Browsing by Issue Date, starting with "2021-11-18"
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- Reporting of secondary findings in clinical genomic sequencing: national guidelines are requiredPublication . Theisen, Patrícia; Rodrigues, Pedro; Gonçalves, JoãoIntroduction: The rapid and growing integration of exome and genome sequencing into clinical genetic diagnosis raises awareness regarding the identification of variants of potential clinical value unrelated to the primary reason for testing (secondary findings, SF). SF pose major challenges, as multiple issues (medical, legal, ethical, economic) and different contexts (e.g. paediatric and prenatal diagnosis, patient and family management, research in rare diseases) must be considered, highlighting the importance to promote standardized reporting of SF. We aim to bring to the consideration of the Portuguese Society of Human Genetics (SPGH) the urgent need for issuing national guidelines for reporting SF from clinical sequencing. Methodology: Consultation and review of guidelines for reporting SF in clinical exome and genome sequencing from different organizations, focusing on the ones issued by the American College of Medical Genetics and Genomics (ACMG)1, the European Society of Human Genetics (ESHG)2 and the French Society of Predictive and Personalized Medicine (SFMPP)3. Results: The ACMG recently published SF v3.0 list4 includes 73 clinically actionable genes mainly related to cancer and cardiovascular phenotypes, for which causal SF should be reported unless patients opted out. The ESHG recommends a more cautious approach, stating that genomic analysis should be as targeted as possible for the time being because a broader analysis raises complex issues in clinical practice. The SFMPP restricts its guidelines for reporting pathogenic SF to a list of 36 actionable cancer genes, requiring a double consent from the patient. Discussion: Considering the diversity of approaches and the complexity involved in reporting SF, we propose that the SPGH should promote the creation of a multidisciplinary workgroup involving all the stakeholders to put forth official national guidelines for reporting SF in clinical sequencing. References 1. Miller DT et al. Genet Med. 2021;23:1381. 2. de Wert G et al. Eur J Hum Genet. 2021;29:365. 3. Pujol P et al. Eur J Hum Genet. 2018;26:1732. 4. Miller DT et al. Genet Med. 2021; 23:1391.
- Internal ribosome entry site (IRES)-mediated translation as a putative candidate mechanism to mRNA-based therapiesPublication . Neto, Maria Inês; Lacerda, Rafaela; Romão, LuísaUntranslated regions in the messenger RNA (mRNA) are susceptible to the interaction of regulatory elements including proteins or non-coding RNA molecules, being an important hotspot to the study of gene expression regulation. Internal Ribosome Entry Sites (IRES) are secondary structures usually located on the 5’UTR of an mRNA molecule that can recruit the ribosome during initiation of protein synthesis without the involvement of the cap structure. This mechanism tends to appear when the cell is under stress conditions, which might include the presence of oncogenes, growth factors or proteins involved in programmed cell death. This work focuses on the human AGO1, an important key player for RNA-mediated gene silencing, and whether its 5’UTR is capable of driving cap-independent translation initiation. To achieve this goal, a construct containing the 5’UTR of human AGO1 was cloned, taking advantage of a bicistronic vector containing two reporter genes, Renilla luciferase (RLuc) and firefly luciferase (FLuc), the last one cloned downstream from the 5’UTR. We performed luminometry assays to assess the relative translation efficiency of FLuc, which is under the control of AGO1 5’UTR. The results showed that human AGO1 5’UTR mediates a cap-independent eIF4G-dependent mechanism of translation initiation enhanced by a free 5’ end. Also, we saw that this alternative mechanism is maintained, and even enhanced, under stress conditions, such as the knock-down of eukaryotic initiation factor 4E, the protein that directly binds to the cap structure. We are currently investigating what is the minimal sequence required for IRES-mediated translation. Combining these results with emerging RNA-based therapies will be helpful to develop novel strategies to prevent and treat disorders, such as cancer, involving dysregulation of AGO1 translation.
- Learning from mRNA: the relevance of the tumour suppressor protein UPF1’s internal ribosome entry site-mediated translation in tumorigenesisPublication . Lacerda, Rafaela; Menezes, Juliane; Romão, LuísaCrucial in several cellular processes, such as nonsense-mediated mRNA decay, cell cycle progression, and telomere maintenance and homeostasis, Up-frameshift 1 (UPF1) has also been considered a tumour suppressor protein in hepatocellular carcinoma and gastric cancer, as it is underexpressed in the latter and negatively correlated to MALAT1 (long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1) expression. UPF1 overexpression inhibits some cancer-specific features, like proliferation, cell cycle progression, cell migration and invasion, and enhances apoptosis, turning the UPF1/MALAT1 pathway a potential therapeutic target for gastric cancer. Here, we investigate the importance of UPF1 internal ribosome entry site (IRES)-dependent translation in tumorigenesis, specifically in colorectal cancer. Thus, we used a bicistronic system with two reporter genes, in which we cloned UPF1 5’ untranslated region (UTR) upstream the second cistron, whose translation will only occur internally. We transcribed the bicistronic mRNA in vitro and transfected HeLa (cervical cancer), NCM460 (normal intestinal-derived colonocytes) and HC116 (pre-metastatic colorectal carcinoma) cells with such mRNA, along with the positive and negative controls. The results show a significant increase in IRES-mediated translation levels compared to those of the negative control, both in normal conditions and under endoplasmic reticulum stress. Also, internal initiation occurs in the absence of the cap structure. Deletional and mutational analysis of UPF1 5’UTR showed that nucleotides 1–100 (stem loop (SL) I) and 151–275 (SL III) — out of 275 nucleotides — are the minimal required sequences for the IRES to work properly. Also, we used RNA antisense oligonucleotides (ASOs) targeting UPF1 IRES SL I and III, and observed a reduced UPF1 expression. Cellular viability increases in HCT116 cells and decreases in NCM460 cells treated with ASOs targeting SL III and SL I, respectively, whereas apoptosis increases in NCM460 cells and decreases in HCT116 cells treated with ASOs targeting SL I. This may be the dawn of a new RNA-based therapeutic approach regulating colorectal cancer development.
- Characterization of an oncogenic isoform of TP53: Δ160p53Publication . Ramalho, Ana Catarina; Rita, Filipa; López-Iniesta, M.; Lacerda, Rafaela; Romão, Luísa; Candeias, Marco M.The transcription factor p53 is a key cell regulator, having roles in varied cellular processes. Widely known as a tumour suppressor protein, p53 is responsible for signalling the adequate response to DNA damage, oncogenic signalling, or other stress stimuli. The target genes of this protein are involved in cell cycle arrest, senescence, apoptosis, and DNA damage response, among other pathways. Besides the full-length p53 (FLp53), to which these functions are attributed, the TP53 gene encodes for eleven other protein isoforms that result from alternative splicing, internal initiation of translation and transcription from an internal promoter. In striking contrast to FLp53, the N-terminally truncated Δ160p53 exhibits pro-oncogenic traits, although it only differs from FLp53 by the lack of its first 159 amino acids. Δ160p53 promotes cell survival, proliferation, invasion, and adhesion, and it is overexpressed in cancer cells harbouring hotspot p53 mutants. The TP53 gene is frequently mutated in cancer, and hotspot mutants result from single missense mutations that convert p53 in a driver of tumorigenesis. As Δ160p53 presents many of the oncogenic roles attributed to p53 cancer mutants, it is plausible that this isoform could be responsible for the paradoxical mutant p53 functions. However, detailed knowledge on the mode of action of Δ160p53 is still lacking. We have performed additional tests to further characterize the oncogenic traits of this isoform. To evaluate the ability of Δ160p53 to promote anchorage-independent cell growth, we have used the soft agar colony formation assay. Preliminary results show a tendency of Δ160p53 to promote growth, when compared to the control and other isoforms. Our new data complements our previous knowledge on Δ160p53 and reinforces the importance of studying this isoform for therapeutic targeting.
- A pro-inflammatory microenvironment triggers overexpression of tumor-related RAC1B in polarized colorectal cancer cellsPublication . Pereira, Joana; Bessa, Cláudia; Gonçalves, Vânia; Matos, Paulo; Jordan, PeterUnderstand how tumor cells respond to a pro-inflammatory microenvironment with changes in the alternative splicing of RAC1B.
- Autism Spectrum Disorder: contribution of genetic variants involved in the nonsense-mediated mRNA decayPublication . Marques, Ana Rita; Santos, João Xavier; Vilela, Joana; Rasga, Célia; Martiniano, Hugo; Oliveira, Guiomar; Romão, Luísa; Moura Vicente, AstridIntroduction: Autism Spectrum Disorder (ASD) is a neurodevelopmental condition characterized by impairedsocial/communication skills and stereotyped/repetitive behaviors. Genetic factors account for 50-80% of the familialrisk of ASD, but genetic determinants are not fully understood and a role for regulatory processes is plausible. Inthis study, we explored the contribution to ASD etiology of genes involved in an important post-transcriptionalregulatory mechanism implicated in neurodevelopment, the Nonsense-Mediated Decay (NMD). Methods: We first compiled a group of 46 genes encoding NMD factors and regulators. In these genes wesearched for Single Nucleotide Variants (SNVs) and Copy Number Variants (CNVs) in two samples of ASD patients(N=1828 and N=3570, respectively). We observed the frequency of these variants in 60146 controls from gnomADv2.1.1 (for SNVs) and in 10355 controls from the Database of Genomic Variant ( for CNVs). In genes with rarevariants (MAF<1% in controls) predicted to be pathogenic in silico , we further investigated whether these variantsaffect protein domains required for NMD. Results: We identified 270 predicted pathogenic SNVs within 38 genes in 524 ASD patients (28.7% of the total ASDcases) and 38 CNVs located in 18 genes in 38 ASD patients (1% of the ASD cases). Five of these genes, RBM8A , UPF2 , FMR1 , SMG6 and EIF4G1, were previously associated with ASD. We found that 136 variants (122 SNVsand 11 CNVs), in 23 genes, were located within known protein domainsrequired for NMD. These variants, identifiedin 258 ASD patients, may affect proper NMD function and consequently contribute to changes in the expression ofNMD targets. Discussion : In this study we identified genetic variants that may affect NMD function in ASD patients. Since mostNMD targets encode proteins expressed in the brain, we hypothesize that NMD impairment can constitute a riskfactor to ASD pathophysiology. Further studies are needed to better understand the impact of these genetic variantson NMD function and their relevance for ASD.A full understanding of these regulatory mechanisms may constitutean opportunity for the development of therapeutic interventions.
- Can Cell-type specific variability be involved in a rare variant of Unverricht-Lundborg? Investigation with iPSC generated modelsPublication . Duarte, Ana Joana; Ribeiro, Diogo; Moreira, Luciana; Amaral, OlgaHomozygosity for a private synonymous mutation in the cystatin-B gene (CSTB, MIM:601145; c.66G>A; p.Q22Q) was detected in a Portuguese patient with a rare, atypical form of Unverricht-Lundborg disease (ULD, MIM #254800). This apparently silent mutation leads to mis-splicing of CSTB pre-mRNA where a normal and an abnormal transcript were detected. Using iPSCs as a source of different cell types, we intend to clarify if the observed abnormal RNA splicing is cell-type specific, and to characterise the subsequent protein mislocalization. In conclusion, we hope to be able to contribute to the understanding of cell-type specific implications in the pathogenesis of ULD.