Departamento de Genética Humana
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Browsing Departamento de Genética Humana by Sustainable Development Goals (SDG) "03:Saúde de Qualidade"
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- Alternative Splicing at the Crossroad of Inflammatory Bowel Diseases and Colitis-Associated Colon CancerPublication . Matos, Paulo; Jordan, PeterSimple Summary: Patients with ulcerative colitis (UC) face a higher risk of developing colorectal cancer (CRC) due to chronic inflammation, a known promoter of tumour growth. Here, we review the molecular differences between colitis-associated cancer (CAC) and sporadic CRC, with a focus on “alternative splicing”, a mechanism by which the same gene can produce various protein forms. We explore how inflammation triggers changes in this process, increasing cancer risk for UC patients. The revised data emphasize that additional research into these molecular changes could help identify new biomarkers (molecules that indicate disease progression) and pave the way for innovative treatments targeting these alterations. Such advances would improve outcomes and quality of life for patients while contributing to cancer prevention and care.
- CCL2 expression predicts clinical outcomes and regulates E-cadherin and angiogenesis in pituitary tumoursPublication . Silva, Ana Luísa; Barry, Sayka; Lopes-Pinto, Mariana; Joaquim, Rita; Miranda, Catarina; Reis, Fábio; Miranda, Micaella; Matos, Paulo; Suleyman, Oniz; Oliveira, Tiago; López-Presa, Dolores; Borrecho, Gonçalo; Tortosa, Francisco; Faria, Claúdia C.; Korbonits, Márta; Marques, PedroThe crosstalk between tumour cells and microenvironment components in pituitary neuroendocrine tumours (PitNETs), including chemokines, may impact tumour behaviour and clinical outcomes. CCL2 was previously identified as a key chemokine in PitNETs, but its role remains unknown. We aimed to study the role of CCL2 in defining the phenotype and clinical outcomes of PitNETs and in regulating macrophage chemotaxis, epithelial-to-mesenchymal transition (EMT) and angiogenesis. We studied CCL2 and E-cadherin expression, macrophages (CD68 and CD163) and vessels (CD31) in samples from 86 PitNET patients. Higher CCL2 mRNA expression was found in patients who required multimodal and multiple treatments and had active disease at the last follow-up. Higher CCL2 immunoreactivity was observed in patients with larger PitNETs. Among somatotroph tumours, CCL2 mRNA expression correlated with serum IGF-1 at the last follow-up. CCL2 mRNA expression levels correlated negatively with CDH1 expression and with E-cadherin complete membranous staining. In vitro, CCL2 downregulated E-cadherin expression in GH3 cells but did not affect cell morphology or migration. CCL2 expression correlated with the number of vessels, vessel perimeter and vessel area in PitNETs but not with PitNET-infiltrating macrophages. Our data suggest that CCL2 may lead to (or is at least a predictive marker of) poorer clinical outcomes and more difficult-to-treat PitNETs, potentially through its regulatory effects on different tumour-related mechanisms beyond immune cell chemotaxis, including in the activation of the EMT pathway and modulation of angiogenesis in PitNETs. Further studies are needed to corroborate our findings and to validate CCL2 as a potential predictive marker and therapeutic target in PitNETs.
- Comparative analysis of hybrid‑SNP microarray and nanopore sequencing for detection of large‑sized copy number variants in the human genomePublication . Silva, Catarina; Ferrão, José; Marques, Bárbara; Pedro, Sónia; Correia, Hildeberto; Valente, Ana; Rodrigues, António Sebastião; Vieira, LuísBackground: Nanopore sequencing is a technology that holds great promise for identifying all types of human genome variations, particularly structural variations. In this work, we used nanopore sequencing technology to sequence 2 human cell lines at low depth of coverage to call copy number variations (CNV), and compared the results variant by variant with chromosomal microarray (CMA) results. Results: We analysed sequencing data using CuteSV and Sniffles2 variant callers, compared breakpoints based on hybrid-SNP microarray, nanopore sequencing and Sanger sequencing, and analysed CNV coverage. From a total of 48 high confidence variants (truth set), variant calling detected 79% of the truth set variants, increasing to 86% for interstitial CNV. Simultaneous use of the 2 callers slightly increased variant calling. Both callers performed better when calling CNV losses than gains. Variant sizes from CMA and nanopore sequencing showed an excellent correlation, with breakpoints determined by nanopore sequencing differing by only 20 base pairs on average from Sanger sequencing. Nanopore sequencing also revealed that four variants concealed genomic inversions undetectable by CMA. In the 10 CNV not called in nanopore sequencing, 8 showed coverage evidence of genomic loss or gain, highlighting the need to improve SV calling algorithms performance. Conclusions: Nanopore sequencing offers advantages over CMA for structural variant detection, including the identification of multiple variant types and their breakpoints with increased precision. However, further improvements in variant calling algorithms are still needed for nanopore sequencing to become a highly robust and standardized approach for a comprehensive analysis of genomic structural variation.
- A Comparative Overview of the Role of Human Ribonucleases in Nonsense-Mediated mRNA DecayPublication . da Costa, Paulo J.; Menezes, Juliane; Guedes, Raquel; Reis, Filipa P.; Teixeira, Alexandre; Saramago, Margarida; Viegas, Sandra C.; Arraiano, Cecília M.; Romão, LuísaEukaryotic cells possess surveillance mechanisms that detect and degrade defective transcripts. Aberrant transcripts include mRNAs with a premature termination codon (PTC), targeted by the nonsense-mediated decay (NMD) pathway, and mRNAs lacking a termination codon, targeted by the nonstop decay (NSD) pathway. The eukaryotic exosome, a ribonucleolytic complex, plays a crucial role in mRNA processing and turnover through its catalytic subunits PM/Scl100 (Rrp6 in yeast), DIS3 (Rrp44 in yeast), and DIS3L1. Additionally, eukaryotic cells have other ribonucleases, such as SMG6 and XRN1, that participate in RNA surveillance. However, the specific pathways through which ribonucleases recognize and degrade mRNAs remain elusive. In this study, we characterized the involvement of human ribonucleases, both nuclear and cytoplasmic, in the mRNA surveillance mechanisms of NMD and NSD. We performed knockdowns of SMG6, PM/Scl100, XRN1, DIS3, and DIS3L1, analyzing the resulting changes in mRNA levels of selected natural NMD targets by RT-qPCR. Additionally, we examined the levels of different human β-globin variants under the same conditions: wild-type, NMD-resistant, NMD-sensitive, and NSD-sensitive. Our results demonstrate that all the studied ribonucleases are involved in the decay of certain endogenous NMD targets. Furthermore, we observed that the ribonucleases SMG6 and DIS3 contribute to the degradation of all β-globin variants, with an exception for βNS in the former case. This is also the case for PM/Scl100, which affects all β-globin variants except the NMD-sensitive variants. In contrast, DIS3L1 and XRN1 show specificity for β-globin WT and NMD-resistant variants. These findings suggest that eukaryotic ribonucleases are target-specific rather than pathway-specific. In addition, our data suggest that ribonucleases play broader roles in mRNA surveillance and degradation mechanisms beyond just NMD and NSD.
- Comparison of the ABC and ACMG systems for variant classificationPublication . Houge, Gunnar; Bratland, Eirik; Aukrust, Ingvild; Tveten, Kristian; Žukauskaitė, Gabrielė; Sansovic, Ivona; rea-Fernández, Alejandro J.B; Mayer, Karin; Paakkola, Teija; McKenna, Caoimhe; Wright, William; Markovic, Milica Keckarevic; Lildballe, Dorte L.; Konecny, Michal; Smol, Thomas; Alhopuro, Pia; Gouttenoire, Estelle Arnaud; Obeid, Katharina; Todorova, Albena; Jankovic, Milena; Lubieniecka, Joanna M.; Stojiljkovic, Maja; Buisine, Marie-Pierre; Haukanes, Bjørn Ivar; Lorans, Marie; Roomere, Hanno; Petit, François M.; Haanpää, Maria K.; Beneteau, Claire; Pérez, Belén; Plaseska-Karanfilska, Dijana; Rath, Matthias; Fuhrmann, Nico; Ferreira, Bibiana I.; Stephanou, Coralea; Sjursen, Wenche; Maver, Aleš; Rouzier, Cécile; Chirita-Emandi, Adela; Gonçalves, João; Kuek, Wei Cheng David; Broly, Martin; Haer-Wigman, Lonneke; Thong, Meow-Keong; Tae, Sok-Kun; Hyblova, Michaela; Dunnen, Johan T. den; Laner, AndreasThe ABC and ACMG variant classification systems were compared by asking mainly European clinical laboratories to classify variants in 10 challenging cases using both systems, and to state if the variant in question would be reported as a relevant result or not as a measure of clinical utility. In contrast to the ABC system, the ACMG system was not made to guide variant reporting but to determine the likelihood of pathogenicity. Nevertheless, this comparison is justified since the ACMG class determines variant reporting in many laboratories. Forty-three laboratories participated in the survey. In seven cases, the classification system used did not influence the reporting likelihood when variants labeled as “maybe report” after ACMG-based classification were included. In three cases of population frequent but disease-associated variants, there was a difference in favor of reporting after ABC classification. A possible reason is that ABC step C (standard variant comments) allows a variant to be reported in one clinical setting but not another, e.g., based on Bayesian-based likelihood calculation of clinical relevance. Finally, the selection of ACMG criteria was compared between 36 laboratories. When excluding criteria used by less than four laboratories (<10%), the average concordance rate was 46%. Taken together, ABC-based classification is more clear-cut than ACMG-based classification since molecular and clinical information is handled separately, and variant reporting can be adapted to the clinical question and phenotype. Furthermore, variants do not get a clinically inappropriate label, like pathogenic when not pathogenic in a clinical context, or variant of unknown significance when the significance is known.
- Lactate-coated polyurea-siRNA dendriplex: a gene therapy-directed and metabolism-based strategy to impair glioblastoma (GBM)Publication . Martins, Filipa; Arada, Renata; Barros, Hélio; Matos, Paulo; Ramalho, José; Ceña, Valentín; Bonifácio, Vasco D.B.; Gonçalves, Luís G.; Serpa, JacintaGlioblastoma (GBM) is a highly lethal disease with limited treatment options due to its infiltrative nature and the lack of efficient therapy able to cross the protective blood-brain barrier (BBB). GBMs are metabolically characterized by increased glycolysis and glutamine dependence. This study explores a novel metabolism-based therapeutic approach using a polyurea generation 4 dendrimer (PURE) surface functionalized with lactate (LA) (PURE-LA), to take advantage of glucose-dependent monocarboxylate transporters (MCTs) overexpression, loaded with selenium-chrysin (SeChry) and temozolomide (TMZ) or complexed with anti-glutaminase (GLS1) siRNAs to abrogate glutamine dependence. The nanoparticles (PURE-LA) were efficient vehicles for cytotoxic compounds delivery, since SeChry@PURE-LA and TMZ@PURE-LA induced significant cell death in GBM cell lines, particularly in U251, which exhibits higher MCT1 expression. The anti-GLS1 siRNA-dendriplex with PURE-LA (PURE-LA-anti-GLS1-siRNA) knocked down GLS1 in the GBM cell lines. In two in vitro BBB models, these dendriplexes successfully crossed the BBB, decreased GLS1 expression and altered the exometabolome of GBM cell lines, concomitantly with autophagy activation. Our findings highlight the potential of targeting glucose and glutamine pathways in GBM using dendrimer-based nanocarriers, overcoming the BBB and disrupting key metabolic processes in GBM cells. PURE-LA-anti-GLS1-siRNA dendriplexes cross the blood-brain barrier (BBB) and impair glioblastoma (GBM) metabolism. The BBB is formed by a thin monolayer of specialized brain microvascular endothelial cells joined together by tight junctions that selectively control the passage of substances from the blood to the brain. It is a major obstacle in the treatment of GBM, since many chemotherapeutic drugs are unable to penetrate the brain. Therefore, we developed a strategy to overcome this obstacle: a lactate-coated polyurea dendrimer generation 4 (PURE) able to cross the BBB in vitro, that act as a nanocarrier of drugs and siRNA to the GBM cells. PURE-LA are nanoparticles functionalized with lactate (LA) to target MCT1, a lactate transporter highly expressed by GBM cells. Moreover, a complex of this nanoparticle with anti-GLS1 (glutaminase) siRNA (PURE-LA-anti-GLS1-siRNA) was made, to target glutamine metabolism. It efficiently knocked down GLS1. Moreover, PURE-LA loaded with SeChry led to BBB disruption.
- The mechanism of nonsense-mediated mRNA decay and its playersPublication . Subtil, Catarina; Loison, Luísa; Santos, Rafaela LacerdaNonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance mechanism harbouring two functions: identification and degradation of transcripts containing premature translation-termination codons (PTC), preventing deleterious effects in the cell; and downregulation of mRNAs in response to cellular needs, maintaining the quality of gene expression. One-third of gene mutations in human genetic diseases, including cancer, are due to nonsense mutations or frameshift that result in transcripts harbouring nonsense codons and can be eliminated by NMD. The several factors involved in this mechanism may act in diverse ways depending on the set of transcripts to be regulated, contributing to the branching of this pathway. Cytoplasmic DIS3 exosome independent 3′-5′ exoribonuclease 2 (DIS3L2) has been reported as one of the factors to induce NMD targets decay. Therefore, this study aims to enlighten how DIS3L2 functions in NMD: i) analyse the correlation between the distinct branches of NMD and cervical and uterus cancer; ii) investigate which branch guides DIS3L2-mediated degradation; and iii) test which region of the NMD/DIS3L2-targets mediate DIS3L2 degradation through a system of hybrid-genes. For our first aim, we detected no correlation between any of the branches of NMD and uterus and cervical cancer. Each factor acts independently. In our second objective, we analysed the mRNA expression of five transcripts, but none displayed a significant alteration in their expression to infer a correlation between DIS3L2 and a particular NMD branch. Relatively to the last aim, we successfully cloned three out of the four constructs but due to time constrictions we could not continue. Nonetheless, further testing is needed to better understand this mechanism and how transcript degradation is mediated, including the diverse factors needed for its activation, which might be the key to future advanced therapeutic strategies.
- Newborn Screening for Sickle Cell Disease: Results from a Pilot Study in the Portuguese PopulationPublication . Rodrigues, Diogo; Marcão, Ana; Lopes, Lurdes; Ventura, Ana; Faria, Teresa; Ferrão, Anabela; Gonçalves, Carolina; Kjöllerström, Paula; Castro, Ana; Fraga, Sofia; Almeida, Marta; Maia, Tabita; Gomes, João; Lachado, Ana; Guerra, Isabel; Ferreira, Fátima; Trigo, Fernanda; Bento, Celeste; Vilarinho, LauraThe Portuguese Newborn Screening Program currently includes 28 pathologies: congenital hypothyroidism, cystic fibrosis, 24 inborn errors of metabolism, sickle cell disease and spinal muscular atrophy. This pilot study for sickle cell disease newborn screening, including 188,217 samples, was performed between May 2021 and December 2023, with phase I, including 24,130 newborns, in the Lisbon and Setubal districts and phase II, including 164,087 newborns, in the whole country. DBS samples were analyzed through capillary electrophoresis. In phase I, a high birth incidence of sickle cell disease was found (1:928 NBs), resulting from the identification of 24 HbSS and 2 HbSC patients. This birth incidence decreased but remained significant when the pilot study for sickle cell disease newborn screening was expanded to a national level, with the identification of 67 sickle cell disease patients (59 HbSS and 8 HbSC), revealing a birth incidence of 1:2449 NBs. These data suggest that this condition is becoming increasingly relevant in Portugal, thus reflecting a general European trend, where sickle cell disease is already recognized as a public health problem. Therefore, it highlights the importance of its integration into the Portuguese National Newborn Screening Program panel in January 2024, thus allowing the early identification and clinical follow-up of these patients.
- Pesquisa de Deleções/Duplicações em Genes Associados a Cancro Hereditário por MLPA DigitalPublication . Alves, Beatriz Correia; Gonçalves, João; Melo, Maria Joana Lima BarbosaA presença, na linha germinativa, de Variações do Número de Cópias (CNV) em genes de predisposição para cancro hereditário pode aumentar a suscetibilidade a esta doença. A identificação de uma CNV patogénica ou provavelmente patogénica num doente oncológico tem um impacto significativo na gestão clínica do indivíduo afetado e dos seus familiares. Tradicionalmente, a pesquisa de CNV no diagnóstico molecular de cancro hereditário é realizada apenas para alguns genes através do MLPA (Multiplex Ligation-dependent Probe Amplification) convencional. Nos últimos anos, o desenvolvimento de softwares de análise in silico de CNV com base em dados de NGS (Next-Generation Sequencing) representou um avanço significativo, ao possibilitar a pesquisa de deleções e duplicações em múltiplos genes em simultâneo. No entanto, estas ferramentas apresentam ainda limitações. Dada a relevância de uma análise abrangente que integre o maior número possível de genes relevantes no âmbito da patologia em causa, este trabalho teve como principal objetivo implementar uma nova metodologia de pesquisa de CNV em genes associados a cancro hereditário, que combina os princípios do MLPA convencional com a capacidade da NGS de analisar vários genes em simultâneo: o MLPA digital. Neste estudo, foi realizada a pesquisa de CNV por MLPA digital em amostras de doentes com história clínica e familiar de cancro, seguida de validação dos resultados utilizando outras metodologias de genética molecular e classificação das variantes identificadas segundo as recomendações da CanVIG-UK. O MLPA digital demonstrou ser eficaz na deteção de deleções e duplicações em genes associados a cancro hereditário, apresentando um desempenho adequado para a utilização em laboratórios clínicos, com sensibilidade de 100% e especificidade de 98%. A eficácia dos softwares de pesquisa in silico panelcn.MOPS e DRAGEN Enrichment foi confirmada através da concordância entre os resultados destas ferramentas e do MLPA digital. Foram identificadas cinco variantes patogénicas ou provavelmente patogénicas nos genes APC, BRCA1, BRCA2 e CHEK2, que justificam os fenótipos dos doentes. Este estudo demonstra que o MLPA digital é uma alternativa ao MLPA convencional na primeira fase de pesquisa molecular de CNV germinativas em genes associados a cancro hereditário, permitindo a análise de múltiplos genes em várias amostras em simultâneo.
- The role of UPF1 cap-independent translation in colorectal cancerPublication . Lacerda, Rafaela; Menezes,Juliane; Elias, Adriana; Sousa, Sofia de; Romão, LuísaColorectal cancer (CRC) is one of the deadliest diseases worldwide with projections pointing towards an increase for the next two decades. Translation dysregulation of many genes contributes to CRC development, and here we are studying the role of translation dysregulation of up-frameshift 1 (UPF1) in CRC. This protein is involved in many cellular mechanisms such as nonsense-mediated mRNA decay, cell cycle progression, or telomere maintenance and homeostasis. It also works as a tumour suppressor protein in most cancers but not in CRC, in which UPF1 plays an oncogenic role. We used the Xena platform to perform in silico analyses that revealed UPF1 protein overexpressed in CRC, contrary to several other analysed cancers. Besides, UPF1 protein levels are increased in CRC compared to the counterpart normal tissues. Experimentally, we confirmed that UPF1 protein expression is maintained in different CRC cell lines under normal conditions or endoplasmic reticulum (ER) stress. To understand the mechanism underlying such maintenance, we used a bicistronic reporter construct to test whether UPF1 5’ untranslated region (UTR) can mediate alternative translation initiation and we concluded that such sequence drives cap-independent translation initiation, in both normal and stress conditions. Deletional and mutational analyses 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 cap-independent translation initiation mechanism to work properly. Using RNA antisense oligonucleotides (ASOs) targeting UPF1 SL I and III, we observed a reduced UPF1 expression in HCT116 (CRC) cells, supporting the functional role of SL I and SL III in mediating cap-independent translation. Altogether, these results highlight the importance of cap-independent translation initiation in UPF1 expression regulation, in conditions that mimic the tumour microenvironment, and this might be used as a therapeutic target.