Browsing by Author "Cho, Soo-Min"
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- 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.
- Genomics and transcriptomics approach - diagnosis of a challenging case of Niemann-Pick type C (NP-C)Publication . Encarnação, Marisa; Coutinho, Maria Francisca; Cho, Soo-Min; Cardoso, Maria Teresa; Chaves, Paulo; Gaspar, Paulo; Santos, Juliana Inês; Ribeiro, Isaura; Quelhas, Dulce; Lacerda, Lúcia; Leão-Teles, Elisa; Futerman, Anthony H.; Vilarinho, Laura; Alves, SandraNP-C is a neurodegenerative Inherited Metabolic Disease with a heterogeneous clinical presentation, and due to mutations in either the NPC1 or NPC2 genes.We have studied a patient with clinical diagnosis of NP-C but presenting inconclusive results regarding molecular analysis.To better characterize this patient, we have performed NGS-based technologies (targeted DNA sequencing and single cell-RNA sequencing).For the molecular diagnosis we used a NGS gene panel followed by the analysis of cDNA.Latter, we used massively parallel single cell RNA-seq (MARS-Seq) to address gene profiling changes and characterize the pathomechanisms related to specific disease-causing mutations. Using our targeted NGS panel we identified two novel mutations in NPC1 gene.Next, through cDNA analysis of one of the patient parents we were able to understand the impact of the silent mutation.This mutation leads to exon skipping giving origin to an out-of-frame transcript and eliciting the nonsense-mediated decay pathway.Thus, we were not able to easily detect the mutant transcript which turned the molecular diagnosis more challenging. Apparently the presence of the other mutation (a missense mutation) impairs the NPC protein folding leading to its ER retention.The MARS-Seq analysis of this patient showed that a number of upregulated genes are related to the unfold protein response (UPR) and ER stress, which deserves further studies.
- Transcriptomics profiling of Niemann-Pick type C patients: activation of the unfold protein response in a specific casePublication . Encarnação, Marisa; Coutinho, Maria Francisca; Cho, Soo-Min; Cardoso, Maria Teresa; Chaves, Paulo; Gaspar, Paulo; Santos, Juliana Inês; Ribeiro, Isaura; Quelhas, Dulce; Lacerda, Lúcia; Leão-Teles, Elisa; Futerman, Anthony H.; Vilarinho, Laura; Alves, SandraBackground: Niemann-Pick type C (NP-C) is a neurodegenerative Lysosomal Storage Disease (LSD) with a heterogeneous clinical presentation secondary to abnormal intracellular accumulation of cholesterol. We have studied a patient with clinical diagnosis of NP-C but presenting inconclusive results regarding biomarkers testing and molecular analysis. To better characterize this patient, we have performed NGS-based technologies (targeted DNA sequencing and single cell-RNA sequencing). Methods: For the molecular diagnosis we used a NGS gene panel followed by the analysis of cDNA (in the patient and both parents). Latter, we have used massively parallel single cell RNA-seq (MARS-Seq) to address gene profiling changes and better characterize the pathomechanisms related to specific disease-causing mutations in this patient as well as in two NPC patients. The most prominent hits from this transcriptomics analysis were validated by qRT-PCR. Results and Discussion: Using our targeted NGS panel we identified two novel mutations in NPC1 gene (p.V505G; p.V562V). Next, through cDNA analysis of one of the patient parents we were able to understand the impact of the V562V silent mutation located in the middle of the exon 11. This mutation leads to exon 11 skipping giving origin to an out-of-frame transcript and eliciting the nonsense-mediated decay pathway. This mechanism contributed to the almost absence of the mutant transcript in the patient. Thus, we were not able to easily detect it in the sequencing electropherogram of the patient which turned the molecular diagnosis more challenging. By its turns, apparently the presence of the other mutation (the missense V505G) impairs the proper NPC protein folding leading to its ER retention. In fact, the MARS-Seq analysis of this patient 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).