Functional genetic basis of KCNB1 encephalopathy

PROJECT SUMMARY Developmental and epileptic encephalopathies (DEEs) are a group of severe disorders characterized by developmental delay/intellectual disability (DD/ID), infant-onset seizures, electroencephalographic (EEG) abnormalities, and elevated mortality risk. DEEs are primarily due to monogenic variants that arise de novo in the affected child. Although each gene-based etiology is rare, collectively DEEs are estimated at 1 in 2,000 live births and represent a significant public health burden due to lifelong disability. Poor response to available treatments is characteristic of DEEs; thus, there is a significant unmet need for effective therapies. De novo pathogenic variants in genes involved in neurotransmission are a leading cause of DEEs. A growing number of voltage-gated potassium channels have been implicated, including KCNB1 that we first reported as a DEE gene in 2014. Since then, at least 150 cases have been confirmed, and over 400 variants in KCNB1 have been identified via clinical genetic testing. Additionally, the clinical spectrum has expanded. Currently, KCNB1 encephalopathy includes DD/ID accompanied by behavioral and movement disturbances, and epilepsies of varying severity, ranging from infrequent seizures to frequent seizures poorly controlled with available treatments. KCNB1 encodes the alpha subunit of the Kv2.1 voltage-gated potassium channel, which has both classical ion conducting activity and non-canonical function at ER- plasma membrane junctions (EPJ). Kv2.1 classical ion conducting activity is critical for neuronal membrane repolarization during action potential trains. Non-canonical activity occurs at EPJ in neurons where dense Kv2.1 clusters serve as non-conducting signaling hubs and facilitate activity-dependent calcium signaling that is critical for synaptic transmission. Classical and non-canonical activities of Kv2.1 contribute to neuronal excitability; thus, functional characterization of variants should assess both. Yet, functional studies to date focused only on classical ion channel activity and protein expression. Thus far, ~30 variants have been characterized with most having loss-of-function effects. However, our current knowledge is incomplete as surveyed variants were biased toward severe DEE, and clustering and EPJ function have been unexplored. We hypothesize that KCNB1 encephalopathy encompasses a range of functional defects on conducting and/or non-conducting functions. To address our hypothesis, we will: (1) determine whether KCNB1 variants are stably expressed at the plasma membrane using immunocytochemistry/flow cytometry; (2) determine how KCNB1 variants impact classical ion conducting function using high-throughput electrophysiology; and (3) determine the impact of KCNB1 variants on non-canonical function by assessing clustering and ER calcium signaling. Our proposed studies will reveal the spectrum of pathogenic mechanisms by evaluating >450 KCNB1 variants, which will provide important functional evidence to aid clinical genetic interpretation and promote development of mechanism-targeted therapeutic interventions.