Browsing by Author "Rodrigues, Pedro"
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- ABC system used as an add-on to clarify germline variants previously classified as VUS according to ACMG guidelinesPublication . Rodrigues, Pedro; Theisen, Patrícia; Silva, Catarina; Mendonça, Joana; Carpinteiro, Dina; Vieira, Luís; Gonçalves, JoãoThe increasing number of patients screened by NGS to identify germline variants associated with hereditary breast/ovarian cancer (HBOC) syndromes, is leading to a growing number of variants classified as Variants of Uncertain Significance (VUS) according to ACMG guidelines1. Since the ACMG system merges functional and clinical data into a one-dimensional system, it is not always clear how the classification was obtained. The ABC system (ABCs) of variant classification2 splits functional and clinical grading and aims to give a better guide to variant significance. The main goals of this work were i) to apply the ABCs to a group of previously classified ACMG-VUS and ii) to evaluate the potential clinical impact of this review/classification. Germline variants (36 - 29 missense, 1 synonymous and 6 intronic) detected in 5 genes (BRCA1, BRCA2, ATM, CHEK2, PALB2) previously classified as ACMG-VUS, were selected from our database of patients with HBOC, to be reclassified with the ABCs. Variant assessment included: query of clinical and population databases, literature and in silico tools (VEP, HSF, Alamut, Varsome). Eleven variants were classified as Class 0 (functional - fVUS); 17 as class E (functional - HFE (Hypothetical Function Effect), and 8 as Class D (functional - LFE (Likely Functional Effect). fVUS are not clinically graded. Considering that ACMG-VUS are not actionable, it is still an ongoing debate if they should be reported or not. Since the ACMG merges functional and clinical data, it might be difficult for clinicians to understand how VUS classification is achieved. The ABCs allowed us to distinguish between VUS classified due to lack of data from those that might have a functional impact. Class 0 variants (11) should not be reported and class E (17) reporting is optional. The use of ABCs highlighted 8 variants (class D) which might be a susceptibility factor with functional impact and should be reported. Functional and segregation studies are of major importance to clarify the clinical significance of these variants. 1-PMID: 25741868. 2-PMID: 33981013. Support: FCT/MCTES, ToxOmics and Human Health (UIDB/00009/2020). GenomePT(POCI-01-0145-FEDER-022184).
- Antioxidant and antimicrobial properties of PLA based active packaging with pomegranate peels and extractPublication . Andrade, Mariana; Rodrigues, Pedro; Barros, Carolina; Cruz, Vasco; Machado, Ana; Barbosa, Cássia; Coelho, Anabela; Furtado, Rosália; Correia, Cristina; Saraiva, Margarida; Vilarinho, Fernanda; Ramos, Fernando; Sanches Silva, AnaActive food packaging’ primary goal is to extend foodstuffs’ shelf life, through a dynamic and continuous interaction between the package and the packaged food. In an emission active packaging, the objective is the gradual release of antioxidant and/or antimicrobial compounds into the food surface, to delay the natural foods’ degradation. The active compounds can be extracted from several sources, such as aromatic plants, seaweeds, fruits by-products, among others. Since 50% of pomegranate is composed by peels, and since it is mainly consumed in juice and jam form, pomegranate peels may represent a considerable asset for the extraction of such compounds. The principal objective of this work was to evaluate the antioxidant and antimicrobial properties of polylactic-based active packaging incorporated with 3% (w/w) of pomegranate peels (PLA/3PP) and 3% (w/w) of pomegranate peels extract (PLA/3PPE). For the in vitro antioxidant activity evaluation, 9.08 cm2 of films were immersed in the food simulator, ethanol 95% (v/v), at 40 °C for 10 days. Then, the DPPH radical scavenging assay was performed, as well as the total content of phenolic compounds, total content in flavonoids, content in punicalagin (A+B) and ellagic acid were determined. Also, to fully determine the total content in punicalagin (A+B) and ellagic acid, the films were kept in methanol at 25 and 40 °C for 24 h. The antimicrobial activity of the films was evaluated with Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli, in accordance with ISO 22196:2011. Results showed that PLA/3PPE presented a higher antioxidant potential and higher content in phenolic compounds and flavonoids. Only ellagic acid was detected in the active PLA-based films. Regarding the antimicrobial activity, both films presented antimicrobial activity against S. aureus.
- Aplicação da sequenciação de nova geração ao diagnóstico genético do cancro da mama hereditárioPublication . Theisen, Patrícia I.; Silva, Catarina; Pereira-Caetano, Iris; Rodrigues, Pedro; Isidro, Glória; Vieira, Luís; Gonçalves, JoãoIntrodução: O cancro da mama hereditário (CMH) constitui 5-10% dos casos de cancro da mama, dos quais cerca de 30% se devem a mutações germinais de elevada penetrância nos genes BRCA1 e BRCA2. Embora tenham sido identificadas mutações noutros genes de suscetibilidade para cancro da mama, estas são raras, pelo que a análise sequencial de múltiplos genes se torna ineficaz e dispendiosa pelos métodos convencionais(1,2). A determinação da causa genética subjacente ao CMH permite não só identificar os indivíduos com risco aumentado de cancro da mama, como oferecer uma medicina personalizada, mais eficaz na redução da incidência de cancro da mama assim como da morbilidade e mortalidade associadas(3). A sequenciação de nova geração (NGS) veio possibilitar a pesquisa de mutações em múltiplos genes em simultâneo, permitindo um diagnóstico molecular rápido, eficaz e com custo inferior ao da sequenciação de Sanger. Objetivos: Otimizar o diagnóstico molecular do CMH através da implementação de um protocolo de NGS baseado num painel abrangente de genes de suscetibilidade para cancro da mama. A 1ª fase de concretização deste objetivo consistiu em validar a utilização da NGS na deteção de mutações nos genes BRCA1, BRCA2 e TP53. Material e métodos: Foram analisadas por NGS 12 amostras de doentes com cancro da mama, previamente sequenciadas pelo método de Sanger para os genes BRCA1, BRCA2 e TP53. As bibliotecas de sequências-alvo foram preparadas a partir de DNA genómico pelo método de captura por hibridação, num protocolo que integrou o Trusight Cancer Sequencing Panel e o kit TruSight Rapid Capture (Illumina), e sequenciadas numa plataforma MiSeq com leituras paired-end de 150 bp. A análise bioinformática incluiu os softwares MiSeq Reporter, VariantStudio e Isaac Enrichment (Illumina). Resultados: Foram detetadas por NGS um total de 97 variantes de sequência nos genes BRCA1, BRCA2 e TP53, das quais 35 são variantes únicas (33 single nucleotide variants e 2 deleções), numa concordância de 100% com a sequenciação de Sanger. Conclusões: Estes resultados preliminares comprovam a eficiência da NGS na deteção de variantes em 3 genes de elevada penetrância para cancro da mama e abrem caminho para a oferta de um painel de genes de suscetibilidade para cancro da mama que permitirá um diagnóstico molecular do CMH mais abrangente, rápido e com custos reduzidos relativamente à sequenciação de Sanger. Bibiografia 1. Tung N, Batteli C, Allen B et al. (2015). Frequency of mutations in individuals with breast cancer referred for BRCA1 and BRCA2 testing using next-generation sequencing with a 25-gene panel. Cancer, 121: 25-33. 2. Apostolou P, Fostira F (2013). Hereditary breast cancer: the era of new susceptibility genes. BioMed Res Int, 2013: 747318. 3. Ellsworth R, Decewicz D, Shriver C, Ellsworth D (2010). Breast cancer in the personal genomics era. Curr Genomics, 11: 146-161.
- BRCA1 and BRCA2 variants identified in patients with a personal/familial history of hereditary breast/ovarian cancers and other hereditary cancer syndromes: challenges related with variants of uncertain significancePublication . Rodrigues, Pedro; Theisen, Patrícia; Silva, Catarina; Carpinteiro, Dina; Mendonça, Joana; Vieira, Luís; Gonçalves, JoãoIntroduction: Screening for BRCA1 and BRCA2 variants (Vs) in patients with Hereditary Breast/Ovarian Cancer (HBOC) or other Hereditary Cancer Syndromes (HCS) is performed using next-generation sequencing (NGS), allowing detection of a high number and types of Vs. The growing use of PARP inhibitors (PARPi) in the treatment of patients with homologous recombination-deficient tumors contributes to an increasing number of patients being screened for BRCA Vs even when family history of HBOC/HCS is absent. These approaches result in a growing number of identified Vs that need to be classified. The goals of this study, apart from identifying pathogenic and likely pathogenic Vs, were to identify uncertain significance Vs (VUS) and bring to discussion their uncertainties and impact on patients and family members. Methodology: BRCA1 and BRCA2 were analyzed in 207 patients mainly with HBOC/HCS, using TruSight® Cancer and MiSeq. Annotation was performed with Variant Interpreter, VEP, HSF, IGV, Alamut and Varsome. Vs were divided in 3 groups (G) according to allele frequency (AF) in population databases (G1:AF>5%, G2:1%≤AF≤5% and G3:AF<1%) and classified according to ACMG-AMP guidelines. Results: In BRCA1 and BRCA2, 45 and 96 Vs were detected, respectively. While in BRCA1 G3, we detected 6 pathogenic (P) Vs and 9 VUS, in BRCA2 G3, we found 9P Vs, 2 Likely Pathogenic (LP), and 15 VUS. We highlight that in G3, VUS were more frequent than P and LP Vs. Discussion: Among G3, 28% of BRCA1 and 25% of BRCA2 Vs were VUS. VUS give rise to difficulties related to management of patients and families. Functional studies of missense or putatively affecting splicing VUS are of major importance to assess their biopathologic impact, as some of them may be hypomorphic and reclassified as P/LP. Accordingly, some VUS may have impact in therapeutic decisions (e.g. PARPi) as well as in patient’s cancer-risk management protocols, including appropriate genetic counselling and VUS screening in selected family members. We predict that new challenges related to VUS will emerge.
- Cancro da mama hereditário: dupla heterozigotia para variantes patogénicas nos genes BRCA1 e ATMPublication . Theisen, Patrícia; Rodrigues, Pedro; Silva, Catarina; Ribeiro, Leonor; Carreiro, Helena; Gervásio, Helena; Vieira, Luís; Gonçalves, JoãoIntrodução/objetivos: O cancro da mama hereditário (CMH) representa 5-10% dos casos de cancro da mama, destes ~30% devem-se a variantes patogénicas germinativas nos genes BRCA1 e BRCA2. Vários outros genes de suscetibilidade para CMH têm sido identificados, com diferentes graus de penetrância. A determinação da causa genética subjacente ao CMH permite identificar os indivíduos com risco aumentado de desenvolver tumores, os quais podem beneficiar de medicina personalizada, e também oferecer o teste genético preditivo a familiares em 1º grau, após aconselhamento genético. A sequenciação de nova geração (NGS) veio revolucionar o diagnóstico molecular do CMH proporcionando alta eficiência e baixos custos, usando painéis multigénicos. Neste trabalho, utilizou-se a NGS para sequenciar um painel de genes de suscetibilidade para CMH numa doente com história pessoal de cancro da mama e ovário e história familiar de cancro do lado materno e paterno. Métodos: NGS com preparação das bibliotecas de sequências-alvo a partir de DNA genómico (protocolo Trusight Cancer - TruSight Rapid Capture, Illumina) e sequenciação no MiSeq (Illumina). Análise bioinformática efetuada com os programas MiSeq Reporter, Enrichment e Variant Studio (Illumina), Alamut Visual e Integrative Genomics Viewer. As variantes patogénicas identificadas por NGS nos genes BRCA1 e ATM foram confirmadas por sequenciação de Sanger. Resultados: Foi identificado um caso raro de dupla heterozigotia para as variantes patogénicas BRCA1: c.2037delinsCC, p.(Lys679Asn*4) e ATM: c.3802delG, p.(Val1268*). A pesquisa destas variantes num familiar com cancro da mama e em dois familiares em 1º grau saudáveis, após aconselhamento genético, permitiu diagnosticá-los como portadores de uma ou de ambas as variantes. Conclusões: A NGS de um painel de genes de suscetibilidade para CMH permitiu identificar um caso raro de dupla heterozigotia para variantes patogénicas nos genes BRCA1 e ATM. Esta abordagem analítica foi crucial pois possibilitou um aconselhamento genético orientado em função das variantes identificadas e do risco de desenvolvimento de tumores associados a uma ou a ambas as variantes. Este caso demonstra a vantagem da sequenciação de um painel multigénico face à análise molecular limitada aos genes BRCA1 e BRCA2.
- Diagnóstico molecular das síndromes de cancro hereditário da mama-ovário e colorretal: a experiência do Departamento de Genética Humana do INSA (1994-2025)Publication . Theisen, Patrícia; Rodrigues, Pedro; Isidro, Glória; Gonçalves, João
- Diagnóstico molecular de cancros hereditários por sequenciação de nova geração: cancro da mama e cancro colorretalPublication . Theisen, Patrícia; Silva, Catarina; Caetano, Iris Pereira; Rodrigues, Pedro; Isidro, Glória; Vieira, Luís; Gonçalves, JoãoO cancro da mama e o cancro colorretal constituem duas das principais causas de morte a nível mundial. Entre 5 a 10% destes casos estão associados a variantes germinais/hereditárias em genes de suscetibilidade para cancro. O objetivo deste trabalho consistiu em validar a utilização da sequenciação de nova geração (NGS) para identificar variantes previamente detetadas pelo método de Sanger em diversos genes de suscetibilidade para cancro da mama e colorretal. Foram sequenciadas por NGS 64 amostras de DNA de utentes com suspeita clínica de predisposição hereditária para cancro da mama ou colorretal, utilizando o painel de sequenciação TruSight Cancer e a plataforma MiSeq (Illumina). Estas amostras tinham sido previamente sequenciadas pelo método de Sanger para os genes BRCA1, BRCA2, TP53, APC, MUTYH, MLH1, MSH2 e STK11. A análise bioinformática dos resultados foi realizada com os softwares MiSeq Reporter, VariantStudio, Isaac Enrichment (Illumina) e Integrative Genomics Viewer (Broad Institute). A NGS demonstrou elevada sensibilidade e especificidade analíticas para a deteção de variantes de sequência em 8 genes de suscetibilidade para cancro colorretal e da mama, uma vez que permitiu identificar a totalidade das 412 variantes (93 únicas, incluindo 27 variantes patogénicas) previamente detetadas pelo método de Sanger. A utilização de painéis de sequenciação de genes de predisposição para cancro por NGS vem possibilitar um diagnóstico molecular mais abrangente, rápido e custo-eficiente, relativamente às metodologias convencionais.
- Effect of pomegranate peels and extract in barrier, optical and mechanical properties of polylactic acid-based active packagingPublication . Andrade, Mariana; Vilarinho, Fernanda; Rodrigues, Pedro; Barros, Carolina; Cruz, Vasco; Machado, Ana Vera; Barbosa, Cássia; Ramos, Fernando; Sanches Silva, AnaBeing more than 50 % of pomegranate (Punica granatum L.) constituted by non-edible parts, namely peels (50%) and seeds (10%), pomegranate is an excellent source of by-products. Its peels and seeds present excellent antioxidant and antimicrobial activities and a high content of phenolic compounds, namely ellagitannins. This work aimed to evaluate the mechanical and optical properties of two polylactic acid (PLA)-based active packaging with 3 wt.% pomegranate peels (3PP) or 3 wt.% pomegranate peel extract (3PPE). All the samples were produced on a laboratory scale with techniques and processing conditions used in industry. The production of packaging with flexible films is mainly carried out by tubular film extrusion. With this processing method it is possible to produce samples with molecular orientation and reduced thickness equal to that of the packages currently on the market. The structural and morphological characterization of the films were evaluated by FTIR and SEM, and the color by UV-vis. Water vapor transmission and mechanical properties were also measured. The color was measured by Shimadzu UV2401PC reflectance spectrophotometer. Water vapor transmission, oxygen permeability and mechanical properties were also measured. The FTIR and SEM results indicate the incorporation of the pomegranate peels and peels extract in the PLA matrix, where PLA/3PPE showed better particle homogenization than the PLA/3PP. Regarding the color variations, the PLA/3PPE presented higher variations in terms of L*, a*, and b*. The incorporation of pomegranate derivates has a negative effect on the tensile strength and Young modulus, but a significant increase of the elongation at break for PLA/3PPE. The PLA film's water vapor barrier properties do not suffer any alteration with the incorporation of pomegranate extract.
- Extending beef meat shelf life: an approach using active food packaging with pomegranate extract and peelsPublication . Andrade, Mariana; Rodrigues, Pedro; Barros, Carolina; Cruz, Vasco; Machado, Ana; Barbosa, Cássia; Coelho, Anabela; Furtado, Rosália; Correia, Cristina; Saraiva, Margarida; Vilarinho, Fernanda; Ramos, Fernando; Sanches Silva, AnaMore than 50% of the pomegranate fruit (Punica granatum L.) is not considered edible, being composed by peels (50%) and seeds (10%). Pomegranate peels represent an excellent source of bioactive compounds with excellent antioxidant and antimicrobial activities due to the peels’ high content in phenolic compounds, specially punicalagin (A+B) and ellagic acid. The aim of the present study was to evaluate the potential effects of a polylactic acid (PLA) based active packaging incorporated with pomegranate peels and an ethanolic extract from pomegranate peels in the shelf-life extension of fresh meat. Two PLA-based active packaging incorporating 3% (w/w) of pomegranate peels (PLA/3PP) and 3% (w/w) of pomegranate peels extract (PLA/3PPE) were used to pack beef meat. The samples were stored at 4 °C, for a maximum of 11 days. The meats’ lipid oxidation and microbial contamination was evaluated at the end of 1, 4, 6, 8 and 11 storage days. Lipid oxidation was measured by the Thiobarbituric Acid Reactive Substances assay (TBARS)[1]. Meat’s total microorganisms count at 30 °C was performed using the automated method TEMPO® Aerobic Count- AFNOR BIO 12/35–05/13. Both PLA active packaging significantly delayed meat’s lipid oxidation and microbial growth. The meat with PLA/3PP and PLA/3PPE presented a difference up to 3 log CFU/g on the 11th storage day. Even though both active packaging were able to inhibit meat’s lipid oxidation, these results showed that PLA/3PP was more efficient, providing insights for the use of this bio-waste towards environmental sustainability.
- Genetic testing for germline variants in homologous recombination repair genes, other than BRCA1 and BRCA2, in patients with suspected hereditary cancer syndromesPublication . Arnaut, Daniela; Rodrigues, Pedro; Theisen, Patrícia; Carpinteiro, Dina; Vieira, Luís; Gonçalves, JoãoHomologous recombination repair (HRR) is the cellular mechanism for error-free repair of DNA double-strand breaks. Pathogenic germline variants in BRCA1 and BRCA2 lead to HRR deficiency associated with breast, ovarian, prostate, pancreatic cancers and are sensitive to PARP inhibitors (PARPi). Defects in HRR genes beyond BRCA1/2 could also result in HRR deficiency and sensitize the tumor to PARPi, thus expanding the subset of patients that can benefit from these targeted therapy cancer drugs. We studied 56 DNA samples obtained from patients with personal and family history of cancer. Genes involved in HRR (ATM, BAP1, BLM, BRIP1, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, NBN, PALB2, RAD51C, RAD51D) were analysed by NGS using TruSight® Hereditary Cancer. Sequence alignment and annotation included DRAGEN Enrichment and Variant Interpreter - Illumina®. Variant classification, according to ACMG-AMP 1, was based on VEP, HSF, Alamut, VarSome and several databases (ex. HGMD, gnomAD, dbSNP). Variants of uncertain significance (VUS) were also classified with the stepwise ABC system2. All pathogenic/likely pathogenic SNVs and CNVs were confirmed by Sanger sequencing or MLPA. We identified 156 SNVs and one CNV, of these 125 were benign/likely benign. Seven clinically actionable variants were found in 10.7% of the patients: 3 pathogenic variants in FANCA, FANCD2 and FANCI give rise to premature stop codons and one pathogenic CNV in FANCA (deletion of exons 38 and 39); 2 likely pathogenic variants in BLM and FANCI affecting splicing and one frameshift in FANCG. Classification of 18 VUS with the ABC system resulted in: 8 class 0 (normal finding), 7 class E (potential interest) and 3 class D (low penetrance) variants. In addition, 7 SNVs were classified as hypomorphic alleles. This study confirmed: i) the importance of extending the molecular study beyond BRCA1/2 to other genes involved in HRR, ii) some variants require functional/family studies to establish their pathogenicity, and iii) these genes could potentially be considered for specific and clinical studies involving PARPi therapy.
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