Personalized structural biology reveals the molecular mechanisms underlying heterogeneous epileptic phenotypes caused by de novo KCNC2 variants

Souhrid Mukherjee; Thomas A Cassini; Ningning Hu; Tao Yang; Bian Li; Wangzhen Shen; Christopher W Moth; David C Rinker; Jonathan H Sheehan; Joy D Cogan; Undiagnosed Diseases Network; John H Newman; Rizwan Hamid; Robert L Macdonald; Dan M Roden; Jens Meiler; Georg Kuenze; John A Phillips; John A Capra

doi:10.1016/j.xhgg.2022.100131

Personalized structural biology reveals the molecular mechanisms underlying heterogeneous epileptic phenotypes caused by de novo KCNC2 variants

HGG Adv. 2022 Jul 19;3(4):100131. doi: 10.1016/j.xhgg.2022.100131. eCollection 2022 Oct 13.

Authors

Souhrid Mukherjee^{1

2}, Thomas A Cassini³, Ningning Hu^{4

5}, Tao Yang⁶, Bian Li^{1

6

2}, Wangzhen Shen⁴, Christopher W Moth², David C Rinker^{7

2}, Jonathan H Sheehan^{2

8}, Joy D Cogan⁹; Undiagnosed Diseases Network; John H Newman¹⁰, Rizwan Hamid⁹, Robert L Macdonald^{6

4}, Dan M Roden^{6

11

12}, Jens Meiler^{7

6

2

11

13

14

15}, Georg Kuenze^{7

2

13}, John A Phillips⁹, John A Capra^{1

2

11

12

16}

Affiliations

¹ Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA.
² Center for Structural Biology, Vanderbilt University, Nashville, TN 37235, USA.
³ Department of Internal Medicine, National Institutes of Health Clinical Center, Bethesda, MD 20814, USA.
⁴ Department of Neurology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
⁵ Department of Genetic Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
⁶ Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
⁷ Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA.
⁸ John T. Milliken Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
⁹ Department of Pediatrics, Division of Medical Genetics and Genomic Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
¹⁰ Pulmonary Hypertension Center, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
¹¹ Department of Biomedical Informatics, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
¹² Vanderbilt Genetics Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
¹³ Institute for Drug Discovery, Leipzig University Medical School, Leipzig, SAC 04103, Germany.
¹⁴ Department of Chemistry, Leipzig University, Leipzig, SAC 04109, Germany.
¹⁵ Department of Computer Science, Leipzig University, Leipzig, SAC 04109, Germany.
¹⁶ Bakar Computational Health Sciences Institute and Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, CA 94143, USA.

Abstract

Whole-exome sequencing (WES) in the clinic has identified several rare monogenic developmental and epileptic encephalopathies (DEE) caused by ion channel variants. However, WES often fails to provide actionable insight for rare diseases, such as DEEs, due to the challenges of interpreting variants of unknown significance (VUS). Here, we describe a "personalized structural biology" (PSB) approach that leverages recent innovations in the analysis of protein 3D structures to address this challenge. We illustrate this approach in an Undiagnosed Diseases Network (UDN) individual with DEE symptoms and a de novo VUS in KCNC2 (p.V469L), the Kv3.2 voltage-gated potassium channel. A nearby KCNC2 variant (p.V471L) was recently suggested to cause DEE-like phenotypes. Computational structural modeling suggests that both affect protein function. However, despite their proximity, the p.V469L variant is likely to sterically block the channel pore, while the p.V471L variant is likely to stabilize the open state. Biochemical and electrophysiological analyses demonstrate heterogeneous loss-of-function and gain-of-function effects, as well as differential response to 4-aminopyridine treatment. Molecular dynamics simulations illustrate that the pore of the p.V469L variant is more constricted, increasing the energetic barrier for K⁺ permeation, whereas the p.V471L variant stabilizes the open conformation. Our results implicate variants in KCNC2 as causative for DEE and guide the interpretation of a UDN individual. They further delineate the molecular basis for the heterogeneous clinical phenotypes resulting from two proximal pathogenic variants. This demonstrates how the PSB approach can provide an analytical framework for individualized hypothesis-driven interpretation of protein-coding VUS.

Keywords: DEE; KCNC2; Undiagnosed Diseases Network; de novo variant; developmental and epileptic encephalopathy; electrophysiology; molecular dynamics simulations; personalized structural biology; rare disease; variant interpretation.