Expanded clinical phenotype spectrum correlates with variant function in SCN2A-related disorders.

SCN2A-related disorders secondary to altered function in the voltage-gated sodium channel NaV1.2 are rare with clinically heterogeneous expressions that include epilepsy, autism, and multiple severe to profound impairments and other conditions. To advance understanding of the clinical phenotypes and their relation to channel function, 81 patients (36, 44% female, median age 5.4 years) with 69 unique SCN2A variants were systematically phenotyped and their NaV1.2 channel function systematically assessed. Participants were recruited through the FamileSCN2A Foundation. Primary phenotype (epilepsy of neonatal-onset, N=27; infant onset, N=18; and later onset N=24; and autism without seizures, (N=12) was strongly correlated with a non-seizure severity index (p=0.002), which was based on presence of severe impairments in gross motor, fine motor, communication abilities, gastrostomy tube dependence, and diagnosis of cortical visual impairment and scoliosis. Non-seizure severity was greatest in the neonatal-onset group and least in the autism group (p=0.002). Children with the lowest severity indices were still severely impaired, as reflected by an average Vineland adaptive behavior composite score of 49.5 (>3 SD below the test's norm-referenced mean). Epileptic spasms were significantly more common in infant onset (67%) than in neonatal (22%) or later-onset (29%) epilepsy (p=0.007). Primary phenotype also strongly correlated with variant function (p<0.0001); gain of function and mixed function variants predominated in neonatal-onset epilepsy, shifting to moderate loss of function in infant-onset epilepsy, and severe and complete loss of function in later-onset epilepsy and autism groups. Exploratory cluster analysis identified five groups representing (1) primarily later-onset epilepsy with moderate loss of function variants and low severity indices, (2) mostly infant-onset epilepsy with moderate loss of function variants but higher severity indices, (3) late-onset and autism only with the lowest severity indices (mostly 0) and severe/complete loss of function variants. Two exclusively neonatal clusters were distinguished from each other largely on non-seizure severity scores and secondarily on variant function. The relation between primary phenotype and variant function emphasizes the role of developmental factors in the differential clinical expression of SCN2A variants based on their effects on NaV1.2 channel function. The non-seizure severity of SCN2A disorders depends on a combination of the age at seizure onset (primary phenotype) and variant function. As precision therapies for SCN2A-related disorders advance toward clinical trials, knowledge of the relationship between variant function and clinical disease expression will be valuable for identifying appropriate patients for these trials and in selecting efficient clinical outcomes.

A novel transcription factor combination for direct reprogramming to a spontaneously contracting human cardiomyocyte-like state.

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+ cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach.

Sox10-cre BAC transgenes reveal temporal restriction of mesenchymal cranial neural crest and identify glandular Sox10 expression.

Diversity of neural crest derivatives has been studied with a variety of approaches during embryonic development. In mammals Cre-LoxP lineage tracing is a robust means to fate map neural crest relying on cre driven from regulatory elements of early neural crest genes. Sox10 is an essential transcription factor for normal neural crest development. A variety of efforts have been made to label neural crest derivatives using partial Sox10 regulatory elements to drive cre expression. To date published Sox10-cre lines have focused primarily on lineage tracing in specific tissues or during early fetal development. We describe two new Sox10-cre BAC transgenes, constitutive (cre) and inducible (cre/ERT2), that contain the complete repertoire of Sox10 regulatory elements. We present a thorough expression profile of each, identifying a few novel sites of Sox10 expression not captured by other neural crest cre drivers. Comparative mapping of expression patterns between the Sox10-cre and Sox10-cre/ERT2 transgenes identified a narrow temporal window in which Sox10 expression is present in mesenchymal derivatives prior to becoming restricted to neural elements during embryogenesis. In more caudal structures, such as the intestine and lower urinary tract, our Sox10-cre BAC transgene appears to be more efficient in labeling neural crest-derived cell types than Wnt1-cre. The analysis reveals consistent expression of Sox10 in non-neural crest derived glandular epithelium, including salivary, mammary, and urethral glands of adult mice. These Sox10-cre and Sox10-cre/ERT2 transgenic lines are verified tools that will enable refined temporal and cell-type specific lineage analysis of neural crest derivatives as well as glandular tissues that rely on Sox10 for proper development and function.

Cryptic prokaryotic promoters explain instability of recombinant neuronal sodium channels in bacteria.

Mutations in genes encoding the human-brain-expressed voltage-gated sodium (NaV) channels NaV1.1, NaV1.2, and NaV1.6 are associated with a variety of human diseases including epilepsy, autism spectrum disorder, familial migraine, and other neurodevelopmental disorders. A major obstacle hindering investigations of the functional consequences of brain NaV channel mutations is an unexplained instability of the corresponding recombinant complementary DNA (cDNA) when propagated in commonly used bacterial strains manifested by high spontaneous rates of mutation. Here, using a combination of in silico analysis, random and site-directed mutagenesis, we investigated the cause for instability of human NaV1.1 cDNA. We identified nucleotide sequences within the NaV1.1 coding region that resemble prokaryotic promoter-like elements, which are presumed to drive transcription of translationally toxic mRNAs in bacteria as the cause of the instability. We further demonstrated that mutations disrupting these elements mitigate the instability. Extending these observations, we generated full-length human NaV1.1, NaV1.2, and NaV1.6 plasmids using one or two introns that interrupt the latent reading frames along with a minimum number of silent nucleotide changes that achieved stable propagation in bacteria. Expression of the stabilized sequences in cultured mammalian cells resulted in functional NaV channels with properties that matched their parental constructs. Our findings explain a widely observed instability of recombinant neuronal human NaV channels, and we describe re-engineered plasmids that attenuate this problem.

KNa1.1 gain-of-function preferentially dampens excitability of murine parvalbumin-positive interneurons.

KCNT1 encodes the sodium-activated potassium channel KNa1.1, expressed preferentially in the frontal cortex, hippocampus, cerebellum, and brainstem. Pathogenic missense variants in KCNT1 are associated with intractable epilepsy, namely epilepsy of infancy with migrating focal seizures (EIMFS), and sleep-related hypermotor epilepsy (SHE). In vitro studies of pathogenic KCNT1 variants support predominantly a gain-of-function molecular mechanism, but how these variants behave in a neuron or ultimately drive formation of an epileptogenic circuit is an important and timely question. Using CRISPR/Cas9 gene editing, we introduced a gain-of-function variant into the endogenous mouse Kcnt1 gene. Compared to wild-type (WT) littermates, heterozygous and homozygous knock-in mice displayed greater seizure susceptibility to the chemoconvulsants kainate and pentylenetetrazole (PTZ), but not to flurothyl. Using acute slice electrophysiology in heterozygous and homozygous Kcnt1 knock-in and WT littermates, we demonstrated that CA1 hippocampal pyramidal neurons exhibit greater amplitude of miniature inhibitory postsynaptic currents in mutant mice with no difference in frequency, suggesting greater inhibitory tone associated with the Kcnt1 mutation. To address alterations in GABAergic signaling, we bred Kcnt1 knock-in mice to a parvalbumin-tdTomato reporter line, and found that parvalbumin-expressing (PV+) interneurons failed to fire repetitively with large amplitude current injections and were more prone to depolarization block. These alterations in firing can be recapitulated by direct application of the KNa1.1 channel activator loxapine in WT but are occluded in knock-in littermates, supporting a direct channel gain-of-function mechanism. Taken together, these results suggest that KNa1.1 gain-of-function dampens interneuron excitability to a greater extent than it impacts pyramidal neuron excitability, driving seizure susceptibility in a mouse model of KCNT1-associated epilepsy.

Nilotinib-induced alterations in endothelial cell function recapitulate clinical vascular phenotypes independent of ABL1.

Nilotinib is a highly effective treatment for chronic myeloid leukemia but has been consistently associated with the development of nilotinib-induced arterial disease (NAD) in a subset of patients. To date, which cell types mediate this effect and whether NAD results from on-target mechanisms is unknown. We utilized human induced pluripotent stem cells (hiPSCs) to generate endothelial cells and vascular smooth muscle cells for in vitro study of NAD. We found that nilotinib adversely affects endothelial proliferation and migration, in addition to increasing intracellular nitric oxide. Nilotinib did not alter endothelial barrier function or lipid uptake. No effect of nilotinib was observed in vascular smooth muscle cells, suggesting that NAD is primarily mediated through endothelial cells. To evaluate whether NAD results from enhanced inhibition of ABL1, we generated multiple ABL1 knockout lines. The effects of nilotinib remained unchanged in the absence of ABL1, suggesting that NAD results from off- rather than on-target signaling. The model established in the present study can be applied to future mechanistic and patient-specific pharmacogenomic studies.

Independent compartmentalization of functional, metabolic, and transcriptional maturation of hiPSC-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) recapitulate numerous disease and drug response phenotypes, but cell immaturity may limit their accuracy and fidelity as a model system. Cell culture medium modification is a common method for enhancing maturation, yet prior studies have used complex media with little understanding of individual component contribution, which may compromise long-term hiPSC-CM viability. Here, we developed high-throughput methods to measure hiPSC-CM maturation, determined factors that enhanced viability, and then systematically assessed the contribution of individual maturation medium components. We developed a medium that is compatible with extended culture. We discovered that hiPSC-CM maturation can be sub-specified into electrophysiological/EC coupling, metabolism, and gene expression and that induction of these attributes is largely independent. In this work, we establish a defined baseline for future studies of cardiomyocyte maturation. Furthermore, we provide a selection of medium formulae, optimized for distinct applications and priorities, that promote measurable attributes of maturation.

Molecular and cellular context influences SCN8A variant function.

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

Epilepsy-associated SCN2A (Na V 1.2) Variants Exhibit Diverse and Complex Functional Properties.

Pathogenic variants in neuronal voltage-gated sodium (Na V ) channel genes including SCN2A , which encodes Na V 1.2, are frequently discovered in neurodevelopmental disorders with and without epilepsy. SCN2A is also a high confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A variants yielded a paradigm in which predominantly gain-of-function (GoF) variants cause epilepsy whereas loss-of-function (LoF) variants are associated with ASD and ID. However, this framework is based on a limited number of functional studies conducted under heterogenous experimental conditions whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of more than 30 SCN2A variants using automated patch clamp recording to assess the analytical validity of this approach and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common population variants using two distinct alternatively spliced forms of Na V 1.2 that were heterologously expressed in HEK293T cells. Multiple biophysical parameters were assessed on 5,858 individual cells. We found that automated patch clamp recording provided a valid high throughput method to ascertain detailed functional properties of Na V 1.2 variants with concordant findings for a subset of variants that were previously studied using manual patch clamp. Additionally, many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-function properties that are difficult to classify overall by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of a larger number of variants, greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor valuable for accurate assessment of Na V channel variant dysfunction. Together, this approach will enhance our ability to discern relationships between variant channel dysfunction and neurodevelopmental disorders.

Epilepsy-associated SCN2A (NaV1.2) variants exhibit diverse and complex functional properties.

Pathogenic variants in voltage-gated sodium (NaV) channel genes including SCN2A, encoding NaV1.2, are discovered frequently in neurodevelopmental disorders with or without epilepsy. SCN2A is also a high-confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A variants yielded a paradigm in which predominantly gain-of-function variants cause neonatal-onset epilepsy, whereas loss-of-function variants are associated with ASD and ID. However, this framework was derived from a limited number of studies conducted under heterogeneous experimental conditions, whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of SCN2A variants using automated patch-clamp recording to demonstrate the validity of this method and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common variants using two alternatively spliced isoforms of NaV1.2 expressed in HEK293T cells. Automated patch-clamp recording provided a valid high throughput method to ascertain detailed functional properties of NaV1.2 variants with concordant findings for variants that were previously studied using manual patch clamp. Many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-functions that are difficult to classify by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of variants with greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor. This approach offers an enhanced ability to discern relationships between channel dysfunction and neurodevelopmental disorders.

Sex and Gene Influence Arrhythmia Susceptibility in Murine Models of Calmodulinopathy.

BACKGROUND: Pathogenic variants in genes encoding CaM (calmodulin) are associated with a life-threatening ventricular arrhythmia syndrome (calmodulinopathy). The in vivo consequences of CaM variants have not been studied extensively and there is incomplete understanding of the genotype-phenotype relationship for recurrent variants. We investigated effects of different factors on calmodulinopathy phenotypes using 2 mouse models with a recurrent pathogenic variant (N98S) in Calm1 or Calm2. METHODS: Genetically engineered mice with heterozygous N98S pathogenic variants in Calm1 or Calm2 were generated. Differences between the sexes and affected genes were assessed using multiple physiological assays at the cellular and whole animal levels. Statistical significance among groups was evaluated using 1-way ANOVA or the Kruskal-Wallis test when data were not normally distributed. RESULTS: Calm1N98S/+ (Calm1S/+) or Calm2N98S/+ (Calm2S/+) mice exhibited sinus bradycardia and were more susceptible to arrhythmias after exposure to epinephrine and caffeine. Male Calm1S/+ mice had the most severe arrhythmia phenotype with evidence of early embryonic lethality, greater susceptibility for arrhythmic events, frequent premature beats, corrected QT prolongation, and more heart rate variability after epinephrine and caffeine than females with the same genotype. Calm2 S/+ mice exhibited a less severe phenotype, with female Calm2 S/+ mice having the least severe arrhythmia susceptibility. Flecainide was not effective in preventing arrhythmias in heterozygous CaM-N98S mice. Intracellular Ca2+ transients observed in isolated ventricular cardiomyocytes from male heterozygous CaM-N98S mice had lower peak amplitudes and slower sarcoplasmic reticulum Ca2+ release following in vitro exposure to epinephrine and caffeine, which were not observed in cardiomyocytes from heterozygous female CaM-N98S mice. CONCLUSIONS: We report heterogeneity in arrhythmia susceptibility and cardiomyocyte Ca2+ dynamics among male and female mice heterozygous for a recurrent pathogenic variant in Calm1 or Calm2, illustrating a complex calmodulinopathy phenotype in vivo. Further investigation of sex and genetic differences may help identify the molecular basis for this heterogeneity.

Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing.

It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.

A Novel Transcription Factor Combination for Direct Reprogramming to a Spontaneously Contracting Human Cardiomyocyte-like State.

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4,960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD , SMAD6 , and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2 + cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach. HIGHLIGHTS: Using network-based algorithm Mogrify, acoustic liquid handling, and high-content kinetic imaging cytometry we screened the effect of 4,960 unique transcription factor combinations. Using 24 patient-specific human fibroblast samples we identified the combination of MYOCD , SMAD6 , and TBX20 (MST) as the most successful direct reprogramming combination. MST cocktail results in reprogrammed cells with spontaneous contraction, cardiomyocyte-like calcium transients, and expression of cardiomyocyte associated genes.

Molecular and Cellular Context Influences SCN8A Variant Function.

Pathogenic variants in SCN8A , which encodes the voltage-gated sodium (Na V ) channel Na V 1.6, are associated with neurodevelopmental disorders including epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by the use of a neonatal-expressed alternatively spliced isoform of Na V 1.6 (Na V 1.6N), and engineered mutations to render the channel tetrodotoxin (TTX) resistant. In this study, we investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in two developmentally regulated splice isoforms (Na V 1.6N, Na V 1.6A). We employed automated patch clamp recording to enhance throughput, and developed a novel neuronal cell line (ND7/LoNav) with low levels of endogenous Na V current to obviate the need for TTX-resistance mutations. Expression of Na V 1.6N or Na V 1.6A in ND7/LoNav cells generated Na V currents that differed significantly in voltage-dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared to the corresponding wild-type (WT) channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

Functional evaluation of human ion channel variants using automated electrophysiology.

Patch clamp recording enabled a revolution in cellular electrophysiology, and is useful for evaluating the functional consequences of ion channel gene mutations or variants associated with human disorders called channelopathies. However, due to massive growth of genetic testing in medical practice and research, the number of known ion channel variants has exploded into the thousands. Fortunately, automated methods for performing patch clamp recording have emerged as important tools to address the explosion in ion channel variants. In this chapter, we present our approach to harnessing automated electrophysiology to study a human voltage-gated potassium channel gene (KCNQ1), which harbors hundreds of mutations associated with genetic disorders of heart rhythm including the congenital long-QT syndrome. We include protocols for performing high efficiency electroporation of heterologous cells with recombinant KCNQ1 plasmid DNA and for automated planar patch recording including data analysis. These methods can be adapted for studying other voltage-gated ion channels.

Functional and pharmacological evaluation of a novel SCN2A variant linked to early-onset epilepsy.

OBJECTIVE: We identified a novel de novo SCN2A variant (M1879T) associated with infantile-onset epilepsy that responded dramatically to sodium channel blocker antiepileptic drugs. We analyzed the functional and pharmacological consequences of this variant to establish pathogenicity, and to correlate genotype with phenotype and clinical drug response. METHODS: The clinical and genetic features of an infant boy with epilepsy are presented. We investigated the effect of the variant using heterologously expressed recombinant human NaV 1.2 channels. We performed whole-cell patch clamp recording to determine the functional consequences and response to carbamazepine. RESULTS: The M1879T variant caused disturbances in channel inactivation including substantially depolarized voltage dependence of inactivation, slower time course of inactivation, and enhanced resurgent current that collectively represent a gain-of-function. Carbamazepine partially normalized the voltage dependence of inactivation and produced use-dependent block of the variant channel at high pulsing frequencies. Carbamazepine also suppresses resurgent current conducted by M1879T channels, but this effect was explained primarily by reducing the peak transient current. Molecular modeling suggests that the M1879T variant disrupts contacts with nearby residues in the C-terminal domain of the channel. INTERPRETATION: Our study demonstrates the value of conducting functional analyses of SCN2A variants of unknown significance to establish pathogenicity and genotype-phenotype correlations. We also show concordance of in vitro pharmacology using heterologous cells with the drug response observed clinically in a case of SCN2A-associated epilepsy.

Negligible-Cost and Weekend-Free Chemically Defined Human iPSC Culture.

Human induced pluripotent stem cell (hiPSC) culture has become routine, yet the cost of pluripotent cell media, frequent medium changes, and the reproducibility of differentiation have remained restrictive. Here, we describe the formulation of a hiPSC culture medium (B8) as a result of the exhaustive optimization of medium constituents and concentrations, establishing the necessity and relative contributions of each component to the pluripotent state and cell proliferation. The reagents in B8 represent only 3% of the costs of commercial media, made possible primarily by the in-lab generation of three E. coli-expressed, codon-optimized recombinant proteins: fibroblast growth factor 2, transforming growth factor ß3, and neuregulin 1. We demonstrate the derivation and culture of 34 hiPSC lines in B8 as well as the maintenance of pluripotency long term (over 100 passages). This formula also allows a weekend-free feeding schedule without sacrificing capacity for differentiation.

High-Throughput Functional Evaluation of KCNQ1 Decrypts Variants of Unknown Significance.

BACKGROUND: The explosive growth in known human gene variation presents enormous challenges to current approaches for variant classification that have implications for diagnosis and treatment of many genetic diseases. For disorders caused by mutations in cardiac ion channels as in congenital arrhythmia syndromes, in vitro electrophysiological evidence has high value in discriminating pathogenic from benign variants, but these data are often lacking because assays are cost, time, and labor intensive. METHODS: We implemented a strategy for performing high-throughput functional evaluations of ion channel variants that repurposed an automated electrophysiological recording platform developed previously for drug discovery. RESULTS: We demonstrated the success of this approach by evaluating 78 variants in KCNQ1, a major gene involved in genetic disorders of cardiac arrhythmia susceptibility. We benchmarked our results with traditional electrophysiological approaches and observed a high level of concordance. This strategy also enabled studies of dominant-negative behavior of variants exhibiting severe loss-of-function. Overall, our results provided functional data useful for reclassifying >65% of the studied KCNQ1 variants. CONCLUSIONS: Our results illustrate an efficient and high-throughput paradigm linking genotype to function for a human cardiac ion channel that will enable data-driven classification of large numbers of variants and create new opportunities for precision medicine.