Detection of elusive DNA copy-number variations in hereditary disease and cancer through the use of noncoding and off-target sequencing reads.

Copy-number variants (CNVs) play a substantial role in the molecular pathogenesis of hereditary disease and cancer, as well as in normal human interindividual variation. However, they are still rather difficult to identify in mainstream sequencing projects, especially involving exome sequencing, because they often occur in DNA regions that are not targeted for analysis. To overcome this problem, we developed OFF-PEAK, a user-friendly CNV detection tool that builds on a denoising approach and the use of "off-target" DNA reads, which are usually discarded by sequencing pipelines. We benchmarked OFF-PEAK on data from targeted sequencing of 96 cancer samples, as well as 130 exomes of individuals with inherited retinal disease from three different populations. For both sets of data, OFF-PEAK demonstrated excellent performance (>95% sensitivity and >80% specificity vs. experimental validation) in detecting CNVs from in silico data alone, indicating its immediate applicability to molecular diagnosis and genetic research.

Disease-specific variant interpretation highlighted the genetic findings in 2325 Japanese patients with retinitis pigmentosa and allied diseases.

BACKGROUND: As gene-specific therapy for inherited retinal dystrophy (IRD) advances, unified variant interpretation across institutes is becoming increasingly important. This study aims to update the genetic findings of 86 retinitis pigmentosa (RP)-related genes in a large number of Japanese patients with RP by applying the standardised variant interpretation guidelines for Japanese patients with IRD (J-IRD-VI guidelines) built upon the American College of Medical Genetics and Genomics and the Association for Molecular Pathology rules, and assess the contribution of these genes in RP-allied diseases. METHODS: We assessed 2325 probands with RP (n=2155, including n=1204 sequenced previously with the same sequencing panel) and allied diseases (n=170, newly analysed), including Usher syndrome, Leber congenital amaurosis and cone-rod dystrophy (CRD). Target sequencing using a panel of 86 genes was performed. The variants were interpreted according to the J-IRD-VI guidelines. RESULTS: A total of 3564 variants were detected, of which 524 variants were interpreted as pathogenic or likely pathogenic. Among these 524 variants, 280 (53.4%) had been either undetected or interpreted as variants of unknown significance or benign variants in our earlier study of 1204 patients with RP. This led to a genetic diagnostic rate in 38.6% of patients with RP, with EYS accounting for 46.7% of the genetically solved patients, showing a 9% increase in diagnostic rate from our earlier study. The genetic diagnostic rate for patients with CRD was 28.2%, with RP-related genes significantly contributing over other allied diseases. CONCLUSION: A large-scale genetic analysis using the J-IRD-VI guidelines highlighted the population-specific genetic findings for Japanese patients with IRD; these findings serve as a foundation for the clinical application of gene-specific therapies.

Sex Differences in Cardiovascular Disease-Related Hospitalization and Mortality in Japan　- Analysis of Health Records From a Nationwide Claim-Based Database, the Japanese Registry of All Cardiac and Vascular Disease (JROAD).

BACKGROUND: The prevalence of cardiovascular disease (CVD) is rising in Japan with its aging population, but there is a lack of epidemiological data on sex differences in CVD, including acute coronary syndrome (ACS), acute heart failure (AHF), and acute aortic disease.Methods and Results: This retrospective study analyzed data from 1,349,017 patients (January 2012-December 2020) using the Japanese Registry Of All Cardiac and Vascular Diseases database. ACS patients were youngest on average (70.5±12.9 years) and had the lowest female proportion (28.9%). AHF patients had the oldest mean age (79.7±12.0 years) and the highest proportion of females (48.0%). Acute aortic disease had the highest in-hospital mortality (26.1%), followed by AHF (11.5%) and ACS (8.9%). Sex-based mortality differences were notable in acute aortic disease, with higher male mortality in Stanford Type A acute aortic dissection (AAD) with surgery (males: 14.2% vs. females: 10.4%, P<0.001) and similar rates in Type B AAD (males: 6.2% vs. females: 7.9%, P=0.52). Aging was a universal risk factor for in-hospital mortality. Female sex was a risk factor for ACS and acute aortic disease but not for AHF or Types A and B AAD. CONCLUSIONS: Sex-based disparities in the CVD-related hospitalization and mortality within the Japanese national population have been highlighted for the first time, indicating the importance of sex-specific strategies in the management and understanding of these conditions.

Common Ancestry-Specific Ion Channel Variants Predispose to Drug-Induced Arrhythmias.

BACKGROUND: Multiple reports associate the cardiac sodium channel gene (SCN5A) variants S1103Y and R1193Q with type 3 congenital long QT syndrome and drug-induced long QT syndrome. These variants are too common in ancestral populations to be highly arrhythmogenic at baseline, however: S1103Y allele frequency is 8.1% in African Americans and R1193Q 6.1% in East Asians. R1193Q is known to increase late sodium current (INa-L) in cardiomyocytes derived from induced pluripotent stem cells but the role of these variants in modulating repolarization remains poorly understood. METHODS: We determined the effect of S1103Y on QT intervals among African-American participants in a large electronic health record. Using cardiomyocytes derived from induced pluripotent stem cells carrying naturally occurring or genome-edited variants, we studied action potential durations (APDs) at baseline and after challenge with the repolarizing potassium current (IKr) blocker dofetilide and INa-L and IKr at baseline. RESULTS: In 1479 African-American participants with no confounding medications or diagnoses of heart disease, QT intervals in S1103Y carriers was no different from that in noncarriers. Baseline APD was no different in cells expressing the Y allele (SY, YY cells) compared with isogenic cells with the reference allele (SS cells). However, INa-L was increased in SY and YY cells and the INa-L blocker GS967 shortened APD in SY/YY but not SS cells (P<0.001). IKr was increased almost 2-fold in SY/YY cells compared with SS cells (tail current: 0.66±0.1 versus 1.2±0.1 pA/pF; P<0.001). Dofetilide challenge prolonged APD at much lower concentrations in SY (4.1 nmol/L [interquartile range, 1.5-9.3]; n=11) and YY (4.2 nmol/L [1.7-5.0]; n=5) than in SS cells (249 nmol/L [22.3-2905]; n=14; P<0.001 and P<0.01, respectively) and elicited afterdepolarizations in 8/16 SY/YY cells but only in 1/14 SS cells. R1193Q cells similarly displayed no difference in baseline APD but increased IKr and increased dofetilide sensitivity. CONCLUSIONS: These common ancestry-specific variants do not affect baseline repolarization, despite generating increased INa-L. We propose that increased IKr serves to maintain normal repolarization but increases the risk of manifest QT prolongation with IKr block in variant carriers. Our findings emphasize the need for inclusion of diverse populations in the study of adverse drug reactions.

Arrhythmia Variant Associations and Reclassifications in the eMERGE-III Sequencing Study.

BACKGROUND: Sequencing Mendelian arrhythmia genes in individuals without an indication for arrhythmia genetic testing can identify carriers of pathogenic or likely pathogenic (P/LP) variants. However, the extent to which these variants are associated with clinically meaningful phenotypes before or after return of variant results is unclear. In addition, the majority of discovered variants are currently classified as variants of uncertain significance, limiting clinical actionability. METHODS: The eMERGE-III study (Electronic Medical Records and Genomics Phase III) is a multicenter prospective cohort that included 21 846 participants without previous indication for cardiac genetic testing. Participants were sequenced for 109 Mendelian disease genes, including 10 linked to arrhythmia syndromes. Variant carriers were assessed with electronic health record-derived phenotypes and follow-up clinical examination. Selected variants of uncertain significance (n=50) were characterized in vitro with automated electrophysiology experiments in HEK293 cells. RESULTS: As previously reported, 3.0% of participants had P/LP variants in the 109 genes. Herein, we report 120 participants (0.6%) with P/LP arrhythmia variants. Compared with noncarriers, arrhythmia P/LP carriers had a significantly higher burden of arrhythmia phenotypes in their electronic health records. Fifty-four participants had variant results returned. Nineteen of these 54 participants had inherited arrhythmia syndrome diagnoses (primarily long-QT syndrome), and 12 of these 19 diagnoses were made only after variant results were returned (0.05%). After in vitro functional evaluation of 50 variants of uncertain significance, we reclassified 11 variants: 3 to likely benign and 8 to P/LP. CONCLUSIONS: Genome sequencing in a large population without indication for arrhythmia genetic testing identified phenotype-positive carriers of variants in congenital arrhythmia syndrome disease genes. As the genomes of large numbers of people are sequenced, the disease risk from rare variants in arrhythmia genes can be assessed by integrating genomic screening, electronic health record phenotypes, and in vitro functional studies. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier; NCT03394859.

High-Throughput Reclassification of SCN5A Variants.

Partial or complete loss-of-function variants in SCN5A are the most common genetic cause of the arrhythmia disorder Brugada syndrome (BrS1). However, the pathogenicity of SCN5A variants is often unknown or disputed; 80% of the 1,390 SCN5A missense variants observed in at least one individual to date are variants of uncertain significance (VUSs). The designation of VUS is a barrier to the use of sequence data in clinical care. We selected 83 variants: 10 previously studied control variants, 10 suspected benign variants, and 63 suspected Brugada syndrome-associated variants, selected on the basis of their frequency in the general population and in individuals with Brugada syndrome. We used high-throughput automated patch clamping to study the function of the 83 variants, with the goal of reclassifying variants with functional data. The ten previously studied controls had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wild-type-like function. 22 suspected BrS variants had loss of channel function (<10% normalized peak current) and 22 variants had partial loss of function (10%-50% normalized peak current). The previously unstudied variants were initially classified as likely benign (n = 2), likely pathogenic (n = 10), or VUSs (n = 61). After the patch clamp studies, 16 variants were benign/likely benign, 45 were pathogenic/likely pathogenic, and only 12 were still VUSs. Structural modeling identified likely mechanisms for loss of function including altered thermostability and disruptions to alpha helices, disulfide bonds, or the permeation pore. High-throughput patch clamping enabled reclassification of the majority of tested VUSs in SCN5A.

Continuous Bayesian variant interpretation accounts for incomplete penetrance among Mendelian cardiac channelopathies.

PURPOSE: The congenital Long QT Syndrome (LQTS) and Brugada Syndrome (BrS) are Mendelian autosomal dominant diseases that frequently precipitate fatal cardiac arrhythmias. Incomplete penetrance is a barrier to clinical management of heterozygotes harboring variants in the major implicated disease genes KCNQ1, KCNH2, and SCN5A. We apply and evaluate a Bayesian penetrance estimation strategy that accounts for this phenomenon. METHODS: We generated Bayesian penetrance models for KCNQ1-LQT1 and SCN5A-LQT3 using variant-specific features and clinical data from the literature, international arrhythmia genetic centers, and population controls. We analyzed the distribution of posterior penetrance estimates across 4 genotype-phenotype relationships and compared continuous estimates with ClinVar annotations. Posterior estimates were mapped onto protein structure. RESULTS: Bayesian penetrance estimates of KCNQ1-LQT1 and SCN5A-LQT3 are empirically equivalent to 10 and 5 clinically phenotype heterozygotes, respectively. Posterior penetrance estimates were bimodal for KCNQ1-LQT1 and KCNH2-LQT2, with a higher fraction of missense variants with high penetrance among KCNQ1 variants. There was a wide distribution of variant penetrance estimates among identical ClinVar categories. Structural mapping revealed heterogeneity among "hot spot" regions and featured high penetrance estimates for KCNQ1 variants in contact with calmodulin and the S6 domain. CONCLUSIONS: Bayesian penetrance estimates provide a continuous framework for variant interpretation.

Comprehensive genetic screening for vascular Ehlers-Danlos syndrome through an amplification-based next-generation sequencing system.

Vascular Ehlers-Danlos syndrome (vEDS) is a hereditary connective tissue disorder (HCTD) characterized by arterial dissection/aneurysm/rupture, sigmoid colon rupture, or uterine rupture. Diagnosis is confirmed by detecting heterozygous variants in COL3A1. This is the largest Asian case series and the first to apply an amplification-based next-generation sequencing through custom panels of causative genes for HCTDs, including a specific method of evaluating copy number variations. Among 429 patients with suspected HCTDs analyzed, 101 were suspected to have vEDS, and 33 of them (32.4%) were found to have COL3A1 variants. Two patients with a clinical diagnosis of Loeys-Dietz syndrome and/or familial thoracic aortic aneurysm and dissection were also found to have COL3A1 variants. Twenty cases (57.1%) had missense variants leading to glycine (Gly) substitutions in the triple helical domain, one (2.9%) had a missense variant leading to non-Gly substitution in this domain, eight (22.9%) had splice site alterations, three (8.6%) had nonsense variants, two (5.7%) had in-frame deletions, and one (2.9%) had a multi-exon deletion, including two deceased patients analyzed with formalin-fixed and paraffin-embedded samples. This is a clinically useful system to detect a wide spectrum of variants from various types of samples.

Non-missense variants of KCNH2 show better outcomes in type 2 long QT syndrome.

AIMS: More than one-third of type 2 long QT syndrome (LQT2) patients carry KCNH2 non-missense variants that can result in haploinsufficiency (HI), leading to mechanistic loss-of-function. However, their clinical phenotypes have not been fully investigated. The remaining two-thirds of patients harbour missense variants, and past studies uncovered that most of these variants cause trafficking deficiency, resulting in different functional changes: either HI or dominant-negative (DN) effects. In this study, we examined the impact of altered molecular mechanisms on clinical outcomes in LQT2 patients. METHODS AND RESULTS: We included 429 LQT2 patients (234 probands) carrying a rare KCNH2 variant from our patient cohort undergoing genetic testing. Non-missense variants showed shorter corrected QT (QTc) and less arrhythmic events (AEs) than missense variants. We found that 40% of missense variants in this study were previously reported as HI or DN. Non-missense and HI-groups had similar phenotypes, while both exhibited shorter QTc and less AEs than the DN-group. Based on previous work, we predicted the functional change of the unreported variants-whether they cause HI or DN via altered functional domains-and stratified them as predicted HI (pHI)- or pDN-group. The pHI-group including non-missense variants exhibited milder phenotypes compared to the pDN-group. Multivariable Cox model showed that the functional change was an independent risk of AEs (P = 0.005). CONCLUSION: Stratification based on molecular biological studies enables us to better predict clinical outcomes in the patients with LQT2.

Association between postoperative delirium and heart rate variability in the intensive care unit and readmissions and mortality in elderly patients with cardiovascular surgery.

This study aimed to investigate the relationship between heart rate variability (HRV), a parameter of the autonomic nervous system activity (ANSA), and postoperative delirium and postoperative events. This retrospective cohort study included elderly patients aged 65 years or older who were admitted to the intensive care unit (ICU) after cardiovascular surgery. ANSA was measured using HRV parameters for 1 h at daytime and 1 h at night-time before ICU discharge. The primary endpoint was the effect of HRV parameters and delirium on mortality and readmission rates within 1 year after discharge, and the secondary endpoint was the association between HRV parameters and delirium. Cox proportional hazards models were used to examine the association between HRV parameters and postoperative events by adjusting for delirium and pre and postoperative information. A total of 71 patients, 39 without delirium and 32 with delirium, met the inclusion criteria. The incidence of death and readmission within 1 year was significantly higher in the delirium group and in the group with higher daytime HF (high frequency power) and r-MSSD (square root of the squared mean of the difference of successive NN intervals), parameters of the parasympathetic nervous system activity (PNSA), than that in other groups. Furthermore, the delirium group had significantly higher HF and r-MSSD than the nondelirium group. Even after adjusting for confounding factors in the multivariate analysis, a trend of higher daytime HF and r-MSSD was observed, indicating a significant effect on the occurrence of combined events within 1 year of discharge. ICU delirium has been associated with higher daytime HF and r-MSSD, parameters of PNSA. ICU delirium was a prognostic factor, and increased daytime PNSA may worsen the prognosis of elderly patients after cardiovascular surgery.

Likely pathogenic structural variants in genetically unsolved patients with retinitis pigmentosa revealed by long-read sequencing.

Despite the successful identification of causative genes and genetic variants of retinitis pigmentosa (RP), many patients have not been molecularly diagnosed. Our recent study using targeted short-read sequencing showed that the proportion of carriers of pathogenic variants in EYS, the cause of autosomal recessive RP, was unexpectedly high in Japanese patients with unsolved RP. This result suggested that causative genetic variants, which are difficult to detect by short-read sequencing, exist in such patients. Using long-read sequencing technology (Oxford Nanopore), we analysed the whole genomes of 15 patients with RP with one heterozygous pathogenic variant in EYS detected in our previous study along with structural variants (SVs) in EYS and another 88 RP-associated genes. Two large exon-overlapping deletions involving six exons were identified in EYS in two patients with unsolved RP. An analysis of an independent patient set (n=1189) suggested that these two deletions are not founder mutations. Our results suggest that searching for SVs by long-read sequencing in genetically unsolved cases benefits the molecular diagnosis of RP.

Central extracorporeal membrane oxygenation with left-ventricular vent for fulminant myocarditis: a retrospective study.

PURPOSE: Fulminant myocarditis presents as acute severe heart failure and requires mechanical cardiocirculatory support. Left-ventricular (LV) decompression is necessary for the successful recovery of these patients. This retrospective study aimed to evaluate the functional outcomes of providing central extracorporeal membrane oxygenation (ECMO) with LV decompression for the treatment of refractory fulminant myocarditis. METHODS: Between January 2015 and February 2021, seven consecutive fulminant myocarditis patients (mean age: 41.1 ± 26.1 years) received central ECMO support with transapical LV decompression, with an 18 French cannula integrated into the ECMO circuit in a Y-fashion. The baseline characteristics and postoperative outcomes of the patients were collected. RESULTS: On admission, all patients received prior peripheral ECMO, and 85.7% (6/7) of patients received prior intra-aortic balloon pumping. However, all patients had refractory cardiogenic shock that failed prior to decompression. Six patients recovered successfully after a mean ECMO support of 20.0 ± 11.5 days and five patients had no recurrence of cardiac decompensation. The mean ICU and mean hospital stays were 36.7 ± 23.5 days and 60.6 ± 24.9 days, respectively. Hospital mortality was 28.6% (2/7). Two patients died due to sepsis and stroke during hospitalization. CONCLUSIONS: Central ECMO with an LV vent was effective for fulminant myocarditis refractory to percutaneous cardiopulmonary support therapy and other therapies.

Dominant negative effects of SCN5A missense variants.

PURPOSE: Up to 30% of patients with Brugada syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A encoding for the protein NaV1.5. Recent studies suggested that NaV1.5 can dimerize, and some variants exert dominant negative effects. In this study, we sought to explore the generality of missense variant NaV1.5 dominant negative effects and their clinical severity. METHODS: We identified 35 LoF variants (<10% of wild type [WT] peak current) and 15 partial LoF variants (10%-50% of WT peak current) that we assessed for dominant negative effects. SCN5A variants were studied in HEK293T cells, alone or in heterozygous coexpression with WT SCN5A using automated patch clamp. To assess the clinical risk, we compared the prevalence of dominant negative vs putative haploinsufficient (frameshift, splice, or nonsense) variants in a BrS consortium and the Genome Aggregation Database population database. RESULTS: In heterozygous expression with WT, 32 of 35 LoF and 6 of 15 partial LoF variants showed reduction to <75% of WT-alone peak current, showing a dominant negative effect. Individuals with dominant negative LoF variants had an elevated disease burden compared with the individuals with putative haploinsufficient variants (2.7-fold enrichment in BrS cases, P = .019). CONCLUSION: Most SCN5A missense LoF variants exert a dominant negative effect. This class of variant confers an especially high burden of BrS.

Loss-of-function mutations in cardiac ryanodine receptor channel cause various types of arrhythmias including long QT syndrome.

AIMS: Gain-of-function mutations in RYR2, encoding the cardiac ryanodine receptor channel (RyR2), cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Whereas, genotype-phenotype correlations of loss-of-function mutations remains unknown, due to a small number of analysed mutations. In this study, we aimed to investigate their genotype-phenotype correlations in patients with loss-of-function RYR2 mutations. METHODS AND RESULTS: We performed targeted gene sequencing for 710 probands younger than 16-year-old with inherited primary arrhythmia syndromes (IPAS). RYR2 mutations were identified in 63 probands, and 3 probands displayed clinical features different from CPVT. A proband with p.E4146D developed ventricular fibrillation (VF) and QT prolongation whereas that with p.S4168P showed QT prolongation and bradycardia. Another proband with p.S4938F showed short-coupled variant of torsade de pointes (scTdP). To evaluate the functional alterations in these three mutant RyR2s and p.K4594Q previously reported in a long QT syndrome (LQTS), we measured Ca2+ signals in HEK293 cells and HL-1 cardiomyocytes as well as Ca2+-dependent [3H]ryanodine binding. All mutant RyR2s demonstrated a reduced Ca2+ release, an increased endoplasmic reticulum Ca2+, and a reduced [3H]ryanodine binding, indicating loss-of-functions. In HL-1 cells, the exogenous expression of S4168P and K4594Q reduced amplitude of Ca2+ transients without inducing Ca2+ waves, whereas that of E4146D and S4938F evoked frequent localized Ca2+ waves. CONCLUSION: Loss-of-function RYR2 mutations may be implicated in various types of arrhythmias including LQTS, VF, and scTdP, depending on alteration of the channel activity. Search of RYR2 mutations in IPAS patients clinically different from CPVT will be a useful strategy to effectively discover loss-of-function RYR2 mutations.

Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts.

Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufacturing and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy, few immunological studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 weeks with no evidence of immune rejection in monkeys treated with clinically relevant doses of methylprednisolone and tacrolimus, and showed electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Additionally, transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary.

Left Coronary Cusp with Fenestration Adherent to the Aortic Wall.

An aortic valve with a coronary cusp adherent to the aortic wall is a rare anomaly. Furthermore, an adherent coronary cusp with fenestration is even rarer. Here, we report a case of aortic valve regurgitation with an adherent and fenestrated left coronary cusp. A 45-year-old man with complaints of dyspnea on exertion was hospitalized. Clinical examination revealed severe aortic valve regurgitation associated with poor cardiac function. He had a history of cardiac murmur present since childhood; however, the details of his cardiac history are unknown. During surgery, a fenestrated left coronary cusp adherent to the aortic wall was observed. Following resection of all aortic cusps, we performed an aortic valve replacement with a mechanical valve. The postoperative recovery was uneventful. In this case, the left coronary artery was perfused by a small fenestration in the adherent coronary cusp. In such cases, the adherent coronary cusp should be released to alleviate the possibility of sudden death and ischemic myocarditis.

[Successful Removal of Intrapericardial Needle using Needle Guidance System].

A 41-year-old man stuck himself with needle through his pericardium during suicide attempt. Chest radiography revealed several needles in the bilateral lung fields as well. Computed tomography (CT) and echocardiography showed massive pericardial effusion and a needle penetrating the pericardium. The patient was initially treated conservatively, including pericardial drainage, and, seven days later, we removed the needle using syngo Needle Guidance in hybrid operating room. The length of skin incision was only 2 cm, and the postoperative course was uneventful. No previous studies, to the best of our knowledge, have shown the use of syngo Needle Guidance to remove a needle in the pericardial cavity. This surgical procedure is minimally invasive for the patient.

Triiodothyronine and dexamethasone alter potassium channel expression and promote electrophysiological maturation of human-induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising tool for disease modeling and drug development. However, hiPSC-CMs remain functionally immature, which hinders their utility as a model of human cardiomyocytes. OBJECTIVE: To improve the electrophysiological maturation of hiPSC-CMs. METHODS AND RESULTS: On day 16 of cardiac differentiation, hiPSC-CMs were treated with 100 nmol/L triiodothyronine (T3) and 1 µmol/L Dexamethasone (Dex) or vehicle for 14 days. On day 30, vehicle- and T3 + Dex-treated hiPSC-CMs were dissociated and replated either as cell sheets or single cells. Optical mapping and patch-clamp technique were used to examine the electrophysiological properties of vehicle- and T3 + Dex-treated hiPSC-CMs. Compared to vehicle, T3 + Dex-treated hiPSC-CMs had a slower spontaneous beating rate, more hyperpolarized resting membrane potential, faster maximal upstroke velocity, and shorter action potential duration. Changes in spontaneous activity and action potential were mediated by decreased hyperpolarization-activated current (If) and increased inward rectifier potassium currents (IK1), sodium currents (INa), and the rapidly and slowly activating delayed rectifier potassium currents (IKr and IKs, respectively). Furthermore, T3 + Dex-treated hiPSC-CM cell sheets (hiPSC-CCSs) exhibited a faster conduction velocity and shorter action potential duration than the vehicle. Inhibition of IK1 by 100 µM BaCl2 significantly slowed conduction velocity and prolonged action potential duration in T3 + Dex-treated hiPSC-CCSs but had no effect in the vehicle group, demonstrating the importance of IK1 for conduction velocity and action potential duration. CONCLUSION: T3 + Dex treatment is an effective approach to rapidly enhance electrophysiological maturation of hiPSC-CMs.

Homology-Based Interactions between Small RNAs and Their Targets Control Dominance Hierarchy of Male Determinant Alleles of Self-Incompatibility in Arabidopsis lyrata.

Self-incompatibility (SI) is conserved among members of the Brassicaceae plant family. This trait is controlled epigenetically by the dominance hierarchy of the male determinant alleles. We previously demonstrated that a single small RNA (sRNA) gene is sufficient to control the linear dominance hierarchy in Brassica rapa and proposed a model in which a homology-based interaction between sRNAs and target sites controls the complicated dominance hierarchy of male SI determinants. In Arabidopsis halleri, male dominance hierarchy is reported to have arisen from multiple networks of sRNA target gains and losses. Despite these findings, it remains unknown whether the molecular mechanism underlying the dominance hierarchy is conserved among Brassicaceae. Here, we identified sRNAs and their target sites that can explain the linear dominance hierarchy of Arabidopsis lyrata, a species closely related to A. halleri. We tested the model that we established in Brassica to explain the linear dominance hierarchy in A. lyrata. Our results suggest that the dominance hierarchy of A. lyrata is also controlled by a homology-based interaction between sRNAs and their targets.

Genetic characteristics of retinitis pigmentosa in 1204 Japanese patients.

BACKGROUND: The genetic profile of retinitis pigmentosa (RP) in East Asian populations has not been well characterised. Therefore, we conducted a large-scale sequencing study to investigate the genes and variants causing RP in a Japanese population. METHODS: A total of 1209 Japanese patients diagnosed with typical RP were enrolled. We performed deep resequencing of 83 known causative genes of RP using next-generation sequencing. We defined pathogenic variants as those that were putatively deleterious or registered as pathogenic in the Human Gene Mutation Database or ClinVar database and had a minor allele frequency in any ethnic population of ≤0.5% for recessive genes or ≤0.01% for dominant genes as determined using population-based databases. RESULTS: We successfully sequenced 1204 patients with RP and determined 200 pathogenic variants in 38 genes as the cause of RP in 356 patients (29.6%). Variants in six genes (EYS, USH2A, RP1L1, RHO, RP1 and RPGR) caused RP in 65.4% (233/356) of those patients. Among autosomal recessive genes, two known founder variants in EYS [p.(Ser1653fs) and p.(Tyr2935*)] and four East Asian-specific variants [p.(Gly2752Arg) in USH2A, p.(Arg658*) in RP1L1, p.(Gly2186Glu) in EYS and p.(Ile535Asn) in PDE6B] and p.(Cys934Trp) in USH2A were found in ≥10 patients. Among autosomal dominant genes, four pathogenic variants [p.(Pro347Leu) in RHO, p.(Arg872fs) in RP1, p.(Arg41Trp) in CRX and p.(Gly381fs) in PRPF31] were found in ≥4 patients, while these variants were unreported or extremely rare in both East Asian and non-East Asian population-based databases. CONCLUSIONS: East Asian-specific variants in causative genes were the major causes of RP in the Japanese population.