Molecular genetic analyses in hereditary cardiac arrhythmia syndromes

The present thesis describes the value and application of NGS in genetic investigations of sudden unexpected death cases and inherited arrhythmia syndromes using targeted sequencing. It represents the conceptualization, establishment and optimization of an NGS workflow for challenging heterogeneous - forensic and diagnostic - sample material. Furthermore, this work illustrates the need of characterization experiments of variants with unknown functional relevance

"Re-evaluation of variants of uncertain significance in patients with hereditary arrhythmogenic disorders"

Abstract Background Genetic diagnostics support the diagnosis of hereditary arrhythmogenic diseases, but variants of uncertain significance (VUS) complicate matters, emphasising the need for regular reassessment. Our study aims to reanalyse rare variants in different genes in order to decrease VUS diagnoses and thus improve risk stratification and personalized treatment for patients with arrhythmogenic disorders. Methods Genomic DNA was analysed using Sanger sequencing and next-generation sequencing (NGS). The Data was evaluated using various databases and in silico prediction tools and classified according to current ACMG standards by two independent experts. Results We identified 53 VUS in 30 genes, of which 17 variants (32%) were reclassified. 13% each were downgraded to likely benign (LB) and benign (B) and 6% were upgraded to likely pathogenic (LP). Reclassifications mainly occurred among variants initially classified in 2017–2019, with rates ranging from 50 to 60%. Conclusion The results support the assumption that regular reclassification of VUS is important, as it provides new insights for genetic diagnostics, that benefit patients and guide therapeutic approach

Genetic analysis of sudden unexpected death cases: evaluation of library preparation methods to handle heterogeneous sample material

Over the past years, next-generation sequencing (NGS) technologies revolutionized the possibilities in a broad range of application areas. Also in the field of forensic genetics, NGS continuously gained in importance and attentiveness. A significant number of sudden cardiac deaths (SCD) in the young is due to heritable arrhythmia syndromes emphasizing the need of examining the genetic basis in these cases also with regard to the identification of relatives and/or patients being at risk. As a result, high-throughput methods became of increasing value in molecular autopsy investigations enabling the analysis of a broad spectrum of genes. Most standard protocols are optimized for high-quality samples and frequently not directly applicable to challenging forensic sample material. In the present study, we intended to examine a comprehensive gene panel associated with SCD and inherited arrhythmogenic disorders. We compared three different hybridization-based library preparation technologies in order to implement a suitable NGS workflow for heterogeneous, forensic as well as diagnostic sample material. The results obtained indicated, that the Illumina technologies Nextera DNA Flex and TruSeq were compatible with samples exhibiting varying levels of degradation. In comparison, the TruSight method also resulted in good sequencing data, but seemed to be more dependent on DNA integrity. The preparation protocols evaluated in our study are not restricted to molecular autopsy investigations and might be helpful for and transferrable to further forensic research applications

Post-mortem genetic investigation of cardiac disease–associated genes in sudden infant death syndrome (SIDS) cases

The sudden infant death syndrome (SIDS) is one of the leading causes of postneonatal infant death. It has been shown that there exists a complex relationship between SIDS and inherited cardiac disease. Next-generation sequencing and surveillance of cardiac channelopathy and cardiomyopathy genes represent an important tool for investigating the cause of death in SIDS cases. In the present study, targeted sequencing of 80 genes associated with genetic heart diseases in a cohort of 31 SIDS cases was performed. To determine the spectrum and prevalence of genetic heart disease associated mutations as a potential monogenic basis for SIDS, a stringent variant classification was applied and the percentage of rare (minor allele frequency ≤ 0.2%) and ultra-rare variants (minor allele frequency ≤ 0.005%) in these genes was assessed. With a minor allele frequency of ≤ 0.005%, about 20% of the SIDS cases exhibited a variant of uncertain significance (VUS), but in only 6% of these cases, gene variants proved to be “potentially informative.” The present study shows the importance of careful variant interpretation. Applying stringent criteria misinterpretations are avoided, as the results of genetic analyses may have an important impact of the family members involved

Clinical utility gene card for: Long-QT syndrome

Background/objectives!#!In the recently introduced GLIM diagnosis of malnutrition (Global Leadership Initiative on Malnutrition), details of how to classify inflammation as an etiologic criterion are lacking. This study aimed to determine at what level of serum C-reactive protein (CRP) the risk of low food intake increases in acutely ill older hospitalized patients.!##!Subjects/methods!#!A total of 377 patients, who were consecutively admitted to a geriatric acute care ward, were analyzed. Nutritional intake was determined using the food intake item of Nutritional Risk Screening and the plate diagram method and patients were grouped into three categories as >75%, 50-75% and ≤50% of requirements. CRP was analyzed according to standard procedures and patients were classified into different CRP groups as follows: 0.0-0.99 mg/dl, 1.0-1.99 mg/dl, 2.0-2.99 mg/dl, 3.0-4.99 mg/dl, 5.0-9.99 mg/dl and ≥10.0 mg/dl.!##!Results!#!Of the total population (mean age of 82.2 ± 6.6 years; 241 females), 82 (22%) had intake <50% of requirements and 126 (33%) demonstrated moderate to severe inflammation. Patients with food intake <50% of requirements had a significantly higher median CRP level compared to patients with food intake >75% of requirements (P < 0.001). The group with serum-CRP levels above 3.0 mg/dl had a markedly higher proportion of patients with low food intake; i.e., <50% and <75% of the requirements.!##!Conclusion!#!A serum-CRP of 3.0 mg/dl appears to be a reasonable threshold of acute inflammation leading to reduced food intake to serve as an orientation with regard to the inflammation criterion of the GLIM diagnosis in acutely ill older patients

Characterization of an N-terminal Na v 1.5 channel variant - a potential risk factor for arrhythmias and sudden death?

BACKGROUND Alterations in the SCN5A gene encoding the cardiac sodium channel Na1.5 have been linked to a number of arrhythmia syndromes and diseases including long-QT syndrome (LQTS), Brugada syndrome (BrS) and dilative cardiomyopathy (DCM), which may predispose to fatal arrhythmias and sudden death. We identified the heterozygous variant c.316A > G, p.(Ser106Gly) in a 35-year-old patient with survived cardiac arrest. In the present study, we aimed to investigate the functional impact of the variant to clarify the medical relevance. METHODS Mutant as well as wild type GFP tagged Na1.5 channels were expressed in HEK293 cells. We performed functional characterization experiments using patch-clamp technique. RESULTS Electrophysiological measurements indicated, that the detected missense variant alters Nav1.5 channel functionality leading to a gain-of-function effect. Cells expressing S106G channels show an increase in Na1.5 current over the entire voltage window. CONCLUSION The results support the assumption that the detected sequence aberration alters Na1.5 channel function and may predispose to cardiac arrhythmias and sudden cardiac death

Variant interpretation in molecular autopsy: a useful dilemma

Sudden cardiac death (SCD) in adolescents and young adults may be the first manifestation of an inherited arrhythmic syndrome. Thus identification of a genetic origin in sudden death cases deemed inconclusive after a comprehensive autopsy and may help to reduce the risk of lethal episodes in the remaining family. Using next-generation sequencing (NGS), a large number of variants of unknown significance (VUS) are detected. In the majority of cases, there is insufficient evidence of pathogenicity, representing a huge dilemma in current genetic investigations. Misinterpretation of such variants may lead to inaccurate genetic diagnoses and/or the adoption of unnecessary and/or inappropriate therapeutic approaches. In our study, we applied current (ACMG) recommendations for variant classification in post-mortem genetic screening of a cohort of 56 SCD victims. We identified a total 53 rare protein-altering variants (MAF < 0.2%) classified as VUS or worse. Twelve percent of the cases exhibited a clinically actionable variant (pathogenic, likely pathogenic or VUS - potentially pathogenic) that would warrant cascade genetic screening in relatives. Most of the variants detected by means of the post-mortem genetic investigations were VUS. Thus, genetic testing by itself might be fairly meaningless without supporting background data. This data reinforces the need for an experienced multidisciplinary team for obtaining reliable and accountable interpretations of variant significance for elucidating potential causes for SCDs in the young. This enables the early identification of relatives at risk or excludes family members as genetic carriers. Also, development of adequate forensic guidelines to enable appropriate interpretation of rare genetic variants is fundamental