Interpretation and actionability of genetic variants in cardiomyopathies: a position statement from the European Society of Cardiology Council on cardiovascular genomics

This document describes the contribution of clinical criteria to the interpretation of genetic variants using heritable Mendelian cardiomyopathies as an example. The aim is to assist cardiologists in defining the clinical contribution to a genetic diagnosis and the interpretation of molecular genetic reports. The identification of a genetic variant of unknown or uncertain significance is a limitation of genetic testing, but current guidelines for the interpretation of genetic variants include essential contributions from clinical family screening that can establish a de novo assignment of the variant or its segregation with the phenotype in the family. A partnership between clinicians and patients helps to solve major uncertainties and provides reliable and clinically actionable information

HELPER: UNA PIATTAFORMA BIOINFORMATICA PER LA PERSONALIZZAZIONE DELLE PIPELINE NGS

Le tecnologie NGS hanno rivoluzionato il mondo della genetica e della medicina, influenzando fortemente la diagnosi delle malattie ereditarie. Il grande numero di applicazioni, sia di diagnostica che di ricerca, ha generato la necessità di adattare l’analisi dei dati prodotti da queste tecnologie per ottimizzare la risposta ai problemi specifici. Il processo di analisi è implementato tramite trasformazioni consecutive dei dati genetici (pipeline) utilizzando un grande numero di tool e software bioinformatici. Spesso le performance dei diversi tool dipendono dal tipo dei dati in ingresso e l’integrazione dei software adatti ai diversi tipi di dati è diventato un passaggio critico per la qualità delle informazioni prodotte. Inoltre, l’utilizzo dei tool, la loro configurazione, la progettazione di pipeline robuste e lo sviluppo di nuove soluzioni di analisi, sono processi complessi che richiedono competenze di coding e la conoscenza dell’esteso panorama bioinformatico. I laboratori che non dispongono di personale specializzato in applicazioni bioinformatiche, possono incontrare difficoltà nell’ottimizzazione del workflow analitico, che spesso viene affidato a software commerciali che applicano uguali regole e sistemi a tutti i geni indistintamente. Durante il percorso di dottorato di ricerca effettuato presso il Centro malattie genetiche cardiovascolari dell’ospedale San Matteo di Pavia, è stata sviluppata la piattaforma Helper. Helper è nata per la progettazione e l’adattamento semplificato delle pipeline bioinformatiche dedicate all’analisi di dati NGS derivati da applicazioni di targeted sequencing. Helper è dotato di una semplice interfaccia grafica mirata a facilitare l’esperienza di sviluppo dei processi analitici bioinformatici anche per chi non possiede particolari conoscenze di sviluppo di codice. Tramite Helper è possibile scegliere quali step effettuare nel workflow di analisi e quali evitare, quali tools e software utilizzare in ogni step selezionato, e con quali argomenti settare i tool utilizzati. Helper permette inoltre di utilizzare le pipeline, progettate ed effettuare l’analisi dei dati NGS, ed è modificabile in base all’esperimento di sequenziamento dal quale derivano i campioni e in base al tipo e all’organizzazione dei campioni. Helper può essere utilizzato sia su workstation, sia su un comune PC, dimostrandosi compatibile con i tempi di analisi dei laboratori di genetica anche in presenza di soluzioni a bassa capacità computazionale. Nel workflow di analisi genetica, Helper è dedicato a quella che è definita come analisi secondaria, che trasforma i dati NGS grezzi in un set di varianti utili all’interpretazione del test genetico. Il lavoro di tesi si è proposto inoltre di introdurre due questioni fondamentali per la diagnosi genetica. La prima è rappresentata dal problema della classificazione patogenica delle varianti identificate dall’analisi bioinformatica. La classificazione delle varianti è un processo delicato a causa della difficoltà esistenti nel trovare regole uniformi e robuste da applicare a tutti i difetti genici. In questa tesi viene proposto un sistema di classificazione per le varianti del gene DES, che prende in considerazione le caratteristiche specifiche del gene che codifica per la proteina di Desmina. Il secondo è l’identificazione dei geni responsabili di un determinato fenotipo, necessaria per l’ottimizzazione del test diagnostico e per la gestione dei pazienti. In questo contesto viene approfondito il problema dei tumori ereditari della mammella e dell’ovaio, tramite lo studio dei risultati di analisi del database genetico sviluppato presso il San Matteo per il monitoraggio delle cause genetiche delle patologie oncologiche.Next generation sequencing (NGS) technologies have revolutionized the world of genetics and medicine, strongly influencing the diagnosis of hereditary diseases. The large number of applications, both diagnosis, and research, has generated the need to adapt the analysis of the data produced by these technologies to optimize the clinical path of many human diseases. The analysis process is implemented through consecutive modifications of the genetic data (pipeline) using bioinformatics tools and software. Often, the performance of the different tools depends on the type of input data; the integration of software suitable for different types of data is a critical step for the quality of the information produced. Furthermore, the use of bioinformatics tools, their configuration, the design of robust pipelines, and the development of new analysis solutions is a complex process that requires coding skills and knowledge of the wide range of existing tools. In this context, bioinformaticians achieved a key role within genetics laboratories, thanks to the skills of developing computer systems combined with the integration of knowledge on target biology systems and related applications; these “in house” tailored activities favor the adaptation of the analyses to each specific questions/objectives. Laboratories using outsourcing analysis tools or entrusting to commercial software that apply the same rules and systems to all genes, without distinction, often face difficult optimization of the analytical workflow. Hence, the growing need for simple and fast tools that can support professionals with limited computer skills in the design of customized pipelines and their use to analyze NGS data. During the PhD course carried out at the Center for Cardiovascular Genetic Diseases of the San Matteo Hospital in Pavia, the Helper platform was developed. Helper was born for the design and simplified adaptation of bioinformatics pipelines for the analysis of NGS data derived from targeted sequencing applications. Helper is equipped with a simple graphic interface aimed at facilitating the development experience of bioinformatics analytical processes even for professionals who do not have coding knowledge. Helper allows the selection of: the steps to carry out (or to avoid) in the analysis workflow; the tools and software to use in each selected step; the arguments to set the tools employed in each application. Helper further allows the use of the pipelines, the design and carrying out of the analysis of NGS data; it can be modified based on the sequencing experiment from which the samples are derived, and on the basis of the organization of the samples. Helper can be used both on a workstation and on a common PC, proving to be compatible with the analysis times of the genetics laboratories even in the presence of solutions with low computational capacity. In the genetic analysis workflow, Helper is part of the process of translating raw NGS data into a set of variants useful for the interpretation of the genetic test. The thesis finally aimed at addressing two fundamental questions for genetic diagnosis. The first question addresses the complex issue of the variant classification as identified by bioinformatics analysis. The classification of genetic variants is a process that reflects difficulties in finding uniform and robust rules shared by all genes. In this thesis, a classification system is proposed for the variants of the DES gene, which takes into consideration the specific characteristics of the gene encoding the Desmin protein. The second question addressed the identification of the genes responsible for a specific phenotype, necessary for the optimization of the diagnostic test and for patient management. In this context, hereditary breast and ovarian tumors is investigated through the study of the results of the analysis of the genetic database developed at San Matteo for monitoring the genetic basis of oncological diseases

Macrophages and Monocytes: “Trojan Horses” in COVID-19

We aimed to explore whether variants of SARS-CoV-2 (Chinese-derived strain (D614, lineage A), Italian strain PV10734 (D614G, lineage B.1.1) and Alpha strain (lineage B.1.1.7)) were able to infect monocytes (MN) and monocyte-derived macrophages (MDM) and whether these infected cells may, in turn, be vectors of infection. For this purpose, we designed an in vitro study following the evolution of MN and MDM infection at different time points in order to confirm whether these cells were permissive for SARS-CoV-2 replication. Finally, we investigated whether, regardless of viral replication, the persistent virus can be transferred to non-infected cells permissive for viral replication. Thus, we co-cultured the infected MN/MDM with permissive VERO E6 cells verifying the viral transmission. This is a further in vitro demonstration of the important role of MN and MDM in the dissemination of SARS-CoV-2 and evolution of the COVID-19 disease

Pathologic Findings at Risk Reducing Surgery in <i>BRCA</i> and Non-<i>BRCA</i> Mutation Carriers: A Single-Center Experience

Risk-reducing surgery (RRS) is recommended in BRCA-mutated carriers because of their increased risk of developing ovarian cancer, while its role is still discussed for women harboring mutations in non-BRCA homologous repair genes. The aim of this study was to retrospectively evaluate the occurrence of pathological findings in a high-risk population undergoing RRS in San Matteo Hospital, Pavia between 2012 and 2022, and correlate their genetic and clinical outcomes, comparing them with a control group. The final cohort of 190 patients included 85 BRCA1, 63 BRCA2, 11 CHEK2, 7 PALB2, 4 ATM, 1 ERCC5, 1 RAD51C, 1 CDH1, 1 MEN1, 1 MLH1 gene mutation carriers and 15 patients with no known mutation but with strong familial risk. Occult invasive serous carcinoma (HGSC) and serous tubal intraepithelial carcinoma (STIC) were diagnosed in 12 (6.3%) women, all of them BRCA carriers. No neoplastic lesion was diagnosed in the non-BRCA group, in women with familial risk, or in the control group. Oral contraceptive use and age ≤45 at surgery were both found to be favorable factors. While p53 signature and serous tubal intraepithelial lesion (STIL) were also seen in the control group and in non-BRCA carriers, STIC and HGSC were only found in BRCA1/2 mutation carriers

Interpretation and actionability of genetic variants in cardiomyopathies: a position statement from the European Society of Cardiology Council on cardiovascular genomics

This document describes the contribution of clinical criteria to the interpretation of genetic variants using heritable Mendelian cardiomyopathies as an example. The aim is to assist cardiologists in defining the clinical contribution to a genetic diagnosis and the interpretation of molecular genetic reports. The identification of a genetic variant of unknown or uncertain significance is a limitation of genetic testing, but current guidelines for the interpretation of genetic variants include essential contributions from clinical family screening that can establish a de novo assignment of the variant or its segregation with the phenotype in the family. A partnership between clinicians and patients helps to solve major uncertainties and provides reliable and clinically actionable information

Interpretation and actionability of genetic variants in cardiomyopathies: a position statement from the European Society of Cardiology Council on cardiovascular genomics

This document describes the contribution of clinical criteria to the interpretation of genetic variants using heritable Mendelian cardiomyopathies as an example. The aim is to assist cardiologists in defining the clinical contribution to a genetic diagnosis and the interpretation of molecular genetic reports. The identification of a genetic variant of unknown or uncertain significance is a limitation of genetic testing, but current guidelines for the interpretation of genetic variants include essential contributions from clinical family screening that can establish a de novo assignment of the variant or its segregation with the phenotype in the family. A partnership between clinicians and patients helps to solve major uncertainties and provides reliable and clinically actionable information

Prevalence and complications of aberrant subclavian artery in patients with heritable and nonheritable arteriopathies

Background: An aberrant subclavian artery (ASA) (or lusoria) is the most common congenital anomaly of the aortic arch (0.5%-2.2%; female-to-male ratio 2:1 to 3:1). ASA can become aneurysmal and result in dissection, involving Kommerell\u27s diverticulum when present and the aorta. Data of its significance in genetic arteriopathies are not available. Objectives: The purpose of this study was to assess the prevalence and complications of ASA in gene-positive and -negative nonatherosclerotic arteriopathies. Materials: The series includes 1,418 consecutive patients with gene-positive (n = 854) and gene-negative arteriopathies (n = 564) diagnosed as part of institutional work-up for nonatherosclerotic syndromic and nonsyndromic arteriopathies. Comprehensive evaluation includes genetic counseling, next-generation sequencing multigene testing, cardiovascular and multidisciplinary assessment, and whole-body computed tomography angiography. Results: ASA was found in 34 of 1,418 cases (2.4%), with a similar prevalence in gene-positive (n = 21 of 854, 2.5%) and gene-negative (n = 13 of 564, 2.3%) arteriopathies. Of the former 21 patients, 14 had Marfan syndrome, 5 had Loeys-Dietz syndrome, 1 had type-IV Ehlers-Danlos syndrome, and 1 had periventricular heterotopia type 1. ASA did not segregate with genetic defects. Dissection occurred in 5 of 21 patients with genetic arteriopathies (23.8%; 2 Marfan syndrome and 3 Loeys-Dietz syndrome), all with associated Kommerell\u27s diverticulum. No dissections occurred in gene-negative patients. At baseline, none of the 5 patients with ASA dissection fulfilled criteria for elective repair according to guidelines. Conclusions: The risk of complications of ASA is higher in patients with genetic arteriopathies and is difficult to predict. In these diseases, imaging of the supra-aortic trunks should enter baseline investigations. Determination of precise indications for repair can prevent unexpected acute events such as those described