Browsing by Author "Silva, Catarina"
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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).
- Accreditation under the International Standard ISO 15189: Experience of a Genetics Laboratory in DNA SequencingPublication . Silva, Catarina; Cardoso, Ana; A Sampaio, Daniel; Carpinteiro, Dina; Mendonça, Joana; Duarte, Sílvia; Barreiro, Paula; Torgal, Helena; Isidro, Glória; Vieira, LuísIntroduction: Health care is to some extent influenced by the results of laboratory tests. In order to provide the best care for the patient, laboratories must seek to achieve high levels of quality and competence. International Standard ISO 15189 specifies these requirements and may be used by laboratories to perform accredited genetic tests of materials derived from the human body. Here we describe the procedures to establish Accreditation of DNA sequencing in our laboratory and the first Accreditation of its kind in Portugal. Methods: Our laboratory started to prepare to comply with ISO 15189 Accreditation requirements for DNA sequencing in 2010. Documents describing administrative and technical procedures of the sequencing workflow including sample registries, laboratory protocols, operation and maintenance of equipments, as well as preparation and use of reagents were produced. Regular examination of laboratory equipments by an external entity was implemented to confirm compliance with working requirements. Requisites for personnel training and demonstration of competence were also implemented. The laboratory participated regularly in the DNA sequencing scheme organized by the European Molecular Genetics Quality Network (EMQN). Results: The laboratory obtained formal recognition by Instituto Português de Acreditação (IPAC) in May 2014. A maximum genotyping score for DNA sequencing has been obtained in the external quality assessment scheme since 2010. Sequencing quality measured in terms of the quality read overlap metrics is currently of approximately 96% according to the EMQN scheme. The laboratory processes and analyzes an average of 28.750 samples per year. Discussion: Accreditation of a genetic test under ISO 15189 is a highly demanding and laborious task for a genetic laboratory. However, it is an important step in order to guarantee the highest quality and reproducibility of genetic test results.
- 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.
- Avaliação do desempenho de uma core-facility de sequenciação genómica especializada em saúde públicaPublication . Vieira, Luís; Silva, Catarina; Duarte, Sílvia; Mendonça, Joana; Carpinteiro, Dina; Sampaio, Daniel A.; Ferrão, José; Santos, Daniela; Machado, Miguel; Isidro, Joana; Barreiro, Paula; Isidro, GlóriaA Unidade de Tecnologia e Inovação (UTI) do Departamento de Genética Humana foi criada em 2009 pelo despacho normativo n.º 15/2009. Apesar de estar integrada num departamento técnico científico, esta unidade constituiu-se desde logo como core-facility de sequenciação genómica do Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA). Este papel envolve uma gestão contínua de prioridades dos serviços a prestar aos utilizadores, no âmbito da resposta a diferentes problemas de saúde pública, aliada a uma preocupação permanente com a qualidade dos resultados e os tempos de resposta. Neste trabalho, apresentamos os resultados da avaliação do desempenho da UTI, desde a introdução da tecnologia de Next-Generation Sequencing (NGS) em 2013, em termos de: (i) métricas de produção da Unidade, (ii) impacto dos resultados publicados no âmbito de colaborações científicas com os grupos de investigação do INSA ou de entidades externas e de (iii) avaliação dos serviços através de um inquérito dirigido aos utilizadores. Até final de 2021, o número de ensaios de NGS e de citações dos trabalhos publicados cresceram, por ano, 39% e 61%, respetivamente. Os utilizadores avaliaram de forma muito positiva os serviços prestados pela UTI em 2021. Globalmente, estes resultados demonstram que o modelo de trabalho de "core- -facility" exercido pela UTI é uma mais-valia na resposta aos problemas da saúde pública em Portugal.
- Base molecular da hemocromatose hereditária não-clássica em PortugalPublication . Faria, Ricardo; Silva, Bruno; Silva, Catarina; Loureiro, Pedro; Queiroz, Ana; Esteves, Jorge; Mendes, Diana; Fleming, Rita; Vieira, Luís; Gonçalves, João; Lavinha, João; Faustino, PaulaA Hemocromatose Hereditária (HH) é uma doença autossómica recessiva caracterizada pela absorção excessiva de ferro a nível intestinal e sua acumulação em órgãos vitais, podendo originar cardiomiopatia, cirrose e carcinoma hepatocelular. O correspondente diagnóstico molecular é obtido pela associação com genótipos específicos no gene HFE (homozigotia para p.Cys282Tyr ou heterozigotia composta p.Cys282Tyr/p.His63Asp). Contudo, nos países do sul da Europa, cerca de um terço dos doentes com diagnóstico clínico de HH não apresenta os referidos genótipos. Para identificar a base molecular da HH não-clássica em Portugal usaram-se metodologias de pesquisa geral de variantes genéticas (SSCP e dHPLC), Next-Generation Sequencing (NGS) e sequenciação de Sanger, cobrindo seis genes relacionados com o metabolismo do ferro em 303 doentes. Identificaram-se 69 variantes diferentes e de vários tipos, por ex. missense, nonsense, de splicing, que perturbam a transcrição do gene ou a regulação da tradução do mRNA. Seguidamente, realizaram-se estudos in silico e in vitro para esclarecer o significado etiológico de algumas das novas variantes. Concluiu-se que a base molecular desta patologia é bastante heterogénea e que a NGS é uma ferramenta adequada para efetuar a análise simultânea dos vários genes num grande número de amostras. Contudo, o estabelecimento da relevância clínica de algumas variantes requer a realização de estudos funcionais.
- 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.
- 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.
- Concordance between variants detected by clinical exome, gene panel and Sanger sequencingPublication . Mendonça, Joana; Silva, Catarina; Theisen, Patrícia; Gonçalves, João; Vieira, LuísIntroduction:Exome sequencing (ES) is becoming a preferred methodology for detecting DNA changes in genetic diseases with no known molecular cause or no definitive diagnosis. This results from the fact that next-generation sequencing technology allows a greater number of bases to be sequenced at an increasingly lower cost. However, sequencing a high number of genes requires an evaluation of the analytical performance of ES before it is used in the clinical setting. Methods: Fifteen genomic DNA samples were used to prepare sequencing libraries with the TruSight One Sequencing Panel (Illumina) consisting of 4813 disease-associated genes ('clinical exome'), according to the manufacturer's procedures. Libraries were sequenced on the MiSeq (Illumina) and the results were analyzed using the MiSeq Reporter and IGV. Variants identified in ES were compared with those validated previously in a subset of genes using the TruSight Cancer gene panel (Illumina) and Sanger sequencing. This study was conducted in 2 phases. In the first, the clinical exome of 9 samples was sequenced and the variants obtained were compared with known variants in 8 genes. In the second phase, 6 samples were sequenced and the variants in 8 genes were analyzed without prior knowledge of the results obtained in the other methods. Furthermore, it was not known that one of these samples had been sequenced in the first phase of the study. Results: In the first phase, ES identified all the exonic (n=41) and intronic flanking (n=15) variants validated in the MSH2, MLH1, APC, MUTYH, BRCA1, BRCA2, STK11 and TP53 genes, while no additional changes have been detected. In the second phase, ES detected a total of 50 variants in MSH2, MLH1, APC, BRCA1, BRCA2, TP53, CDH1 and ATM genes which were found to include each of the 46 variants previously validated and 4 additional changes located outside the genomic regions defined in the gene panel. The same 15 exonic variants were identified in the sample independently processed and sequenced in both phases. Taken together, 87 variants were independently identified using different sequencing approaches. Discussion: The results of this work showed a complete agreement between variants identified by clinical exome, gene panel and Sanger sequencing. Moreover, these results support the notion that the clinical exome panel can also be used as a set of sub-panels of genes applicable to different genetic diseases.
- Detection of copy number variants in the human genome: Is long-read sequencing an alternative to genomic microarrays?Publication . Silva, Catarina; Ferrão, José; Marques, Barbara; Pedro, Sónia; Correia, Hildeberto; Rodrigues, António Sebastião; Vieira, LuísIntroduction: Copy number variations (CNVs) represent ~13% of the human genome and can harbour important genes and regulatory elements. High-resolution whole genome microarray (MA) analysis is the gold standard tool for detection of CNVs associated with genetic disorders. While short-read sequencing (SRS) can address SV detection, the use of long-read sequencing as proven to overcome SRS mapping inaccuracy in highly repetitive DNA regions and improve genome contiguity. We applied whole genome nanopore sequencing (NS) to call CNVs and compared the results with those obtained by microarray. Methodology: Genomic DNA from 2 cell lines (EOL-1 and 697) were processed using the CytoSan HD Array (Affymetrix) and ChAS software (ThermoFisher). A minimum CNV calling size threshold of 35 Kb was used. DNA was also sequenced on the MinION device (Oxford Nanopore Technologies) following a rapid library preparation method. Sequencing data were basecalled using Guppy, mapped with LRA, and SVs called using both CuteSV and Sniffles2. Sanger sequencing was performed to demonstrate breakpoint positions for 3 CNVs. R packages were used to perform comparisons between MA and NS data. Results: A total of 49 CNVs were confirmed after curated MA analysis in both cell lines, ranging in size from 35 Kb to 79 Mb. From those, 43 CNVs (87.7%) were called in nanopore data by either one (4 CNVs) or both (39 CNVs) callers with a mean whole genome coverage of ~12X. Six of 43 CNVs were called as inversions instead. In 3 CNVs the size of the variant was found to be smaller (ranging from ~5 to 22 Kb) than the threshold of MA analysis. The correlation between CNV sizes obtained with MA and NS was of 0.71 with Sniffles2 and 0.74 with CuteSV, whereas the correlation between callers was of 0.99. The breakpoint precision obtained for NS was much higher (ranging for CuteSV from 2 to 42 bp; and for Sniffles2 from 0 to 87 bp) than the one obtained for MA (ranging from 774 to 7618 bp). Conclusions: NS technology proved to be technically effective in the detection of CNVs of different types and sizes and thus posing itself as an alternative to MA in the detection of pathogenic SVs associated with genetic diseases. However, NS data analysis requires fine-tuning of the analysis conditions as well as the use of different methods, for greater reliability of results in a clinical context.