Alfred L. George, Jr., M.D.
Jen Pan, Ph.D.
For the channelopathies, in vitro functional assessments of ion channel function by electrophysiological recordings using the patch clamp technique applied to heterologously expressed, recombinant channels has been the cornerstone of research determining the pathogenicity of variants and establishing genotype-phenotype relationships. Although patch clamp recording has been a revolutionary technique in ion channel biology, the technique in its typical embodiment has limited throughput and is extremely time and labor intensive. Hence, only highly expert laboratories have access to this technology, and there is heterogeneity of operator skill, methods and equipment creating non-standardization that hampers reproducibility. Automated patch clamp recording platforms have evolved considerably in the past several years and offer new opportunities to increase throughput, tackle larger scale projects and promote standardization. The most successful technological breakthrough has been development of planar patch clamp, which enables parallelization of recording in multi-well plates.
Project 1 is a large-scale endeavor exploiting advanced, automated patch clamp recording at two academic centers (Northwestern University, Broad Institute). Both Northwestern and the Broad Institute have the same automated patch clamp instruments (Nanion Syncropatch) with capacity to run either one (Broad) or two (Northwestern) 384-well planar patch plates. Together, we have a single run capacity of 1,152 wells, which translates into the capability to record simultaneously from >1000 cells at a time. Given that multiple runs are possible within a single day, this capacity translates into an industrial scale operation that will generate an amount of data that is inconceivable using manual electrophysiology. Indeed, a single 384-well automated patch clamp experiment, which takes 40 minutes, produces more useable data than 12 electrophysiologists working an entire day. Our combined capacity to run multiple 384-well plates per day would translate into an army of 70-80 postdocs working continuously. This enormous capacity for electrophysiological recording will enable us to determine the functional consequences of up to 1000 epilepsy-associated ion channel variants within the 5-year time frame of this Center. Preliminary studies have established a strong concordance between automated patch clamp and traditional manual patch clamp recording methods for several ion channel genes providing a high level of confidence that the data generated with have high technical validity. Adoption of this new high throughput approach for functional evaluation of genetic variants associated with epilepsy will have a transformative impact on the field and represents an especially innovative aspect of our Center proposal.
In Aim 1 we are determining the function of high priority variants in NaV and KV genes (SCN1A, SCN2A, SCN3A, SCN8A, KCNQ2, KCNQ3, KCNB1) that together represent >30% of all reported variants in epilepsy and related neurodevelopmental disorders. We have the expertise to also investigate variants in voltage-gated calcium channel genes, some of which are emerging epilepsy genes. Our efforts will enable the functional evaluation of approximately half of all currently known epilepsy-associated ion channel variants and establish a standardized experimental platform that can be exploited for studying new ion channel gene variants in the future (Aim 1). Additional functional and pharmacological studies of prototypical human variants will be performed in the orthologous murine cDNAs to validate and prioritize variants for generating mouse models (Project 3). This is important because function and drug responses can be species specific.
Aim 1 interfaces with the Mutagenesis and Cell Expression Core (Core B) to generate, validate and heterologously express the variants. Collaboration with the Variant Prioritization and Curation Core (Core A) establishes which variants have highest priority for functional evaluation. We also work with Core A to determine the impact of functional annotations on the classification and interpretation of variants within a standardized diagnostic framework, and to curate these data in publically accessible databases (e.g., ClinVar).
In Aim 2, using the same automated electrophysiological platform, we are investigating the responses of prototypical variants in each gene to antiepileptic drugs as a proof-of-concept for prioritizing anticonvulsant medication based on genotype. In Aim 3, we are undertaking a pilot study utilizing saturation mutagenesis coupled with a cell survival assay to identify all potential missense loss-of-function variants within a discrete mutation 'hot spot' region of SCN1A. This pilot study addresses the challenge caused by the massive and growing number of variants in this gene by experimentally determining all possible loss-of-function variants in a preemptive manner. A similar strategy would be applicable to other NaV channel genes in the future.
Project 1 offers an opportunity for a truly transformative impact on the field by investigating the genes with the greatest collective variant burden among the monogenic epilepsies. Results from Project 1 will directly contribute to improving the accuracy of genetic testing in epilepsy, contribute to decrypting a massive number of variants with unknown significance, and standardizing a high throughput strategy for evaluating the functional consequences of other ion channel variants in the future.
Highlights of Project 1:
• Massively parallel functional assay platforms (automated patch clamp recording)
• Functional evaluation of up to 1,000 variants will reduce VUS burden for ion channel genes
• Novel strategy (saturation mutagenesis) to predetermine all possible LOF variants in SCN1A
Parthasarathy S, et al. 2022. A recurrent de novo splice site variant involving DNM1 exon 10a causes developmental and epileptic encephalopathy through a dominant-negative mechanism. Am J Hum Genet (Dec 2022)
Seiffert S, et al. 2022. Modulating effects of FGF12 variants on Na1.2 and Na1.6 being associated with developmental and epileptic encephalopathy and Autism spectrum disorder: A case series. EBioMedicine (Sep 2022)