RESUMO
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.
Assuntos
Modelos Genéticos , Peixe-Zebra , Humanos , Animais , Peixe-Zebra/genética , Síndrome , Arritmias Cardíacas/genética , Miócitos CardíacosRESUMO
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.
Assuntos
Síndrome de Brugada , Canal de Sódio Disparado por Voltagem NAV1.5 , Bélgica/epidemiologia , Eletrocardiografia , Humanos , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/genética , FenótipoRESUMO
Truncating variants in TTN (TTNtv) are present in 15-25 % of patients with idiopathic dilated cardiomyopathy. Interestingly, the pathogenicity of TTNtv seems to be linked to their location within the gene. More proximal I-band TTNtv (TTNtvI) harbour less pathogenic potential than distant A-band TTNtv (TTNtvA). We created isogenic human induced pluripotent stem cell lines (hiPSC) with TTNtvI and TTNtvA using CRISPR/Cas9, for the investigation of the pathomechanism in hiPSC-derived cardiomyocytes (hiPSC-CMs). Exon 48 (E48), located in the I-band, and exon 357 (E357), located in the A-band were targeted.
RESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Humanos , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Peixe-Zebra/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
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.
Assuntos
Canal de Potássio KCNQ1 , Síndrome do QT Longo , Animais , Cricetinae , Alelos , Bélgica , Cricetulus , Canal de Potássio KCNQ1/genética , Humanos , Síndrome de Jervell-Lange Nielsen/genética , Síndrome do QT Longo/genéticaRESUMO
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.
Assuntos
Células-Tronco Pluripotentes Induzidas , Potenciais de Ação , Diferenciação Celular , Fenômenos Eletrofisiológicos , Humanos , Miócitos CardíacosRESUMO
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.