NGS Panels applied to Hereditary Cancer Syndromes

Cancer is among the leading causes of morbidity and mortality worldwide (Okur et al, 2017). Germline pathogenic variants for monogenic, highly penetrant cancer susceptibility genes are observed in 5%–10% of all cancers (Lu et al, 2014). Hereditary cancers due to monogenic causes are characterized by earlier age of onset, other associated cancers, and often a family history of specific cancers. From the clinical perspective, it is important to recognize the affected individuals to provide them the best clinical management (Hennessy et al, 2010; Ledermann et al, 2014; Pennington et al, 2014) and to identify at-risk family members who will benefit from predictive genetic testing and enhanced surveillance, including early detection and/or risk reduction measures (Kurian et al, 2010; Okur et al, 2017). Germline variants identified in major cancer susceptibility genes associated with hereditary breast or ovarian cancer (HBOC) or hereditary colorectal cancer (HCRC), also account for 5-10% of the patients with these cancers. In the last years, new susceptibility genes, with different penetrance degrees, have been identified. Variants in any of those genes are rare and classical methodologies (e.g. Sanger sequencing - SS) are time consuming and expensive. Next-generation sequencing (NGS) has several advantages compared to SS, including the simultaneous analysis of many samples and sequencing of a large set of genes, higher sensitivity (down to 1% vs 15-20% in SS), lower cost and faster turnaround time, reasons that make NGS the best approach for molecular diagnosis. It is possible nowadays to choose between whole-genome sequencing (WGS), whole-exome sequencing (WES) and NGS limited to a set of genes (NGS-Panel). In cases where a suspected genetic disease or condition has been identified, targeted sequencing of specific genes or genomic regions is preferred (Grada et al, 2013). For that reason, we use NGS-Panel approach using TruSight Cancer (Illumina) to sequence DNA extracted from blood samples of patients with personal and/or familiar history of cancer. This hereditary cancer gene panel sequences 94 genes associated with both common (e.g., breast, colorectal) and rare hereditary cancers and allows the creation of virtual gene panels according to each phenotype or disease under study. NGS workflow analysis (Figure 1) includes five steps: quality assessment of raw data, read alignment to a reference genome, variant identification/calling, variant annotation and data visualization (Pabinger et al, 2013). The establishment of the most appropriate bioinformatics pipeline is crucial in order to achieve the best results. NGS data allows the identification of several types of variants like single nucleotide variants (SNVs), small insertions/deletions, inversions and also copy number variants (CNVs).FCT - UID/BIM/0009/2016info:eu-repo/semantics/publishedVersio

Presumed TP53 mosaicism: variants detected using a NGS hereditary cancer multigene panel

Aims/Context: NGS multigene panels are routinely used to identify germline pathogenic variants in cancer susceptibility genes. In addition, NGS allows the identification of low-level mosaicism events that may not be detectable by conventional Sanger sequencing. We describe two cases of presumed TP53 mosaic variants detected by NGS on blood-derived DNA, and confirmed by ARMS-PCR and Sanger sequencing. Case 1: female, 87 years old, colon cancer at 83 and metachronous breast cancer at 86, no history of familial cancer. Case 2: female, 75 years old, ovarian cancer at 71, local relapse at 74. Methods: NGS using TruSight® Cancer Sequencing Panel and TruSight® Rapid Capture kit (Illumina) and paired-end sequencing on MiSeq® platform (Illumina). Bioinformatic analysis with MiSeq Reporter, Enrichment, VariantStudio, VEP, Alamut Visual, VarAFT, VarSome and IGV. ARMS-PCR and Sanger sequencing were used to confirm the TP53 variants. Results and Conclusions: Two cases of presumed TP53 mosaic variants were studied. Case 1: the missense alteration TP53: c.764T>G, p.(Ile255Ser) was detected with a variant allele frequency (VAF) of 26% (39/150 reads). This variant is described in ClinVar as a somatic alteration, classified as likely pathogenic. It is not reported in gnomAD and VarSome software classified it as a variant of uncertain significance. Case 2: missense variant TP53: c.524G>A, p.(Arg175His) detected with a VAF of 15% (10/58 reads). This variant is described as pathogenic in HGMD Professional, LOVD and ClinVar, in association with Li-Fraumeni syndrome. These two cases seem to represent TP53 mosaicism, supported by: i) VAF lower than 30%, ii) detection at the sensitivity limit of Sanger sequencing and iii) confirmation by ARMS-PCR. Confirming this hypothesis by studying tumor and other tissue samples and offspring analysis (underway in both cases), is essential for disease diagnosis, assessing recurrence risk and genetic counseling. The hypothesis of acquired aberrant clonal expansion limited to the hematologic compartment, versus a germline variant should be considered in similar cases, and confirmatory methodologies are mandatory.info:eu-repo/semantics/publishedVersio

Concordance between variants detected by clinical exome, gene panel and Sanger sequencing

Introduction: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.N/

Importância do estudo multigénico no diagnóstico molecular de doenças raras por sequenciação de nova geração

Introdução: A sequenciação de nova geração (NGS) revolucionou o diagnóstico molecular das doenças raras (DR) proporcionando a análise de um maior número de genes, resultados mais rápidos e custos reduzidos. A NGS usando diferentes abordagens, possibilita o estudo do genoma (WGS), do exoma (WES), de genes individuais ou de painéis multigénicos (NGS-PMG). Objetivos: Aplicação de NGS-PMG no estudo de doenças raras específicas. Métodos: Preparação de bibliotecas de sequências-alvo a partir de DNA genómico, utilizando a captura com sondas (Trusight Cancer-Rapid Capture, Illumina) ou amplicões (painéis customizados - AmpliSeq, Illumina). Sequenciação no MiSeq (Illumina) e análise bioinformática usando software da Illumina e programas disponíveis on line. Validação de alterações pontuais por sequenciação de Sanger. Resultados: A utilização da metodologia Trusight-Cancer, que inclui a sequenciação de 94 genes de suscetibilidade para cancro hereditário (CH), permite-nos a análise seletiva de PMG, aplicados ao estudo de por ex., síndrome de Lynch, Polipose Adenomatose Familiar, cancro Hereditário da Mama e Ovário). A aplicação de PMG por AmpliSeq a um painel de genes desenhado in silico e específico de patologias do desenvolvimento sexual (PDS), permite-nos a análise molecular de 40 genes associados a hipogonadismo, Síndrome de Kallmann, insensibilidade aos androgénios, androgenização precoce, ciliopatias, falância ovárica prematura. Os resultados obtidos (sem falsos negativos), permitiram detetar diferentes tipos de alterações moleculares em doentes com diferentes DR, proporcionando em muitos um diagnóstico definitivo. Destaca-se que a estratégia de NGS implementada para o CH, integra também a avaliação de variações do número de cópias (deleções e duplicações) e a validação destes resultados por metodologia diferente - MLPA. Conclusões: A utilização de NGS-PMG possibilitando a identificação de novas variantes em novos genes, permite confirmar o diagnóstico clínico e contribuir para um melhor acompanhamento dos doentes e prevenção da doença genética hereditária. No caso de doenças cujo fenótipo não permite inferir um PMG a analisar, o alargamento ao WES ou ao WGS, para além de poder contribuir para o diagnóstico, gerará novo conhecimento e eventualmente o desenvolvimento de terapias inovadores.Trabalho parcialmente financiado pela Fundação para a Ciência e a Tecnologia, Projeto: UID/BIM/0009/2016info:eu-repo/semantics/publishedVersio

Genetic testing for germline variants in homologous recombination repair genes, other than BRCA1 and BRCA2, in patients with suspected hereditary cancer syndromes

Homologous 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.Funding: FCT/MCTES, Projects - ToxOmics and Human Health (UIDB/00009/2020) and GenomePT (POCI-01-0145-FEDER-022184).info:eu-repo/semantics/publishedVersio

ABC system used as an add-on to clarify germline variants previously classified as VUS according to ACMG guidelines

The 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).FCT/MCTES, ToxOmics and Human Health (UIDB/00009/2020). GenomePT(POCI-01-0145-FEDER-022184).info:eu-repo/semantics/publishedVersio

Validação e implementação do diagnóstico molecular em cancro coloretal hereditário (ccrh) por sequenciação de nova geração

A análise molecular tradicional na síndrome de Lynch, associada a alterações nos genes MLH1, MSH2, MSH6, PMS2 e EPCAM, na polipose adenomatosa familiar (FAP), causada por alterações no gene APC e na polipose associada a alterações no gene MUTYH, é baseada na PCR e na sequenciação pelo método de Sanger, tem custos elevados e é demorada, pelo que é relevante a sua substituição pela sequenciação de nova geração (NGS) permitindo esta, a análise simultânea de múltiplos genes, com custos mais reduzidos e num menor intervalo de tempo. Tendo por objetivo disponibilizar a análise molecular por NGS para CCRH, procedeu-se à sua validação para os genes MLH1, MSH2, APC, MUTYH, STK11, em 26 amostras, previamente analisados pelo método de Sanger. Utilizou-se a tecnologia da Illumina que compreende o Trusight Cancer Sequencing Panel (que possibilita a análise de 94 genes), o kit TruSight Rapid Capture, o sequenciador MiSeq e a respetiva análise bioinformática com os softwares apropriados. Foram detetadas por NGS 78 alterações nos genes anteriormente referidos, 32 alterações correspondem a variantes únicas [5 deleções de tamanho variável (2 a 17 nucleótidos), uma inserção-deleção e 26 alterações envolvendo um único nucleótido], tendo todas as alterações sido previamente identificadas pelo método de Sanger. Os resultados obtidos demonstram a elevada sensibilidade e especificidade da NGS na deteção de alterações nos genes em causa permitindo, assim, disponibilizar a NGS para um painel alargado de genes de suscetibilidade para CCRH, permitindo um diagnóstico molecular mais abrangente, rápido e mais económico relativamente à sequenciação de Sanger

Validation of next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer

Introduction: Molecular diagnosis of hereditary breast and ovarian cancer (HBOC) has been mostly based on the identification of germline inactivating mutations in the high-penetrant genes BRCA1 and BRCA2. Although several other HBOC susceptibility genes have been identified, mutations in any of those are rare, rendering sequential genetic testing with standard methodologies time consuming and expensive. Next-generation sequencing (NGS) gene panels allow the simultaneous sequencing of multiple HBOC susceptibility genes at a lower cost. The aim of this work was to validate the use of an NGS cancer susceptibility gene panel for the identification of mutations previously detected by Sanger sequencing in the BRCA1, BRCA2 and TP53 genes. Methods: 20 samples from patients with personal/family history of breast cancer were sequenced on a MiSeq using the Trusight Cancer Sequencing Panel (Illumina). Bioinformatic analysis of NGS data included the MiSeq Reporter, VariantStudio and Isaac Enrichment tools (Illumina). Results: NGS successfully identified all 204 variants (38 unique, including 2 deletions and a splice variant) previously detected by Sanger sequencing in the BRCA1, BRCA2 and TP53 genes. Until now, no false-negative or false-positive results were obtained. Discussion: These results demonstrate the high analytical sensitivity and specificity obtained with NGS for the detection of sequence variants in 3 HBOC high-penetrant genes. These validation assays open the way to the definition of a clinically useful multigene panel for HBOC susceptibility based on the Trusight Cancer Sequencing Panel. This will allow a comprehensive and cost-effective molecular diagnosis of HBOC with a shorter turnaround time when compared to standard methodologies. In addition, with appropriate genetic counselling and specialized clinical surveillance, families with HBOC will benefit from these new technologies which have high impact in public health

Inherited Colorectal Cancer - validation of molecular diagnosis by Next Generation Sequencing

Introduction:Colorectal cancer is the third major cause of cancer related deaths worldwide. Around 5% of these cases are due to Inherited Colorectal Cancer (ICC) associated with highly penetrant single-gene mutations. Conventional molecular analysis of patients with ICC is well established and usually comprises PCR followed by Sanger sequencing of different genes with autosomal dominant inheritance - MLH1, MSH2, MSH6, PMS2 and EPCAM-3’ deletions in Lynch syndrome, APC in Familial Adenomatous Polyposis (FAP), or the study of an autosomal recessive condition with colorectal polyps associated with MUTYH variants. As standard molecular methodologies have high costs and are time consuming, they are progressively being replaced by Next Generation Sequencing (NGS), which allows the analysis of multiple genes simultaneously and with lower costs compared to Sanger sequencing. In order to validate NGS analysis for a set of genes associated with ICC, we performed NGS in 26 DNA samples from patients previously analysed by Sanger sequencing. Methods: NGS was performed using the Trusight Cancer Sequencing Panel and the MiSeq sequencer (Illumina), followed by bioinformatic analysis of the MLH1, MSH2, APC, MUTYH and STK11 genes using the MiSeq Reporter, VariantStudio and Isaac Enrichment tools. Results: Data analysis revealed 77 variants (31 unique, comprising 4 deletions, 1 insertion, 2 indels and 24 single nucleotide variants). Of these, 76 variants were previously identified by Sanger sequencing. NGS produced a false positive result associated with low coverage in STK11 (c.375-49G>A). Discussion: Results obtained by NGS are consistent with Sanger sequencing and showed high analytical sensitivity and specificity. Therefore after this initial validation, with high repeatability, conventional molecular analysis can be replaced by NGS, allowing us to offer the possibility to screen more genes, at lower costs and with a shorter turnaround time

Validação da sequenciação de nova geração (NGS) no diagnóstico molecular de formas hereditárias de cancro da mama e colorretal

Introdução: O cancro da mama e o cancro colorretal constituem, entre as patologias oncológicas, duas das principais causas de morte. Cinco a 10% destes casos estão associados a alterações germinais em genes reconhecidamente associados a suscetibilidade para desenvolvimento de formas hereditárias de cancro da mama e de cancro colorretal. Objetivos: O presente trabalho teve como objetivo validar a metodologia de sequenciação de nova geração (NGS), por comparação com os resultados obtidos previamente pelo método de Sanger, para diversas variantes presentes em diferentes genes - BRCA1, BRCA2, TP53, APC, MUTYH, MLH1, MSH2 e STK11 - que conferem suscetibilidade para desenvolvimento de cancro da mama e/ou colorretal. Métodos: 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 (análise de 94 genes) e a plataforma MiSeq (Illumina). A análise bioinformática dos resultados foi realizada com recurso aos softwares MiSeq Reporter, VariantStudio e Isaac Enrichment (Illumina).N/