RESUMEN
Genetic testing for inherited arrhythmias and discriminating pathogenic or benign variants from variants of unknown significance (VUS) is essential for gene-based medicine. KCNQ1 is a causative gene of type 1 long QT syndrome (LQTS), and approximately 30% of the variants found in type 1 LQTS are classified as VUS. We studied the role of zebrafish cardiac arrhythmia model in determining the clinical significance of KCNQ1 variants. We generated homozygous kcnq1 deletion zebrafish (kcnq1del/del) using the CRISPR/Cas9 and expressed human Kv7.1/MinK channels in kcnq1del/del embryos. We dissected the hearts from the thorax at 48 h post-fertilization and measured the transmembrane potential of the ventricle in the zebrafish heart. Action potential duration was calculated as the time interval between peak maximum upstroke velocity and 90% repolarization (APD90). The APD90 of kcnq1del/del embryos was 280 ± 47 ms, which was significantly shortened by injecting KCNQ1 wild-type (WT) cRNA and KCNE1 cRNA (168 ± 26 ms, P < 0.01 vs. kcnq1del/del). A study of two pathogenic variants (S277L and T587M) and one VUS (R451Q) associated with clinically definite LQTS showed that the APD90 of kcnq1del/del embryos with these mutant Kv7.1/MinK channels was significantly longer than that of Kv7.1 WT/MinK channels. Given the functional results of the zebrafish model, R451Q could be reevaluated physiologically from VUS to likely pathogenic. In conclusion, functional analysis using in vivo zebrafish cardiac arrhythmia model can be useful for determining the pathogenicity of loss-of-function variants in patients with LQTS.
Asunto(s)
Síndrome de QT Prolongado , Pez Cebra , Animales , Humanos , Canal de Potasio KCNQ1/genética , Síndrome de QT Prolongado/genética , Mutación , ARN Complementario , Virulencia , Pez Cebra/genéticaRESUMEN
The Effect of Diet on Cardiovascular Disease, Heart Disease, and Blood Vessels [...].
Asunto(s)
Enfermedades Cardiovasculares/prevención & control , Dieta , Ácidos Grasos Omega-3/farmacología , Animales , Cardiotónicos/administración & dosificación , Cardiotónicos/farmacología , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/genética , HDL-Colesterol/fisiología , Ácidos Grasos Omega-3/administración & dosificación , Fructosa/efectos adversos , Microbioma Gastrointestinal/fisiología , Factores de Riesgo de Enfermedad Cardiaca , Cardiopatías/etiología , Cardiopatías/prevención & control , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Hipertensión/etiología , Estilo de Vida , Lipoproteínas HDL/fisiología , Obesidad/complicaciones , Ratas , Factores de Riesgo , Cloruro de Sodio Dietético/efectos adversos , Ácidos Grasos trans/efectos adversos , Enfermedades Vasculares/etiología , Enfermedades Vasculares/prevención & controlRESUMEN
Gene correction of induced pluripotent stem cells (iPSCs) has therapeutic potential for treating homozygous familial hypercholesterolemia (HoFH) associated with low-density lipoprotein (LDL) receptor (LDLR) dysfunction. However, few data exist regarding the functional recovery and immunogenicity of LDLR gene-corrected iPSC-derived hepatocyte-like cells (HLCs) obtained from an HoFH patient. Therefore, we generated iPSC-derived HLCs from an HoFH patient harbouring a point mutation (NM_000527.4:c.901 G > T) in exon 6 of LDLR, and examined their function and immunogenicity. From the patient's iPSCs, one homozygous gene-corrected HoFH-iPSC clone and two heterozygous clones were generated using the CRISPR/Cas9 method. Both types of iPSC-derived HLCs showed recovery of the function of LDL uptake in immunofluorescence staining analysis. Furthermore, these gene-corrected iPSC-derived HLCs showed little immunogenicity against the patient's peripheral blood mononuclear cells in a cell-mediated cytotoxicity assay. These results demonstrate that LDL uptake of iPSC-derived HLCs from HoFH can be restored by gene correction without the appearance of further immunogenicity, suggesting that gene-corrected iPSC-derived HLCs are applicable to the treatment of HoFH.
Asunto(s)
Terapia Biológica/métodos , Terapia Genética/métodos , Hepatocitos/citología , Hiperlipoproteinemia Tipo II/inmunología , Células Madre Pluripotentes Inducidas/fisiología , Lipoproteínas LDL/metabolismo , Diferenciación Celular , Línea Celular , Células Cultivadas , LDL-Colesterol/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Citotoxicidad Inmunológica , Hepatocitos/metabolismo , Homocigoto , Humanos , Hiperlipoproteinemia Tipo II/genética , Células Madre Pluripotentes Inducidas/trasplante , Lipoproteínas LDL/genética , Mutación/genéticaRESUMEN
BACKGROUND: Few rare variants in atrial fibrillation (AF)-associated genes have been functionally characterized to identify a causal relationship between these variants and development of AF. We here sought to determine the clinical effect of rare variants in AF-associated genes in patients with lone AF and characterized these variants electrophysiologically and bioinformatically. METHODS AND RESULTS: We screened all coding regions in 12 AF-associated genes in 90 patients with lone AF, with an onset of 47±11 years (66 men; mean age, 56±13 years) by high-resolution melting curve analysis and DNA sequencing. The potassium and sodium currents were analyzed using whole-cell patch clamping. In addition to using 4 individual in silico prediction tools, we extended those predictions to an integrated tool (Combined Annotation Dependent Depletion). We identified 7 rare variants in KCNA5, KCNQ1, KCNH2, SCN5A, and SCN1B genes in 8 patients: 2 of 8 probands had a family history of AF. Electrophysiological studies revealed that 2 variants showed a loss-of-function, and 4 variants showed a gain-of-function. Five of 6 variants with electrophysiological abnormalities were predicted as pathogenic by Combined Annotation Dependent Depletion scores. CONCLUSIONS: In our cohort of patients with lone AF, 7 rare variants in cardiac ion channels were identified in 8 probands. A combination of electrophysiological studies and in silico predictions showed that these variants could contribute to the development of lone AF, although further in vivo study is necessary to confirm these results. More than half of AF-associated rare variants showed gain-of-function behavior, which may be targeted using genotype-specific pharmacological therapy.