hERG Research Articles

EN-510-02 SUPPRESSION AND REPLACEMENT GENE THERAPY FOR KCNH2-MEDIATED ARRHYTHMIAS

KCNH2-mediated arrhythmias are caused by either loss-of-function (type 2 long QT syndrome, LQT2) or gain-of-function (type 1 short QT syndrome, SQT1) pathogenic variants in the KCNH2-encoded Kv11.1 potassium channel which is essential for the rapid delayed rectifier current (IKr) that contributes to the cardiac action potential (AP). No current therapies target the molecular cause of either LQT2 or SQT1. To rescue the pathologic phenotype in cell models of LQT2 and SQT1 using our novel gene therapy. A dual-component “suppression-and-replacement” (SupRep) KCNH2 gene therapy was created by cloning into a single construct a custom-designed KCNH2 shRNA that produces ∼80% knockdown (suppression) and a “shRNA-immune” (shIMM) KCNH2 cDNA (replacement). Patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and their CRISPR-Cas9 variant-corrected isogenic control (IC) iPSC-CMs were made for 3 LQT2- (G604S, G628S, N633S) and 1 SQT1- (N588K) causative variants. All 4 variant lines were treated with KCNH2-SupRep or non-targeting control shRNA (sham). FluoVolt voltage dye was used to measure the APD at 90% repolarization (APD90). KCNH2-SupRep achieved mutation-independent rescue of the pathologic phenotype in both LQT2 and SQT1. For LQT2-causative variants, treatment with KCNH2-SupRep resulted in shortening of the pathologically prolonged APD90 to near curative (IC-like) APD90 levels (G604S IC, 471±25ms; G628S IC, 429±16ms; N633S IC, 405±55ms) compared to treatment with sham (G604S: SupRep-treated, 452±76ms vs. sham-treated, 550±41ms, p<0.0001; G628S: SupRep-treated, 491±38ms vs. sham-treated, 674±20ms, p<0.0001; N633S: SupRep-treated, 399±105ms vs. sham-treated, 577±39ms, p<0.0001). Conversely, for the SQT1-causative N588K, treatment with KCNH2-SupRep resulted in therapeutic prolongation of the pathologically shortened APD90 (IC: 429±16ms; SupRep-treated: 396±61ms; sham-treated: 274±12ms). We provide the first proof-of-principle gene therapy for correction of both LQT2 and SQT1. Akin to our sentinel discovery of SupRep gene therapy for LQT1, KCNH2-SupRep gene therapy successfully corrected/normalized the pathologic APD90, thereby eliminating the pathognomonic feature of both LQT2 and SQT1.

Clinical characteristics of patients with various genetic types of long QT syndrome

The aim of the study is to evaluate clinical characteristics, including adverse events and outcomes, in patients with various genetic types of long QT syndrome (LQTS).Material and methods. We examined 24 patients with a clinical diagnosis of LQTS, observed in the for 5 years. The clinical and instrumental study included registration of electrocardiography (ECG), Holter monitoring, collection of a genealogical history with an ECG assessment of all family members and identification of cases of sudden cardiac death (SCD) in the family or the presence of a family form of the disease, echocardiography and cardiac magnetic resonance imaging to exclude structural changes in the myocardium. The search for mutations in the coding sequences of genes associated with the development of channelopathy and other hereditary heart rhythm disorders was carried out by next generation sequencing (NGS).Results. Mutations in 4 genes associated with LQTS (KCNQ1, KCNH2, CACNA1C, ANK2) were detected in 18 out of 24 (75.0%) patients. Mutations in the KCNQ1, KCNH2 and CACNA1C genes were detected in 14 (58.0%) patients. In 4 out of 24 (17%) patients, two or more variants of clinical significance (VUS) were detected in the genes associated with LQTS and hereditary arrhythmias, 6 patients had no genetic changes. The most severe form of the disease with pronounced clinical manifestations and episodes of clinical death with subsequent resuscitation measures, as well as a significant increase in the QTc interval exceeding 500 ms, was observed in patients with LQT2 and multiple mutations. Implantation of a cardioverter-defibrillator (CD) was required in 14 (58.3%) patients, including 11 (78.56%) - for secondary prevention of SCD and 3 (21.4%) - for primary prevention.Conclusion. A comparative analysis between different genetic types of LQTS (LQT1; LQT2; patients with multiple VUS) showed that in patients with LQT1 syndrome, despite the early manifestation of the disease and the presence of syncopal conditions, life-threatening arrhythmias, SCD and the frequency of CD implantation were significantly less often recorded than in other LQTS. The most severe form of the disease with pronounced clinical manifestations, episodes of clinical death with subsequent resuscitation and CD implantation was observed both in the group of probands with LQT2 and in patients with several nucleotide variants (VUS), one of which was in the CACNA1C or ANK2 genes.

A CLINICAL CASE OF NON-COMPACTION CARDIOMYOPATHY WITH CONCOMITANT MYOPATHIC SYNDROME AND MUTATIONS IN THE LMNA AND KCNH2 GENES

A clinical case of a patient with non-compaction cardiomyopathy, early development of life-threatening arrhythmias and conduction disorders, myopathic syndrome and mutations in the LMNA and KCNH2 genes is presented. The issues of diagnostics based on imaging technologies, complex differential diagnostics of non-compaction cardiomyopathy and dilated cardiomyopathy, as well as the basic principles of treatment are discussed. We also present the main provisions of European and American experts on the concept of isolation of lamina-associated cardiomyopathies for mandatory molecular genetic testing and, if identified, early implantation of ICD for the prevention of SCD.

Spectrum of mutations and their phenotypic manifestations in children and adults with long QT syndrome

Aim. To determine the spectrum of mutations in the genes responsible for the long QT syndrome (LQTS) and study their phenotypic manifestations in patients with LQTS in different age groups.Materials and methods. The study included 35 unrelated probands with a clinical diagnosis of LQTS: 23 adults (8 men) and 12 children (9 boys). There were following clinical features: syncope — 54%, positive family history for SCD — 29%, implanted cardioverter defibrillator (ICD) — 46%. All participants underwent 12-lead electrocardiography (ECG), 24-hour Holter monitoring, genealogical analysis, echocardiography and cardiac MRI. The genetic study was performed by nextgeneration sequencing (NGS) using the MiSeq system (Illumina). The quantitative comparison of two unrelated groups was carried out using the nonparametric MannWhitney U-test. The differences were considered significant at p&lt;0,05.Results. In the examined group of 35 probands, 23 genetic variants of pathogenicity class IV and V (hereinafter referred to as) were identified. The molecular genetic variant of the disease was verified in 66% of probands. At the same time, the detection of mutations in the group with early manifestation (children) was significantly higher: 83% (10 out of 12 children) vs 57% in adults (13 out of 23). Rare genetic variants of uncertain significance (VUS, class III pathogenicity) were detected in 4 probands (11%). In the groups of children and adults with LQT1, LQT2 and LQT3, the sex distribution deviated from the 1:1 ratio. Among children, two-thirds were boys, among adults — the same proportion was represented by women. Disease manifestation time, QTc duration and adverse events risk depended on the genetic type of LQTS, intragenic localization of mutations and sex. In children, all 4 missense mutations in the KCNQ1 gene were located in transmembrane domain, and in adults, 4 mutations were in the transmembrane domain and three — in the C-terminal domain of the protein. LQT1 in boys was characterized by early manifestation, while QTc did not exceed 500 ms and there were no adverse outcomes. Two women out of 7 adults with LQT1 with mutations in the transmembrane domain had na ICD (QTc &gt;520 ms). All patients with LQT2 (4 children, 4 adults) had QTc &gt;500 ms. At the same time, 2 children and 3 women had an ICD. LQT3 was diagnosed only in the children subgroup (2 boys, with QTc of 510 ms and QTc of 610 ms); one of them died suddenly despite beta-blocker therapy. Four adult patients, carriers of class III pathogenicity variants, had QTc &lt;500 ms and delayed disease manifestation (after 30 years). Three of them had episodes of clinical death with subsequent resuscitation and implantation of cardioverter defibrillator.Conclusion. The average diagnostic efficiency of mutation identification using NGS in patients with clinically manifest LQTS was 66%. At the same time, mutations were more common in the children’s group. In genotype-positive probands, the risk of adverse outcomes correlated with sex, age and the genetic variant of disease. The greatest number of adverse outcomes was observed in carriers of mutations in both KCNH2 (LQT2) and SCN5A (LQT3) genes. Variants with unknown clinical significance were identified in 4 probands (11%), which potentially allowed to confirm the diagnosis after functional tests.

Abstract 12269: Investigating Arrhythmogenic Potential of a Novel KCNH2 Mutation Leading to Long QT Syndrome

Background: Mutations in the gene KCNH2 have been associated with both Short and Long QT syndrome. In this study, we identified a 1-day old infant that exhibited T-wave alternans coupled with Long QT Syndrome leading to aborted sudden infant death. Methods: Genetic analysis of the patient revealed a mutation in KCNH2 resulting in a proline to leucine substitution at position 632 (P632L). Functional electrophysiological studies were performed with the hERG mutation transiently transfected into either Human Embryonic Kidney (HEK) cells or human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The functional effects of P632L were incorporated into biophysical computational models of hiPSC-CM as well as adult human cardiomyocytes to further assess its arrhythmogenic potential. Results: The male infant displayed multiple repolarization disorders including T-wave alternans and a QTc =510 ms that appeared 1 day after birth. Genetic screening revealed a heterozygous missense mutation (P632L) in KCNH2 in the infant. Patch clamp analysis of HEK cells transiently transfected with P632L mutation showed a complete loss of function of HERG current compared to WT HERG. Transient transfection of P632L into WT hiPSC myocytes resulted in prolongation of the hiPSC action potential compared to untransfected hiPSC myocytes. Co-transfection of WT and non-functional P632L channels into HEK cells resulted in a dramatic loss of current suggesting a dominant negative effect. Implementing the effects of a complete block of HERG current in the hiPSC-CM computer model prolonged the action potential by 45% and depolarized the resting membrane potentials. In adult human myocyte models, the effects of HERG blockade were more severe in Purkinje cells than that in ventricular myocytes (71% vs. 16% APD prolongation, respectively). Conclusions: The mutation P632L causes a complete loss of HERG current and results in QT prolongation. Numerical simulations suggest a more severe phenotype in Purkinje cells than in ventricular myocytes which could be proarrhythmic.

Establishment of a human-induced pluripotent stem cell line, KSCBi014-A, from a long QT syndrome type 2 patient harboring a KCNH2 mutation

Long QT syndrome type 2 (LQT2) is a heart disorder caused by a loss-of-function mutation in the KCNH2 gene that is an essential factor in cardiac repolarization and affects the heart rate. This study has generated a human-induced stem cell line (KSCBi014-A) carrying the KCNH2 (c.453delC) mutation from an LQT2 patient. The non-integrative Sendai virus-mediated induced pluripotent stem cell (iPSC) reprogramming method was used for iPSC line generation. The KSCBi014-A line maintained stem cell-like morphology, normal karyotype, and pluripotency, and could differentiate into three germ layers in vitro.

Derivation and characterization of two human induced pluripotent stem cell lines (NUIGi004-A) and (NUIGi012-A) from two patients with LQT2 disease

Long QT syndrome type 2 (LQT2) is associated with KCNH2, which encodes the α subunit of the ion channel that controls the K+ current in the heart. Mutations of KCNH2 cause loss of Kv11.1 channel function by disrupting subunit folding, assembly, or trafficking of the channel to the cell surface. Here we generated two induced pluripotent stem cell (iPSC) lines from two patients carrying mutation in KCNH2 gene. These iPSCs express the pluripotent markers and have the capacity of differentiation into other cell types. These patient-derived iPSCs are useful for investigating the disease pathology and identifying the therapeutic target.

Melatonin ameliorates cardiac remodelling in fructose-induced metabolic syndrome rat model by using genes encoding cardiac potassium ion channels.

Metabolic syndrome comprises a group of disorders, including cardiac abnormalities. Ventricular arrhythmias observed in metabolic syndrome are due to the impaired ventricular repolarization. This study aims to determine the effects of melatonin on cardiac ventricular repolarization in metabolic syndrome rat model. Sprague-Dawley rats were divided into control (n = 8), melatonin (n = 8), metabolic syndrome (n = 8) and metabolic syndrome + melatonin (n = 8) groups. Fructose (200g/lt/day) was added into the drinking water during 8weeks of rats to induce metabolic syndrome model. In the last two weeks, melatonin (20mg/kg/day) was administered via oral gavage. Blood pressure measurements and ECG recordings were taken at three different times. Blood and left ventricular tissue samples were harvested and the KCNQ1,3 and KCNH2 gene expressions were analysed by qRT-PCR method. We observed insulin resistance, hyperglycemia, dyslipidemia and higher systolic blood pressure in metabolic syndrome group (p < 0.01, for all). Prolonged QT interval was observed in metabolic syndrome group (p < 0.001). The expression levels of the KCNQ genes encoding the Kv7 channel was significantly reduced, however KCNH2 gene which encodes Kv11.1 channel was increased in metabolic syndrome group compared to control group (p < 0.05, p < 0.001, respectively). Melatonin significantly normalised the prolongation on QT interval in metabolic syndrome group (p < 0.001) and the expressions of the KCNQ (p < 0.002) and KCNH2 genes (p = 0.003). The present study revealed that melatonin had ameliorative effects on ventricular repolarization by improving the prolonged QT duration in rats with metabolic syndrome and this effect was generated by the KCNQ and KCNH2 gene families.

Role and mechanism of chaperones calreticulin and ERP57 in restoring trafficking to mutant HERG‑A561V protein.

Long QT syndrome type 2 is caused by a mutation in the human-ether-a-go-go-related gene (HERG) gene encoding the rapidly activating delayed rectifier K-current. HERG is a key cell membrane glycoprotein; however, whether the maturation process of HERG protein involves key molecules derived from the calnexin (CNX)/calreticulin (CRT) cycle and how these molecules work remains unknown. Using western blotting, the present study screened the key molecules CNX/CRT/endoplasmic reticulum protein 57 (ERP57) involved in this cycle, and it was revealed that the protein expression levels of CNX/CRT/ERP57 in wild-type (WT)/A561V cells were increased compared with those in WT cells (n=3; P<0.05). Additionally, a co-immunoprecipitation experiment was used to reveal that the ability of CNX/ERP57/CRT to interact with HERG was significantly increased in A561V and WT/A561V cells (n=3; P<0.05). A plasmid lacking the bb′ domain of ERP57 was constructed and it was demonstrated that the key site of ERP57 binding to CRT and immature HERG protein is the bb′ domain. The whole-cell patch-clamp technique detected that the tail current density increased by 46% following overexpression of CRT and by 53% following overexpression of ERP57 in WT/A561V cells. Overexpression of CRT and ERP57 could increased HERG protein levels on the membrane detected by confocal imaging. Furthermore, overexpression of ERP57 and CRT proteins could restore the HERG-A561V mutant protein trafficking process and rescue the dominant-negative suppression of WT. Overall, ERP57/CRT served a crucial role in the HERG-A561V mutant protein trafficking deficiency and degradation process.

A novel variant in the SLC4A3 gene with high penetrance in a family with short QT Syndrome

Abstract Funding Acknowledgements Type of funding sources: None. Background Short QT syndrome (SQTS) is a rare, autosomal dominant disease causing sudden cardiac death (SCD). Current guidelines recommend genetic testing. Associated variants in KCNQ1, KCNH2, KCNJ2 and SLC4A3 genes have been reported. Purpose We report a family with a variant in the SLC4A3 gene with several presentations of SCD and high clinical penetrance of SQTS. Methods We performed a post-mortem genetic testing in the index patient in whom prior ECG was available. Subsequently, clinical and electrophysiological work-up and cascade screening (CS) of the detected suspected variant was carried out in available relatives. Results The index patient had suffered a SCD at the age of 17 (figure, upper panel, arrow). A previously registered ECG showed a shortened QTc of 340ms (figure, lower panel). Autopsy revealed no structural heart disease. Post-mortem genetic testing revealed variants in the LDB3, MYH7 and a novel heterozygous missense variant, p.(Ser1039Arg) also in the SLC4A3 gene. Although predictive bioinformatic algorithms (AlignGVGD, SIFT, MutationTaster, Polyphen2) showed conflicting classifications, family history was notable for SCD without post-mortem genetic work-up in three second degree relatives (figure, upper panel, patients 207, 208 and 305, age of death 33, 25 and 33 years respectively). CS was performed in first and second degree relatives of the index patient and was highly suggestive for disease association of the variant in the SLC4A3 gene with co-segregation in all clinically affected family members. Only one patient with the variant had a normal QTc (figure, upper panel, patient 202) of 407ms, however this patient was on regular QT-prolonging medication (risperidone and loperamide). Conclusion Genetic testing revealed a novel in the SLC4A3 gene, which was recently implicated in the pathogenesis of the SQTS. Although predictive bioinformatic algorithms yielded conflicting results, CS of family members suggests a likely pathogenicity (class IV) of the variant. Further CS or functional tests are necessary to establish causality. Abstract Figure. ECG of index patient and family tree

Cardiotoxicity assessment using 3D vascularized cardiac tissue consisting of human iPSC-derived cardiomyocytes and fibroblasts

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for cardiac safety assessment but have limitations for the evaluation of drug-induced contractility. Three-dimensional (3D) cardiac tissues are similar to native tissue and valuable for the assessment of contractility. However, a longer time and specialized equipment are required to generate 3D tissues. We previously developed a simple method to generate 3D tissue in a short period by coating the cell surfaces with extracellular matrix proteins. We hypothesized that this 3D cardiac tissue could be used for simultaneous evaluation of drug-induced repolarization and contractility. In the present work, we examined the effects of several compounds with different mechanisms of action by cell motion imaging. Consequently, human ether-a-go-go-related gene (HERG) channel blockers with high arrhythmogenic risk caused prolongation of contraction-relaxation duration and arrhythmia-like waveforms. Positive inotropic drugs, which increase intracellular Ca2+ levels or myocardial Ca2+ sensitivity, caused an increase in maximum contraction speed (MCS) or average deformation distance (ADD) (ouabain, 138% for MCS at 300 nM; pimobendane, 132% for ADD at 3 μM). For negative inotropic drugs, verapamil reduced both MCS and ADD (61% at 100 nM). Thus, this 3D cardiac tissue detected the expected effects of various cardiovascular drugs, suggesting its usefulness for cardiotoxicity evaluation.

Outcomes of post mortem genetic diagnosis in SCD victims and primary prevention of cardiac arrest in relatives: a nationwide multidisciplinary and multicentric collaboration in the Czechia

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Supported by Ministry of Health of the Czech Republic Introduction Post mortem genetic analysis in sudden cardiac death (SCD) represents an important diagnostic tool for the primary prevention of cardiac arrest in victim´s relatives. Purpose To assess the underlying molecular pathogenesis of SCD in a representative Czech cohort and to evaluate the effects of primary prevention of SCD in genetic relatives. Patients and Methods Between 2016 and 2020 we have ascertained 100 SCD cases (29 females/71 males; age range 0-52 years). According to autopsy protocols, cases with SCD were divided into categories of sudden arrhythmic death (SADS), sudden unexplained death (in infants; SUD/SUDI), thoracic aortic aneurysm/dissection and cardiomyopathy hypertrophic, arrhythmic, dilated (HCM, ACM, DCM) and sudden infant death syndrome (SIDS). DNA was isolated from post mortem biopsies / relatives blood and subjected to massively parallel sequencing (Illumina, USA) comprising custom-made candidate gene panel (100 genes). Genetic counselling and cardiological examinations were carried out in 245 family members. Results According to post mortem-established diagnosis, we identified 20 victims with SADS and SUD/SUDI, 11 with HCM and DCM, 19 with ACMG, 8 SIDS cases and 9 acute dissection cases. Most of victims died at sleep or at rest, while only 10/100 victims died during strenuous sport activities. About 50% of SCD victims did not report any apparent cardiac complaints. Highly likely or certain molecular etiology (i.e. based on presence of ACMG.net Class 4 to 5 variants) was disclosed in 19/100 (19%) in RYR2, KCNH2, SCN5A, FLNC (stop), TTN, RBM 20, LMNA/C, PRKAG2, MYBPC3, DSC2, FHL1, TGFBR1 and Col3A1 genes (see Tab). Finally, we identified 52/241 phenotype/genotype positive family members who are at risk of cardiac arrest and were offered corresponding cardiological care. Conclusion Multidisciplinary cooperation, together with centralized and standardized molecular genetic testing, enables the primary prevention of cardiac arrest in relatives of SCD victims. Results of post mortem genetic analysis Post mortem diagnosis Nr. Gender Age (years) Nr. of positive cases (DNA variant class IV or V) Gene Nr. examined relatives/phenotype or genotype positive cases SADS 20 8 females12 males 3-52 5/20 (25%) KCNH2 3x RYR2RANGFR 56/11 SUD/SUDI 20 5 females, 15 males 0-50 1/18 (5%, 2 non informative cases) RYR2 45/9 HCM 11 0 females11 males 14-52 3/11 (27%) MYBPC3FHL1PRKAG2 26/9 DCM 11 3 females8 males 8-48 4/11 (36%) TTN (3x)RBM 20FLNC (stop) 24/7 ACM 19 9 females10 males 17 - 49 4/19 (21%) SCN5AFLNC (stop)DSC2LMNA/C 58/9 SIDS 8 3 females5 males &amp;lt; 1 0/8 - 12/0 Acute dissection 9 1 female8 males 16-49 2/9 (22%) TGFBR1Col3A1 24/7

Regulation of Kv11.1 Isoform Expression by Polyadenylate Binding Protein Nuclear 1.

The Kv11.1 voltage-gated potassium channel, encoded by the KCNH2 gene, conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation to generate two C-terminal Kv11.1 isoforms in the heart. Utilization of a poly(A) signal in exon 15 produces the full-length, functional Kv11.1a isoform, while intron 9 polyadenylation generates the C-terminally truncated, nonfunctional Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO isoforms plays an important role in the regulation of Kv11.1 channel function. In this study, we tested the hypothesis that the RNA polyadenylate binding protein nuclear 1 (PABPN1) interacts with a unique 22 nt adenosine stretch adjacent to the intron 9 poly(A) signal and regulates KCNH2 pre-mRNA alternative polyadenylation and the relative expression of Kv11.1a C-terminal isoforms. We showed that PABPN1 inhibited intron 9 poly(A) activity using luciferase reporter assays, tandem poly(A) reporter assays, and RNA pulldown assays. We also showed that PABPN1 increased the relative expression level of the functional Kv11.1a isoform using RNase protection assays, immunoblot analyses, and patch clamp recordings. Our present findings suggest a novel role for the RNA-binding protein PABPN1 in the regulation of functional and nonfunctional Kv11.1 isoform expression.

A novel mutation in KCNH2 yields loss-of-function of hERG potassium channel in long QT syndrome 2.

Mutations in hERG (human ether-à-go-go-related gene) potassium channel are closely associated with long QT syndromes. By direct Sanger sequencing, we identified a novel KCNH2 mutation W410R in the patient with long QT syndrome 2 (LQT2). However, the electrophysiological functions of this mutation remain unknown. In comparison to hERGWT channels, hERGW410R channels have markedly decreased total and surface expressions. W410R mutation dramatically reduces hERG channel currents (IKr) and shifts its steady-state activation curve to depolarization. Moreover, hERGW410R channels make dominant-negative effects on hERGWT channels. Significantly, we find hERG channel blocker E-4031 could partially rescue the function of hERGW410R channels by increasing the membrane expression. By using in silico model, we reveal that hERGW410R channels obviously elongate the repolarization of human ventricular myocyte action potentials. Collectively, W410R mutation decreases the currents of hERG channel, because of diminished membrane expression of mutant channels, that subsequently leads to elongated repolarization of cardiomyocyte, which might induce the pathogenesis of LQT2. Furthermore, E-4031 could partially rescue the decreased activity of hERGW410R channels. Thus, our work identifies a novel loss-of-function mutation in KCNH2 gene, which might provide a rational basis for the management of LQT2.

A novel KCNH2 frameshift mutation (c.46delG) associated with high risk of sudden death in a family with congenital long QT syndrome type 2

BackgroundThe congenital long QT syndrome type 2 is caused by mutations in KCNH2 gene that encodes the alpha subunit of potassium channel Kv11.1. The carriers of the pathogenic variant of KCNH2 gene manifest a phenotype characterized by prolongation of QT interval and increased risk of sudden cardiac death due to life-threatening ventricular tachyarrhythmias.ResultsA family composed of 17 members with a family history of sudden death and recurrent syncopes was studied. The DNA of proband with clinical manifestations of long QT syndrome was analyzed using a massive DNA sequencer that included the following genes: KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, ANK2, KCNJ2, CACNA1, CAV3, SCN1B, SCN4B, AKAP9, SNTA1, CALM1, KCNJ5, RYR2 and TRDN. DNA sequencing of proband identified a novel pathogenic variant of KCNH2 gene produced by a heterozygous frameshift mutation c.46delG, pAsp16Thrfs*44 resulting in the synthesis of a truncated alpha subunit of the Kv11.1 ion channel. Eight family members manifested the phenotype of long QT syndrome. The study of family segregation using Sanger sequencing revealed the identical variant in several members of the family with a positive phenotype.ConclusionsThe clinical and genetic findings of this family demonstrate that the novel frameshift mutation causing haploinsufficiency can result in a congenital long QT syndrome with a severe phenotypic manifestation and an elevated risk of sudden cardiac death.

Análise in silico do perfil farmacocinético e toxicológico de fármacos em pesquisa para o tratamento da COVID-19

A pesquisa em pauta tem como objetivo o fornecimento de informações sobre os medicamentos em estudo para o tratamento da Covid-19, além de elucidar dados dos parâmetros farmacocinéticos e toxicológicos que possam auxiliar na terapia do paciente acometido com o coronavírus, através de uma análise in silico. Para alcançar os objetivos propostos, utilizou-se de ferramentas in silico, empregando os softwares ACD/Chem Sketch (versão 14.0) e Marvin Sketch. A análise dos parâmetros farmacocinéticos e toxicológicos foi feita através do servidor PreADMET, que realiza a predição dos dados baseando-se na relação estrutura/atividade. Para complementar os dados coletados em ferramentas computacionais realizou-se um levantamento bibliográfico nas principais bases de dados sobre os parâmetros analisados. Conforme as análises dos resultados ADME os medicamentos abiraterona e brequinar são os que apresentam mais parâmetros farmacocinéticos desejáveis. Alguns dos medicamentos observados demonstraram respostas satisfatórias, em especial a cloroquina, que atendeu ao parâmetro de passagem na BHE, uma vez que o coronavírus vem causando manifestações no SNC, porém esta medicação está relacionada com o crescimento de mortes por arritmias. Já os parâmetros toxicológicos apresentam em sua maioria fármacos não mutagênicos e não carcinogênicos e, em relação ao hERG, a maioria classifica-se como médio risco. Os fármacos empregados no tratamento dos pacientes cardiopatas devem ser escolhidos cuidadosamente, pois alguns deles elevam o risco cardíaco. A ivermectina e a nitazoxanida apresentam metabólitos com potencial mutagênico, portanto, devem ser utilizados com cautela.

Two novel variants in the SLC4A3 gene in two families with Short QT Syndrome: the role of cascade screening

Abstract Background Short QT syndrome (SQTS) is a rare, autosomal dominant disease causing sudden cardiac death (SCD). Genetic testing is recommended according to current guidelines. Variants in KCNQ1, KCNH2, KCNJ2 and SLC4A3 genes have been reported in SQTS. Purpose We report implications of genetic testing and cascade screening (CS) in two families with phenotypical presentation of SQTS and novel genetic variants of unknown significance. Methods We performed a thorough clinical and electrophysiological work-up of the index patients of both families. In addition, genetic screening was conducted. Subsequently, segregation analysis of potentially pathogenic variants was carried out in available relatives. Results Index patient 1 presented with a history of recurrent syncope. His ECG showed a shortened QTc of 340ms. Family history was unremarkable. Structural heart disease was excluded by cardiac MRI and coronary angiography. Genetic testing detected a rare heterozygous missense variant in the KCNH2 gene (p.(Arg328Cys), frequency 0.053%), predicted to be pathogenic according to various prediction algorithms (Polyphen, SIFT, Align GVGD, mutation taster). CS of relatives did not confirm this variant as the causative mutation. Reanalysis of whole-exome sequencing data revealed a novel heterozygous missense variant, p.(Arg370Cys) in the recently identified SLC4A3 gene. A variant at the same position has previously been associated with SQTS. CS suggested disease association. The second index patient had a SCD at the age of 17. A previously registered ECG showed a shortened QTc of 340ms. Autopsy revealed no structural heart disease. Post-mortem genetic testing revealed variants in the LDB3, MYH7 and a novel heterozygous missense variant, p.(Ser1039Arg) also in the SLC4A3 gene. Family history was positive for SCD in three 2° relatives. The index patient's father had a positive phenotype with a QTc of 365ms. CS again suggested disease association of the variant in the SLC4A3 gene only. Conclusion Genetic testing revealed two novel variants in the SLC4A3 gene, which was recently implicated in the pathogenesis of the SQTS. Predictive bioinformatic algorithms to assess the pathogenicity of missense variants are of limited relevance, but genetic analysis of additional unaffected and affected family members may be instrumental to identify pathogenic DNA sequence variations. Family tree index patients 1 and 2 Funding Acknowledgement Type of funding source: None

Is mexiletine ready for prime time in patients with Type 2 Long QT Syndrome?

Abstract Background Mexiletine has been proven effective in shortening the duration of ventricular repolarization and reducing the arrhythmic events in type 3 Long QT Syndrome (LQT3). Initial reports indicate that mexiletine might also be effective in patients with type 2 Long QT Syndrome (LQT2, caused by loss-of-functions variants on KCNH2 gene, coding for HERG potassium channel), but this issue has not been investigated in detail. Purpose We quantified the electrocardiographic (ECG) effects of mexiletine in a cohort of LQT2 patients. Methods Twelve-lead ECGs were collected before and after the administration of mexiletine to evaluate the drug's effect on the heart rate-corrected QT interval (QTc). QTc intervals were classified as being (1) at “high-risk” if &amp;gt;500 ms or (2) “normal” if &amp;lt;460 ms, before and after the administration of the drug. KCNH2 variants were defined as (1) trafficking-deficient or (2) non-trafficking-deficient, based on functional studies. Results We tested the maximum tolerated dose of mexiletine in 20 patients (11 males, 55%), who were 17±16 years old at diagnosis, affected by genetically established LQT2. The mean age at the beginning of mexiletine administration was 23±15 years and the mean daily dose administered was 9±2 mg/kg/day. Before mexiletine, the mean QTc interval was 527±53 ms and 10/20 (50%) patients had high-risk QTc values (i.e. QTc &amp;gt;500 ms). After mexiletine, the mean QTc interval shortened to 484±47 ms in the overall population (p=0.001). In the majority of patients (18/20, 90%; Figure 1) QTc interval shortened, with a mean shortening of 42±28 ms, and a high-interindividual variability (range of shortening from 86 ms to 8 ms). Just 2 (10%) patients did not show any reduction of the QTc, despite receiving the highest adult dosages of mexiletine in our cohort (up to 10 mg/kg/day). As compared to baseline conditions, after mexiletine the proportion of patients with high risk QTc values (&amp;gt;500 ms) decreased non-significantly from 50% to 35% (p=0.52). Furthermore, in only 6/20 (30%) patients the QTc normalized (i.e. QTc &amp;lt;460 ms) after the initiation of treatment. The effect of mexiletine was not influenced by gender (p=0.89) or by the functional effect of the KCNH2 mutation (trafficking-deficient vs. non-trafficking-deficient variants, p=0.41). The effect of mexiletine in LQT2 patients was inferior to the one previously observed in a cohort of 34 LQT3 patients, who had similar QTc values at baseline and received similar dosages of mexiletine, but showed a significantly higher reduction of the average QTc interval (63±37 ms in LQT3 vs. 42±28 ms in LQT2, p=0.02). Conclusions Mexiletine induces a reduction of the QTc interval in most LQT2 patients, but many patients remain with high-risk QTc values after receiving the drug. The demonstration that an average QTc shortening of 42 ms is enough to reduce arrhythmic events is necessary, before the introduction of mexiletine in clinical practice for LQT2. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Research and University Dipartimenti di Eccellenza 2018–2022 grant to the Molecular Medicine Department (University of Pavia)

Generation of a human embryonic stem cell line (WAe009-A-43) carrying a Long QT syndrome related mutation in KCNH2 gene

The hereditary Long QT syndrome (LQTS) is a life-threaten channelopathy of the heart characterized by prolonged QT intervals and predisposition to occur polymorphic ventricular tachyarrhythmias. LQTS type 2 is the second most prevalent type of LQTS and more than 200 putative disease-causing mutations have been identified for KCNH2. Herein, we have generated a human embryonic stem cell line (WAe009-A-43) carrying a LQTS related mutation in KCNH2 (WAe009-A-43). The WAe009-A-43 line maintained stem cell like morphology, pluripotency, normal karyotype and could differentiate into all three germ layers in vivo.

First episode of ventricular fibrillation in an 84-year-old man with long-QT type 2 syndrome: A case report

Congenital long QT syndrome (LQTS) is associated with ventricular arrhythmia and an increased risk of sudden cardiac death in young people. However, it is extremely rare for an elderly man to experience ventricular fibrillation (VF) due to congenital LQTS as a first episode. We describe the case of an 84-year-old man who experienced syncope after urination. He had a medical history of hypertension and asthma, but no history of syncope. Electrocardiographic findings in 2017 showed QT prolongation (corrected QT = 505 ms). No medication that could induce QT prolongation was administered. Blood test results on admission showed no electrolyte abnormalities, and there were no abnormal findings on echocardiography. The second episode of loss of consciousness occurred during hospitalization, and electrocardiography revealed incessant torsade de pointes, caused by R-on-T with short-long-short (SLS) sequences due to bradyarrhythmia. Coronary angiography did not detect myocardial ischemia, and an implantable cardioverter-defibrillator was implanted for secondary prevention. Genetic testing revealed a mutation of the KCNH2 gene, indicating LQTS type 2. In summary, we report a rare case of prolonged QT interval with SLS sequences due to sick sinus syndrome triggering VF as the first attack in an elderly patient with LQTS type 2.<Learning objective: Physicians should be aware of the prolongation of QT as a cause of syncope in elderly patients and should pay attention to QT duration. Furthermore, patients with elderly-onset QT prolongation may have a genetic background associated with congenital long QT syndrome (LQTS); therefore, we should not hesitate to perform genetic testing in cases where LQTS is suspected in elderly patients.>