Artificial intelligence-enhanced electrocardiography derived body mass index as a predictor of future cardiometabolic disease.

The electrocardiogram (ECG) can capture obesity-related cardiac changes. Artificial intelligence-enhanced ECG (AI-ECG) can identify subclinical disease. We trained an AI-ECG model to predict body mass index (BMI) from the ECG alone. Developed from 512,950 12-lead ECGs from the Beth Israel Deaconess Medical Center (BIDMC), a secondary care cohort, and validated on UK Biobank (UKB) (n = 42,386), the model achieved a Pearson correlation coefficient (r) of 0.65 and 0.62, and an R2 of 0.43 and 0.39 in the BIDMC cohort and UK Biobank, respectively for AI-ECG BMI vs. measured BMI. We found delta-BMI, the difference between measured BMI and AI-ECG-predicted BMI (AI-ECG-BMI), to be a biomarker of cardiometabolic health. The top tertile of delta-BMI showed increased risk of future cardiometabolic disease (BIDMC: HR 1.15, p < 0.001; UKB: HR 1.58, p < 0.001) and diabetes mellitus (BIDMC: HR 1.25, p < 0.001; UKB: HR 2.28, p < 0.001) after adjusting for covariates including measured BMI. Significant enhancements in model fit, reclassification and improvements in discriminatory power were observed with the inclusion of delta-BMI in both cohorts. Phenotypic profiling highlighted associations between delta-BMI and cardiometabolic diseases, anthropometric measures of truncal obesity, and pericardial fat mass. Metabolic and proteomic profiling associates delta-BMI positively with valine, lipids in small HDL, syntaxin-3, and carnosine dipeptidase 1, and inversely with glutamine, glycine, colipase, and adiponectin. A genome-wide association study revealed associations with regulators of cardiovascular/metabolic traits, including SCN10A, SCN5A, EXOG and RXRG. In summary, our AI-ECG-BMI model accurately predicts BMI and introduces delta-BMI as a non-invasive biomarker for cardiometabolic risk stratification.

Clinical and functional characterisation of a recurrent KCNQ1 variant in the Belgian population.

BACKGROUND: The c.1124_1127delTTCA p.(Ile375Argfs*43) pathogenic variant is the most frequently identified molecular defect in the KCNQ1 gene in the cardiogenetics clinic of the Antwerp University Hospital. This variant was observed in nine families presenting with either Jervell-Lange-Nielsen syndrome or long QT syndrome (LQTS). Here, we report on the molecular, clinical and functional characterization of the KCNQ1 c.1124_1127delTTCA variant. RESULTS: Forty-one heterozygous variant harboring individuals demonstrated a predominantly mild clinical and electrophysiological phenotype, compared to individuals harboring other KCNQ1 pathogenic variants (5% symptomatic before 40 years of age, compared to 24% and 29% in p.(Tyr111Cys) and p.(Ala341Val) variant carriers, respectively, 33% with QTc ≤ 440 ms compared to 10% in p.(Tyr111Cys) and p.(Ala341Val) variant carriers). The LQTS phenotype was most comparable to that observed for the Swedish p.(Arg518*) founder mutation (7% symptomatic at any age, compared to 17% in p.(Arg518*) variant carriers, 33% with QTc ≤ 440 ms compared to 16% in p.(Arg518*) variant carriers). Surprisingly, short tandem repeat analysis did not reveal a common haplotype for all families. One KCNQ1 c.1124_1127delTTCA harboring patient was diagnosed with Brugada syndrome (BrS). The hypothesis of a LQTS/BrS overlap syndrome was supported by electrophysiological evidence for both loss-of-function and gain-of-function (acceleration of channel kinetics) in a heterologous expression system. However, BrS phenotypes were not identified in other affected individuals and allelic KCNQ1 expression testing in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) showed nonsense mediated decay of the c.1124_1127delTTCA allele. CONCLUSIONS: The c.1124_1127delTTCA frameshift variant shows a high prevalence in our region, despite not being confirmed as a founder mutation. This variant leads to a mild LQTS phenotype in the heterozygous state. Despite initial evidence for a gain-of-function effect based on in vitro electrophysiological assessment in CHO cells and expression of the KCNQ1 c.1124_1127delTTCA allele in patient blood cells, additional testing in iPSC-CMs showed lack of expression of the mutant allele. This suggests haploinsufficiency as the pathogenic mechanism. Nonetheless, as inter-individual differences in allele expression in (iPSC-) cardiomyocytes have not been assessed, a modifying effect on the BrS phenotype through potassium current modulation cannot be excluded.

Improved selection of zebrafish CRISPR editing by early next-generation sequencing based genotyping.

Despite numerous prior attempts to improve knock-in (KI) efficiency, the introduction of precise base pair substitutions by the CRISPR-Cas9 technique in zebrafish remains challenging. In our efforts to generate KI zebrafish models of human CACNA1C mutations, we have tested the effect of several CRISPR determinants on KI efficiency across two sites in a single gene and developed a novel method for early selection to ameliorate KI efficiency. We identified optimal KI conditions for Cas9 protein and non-target asymmetric PAM-distal single stranded deoxynucleotide repair templates at both cacna1c sites. An effect of distance to the cut site on the KI efficiency was only observed for a single repair template conformation at one of the two sites. By combining minimally invasive early genotyping with the zebrafish embryo genotyper (ZEG) device and next-generation sequencing, we were able to obtain an almost 17-fold increase in somatic editing efficiency. The added benefit of the early selection procedure was particularly evident for alleles with lower somatic editing efficiencies. We further explored the potential of the ZEG selection procedure for the improvement of germline transmission by demonstrating germline transmission events in three groups of pre-selected embryos.

Inherited Ventricular Arrhythmia in Zebrafish: Genetic Models and Phenotyping Tools.

In the last years, the field of inheritable ventricular arrhythmia disease modelling has changed significantly with a push towards the use of novel cellular cardiomyocyte based models. However, there is a growing need for new in vivo models to study the disease pathology at the tissue and organ level. Zebrafish provide an excellent opportunity for in vivo modelling of inheritable ventricular arrhythmia syndromes due to the remarkable similarity between their cardiac electrophysiology and that of humans. Additionally, many state-of-the-art methods in gene editing and electrophysiological phenotyping are available for zebrafish research. In this review, we give a comprehensive overview of the published zebrafish genetic models for primary electrical disorders and arrhythmogenic cardiomyopathy. We summarise and discuss the strengths and weaknesses of the different technical approaches for the generation of genetically modified zebrafish disease models, as well as the electrophysiological approaches in zebrafish phenotyping. By providing this detailed overview, we aim to draw attention to the potential of the zebrafish model for studying arrhythmia syndromes at the organ level and as a platform for personalised medicine and drug testing.

Morpho-functional comparison of differentiation protocols to create iPSC-derived cardiomyocytes.

Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) offer an attractive platform for cardiovascular research. Patient-specific iPSC-CMs are very useful for studying disease development, and bear potential for disease diagnostics, prognosis evaluation and development of personalized treatment. Several monolayer-based serum-free protocols have been described for the differentiation of iPSCs into cardiomyocytes, but data on their performance are scarce. In this study, we evaluated two protocols that are based on temporal modulation of the Wnt/ß-catenin pathway for iPSC-CM differentiation from four iPSC lines, including two control individuals and two patients carrying an SCN5A mutation. The SCN5A gene encodes the cardiac voltage-gated sodium channel (Nav1.5) and loss-of-function mutations can cause the cardiac arrhythmia Brugada syndrome. We performed molecular characterization of the obtained iPSC-CMs by immunostaining for cardiac specific markers and by expression analysis of selected cardiac structural and ionic channel protein-encoding genes with qPCR. We also investigated cell growth morphology, contractility and survival of the iPSC-CMs after dissociation. Finally, we performed electrophysiological characterization of the cells, focusing on the action potential (AP) and calcium transient (CT) characteristics using patch-clamping and optical imaging, respectively. Based on our comprehensive morpho-functional analysis, we concluded that both tested protocols result in a high percentage of contracting CMs. Moreover, they showed acceptable survival and cell quality after dissociation (>50% of cells with a smooth cell membrane, possible to seal during patch-clamping). Both protocols generated cells presenting with typical iPSC-CM AP and CT characteristics, although one protocol (that involves sequential addition of CHIR99021 and Wnt-C59) rendered iPSC-CMs, which were more accessible for patch-clamp and calcium transient experiments and showed an expression pattern of cardiac-specific markers more similar to this observed in human heart left ventricle samples.

Molecular autopsy and subsequent functional analysis reveal de novo DSG2 mutation as cause of sudden death.

Sudden cardiac death (SCD) is a common cause of death in young adults. In up to 80% of cases a genetic cause is suspected. Next-generation sequencing of candidate genes can reveal the cause of SCD, provide prognostic management, and facilitate pre-symptomatic testing and prevention in relatives. Here we present a proband who experienced SCD in his sleep for which molecular autopsy was performed. We performed a post-mortem genetic analysis of a 49-year-old male who died during sleep after competitive kayaking, using a Cardiomyopathy and Primary Arrhythmia next-generation sequencing panel, each containing 51 candidate genes. Autopsy was not performed. Genetic testing of the proband resulted in missense variants in KCNQ1 (c.1449C > A; p.(Asn483Lys)) and DSG2 (c.2979G > T; p.(Gln993His)), both absent from the gnomAD database. Familial segregation analysis showed de novo occurrence of the DSG2 variant and presence of the KCNQ1 variant in the proband's mother and daughter. KCNQ1 p.(Asn483Lys) was predicted to be pathogenic by MutationTaster. However, none of the KCNQ1 variant carrying family members showed long QTc on ECG or Holter. We further functionally analysed this variant using patch-clamp in a heterologous expression system (Chinese Hamster Ovary (CHO) cells) expressing the KCNQ1 mutant in combination with KCNE1 wild type protein and showed no significant changes in electrophysiological function of Kv7.1. Based on the above evidence, we concluded that the DSG2 p.(Gln993His) variant is the most likely cause of SCD in the presented case, and that there is insufficient evidence that the identified KCNQ1 p.(Asn483Lys) variant would confer risk for SCD in his mother and daughter. Fortunately, the DSG2 variant was not inherited by the proband's two children. This case report indicates the added value of molecular autopsy and the importance of subsequent functional study of variants to inform patients and family members about the risk of variants they might carry.

Clinical characterization of the first Belgian SCN5A founder mutation cohort.

AIMS: We identified the first Belgian SCN5A founder mutation, c.4813 + 3_4813 + 6dupGGGT. To describe the clinical spectrum and disease severity associated with this mutation, clinical data of 101 SCN5A founder mutation carriers and 46 non-mutation carrying family members from 25 Belgian families were collected. METHODS AND RESULTS: The SCN5A founder mutation was confirmed by haplotype analysis. The clinical history and electrocardiographic parameters of the mutation carriers and their family members were gathered and compared. A cardiac electrical abnormality was observed in the majority (82%) of the mutation carriers. Cardiac conduction defects, defined as PR or QRS prolongation on electrocardiogram (ECG), were most frequent, occurring in 65% of the mutation carriers. Brugada syndrome (BrS) was the second most prevalent phenotype identified in 52%, followed by atrial dysrythmia in 11%. Overall, 33% of tested mutation carriers had a normal sodium channel blocker test. Negative tests were more common in family members distantly related to the proband. Overall, 23% of the mutation carriers were symptomatic, with 8% displaying major adverse events. As many as 13% of the patients tested with a sodium blocker developed ventricular arrhythmia. One family member who did not carry the founder mutation was diagnosed with BrS. CONCLUSION: The high prevalence of symptoms and sensitivity to sodium channel blockers in our founder population highlights the adverse effect of the founder mutation on cardiac conduction. The large phenotypical heterogeneity, variable penetrance, and even non-segregation suggest that other genetic (and environmental) factors modify the disease expression, severity, and outcome in these families.

Compound Heterozygous SCN5A Mutations in Severe Sodium Channelopathy With Brugada Syndrome: A Case Report.

Aims: Brugada syndrome (BrS) is an inherited cardiac arrhythmia with an increased risk for sudden cardiac death (SCD). About 20% of BrS cases are explained by mutations in the SCN5A gene, encoding the main cardiac sodium Nav1.5 channel. Here we present a severe case of cardiac sodium channelopathy with BrS caused by SCN5A compound heterozygous mutations. We performed a genetic analysis of SCN5A in a male proband who collapsed during cycling at the age of 2 years. Because of atrial standstill, he received a pacemaker, and at the age of 3 years, he experienced a collapse anew with left-sided brain stroke. A later ECG taken during a fever unmasked a characteristic BrS type-1 pattern. The functional effect of the detected genetic variants was investigated. Methods and Results: Next-generation sequencing allowed the detection of two SCN5A variants in trans: c.4813+3_4813+6dupGGGT-a Belgian founder mutation-and c.4711 T>C, p.Phe1571Leu. A familial segregation analysis showed the presence of the founder mutation in the proband's affected father and paternal aunt and the de novo occurrence of the p.Phe1571Leu. The functional effect of the founder mutation was previously described as a loss-of-function. We performed a functional analysis of the p.Phe571Leu variant in HEK293 cells alone or co-expressed with the ß1-subunit. Compared to the SCN5A wild type, p.Phe1571Leu displayed a hyperpolarizing shift in the voltage dependence of inactivation (loss-of-function), while the activation parameters were unaffected. Using the peptide toxin nemertide α-1, the variant's loss-of-function effect could be restored due to a toxin-dependent reduction of channel inactivation. Conclusion: This is the first report providing support for the pathogenicity of the p.Phe1571Leu SCN5A variant which, together with the c.4813+3_4813+6dupGGGT founder mutation, explains the severity of the phenotype of cardiac sodium channelopathy with BrS in the presented case.