, resulting successfully in the production of an in-frame mRNA. Currently, our objective is to evaluate the therapeutic potential of this approach, both in vitro in C57BL/6 fibroblasts and in vivo in C57BL/6 mice. For this, 18 animals were used, divided into 6 groups: groups 1 and 4 were injected with saline solution, groups 2 and 5 were injected with AO at 25 mg/kg and groups 3 and 6 were injected with AO at 50 mg/kg. All animals were injected by intraperitoneal route and were sacrificed after 4 days (groups 1, 2, 3) or 7 days (groups 4, 5, 6) post-treatment. At the end of the experiment, the organs were collected and frozen at -80ºC, for later RNA extraction, cDNA synthesis and RT-PCR. After results analysis, the exon 19 skipping was not observed using any of the tested doses or incubation periods. So, we can theorize that the doses administered were not sufficient to achieve a response or the AO might have had a high clearance rate. As for the in vitro experience, the C57BL/6 fibroblasts were seeded in 6-well plates and subsequently transfected with concentrations of AO ranging from 10nM to 600nM. After 24h or 48h of incubation, cells were collected and cDNA analysis revealed a full length transcript but also another one of lower molecular weight compatible with exon-skipping. These are preliminary data, so in the near future more experiments will be done.
- Assessing Lysosomal Disorders in the NGS Era: Identification of Novel Rare VariantsPublication . Encarnação, Marisa; Coutinho, Maria Francisca; Silva, Lisbeth; Ribeiro, Diogo; Ouesleti, Souad; Campos, Teresa; Santos, Helena; Martins, Esmeralda; Cardoso, Maria Teresa; Vilarinho, Laura; Alves, SandraLysosomal storage diseases (LSDs) are a heterogeneous group of genetic disorders with variable degrees of severity and a broad phenotypic spectrum, which may overlap with a number of other conditions. While individually rare, as a group LSDs affect a significant number of patients, placing an important burden on affected individuals and their families but also on national health care systems worldwide. Here, we present our results on the use of an in-house customized next-generation sequencing (NGS) panel of genes related to lysosome function as a first-line molecular test for the diagnosis of LSDs. Ultimately, our goal is to provide a fast and effective tool to screen for virtually all LSDs in a single run, thus contributing to decrease the diagnostic odyssey, accelerating the time to diagnosis. Our study enrolled a group of 23 patients with variable degrees of clinical and/or biochemical suspicion of LSD. Briefly, NGS analysis data workflow, followed by segregation analysis allowed the characterization of approximately 41% of the analyzed patients and the identification of 10 different pathogenic variants, underlying nine LSDs. Importantly, four of those variants were novel, and, when applicable, their effect over protein structure was evaluated through in silico analysis. One of the novel pathogenic variants was identified in the GM2A gene, which is associated with an ultra-rare (or misdiagnosed) LSD, the AB variant of GM2 Gangliosidosis. Overall, this case series highlights not only the major advantages of NGS-based diagnostic approaches but also, to some extent, its limitations ultimately promoting a reflection on the role of targeted panels as a primary tool for the prompt characterization of LSD patients.
- Assessing Niemann-Pick Type C (NP-C) through a multi-omics approach genomic and transcriptomic profile of challenging casesPublication . Encarnação, Marisa; Coutinho, Maria Francisca; Cho, Soo-Min; Cardoso, Maria Teresa; Chaves, Paulo; Ribeiro, Isaura; Santos, Juliana Inês; Gaspar, Paulo; Quelhas, Dulce; Lacerda, Lúcia; Leão-Teles, Elisa; Futerman, Anthony H.; Vilarinho, Laura; Alves, SandraNiemann-Pick type C (NP-C) is a neurodegenerative Inherited Metabolic Disease with a heterogeneous clinical presentation, due to mutations in either the NPC1 or NPC2 genes. We studied patients with clinical diagnosis of NP-C but presenting inconclusive results regarding biomarkers testing and molecular analysis. Using NGS- targeted DNA sequencing we have identified some novel putative mutations and subsequent cDNA analysis allowed us to stablish the functional effect of a silent mutation (previously reported as a polymorphism) in the NPC1 splicing process. We demonstrated that this mutation leads to exon skipping, frameshift and premature stop codon and identified it in two NP-C patients from two unrelated Portuguese families. This mutation most likelly leads to a very unstable transcript that was overlooked. Also, to better characterize the pathomechanisms related to specific disease-causing mutations in NP-C patients, we analysed gene expression profiles in cultured skin fibroblasts and compared them to control individuals using Massively Parallel RNA Single-Cell Sequencing (MARS-Seq). The most prominent hits from this transcriptomics analysis were validated by qRT-PCR. The MARS-Seq analysis showed that a number of genes were upregulated and a significant number of the highly enriched genes are related to the unfold protein response (UPR) and Endoplasmic Reticulum (ER) stress, in a specific patient, which deserves further studies. ER stress is a hallmark of many neurodegenerative diseases, including LSD and can be due to misfolded/unfolded proteins as result of a specific missense mutation. Our preliminary results suggest that UPR activation is variable among NP-C patients, and this is likely to depend on the mutation type. Several other factors may contribute to this though, which could explain the heterogeneous presentation of this pathology. Additionally, we have investigated the same patients studied in MARS-Seq at the protein and celular levels. Interestingly, and according to recently published work, we observed that, for the analyzed mutations a significant part of the mutated NPC1 protein was retained/ delayed in the ER.