Ye, Dan

Introduction: Loss-of-function (LOF) variants in the KCNH2 -encoded Kv11.1 potassium channel cause type 2 long QT syndrome (LQT2). To date, hundreds of KCNH2 missense variants (MVs) have been published as “disease-causative”. However, it has been suggested that 10% of rare LQTS variants in the literature may be "false positives”. Objective: To determine previously published KCNH2 variants that are likely false positives and warrant demotion to at least likely benign status. Methods: A list of all case-derived LQT2-associated MVs from six large published compendia was compiled. The frequency of each MV within the Genome Aggregation Database (gnomAD, n = 141,352 individuals) was recorded. Eight in silico variant assessment tools were used to grade each MV. Variants (n=8) absent in gnomAD but predicted “benign” by all 8 tools were considered potential false positives, despite being ultra-rare, and were characterized functionally using whole-cell patch clamp. Results: Overall, 339 KCNH2 MVs were identified among LQTS cases. Of these, 13 (4%) were seen at a minor allele frequency (MAF) > 0.0004 in gnomAD. This MAF cut-off is derived from the frequency of R176W-KCNH2, a well-established albeit weakly penetrant (i.e. < 20%) LQT2-causative MV. Variants seen at MAF > R176W-KCNH2 are unlikely to be LQT2-contributing. However, 253 MVs (75%) were absent in gnomAD. Of these, 8 (4%) MVs (I96V, G187S, A203T, P241L, H254Q, G314S, P935S, and P963T) were predicted by all 8 in silico tools to be benign and had never been characterized functionally. Patch clamp studies did not demonstrate any LOF perturbation for these 8 MVs. Conclusion: This study offers compelling evidence for the demotion from LQT2-causative status to likely benign status for 21 (6%) of the 339 previously published LQT2 MVs based either on i) gnomAD MAF unacceptably high for a 1:8000 disorder (LQT2) for 13 MVs or ii) ultra-rarity (never seen in gnomAD) but in silico variant assessment tools predicting benign impact of the amino acid substitution and in vitro functional validation studies devoid of LOF for 8 additional MVs. This meticulous “pruning” exercise must be conducted for all published variants previously implicated as the monogenic cause for LQTS in particular and all genetic heart diseases in general.

The ability to accurately phenotype cells differentiated from human induced pluripotent stem cells (hiPSCs) is essential for their application in modeling developmental and disease processes, yet also poses a particular challenge without the context of anatomical location. Our specific objective was to determine if single-cell gene expression was sufficient to predict the electrophysiology of iPSC-derived cardiac lineages, to evaluate the concordance between molecular and functional surrogate markers. To this end, we used the genetically encoded voltage indicator ArcLight to profile hundreds of hiPSC-derived cardiomyocytes (hiPSC-CMs), thus identifying patterns of electrophysiological maturation and increased prevalence of cells with atrial-like action potentials (APs) between days 11 and 42 of differentiation. To profile expression patterns of cardiomyocyte subtype-associated genes, single-cell RNA-seq was performed at days 12 and 40 after the populations were fully characterized with the high-throughput ArcLight platform. Although we could detect global gene expression changes supporting progressive differentiation, individual cellular expression patterns alone were not able to delineate the individual cardiomyocytes into atrial, ventricular, or nodal subtypes as functionally documented by electrophysiology measurements. Furthermore, our efforts to understand the distinct electrophysiological properties associated with day 12 versus day 40 hiPSC-CMs revealed that ion channel regulators SLMAP , FGF12 , and FHL1 were the most significantly increased genes at day 40, categorized by electrophysiology-related gene functions. Notably, FHL1 knockdown during differentiation was sufficient to significantly modulate APs toward ventricular-like electrophysiology. Thus, our results establish the inability of subtype-associated gene expression patterns to specifically categorize hiPSC-derived cells according to their functional electrophysiology, and yet, altered FHL1 expression is able to redirect electrophysiological maturation of these developing cells. Therefore, noncanonical gene expression patterns of cardiac maturation may be sufficient to direct functional maturation of cardiomyocytes, with canonical gene expression patterns being insufficient to temporally define cardiac subtypes of in vitro differentiation.

Abstract Cholangiocarcinomas are rare but aggressive cancers, with an increasing incidence and a dismal 5-year survival rate of less than 5%. Mutations in the genes encoding isocitrate dehydrogenase, IDH1 and IDH2, have been reported in ∼70% of gliomas, ∼20% of myeloid leukemias, ∼56% of chondrosarcomas, and ∼10% of melanomas. We discovered IDH1 and IDH2 mutations in 29 of 305 (9%) intrahepatic cholangiocarcinomas. Tumors with mutations in IDH1 or IDH2 had lower 5-hydroxymethylcytosine and higher 5-methylcytosine levels, as well as increased dimethylation of histone H3K79. Mutations in IDH1 or IDH2 were independently associated with longer time to recurrence of cholangiocarcinoma in multivariate analysis (p = 0.046). We identified 2,267 genes that were significantly hypermethylated in tumors with mutations in IDH1 or IDH2. Hypermethylated CpG sites were significantly enriched in CpG shores and in the 1500 bp upstream of annotated transcription start sites, suggesting a global regulation of transcriptional potential. Gene expression profiling of 7 IDH1/2-mutant and 20 IDH1/2-wildtype tumors revealed increased expression of citric acid cycle and oxidative phosphorylation enzymes, as well as decreased expression of cytoskeleton organization genes. Integrated analyses of DNA methylation and gene expression identified interleukin-6 and several of its downstream targets as candidates for silencing by DNA methylation. Conclusions: Cholangiocarcinomas with mutations in IDH1 or IDH2 represent a molecular subclass with distinct signaling pathway alterations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1058. doi:1538-7445.AM2012-1058