Neurodevelopmental and Epilepsy Phenotypes in
Individuals With Missense Variants in the
Voltage-Sensing and Pore Domains of KCNH5

Hannah C Happ, Lynette G Sadleir, Matthew Zemel, Guillem de
Valles-Ibáñez, Michael S Hildebrand, Allyn Mcconkie-Rosell, Marie Mcdonald,
Halie May, Tristan Sands, Vimla Aggarwal, et al.

► To cite this version:

Hannah C Happ, Lynette G Sadleir, Matthew Zemel, Guillem de Valles-Ibáñez, Michael S Hildebrand, et al. Neurodevelopmental and Epilepsy Phenotypes in Individuals With Missense Variants in the Voltage-Sensing and Pore Domains of KCNH5. Neurology, 2023, 100 (6), pp.e603-e615. 10.1212/WNL.0000000000201492. hal-04133606

HAL Id: hal-04133606

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Submitted on 20 Jun 2023

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<figure><img src="image_1.png" /></figure>
# Neurodevelopmental and Epilepsy Phenotypes in Individuals With Missense Variants in the Voltage-Sensing and Pore Domains of KCNH5

Hannah C. Happ, BA,* Lynette G. Sadleir, MBChB, MD,* Matthew Zemel, DO, Guillem de Valles-Ibáñez, PhD, Michael S. Hildebrand, PhD, Allyn McConkie-Rosell, PhD, Marie McDonald, MD, Halie May, CGC, Tristan Sands, MD, Vimla Aggarwal, MBBS, Christopher Elder, MD, Timothy Feyma, MD, Allan Bayat, MD, Rikke S. Møller, PhD, Christina D. Fenger, PhD, Jens Erik Klint Nielsen, MD, Anita N. Datta, MD, Kathleen M. Gorman, MD, Mary D. King, MD, Natalia D. Linhares, PhD, Barbara K. Burton, MD, Andrea Paras, MS, Sian Ellard, PhD, Julia Rankin, MD, Anju Shukla, MBBS, Purvi Majethia, Rory J. Olson, MD, Karthik Muthusamy, MBBS, Lisa A. Schimmenti, MD, Keith Starnes, MD, Lucie Sedláčková, PhD, Katalin Štěrbová, MD, Markéta Vlčková, PhD, Petra Laššuthová, PhD, Alena Jahodová, PhD, Brenda E. Porter, MD, PhD, Nathalie Couque, MD, Estelle Colin, Clément Prouteau, MD, Corinne Collet, Thomas Smol, MD, Roseline Caumes, MD, Fleur Vansenne, MD, PhD, Francesca Bisulli, MD, Laura Licchetta, MD, PhD, Richard Person, PhD, Erin Torti, MS, Kirsty McWalter, CGC, Richard Webster, MD, Elizabeth E. Gerard, MD, Gaetan Lesca, MD, PhD, Pierre Szepetowski, MD, PhD, Ingrid E. Scheffer, PhD, Heather C. Mefford, MD, PhD,† and Gemma L. Carvill, PhD†

Neurology® 2023;100:e603-e615. doi:10.1212/WNL.0000000000201492

**Correspondence**

Dr. Carvill
gemma.carvill@
northwestern.edu

## Abstract

### Background and Objectives

KCNH5 encodes the voltage-gated potassium channel EAG2/Kv10.2. We aimed to delineate the neurodevelopmental and epilepsy phenotypic spectrum associated with de novo KCNH5 variants.

### Methods

We screened 893 individuals with developmental and epileptic encephalopathies for KCNH5 variants using targeted or exome sequencing. Additional individuals with KCNH5 variants were identified through an international collaboration. Clinical history, EEG, and imaging data were analyzed; seizure types and epilepsy syndromes were classified. We included 3 previously published individuals including additional phenotypic details.

From the Ken and Ruth Davee Department of Neurology (K.C.H., E.E.G., G.L.C.), Northwestern University Feinberg School of Medicine, Chicago, IL; University of Otago (L.G.S.), Wellington, New Zealand; University of Washington (M.Z.), Seattle; Department of Medicine (G.d.V.-I., R.W., I.E.S.), Epilepsy Research Centre, The University of Melbourne, Austin Health, Heidelberg, Victoria, Australia; Duke University Medical Center (A.M.-R., M.M.), Durham, NC; Institute for Genomic Medicine (H.M., T.S.), Columbia University Irving Medical Center, New York, NY; Departments of Pathology and Cell Biology (V.A.), and Neurology (C.E.), Columbia University Irving Medical Center, New York, NY; Gillette Children's Specialty Healthcare (T.F.), St. Paul, MN; Department of Epilepsy Genetics and Personalized Medicine (A.B., R.S.M., C.D.F.), Danish Epilepsy Center, Dianalund, Denmark; Institute of Regional Health Research (A.B., R.S.M.), University of Southern Denmark; Amplexa Genetics (C.D.F.), Odense, Denmark; Department of Clinical Medicine (J.E.K.N.), Zealand University Hospital, Roskilde, Denmark; University of British Columbia (A.N.D.), Vancouver, Canada; The Department of Neurology and Clinical Neurophysiology (K.M.G., M.D.K.), Children's Health Ireland at Temple St., Dublin 1, Ireland; School of Medicine and Medical Science (K.M.G., M.D.K.), University College Dublin, Ireland; Genuity Science (N.L.), Dublin, Ireland; Ann & Robert H. Lurie Children's Hospital of Chicago (B.K.B., A.P.), Chicago, IL; Department of Pediatrics (B.K.B., A.P., G.L.C.), Northwestern University Feinberg School of Medicine, Chicago, IL; Exeter Genomics Laboratory (S.E.), Royal Devon University Healthcare NHS Foundation Trust, Exeter, United Kingdom; Institute of Clinical and Biomedical Science (S.E.), University of Exeter, United Kingdom; Department Clinical Genetics (J.R.), Royal Devon University Healthcare NHS Foundation Trust, Exeter, United Kingdom; Department of Medical Genetics (A.S., P.M.), Kasturba Medical College, Manipal, Manipal Academy of Higher Education, India; Center for Individualized Medicine (R.J.O., K.M., L.A.S.), Mayo Clinic, Rochester, MN; Departments of Clinical Genomics (K.M., L.A.S.), and Neurology (K.S.), Mayo Clinic, Rochester, MN; Neurogenetic Laboratory (L.S., P.J.), Department of Pediatric Neurology, Second Faculty of Medicine, Charles University in Prague and Motol University Hospital, Czech Republic; Epilepsy Research Centre Prague—EpiReC Consortium (L.S., K.S., M.V., P.L., A.J.); Motol University Hospital is a full member of the ERN EpICARE; Department of Pediatric Neurology (K.S., A.J.), Second Faculty of Medicine, Charles University in Prague and Motol University Hospital, Czech Republic; Biology and Medical Genetics (M.V.), Second Faculty of Medicine, Charles University in Prague and Motol University Hospital, Czech Republic; Stanford University School of Medicine (B.E.P.), Palo Alto, CA; Laboratoire de Biologie médicale multisites Seqoia-FMG2025 (N.C., C.C.), Laboratoire Génétique Moléculaire Robert-Debré, Paris, France; Service de Génétique (E.C., C.P.), CHU d'Angers, Angers, France; University Lille (T.S.), CHU Lille, ULR7364—RADEME, Institut de Genetique Médicale, France; University Lille (R.C.), CHU Lille, ULR7364—RADEME, Clinique de Genetique, France; Univeristy Medical Center Groningen (F.V.), Groningen, the Netherlands; Department of Biomedical and NeuroMotor Sciences (F.B.), University of Bologna, Italy; IRCCS Istituto delle Scienze Neurologiche di Bologna (F.B., L.L.), Full Member of the ERN EpICARE Bologna, Italy; GeneDx (R.P., E.T., K.M.), Gaithersburg, MD; T.Y. Nelson Department of Neurology and Neurosurgery (R.W.), Children's Hospital at Westmead, Westmead, New South Wales, Australia; Department of Medical Genetics (G.L.), University Hospital of Lyon, Claude Bernard Lyon 1 University, France; INSERM, Aix-Marseille University (P.S.), INMED, France; Department of Neurology (I.E.S.), Royal Children's Hospital, Department of Paediatrics, The University of Melbourne, and Murdoch Children's Research Institute, Parkville, Victoria, Australia; The Florey Institute of Neuroscience and Mental Health (I.E.S.), Victoria, Australia; Center for Pediatric Neurological Disease Research (H.C.M.), St. Jude Children's Research Hospital, Memphis, TN; and Department of Pharmacology (G.L.C.), Northwestern University Feinberg School of Medicine, Chicago, IL.

Go to Neurology.org/N for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.

<footnote>*These authors contributed equally as first authors.</footnote>

<footnote>†These authors contributed equally as senior authors.</footnote>
# Glossary

**ACMG** = American College of Medical Genetics and Genomics; **DEE** = developmental and epileptic encephalopathy; **EAG** = ether-a-go-go; **ID** = intellectual disability; **GOF** = gain of function; **MIP** = molecular inversion probe; **NDD** = neurodevelopmental disorder; **VUS** = variants of uncertain significance.

# Results

We report a cohort of 17 patients, including 9 with a recurrent de novo missense variant p.Arg327His, 4 with a recurrent missense variant p.Arg333His, and 4 additional novel missense variants. All variants were located in or near the functionally critical voltage-sensing or pore domains, absent in the general population, and classified as pathogenic or likely pathogenic using the American College of Medical Genetics and Genomics criteria. All individuals presented with epilepsy with a median seizure onset at 6 months. They had a wide range of seizure types, including focal and generalized seizures. Cognitive outcomes ranged from normal intellect to profound impairment. Individuals with the recurrent p.Arg333His variant had a self-limited drug-responsive focal or generalized epilepsy and normal intellect, whereas the recurrent p.Arg327His variant was associated with infantile-onset DEE. Two individuals with variants in the pore domain were more severely affected, with a neonatal-onset movement disorder, early-infantile DEE, profound disability, and childhood death.

# Discussion

We describe a cohort of 17 individuals with pathogenic or likely pathogenic missense variants in the voltage-sensing and pore domains of Kv10.2, including 14 previously unreported individuals. We present evidence for a putative emerging genotype-phenotype correlation with a spectrum of epilepsy and cognitive outcomes. Overall, we expand the role of EAG proteins in human disease and establish KCNH5 as implicated in a spectrum of neurodevelopmental disorders and epilepsy.

The voltage-gated potassium channels are a large group of transmembrane proteins critical for controlling neuronal excitability and regulating electrophysiologic properties in the brain.¹ The ether-a-go-go (EAG) subfamily consists of 2 members, EAG1/Kv10.1 encoded by KCNH1 and EAG2/Kv10.2 encoded by KCNH5. Pathogenic gain-of-function (GOF) variants in KCNH1 are implicated in Temple-Baraitser and Zimmermann-Laband syndromes²⁻⁴; epilepsy is a common feature of these overlapping syndromes.⁵ Individuals with variants in KCNH5 have been reported in large exome sequencing cohort studies of individuals with neurodevelopmental disorders (NDDs). Of the 3 previously reported individuals, 2 had a recurrent missense variant (p.Arg327His) associated with a developmental and epileptic encephalopathy (DEE) with onset prior to 1 year of age.⁶,⁷ The third had a neonatal-onset DEE and a different de novo missense variant (p.Ile463Thr).⁸ Despite these case reports, the KCNH5-associated phenotypes are not well delineated.

KCNH5 is expressed in excitatory neurons in upper layer IV of the cerebral cortex and the hippocampus.⁹,¹⁰ KCNH5 encodes for Kv10.2, which localizes to the somatodendritic region where it plays a role in controlling the electrical coupling between cell bodies and distal dendrites.¹⁰ Like most potassium channels, it consists of 4 transmembrane domains (S1–S4) that make up the voltage-sensing domain and 2 transmembrane domains and a reentrant loop (S5–S6) that comprise the pore module (Figure 1, A and C). The recurrent Kv10.2 p.Arg327His variant localizes to S4, where voltage-clamp analysis in a heterologous expression system demonstrated a hyperpolarized shift of voltage dependence of activation and

acceleration of activation, consistent with a gain-of-function pathogenic mechanism.¹¹

Here we report a cohort of 17 individuals with de novo or inherited (n = 1) missense variants in the voltage-sensing and pore domains of KCNH5. We describe 14 new patients and provide additional phenotypic information for the 3 published individuals from large cohort studies.⁶⁻⁸ We analyze the spectrum of KCNH5-associated neurodevelopment disorders, particularly the epilepsy phenotypes, and discuss genotype-phenotype correlations.

# Methods

## Standard Protocol Approvals, Registrations, and Patient Consents

We performed KCNH5 targeted resequencing in 680 individuals with DEEs and exome sequencing in 213 individuals with DEEs, totaling 893 individuals. All individuals underwent clinical assessment, and medical records, EEG, and imaging data were obtained. Seizure types and epilepsy syndromes were classified according to the ILAE classification.¹²,¹³ Individuals with DEEs had been previously tested for pathogenic variants in the majority of known DEE genes (¹⁴ and unpublished data). The study was approved by the Austin Health Human Research Ethics Committee (H2007/02961), the New Zealand Health and Disability Ethics Committee (NTY/12/06/053/AM15), and the University Hospital of Lyon and Aix-Marseille University in France Ethics Committee (05/78, CPP Strasbourg Alsace 1). Written informed consent was provided by the
<figure><img src="image_2.png" /><figcaption>**Figure 1** Location and Conservation of the 6 Unique De Novo KCNH5 Missense Variants</figcaption></figure>

(A) Schematic of Kv10.2 (EAG2), modified from Protter³⁵ plot of Q8NCM2 (KCNH5_Human). Of the 6 transmembrane domains, S1-S4 make up the voltage-sensing domain, and S5-6 form the pore helix. The KCNH5 patient-specific variants identified in this study are indicated by their amino acid change. (B) Graph of missense tolerance ratio (MTR; y-axis) and protein position (x-axis), with variants indicated. MTR³⁶ is a measure of protein-encoding cDNA sequence intolerance to missense variants. Variant positions with a value greater than 1 (blue line) is considered neutral; values below 1 are under constraint. Two variants (p.Arg327His and p.Arg333His) are in the 5th percentile of least tolerated missense alterations in the exome. The 5th and 25th percentiles are highlighted in red and yellow, respectively. (C) Locations of variants (color coded) are mapped onto the crystal structure of homotetrameric assembly of Kv10.1 (PDB5K7L); all variants are perfectly conserved between KCNH5 and KCNH1 and are mapped to the corresponding amino acid. The pore domain is highlighted in white, and a single tetrameric subunit is colored light blue. EAG = ether-a-go-go
patient or their parent or legal guardian in the case of minors or
those with intellectual disability (ID).

We identified an additional 14 individuals with KCNH5 variants through GeneMatcher.¹⁵ These individuals were consented using research protocols approved by their local ethics committees. Their clinicians provided clinical information. For the 3 previously published individuals (probands 2, 10, and 15), we obtained additional clinical information. In total, the cohort consists of 19 individuals with KCNH5 missense variants, with 17 having variants localized in or near the voltage-sensing or pore domains.

## KCNH5 Variant Identification and Analysis

We used molecular inversion probes (MIPs) to capture all exons and 5 base pairs of flanking intronic KCNH5 sequence; next-generation sequencing and data analysis were performed as described previously.¹⁴ We resequenced KCNH5, covering 95% of the gene at a depth of 50X (median across the cohort of 680 individuals). Exome sequencing (n = 213) was performed via Epi25.¹⁶ We considered only variants that were nonsynonymous, altered the acceptor/donor splice sites, or indels that disrupted the coding frame. Only variants that were not present in gnomAD¹⁷ and TOPMed¹⁸ were considered for segregation analysis. The variants in the 14 individuals recruited by the matchmaker exchange network¹⁵ were identified via clinical or research genome (n = 2), exome (n = 10), or gene panel (n = 2) sequencing.¹⁶ Library preparation and data analysis were performed using local protocols and computational pipelines. Variants were classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines for variant classification.¹⁹

KCNH1 (EAG1/Kv10.1) and KCNH5 (EAG2/Kv10.2) have 72% identity at the amino acid level. The 3-dimensional structure of Kv10.2 has not yet been resolved; thus, to model the location of each of the identified missense variants, we used the Kv10.2 homology model generated from the Kv10.1 cryo-EM structure (5k7l.1.A) using UCSF ChimeraX.²⁰

## Data Availability

All data from this study are available in the manuscript or supplementary materials.

## Results

### Rare Missense Variants in KCNH5

We identified 2 individuals with the previously reported de novo missense variant p.Arg327His in 2 DEE cohorts (1/680 by MIPs and 1/213 by exome¹⁶). Through our international collaboration, we identified 5 additional individuals with this recurrent variant, which arose de novo in 4 individuals (Table 1). We also identified 5 additional missense variants in the voltage-sensing and pore domains of Kv10.2 (Figure 1). One variant (p.Arg333His) was present in 4 individuals; it arose de novo in 3 and was inherited from an affected mother in the fourth proband. The 4 other de novo missense variants were each observed once in single individuals.

The amino acids at all 6 variant positions (2 recurrent, 4 singletons) were conserved between Kv10 proteins. The missense variants had CADD²¹ scores of 26–32, conferring a high likelihood of deleteriousness (Figure 1B and Table 1). All variants were absent from the general population (gnomAD and TOPMed) and were classified as either pathogenic or likely pathogenic (P/LP) by the ACMG criteria (eTable 1, links.lww.com/WNL/C453).¹⁹

We also identified 2 de novo variants of uncertain significance (VUS) in the N-terminal (p.Leu181Pro) and C-terminal (p.Ile606Thr) of Kv10.2 in 2 individuals with NDDs but without seizures (eAppendix 1, links.lww.com/WNL/C453). These VUS were located outside of the functionally critical pore or voltage-sensing domains. One variant (p.Leu181Pro) did not segregate with the NDD in the family, and the individual with the p.Ile606Thr variant also carried a pathogenic 16p11.2 microdeletion that may explain some or all of their clinical features. Although these variants are included here for completeness, this study focuses on the 17 individuals with variants in the voltage-sensing or pore-forming domains.

### Phenotypic Features in Individuals With KCNH5 Pathogenic or Likely Pathogenic Variants

All 17 individuals with pathogenic or likely pathogenic missense variants in the voltage-sensing or pore domains had epilepsy (Table 1). Moreover, the mother of proband 14 who carries the recurrent p.Arg333His variant also has epilepsy, although detailed clinical information was not available. The median age at seizure onset was 6 months (range: 1–12 months). A wide range of seizure types were seen: focal impaired awareness seizures (8), focal to bilateral tonic-clonic seizures (9), focal motor seizures (8), generalized myoclonic (4), generalized tonic-clonic seizure (2), myoclonic-atonic (1), absence seizure (1), unknown onset tonic-clonic seizures (6), and epileptic spasms (2). Nine (53%) individuals had a DEE. The remainder had focal epilepsy (3), generalized epilepsy (2), or there was inadequate information to make an epilepsy type diagnosis (3). Nine individuals (53%) achieved seizure freedom, with median seizure offset at 2.5 years (range: 3 months to 8 years).

Developmental concerns were noted at a median age of 15 months in 14 individuals (range birth to 3.5 years), predominantly affecting language (11). One child had normal development, and for 2 individuals, aged 61 years, early developmental information was limited. Cognitive outcome for the 10 individuals over 5 years of age ranged from normal (3/10) to mild (3/10), moderate (2/10), severe (1/10), and profound (1/10) ID. Two individuals died at ages 13 months and 9 years, respectively.

There were no consistent or significant dysmorphic features noted in these individuals. Proband 1 was reported to have long eyelashes and mild ptosis, whereas proband 2 had prominent ears, small hands, and a flexion contracture of the 4th and 5th digits of one hand. Significant sleep disorders were not noted in any individual.
<table><caption>**Table 1** Clinical Presentation of Our Cohort and Previously Published Individuals With P/LP KCNH5 Missense Variants</caption><thead><tr><th>Proband<br>(age at<br>study, sex)</th><th>Protein<br>change<br>(CADD)</th><th>Inheritance</th><th>Syndrome</th><th>Age at sz<br>onset:<br>type</th><th>Other sz<br>type(s)</th><th>Sz<br>offset</th><th>Development<br>(age of first<br>concern);<br>regression<br>(age)</th><th>Developmental and<br>behavioral outcome</th><th>EEG</th><th>MRI</th><th>ASMs (positive<br>response underlined)</th></tr></thead><tbody><tr><td>1 (4y, M)</td><td>p.Lys324Glu<br>(27)</td><td>De novo</td><td>DEE</td><td>3m: FMS<br>(Hc)</td><td>FBTCS, SE, and<br>TCS</td><td>N/A</td><td>GDD (18m); no<br>ADHD</td><td>Sev ID (single words,<br>amb, and assisted feeds;<br>ADHD</td><td>3m: N; 10m: FD<br>11m, 18m: N</td><td>3m: N</td><td>LEV, PHT, CLB, VPA, and<br>LTG</td></tr><tr><td>2<sup>Veeramah</sup><br>(13y, M)</td><td>p.Arg327His<br>(32)</td><td>De novo</td><td>FDEE</td><td>6m: TCS</td><td>TCS and FMS<br>(Hc)</td><td>N/A</td><td>GDD (6m); yes<br>(3y)</td><td>Sev ID (NV); ASD</td><td>Freq MFD (sleep<br>activation)</td><td>NP</td><td>VPA (partial), others<br>unknown</td></tr><tr><td>3 (5.5y, M)</td><td>De novo</td><td>DEE: D/EE-<br>SWAS</td><td>5m: FIAS</td><td>FBTCS and<br>FMS (Hc)</td><td></td><td>3y</td><td>Language<br>delay (2.5y);<br>yes (2.5y)</td><td>Mild/Mod ID (sentences<br>and amb); ADHD</td><td>6m: FMS; 9m, 11m: MFD;<br>2.8y CSWS; 3y: MFD<br>(sleep activation); 11y:<br>focal slow</td><td>6m: N, 4y: N</td><td>PB, CBZ, CLB, LEV, STR,<br>VPA, and TPM</td></tr><tr><td>4 (61y, F)</td><td>Parents<br>unavailable</td><td>Epilepsy:<br>unknown<br>type</td><td>6m:<br>Unknown</td><td>GTCS, Abs, and<br>FAMS</td><td></td><td>N/A</td><td>Unknown; no</td><td>N (learning difficulties at<br>school; sentences, amb,<br>and self-feeds)</td><td>56y: focal slow, FD, and<br>GSW</td><td>55y: N</td><td>CBZ, LEV, PB, PHT, TPM,<br>VPA, CBZ, PGB, and LTG</td></tr><tr><td>5 (5y, F)</td><td>De novo</td><td>FDEE</td><td>6m: FS</td><td>FMS and<br>FBTCS</td><td></td><td>N/A</td><td>Language<br>delay (16m); no</td><td>Mod ID (2-word<br>sentences, amb, and<br>self-feeds); autistic trait</td><td>8m: N; 12m: MFD; 2y 9m:<br>slow</td><td>NP</td><td>OXC and LEV</td></tr><tr><td>6 (10y, F)</td><td>De novo</td><td>FDEE</td><td>7m: FIAS</td><td>FBTCS and<br>FMS (Hc)</td><td></td><td>N/A</td><td>Language and<br>behavior delay<br>(3.5y); no</td><td>Mild ID (sentences, amb,<br>and self-feeds);<br>behavioral difficulties</td><td>7m: N; 3y: FD; 5y, 6y, 8y,<br>10y: slow, Freq MFD<br>(sleep activation)</td><td>1y, 3y, 5y: N</td><td>VPA (partial), LEV, LCS<br>(partial), TPM, LTG, CLB,<br>CBZ, ETX, GBP, CBD, and<br>KD</td></tr><tr><td>7 (9y, M)</td><td>De novo</td><td>DEE</td><td>10m:<br>FIAS</td><td>MyAS, My, and<br>TCS</td><td></td><td>6y</td><td>Language<br>delay (15m); no</td><td>Mod ID (sentences, amb,<br>and self-feeds)</td><td>1y: slow; 5y, 6y: Freq MFD<br>(sleep activation)</td><td>10m, 4y: N</td><td>CBZ (partial) and LTG</td></tr><tr><td>8 (3.8y F)</td><td>De novo</td><td>FDEE</td><td>2m: FMS</td><td>FBTCS and<br>FIAS</td><td></td><td>N/A</td><td>Language<br>delay (15m); no</td><td>Sev ID (2-word<br>sentences, amb, and<br>assisted feeds); ASD</td><td>2y 9m: Slow, FD (sleep<br>activation)</td><td>11m, 18m: N</td><td>LEV, LTG, CLB, and CBZ<br>(partial)</td></tr><tr><td>9 (19m, M)</td><td>De novo</td><td>CE</td><td>7m: GTCS</td><td>FBTCS</td><td></td><td>14 m</td><td>Language<br>delay (14m); no</td><td>Language delay (single<br>words, amb, and self-<br>feeds)</td><td>9m: GSW; 11m, 13m: N</td><td>14m: N</td><td>LEV and OXC</td></tr><tr><td>10<sup>Minardi</sup><br>(61y, M)</td><td>Parents<br>unavailable</td><td>DEE</td><td>&lt;1 y: TCS</td><td>None</td><td></td><td>8y</td><td>GDD<br>(unknown); no</td><td>Mild ID (sentences, amb,<br>and self-feeds)</td><td>54, 56-59y: slow, diffuse<br>MFD (sleep activation)</td><td>52y, 53y: multiple<br>nonenhancing white matter<br>abnormalities; arachnoid cyst;<br>mineral deposits in the orbit<br>(eFigure 1, links.lww.com/WNL/<br>C453)</td><td>LEV and LTG</td></tr></tbody></table>

Continued
<table><caption>**Table 1** Clinical Presentation of Our Cohort and Previously Published Individuals With P/LP KCNH5 Missense Variants (continued)</caption><thead><tr><th>Proband<br>(age at<br>study, sex)</th><th>Protein<br>change<br>(CADD)</th><th>Inheritance</th><th>Syndrome</th><th>Age at sz<br>onset:<br>type</th><th>Other sz<br>type(s)</th><th>Sz<br>offset</th><th>Development<br>(age of first<br>concern);<br>regression<br>(age)</th><th>Developmental and<br>behavioral outcome</th><th>EEG</th><th>MRI</th><th>ASMs (positive<br>response underlined)</th></tr></thead><tbody><tr><td>11 (3.5y, F)</td><td>p.Arg333His<br>(30)</td><td>De novo</td><td>FE</td><td>8m: FIAS</td><td>FBTCS</td><td>12m</td><td>N; no</td><td>N (sentences, amb, and<br>self-feeds)</td><td>11m, 3y: N</td><td>13m: N</td><td><u>VPA</u></td></tr><tr><td>12 (7y, F)</td><td></td><td>De novo</td><td>Epilepsy:<br>unknown<br>type</td><td>9m: TCS</td><td>None</td><td>5y</td><td>Language<br>delay (18m); no</td><td>N (sentences, amb, and<br>self-feeds)</td><td>15m: N; 7y: FD</td><td>15m, 7y: N</td><td>LEV, <u>TPM</u>, and <u>ZNS</u></td></tr><tr><td>13 (10y, F)</td><td></td><td>De novo</td><td>FE</td><td>10m:<br>FIAS</td><td>FBTCS</td><td>1y</td><td>Language and<br>gross motor<br>delay (18m); no</td><td>N (sentences, amb, and<br>self-feeds); behavioral<br>difficulties</td><td>12m:, 2y: focal slow, 3y:N</td><td>4y, 9y: frontal atrophy (not<br>progressive); arachnoid cyst;<br>Chiari I malformation (eFigure 2)</td><td><u>VPA</u>, weaned at 4y</td></tr><tr><td>14 (1.3y, F)</td><td></td><td>Inherited<br>from<br>affected<br>mother</td><td>GE</td><td>2m: My</td><td>None</td><td>3m</td><td>Hypotonia<br>(4m); no</td><td>N (single words and<br>walks with support)</td><td>2m: GSW; 3m: N</td><td>4m: N</td><td><u>VPA</u></td></tr><tr><td>15<sup>Imafidon</sup><br>(11m, F)<br>(deceased)</td><td>p.Ile463Thr<br>(28)</td><td>De novo</td><td>DEE: EIDEE</td><td>3m: TCS</td><td>ES and My</td><td>N/A</td><td>Hypotonia,<br>GDD (birth);<br>yes (4m)</td><td>Profound DD</td><td>1d: N; 3m, 5m and 7m:<br>FD; 10m: slow, MFD<br>(continuous)</td><td>9d: N; 3m: delayed myelination<br>and mild cerebral atrophy</td><td>VPA, CLN, and LEV</td></tr><tr><td>16 (9y, F)<br>(deceased)</td><td>p.Thr468Pro<br>(26)</td><td>De novo</td><td>DEE: EIDEE</td><td>1m: FTS</td><td>ES, TCS, and<br>My</td><td>N/A</td><td>Hypotonia,<br>GDD (birth); no</td><td>Profound ID: NV, NA, and<br>G-tube</td><td>1w: N; 1m: FD; 1y: BS; 2y:<br>slow, MFD, GSW; 5y, 6y:<br>MFD, BS</td><td>3w: increased signal posterior<br>fossa; 2y (CT): cerebral atrophy</td><td>VPA, KD, VGB, STR, PN,<br>PLP, TPM, <u>LEV</u> (partial),<br>and <u>PB</u> (partial)</td></tr><tr><td>17 (12y, F)</td><td>p.Phe471Ser<br>(31)</td><td>De novo</td><td>DE + FE</td><td>1y: TCS</td><td>FIAS</td><td>5y</td><td>GDD (9m); no</td><td>Mod ID (sentences, amb,<br>and self-feeds)</td><td>1y, 4y, 7y: FD (sleep<br>activation)</td><td>1y, 3y: N</td><td><u>VPA</u>, weaned at 11y</td></tr></tbody></table>

Abbreviations: Abs = absence seizures; amb = ambulatory; ADHD = attention-deficit hyperactivity disorder; ASD = autism spectrum disorder; ASM = antiseizure medication; BS = burst suppression; CBD = cannabidiol; CBZ = carbamazepine; CE = combined focal and generalized epilepsy; CLB = clobazam; CLN = clonazepam; CSWS = continuous spike and wave in slow wave sleep; d = day; DD = developmental delay; DEE = developmental and epileptic encephalopathy; D/EE-SWAS = developmental and/or epileptic encephalopathy with spike-wave activation in sleep; DE + FE = developmental encephalopathy and focal epilepsy; EIDEE = early infantile DEE; ES = epileptic spasm; ETX = ethosuximide; FAMS = focal aware motor seizure; FBTCS = focal to bilateral tonic-clonic seizure; FD = focal discharge; FDEE = focal DEE; FE = focal epilepsy; FIAS = focal impaired awareness seizure; FMS = focal motor seizure; FMy = focal myoclonic seizure; FTS = focal tonic seizure; Freq = frequent; FS = febrile seizure; GBP = gabapentin; GDD = global developmental delay; GE = generalized epilepsy; GSW = generalized spike and wave; GTCS = generalized tonic-clonic seizure; Hc = hemiclonic; ID = intellectual disability; LEV = levetiracetam; KD = ketogenic diet; LCS = lacosamide; LTG = lamotrigine; m = months; MFD = multifocal discharge; Mod = moderate; My = myoclonic; MyAS = myoclonic-atomic seizure; N = normal; N/A = not applicable; NA = nonambulatory; NP = not performed; NV = nonverbal; OXC = oxcarbazepine; PB = phenobarbital; PGB = pregabalin; PHT = phenytoin; PLP = pyridoxal-5-phosphate; P/LP = pathogenic/likely pathogenic; PM = primary motor; PN = pyridoxine; SE = status epilepticus; Sev = severe; STR = steroid; Sz = seizure; TCS = tonic-clonic seizure; TPM = topiramate; VAF = variant allele frequency; VGB = vigabatrin; VPA = valproate; VUS = variants of uncertain significance; w = weeks; y = years; ZNS = zonisamide.

References indicate previously published.
For the 9 individuals with the recurrent voltage-sensing domain p.Arg327His variant, seizure onset occurred at a median age of 6 months (range 2–10 months). For the 8 individuals for whom an epilepsy syndrome diagnosis could be made, all but one, who was 19 months old, had a DEE (7/8), which was focal in 5 individuals. Proband 3 had the syndrome of epileptic encephalopathy with spike-and-wave activation in sleep, which was associated with language regression.¹² All patients had EEGs, which captured sleep. Sleep activation of epileptiform discharges was noted in 6 other patients but was not associated with developmental regression. The cognitive outcome for those over 5 years of age with this recurrent variant ranged from normal with learning difficulties (1/6) to mild (3/6), moderate (1/6), or severe ID (1/6). For those in whom the complete antiseizure medication history was available, their epilepsy was drug resistant (8/9), with only 4/9 eventually becoming seizure-free. Brain MRI was normal in all apart from proband 10. He had no early imaging, but an MRI performed at 52 years demonstrated nonenhancing periventricular white matter abnormalities suggestive of demyelinating lesions. The lesions were stable on a repeat MRI 6 months later. Note was also made of an arachnoid cyst and mineral deposits in the orbit (eFigure 1, links.lww.com/WNL/C453).

The phenotype was much less severe for the 4 individuals with the second recurrent voltage-sensing domain p.Arg333His variant. This group had onset at a similar age (2–10 months) with either focal or generalized seizures. In contrast, their seizures were drug responsive, particularly to valproate (3/4), and all became seizure-free (aged 3 months to 5 years). One of the 4 individuals had mild motor and language delay at presentation, 1 had mild language delay, and 1 had only mild motor delay. All had normal cognitive development as assessed by their clinician when last seen at ages 1–10 years. MRI was normal apart from proband 13 who had mild frontal cortical atrophy, an arachnoid cyst, and a Chiari I malformation (eFigure 2, links.lww.com/WNL/C453).

Strikingly, probands 15 (p.Ile463Thr) and 16 (p.Thr468Pro), with missense variants in or at the junction of the S6 transmembrane pore-forming domain, had a much more severe phenotype. Both individuals presented with persistent non-epileptic myoclonus on day 1 of life and developed an early-infantile DEE with drug-resistant seizures by 3 months and eventual epileptic spasms. They had very limited developmental progression. Neuroimaging showed cortical atrophy. Proband 15 died at 13 months due to respiratory failure secondary to increased seizure activity. Proband 16 died at 9 years due to pneumonia.

There were 2 additional individuals with likely pathogenic variants and epilepsy. Proband 1, with p.Lys324Glu variant in the voltage-sensing domain, had a similar phenotype to individuals with the nearby recurrent p.Arg327His variant. This individual had a focal DEE with focal seizures beginning at 3 months and developmental delay evident by 18 months, evolving to severe ID. Proband 17 had the p.Phe471Ser variant in the C-terminal

cytoplasmic domain, which was very close to the pore-forming domain. She had moderate ID, drug-responsive focal epilepsy, and a hyperkinetic movement disorder consisting of a tremor predominantly in the upper limbs and exacerbated when she was tired or unwell. She did not have an epileptic encephalopathy and presented a similar epilepsy phenotype to patients with the recurrent p.Arg333His variant. We provide clinical vignettes for 3 patients (probands 6, 13, and 16) to highlight the spectrum of phenotypes associated with KCNH5 pathogenic variants (eAppendix 2, Clinical Vignettes, links.lww.com/WNL/C453).

## Discussion

Potassium channel subunits are one of the most important groups of genes associated with epilepsy, in particular with the most severe group of epilepsies, the DEEs.²² Here, we show that de novo pathogenic variants in KCNH5 in 17 patients are frequently associated with a DEE but may also cause self-limited epilepsies. We describe the phenotypic spectrum, with particular emphasis on the phenotype-genotype correlations and the putative effect of these missense variants on Kv10.2 channel function.

We highlight a genotype-phenotype correlation for variants across the protein (Figure 2). The p.Arg333His variant is associated with the mildest epilepsy phenotype, a drug-responsive self-limited focal or generalized epilepsy with normal cognitive outcome. Conversely, the nearby most common recurrent p.Arg327His variant has a far more severe epilepsy phenotype of DEE. Of interest, the nearby p.Lys324Glu voltage-sensing domain variant was also associated with a DEE. Individuals with these 2 variants predominantly presented with an infantile-onset DEE between 2 and 10 months of age with drug-resistant generalized and focal seizures. Early developmental concerns were noted with cognitive outcome ranging from learning difficulties to severe ID. Finally, 2 individuals with variants in the pore-forming S6 transmembrane domain (p.Ile463Thr and p.Thr468Pro) had an even more severe phenotype of neonatal-onset DEE with onset on day 1 of life resulting in profound disability and death.

The p.Arg327His variant, located in the voltage-sensing S4 transmembrane domain, acts in a GOF manner.¹¹ Multistate structural modeling demonstrated that the p.Arg327His variant favors channel opening, and voltage-clamp experiments showed a hyperpolarized shift of voltage dependence of activation and acceleration of activation.¹¹ These changes in electrophysiologic properties are consistent with the GOF mechanism that is typically observed for several potassium channels implicated in DEEs, such as KCNH1,² KCNT1,²³ and KCNT2.²⁴ A subset of epilepsy-associated variants in KCNA2, KCNB1, KCNQ2, and KCNQ3 have similarly been shown to act in a GOF manner.²⁵⁻²⁷ How these changes lead to epilepsy remains unresolved.²⁸ We hypothesize that the new KCNH5 voltage-sensing and pore domain pathogenic variants described here also have a similar GOF mechanism to the p.Arg327His variant. This is supported by in silico data
<figure><img src="image_3.png" /><figcaption>**Figure 2** Phenotypic Spectrum Associated With KCNH5 Missense Variants</figcaption></figure>

The number of individuals in the phenotype group is represented by circle size, and colors of each circle and star match the variant/phenotype class. The individuals with the p.Lys324Glu and p.Arg327His variants in the S4 transmembrane domain presented with an infantile-onset DEE with drug-resistant generalized and focal seizures, whereas the 4 individuals with the nearby recurrent p.Arg333His variant had drug-responsive seizures and became seizure-free. The 2 individuals with variants in or directly at the junction of the S6 transmembrane domain (p.Ile463Thr and p.Thr468Pro) are the most severely affected with a neonatal-onset movement disorder and early-infantile DEE. The single individual with the nearby p.Phe471Ser variant was less severely affected with moderate ID and drug-responsive seizures. Range is indicated in parentheses where applicable. DE = developmental encephalopathy; DEE = developmental and epileptic encephalopathy.

including the tolerance of KCNH5 to loss-of-function (truncating) variants in the general population. KCNH5 has a probability of loss-of-function intolerant (pLI) score of 0.02²⁹. Epilepsy-associated genes that confer pathogenicity via haploinsufficiency typically have pLI scores ~1, highlighting that loss of function is unlikely to be the pathogenic mechanism for KCNH5 variants. Conversely, missense variants in the gene are not as well tolerated, with a z-score of 2.51 (Figure 1B), suggesting selection against certain missense variants throughout human evolution.

The 2 most severely affected individuals carried missense variants (p.Ile463Thr and p.Thr468Pro) that cluster right at the junction of the S6 transmembrane domain and the early C-terminal cytoplasmic domain that form the channel pore. The 2 missense variants are located within 5 amino acids of each other right at the channel pore. Missense variants are similarly found in and just outside the S6 domain of the EAG1/Kv10 pore encoded by KCNH1 in individuals with Temple-Baraitser syndrome, which includes epilepsy as a key feature. Detailed electrophysiologic studies of these S6 missense variants in KCNH1 showed delayed deactivation and decreased threshold of activation in both human cells and Xenopus oocytes, consistent with a GOF mechanism.²,³ We hypothesize that the pore-forming KCNH5 missense variants act in a similar GOF manner. Like other voltage-gated potassium channels, Kv10.2 comprises 4 subunits that form a homotetramer. Any given tetramer could have between zero

and 4 subunits containing the variant residue, and it is possible that the function of the resulting channels could be differentially disrupted. This principle is a challenge in ion channel physiology generally, and in particular, for epilepsy, new tools and models are currently being developed to address this challenge. Moreover, future studies will also be necessary to confirm the functional effect and determine whether missense pathogenic variants in the most central pore region have a more profound GOF effect on Kv10.2 function than those in the voltage-sensing domain or located further away from the pore.

De novo missense variants in KCNH1/Kv10.1 cause Temple-Baraitser syndrome (TBS) and Zimmermann-Laband syndrome (ZLS).²⁻⁴ Features of both include ID, epilepsy, facial dysmorphism, and nail hypoplasia.³⁰ GOF KCNH1 missense variants have recently been reported in individuals with ID and epilepsy without the additional phenotypic features of TBS/ZLS.³¹,³² Individuals with both syndromic and nonsyndromic KCNH1-associated disorders with epilepsy have both focal and generalized seizures in infancy.⁵ Three of the 6 reported KCNH5 variants (p.Lys324Glu, p.Arg327His, and p.Ile463Thr) affect analogous KCNH1 amino acid residues that have been reported in individuals with KCNH1-associated NDDs,²,⁴,³² further highlighting the importance of these positions on channel function. KCNH1 and KCNH5 are predominantly expressed in the adult CNS (Human Protein Atlas³³ and GTEx³⁴); however, current expression data sets are largely limited to adult tissues.
Particularly as all 6 amino acid residues affected by the nucleotide changes described here are conserved between KCNH1 and KCNH5 (eFigure 3, links.lww.com/WNL/C453), future data sets such as the Developmental Genotype-Tissue Expression initiative will be valuable for evaluating both the multisystem involvement of KCNH1-associated disorders and the CNS-limited KCNHS-related phenotypes.

Overall, we establish KCNHS as a gene associated with epilepsy and neurodevelopmental phenotypes and identify an emerging genotype-phenotype correlation. We describe 17 individuals with pathogenic or likely pathogenic variants in KCNHS, including 4 novel variants. The p.Arg327His variant located in the voltage-sensing domain causes gain of channel function,¹¹ and we hypothesize that the additional KCNHS voltage-sensing and pore domain variants also lead to epilepsy via a similar GOF mechanism. We highlight an intriguing genotype-phenotype correlation with a spectrum of epilepsy and cognitive outcomes. These observations will be further supported by examining the functional effects associated with the location of different variants in Kv10.2, with potential therapeutic implications. Our study expands the role of EAG proteins in human disease, highlighting that KCNHS variants are implicated in a spectrum of NDD and epilepsy phenotypes.

## Accession Numbers

KCNHS mRNA NM_139318.4 and protein NP_647479.2 sequence.

KCNH1 protein NP_758872.1.

## Acknowledgment

The authors thank the patients and their families for participating in our research study. They also thank Jennifer Kearney for her valuable insight on ion channel genetics. They thank the Epi25 principal investigators, local staff from individual cohorts, and all patients with epilepsy who participated in the study for making possible this global collaboration and resource to advance epilepsy genetics research. This work is part of the Centers for Common Disease Genomics (CCDG) program, funded by the National Human Genome Research Institute (NHGRI) and the National Heart, Lung, and Blood Institute (NHLBI). CCDG-funded Epi25 research activities at the Broad Institute, including genomic data generation in the Broad Genomics Platform, are supported by NHGRI grant UM1 HG008895 (PIs: Eric Lander, Stacey Gabriel, Mark Daly, and Sekar Kathiresan). The Genome Sequencing Program efforts were also supported by NHGRI grant 5U01HG009088-02. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The authors thank the Stanley Center for Psychiatric Research at the Broad Institute for supporting the genomic data generation efforts.

## Study Funding

This work was supported by funding from the NIH (NINDS NS069605). H.C. Mefford is a recipient of the Burroughs Wellcome Fund Career Award for Medical Scientists. G.L.

Carvill was funded by the Citizens United for Research in Epilepsy (CURE) Taking Flight Award and NIH (NINDS NS089858). I.E. Scheffer is supported by a National Health and Medical Research Council of Australia (NHMRC) Program grant, Investigator grant, as well as a Centre of Research Excellence and Synergy grant. L.G. Sadleir and G. Valles-Ibáñez were supported by Health Research Council of New Zealand and Cure Kids research grants. L. Sedlackova, K. Sterbova, M. Vlckova, P. Lassuthova, and A. Jahodova are supported by the Ministry of Health of the Czech Republic AZV NU20-04-00279.

## Disclosure

L.G. Sadleir is a consultant for the Epilepsy Consortium and has received travel grants from Seqirus and Nutricia. She has received research grants and consultancy fees from Zynerba Pharmaceuticals. She has served on an Eisai Pharmaceuticals scientific advisory panel. R. Person, E. Torti, and K. McWalter are employees of GeneDx. B.K. Burton has received consulting fees and/or honoraria from Aeglea, Alexion, Applied Therapeutics, BioMarin, Capsida, Denali, Horizon, JCR Pharma, Moderna, SIO, Takeda, and Ultra-genyx and has conducted clinical trials funded by BioMarin, Denali, JCR Pharma, Homology Medicines, Sangamo, and Ultragenyx. I.E. Scheffer has served on scientific advisory boards for BioMarin, Chiesi, Eisai, Encoded Therapeutics, GlaxoSmithKline, Knopp Biosciences, Nutricia, RogCon, Takeda Pharmaceuticals, UCB, and Xenon Pharmaceuticals; has received speaker honoraria from GlaxoSmithKline, UCB, BioMarin, Biocodex, Chiesi, Liva Nova, and Eisai; has received funding for travel from UCB, Biocodex, GlaxoSmithKline, BioMarin, and Eisai; has served as an investigator for Anavex Life Sciences, Cerecin Inc, Cereval Therapeutics, Eisai, Encoded Therapeutics, Epi-Minder Inc, Epygenix, ES Therapeutics, GW Pharma, Marinus, Neurocrine BioSciences, Ovid Therapeutics, Takeda Pharmaceuticals, UCB, Ultragenyx, Xenon Pharmaceutical, Zogenix, and Zynerba; has consulted for Atheneum Partners, Care Beyond Diagnosis, Epilepsy Consortium, Ovid Therapeutics, UCB, and Zynerba Pharmaceuticals; and is a Non-Executive Director of Bellberry Ltd and a Director of the Australian Academy of Health and Medical Sciences and the Australian Council of Learned Academies Limited. She may accrue future revenue on pending patent WO61/010176 (filed: 2008): Therapeutic Compound; has a patent for SCN1A testing held by Bionomics Inc and licensed to various diagnostic companies; and has a patent molecular diagnostic/theranostic target for benign familial infantile epilepsy (BFIE) [PRRT2] 2011904493 & 2012900190 and PCT/AU2012/001321 (TECH ID:2012-009). The remaining authors have nothing relevant to disclose. Go to Neurology.org/N for full disclosures.

## Publication History

Previously published at medRxiv, https://doi.org/10.1101/2022.04.26.22274147. Received by Neurology April 28, 2022. Accepted in final form September 14, 2022. Submitted and externally peer reviewed. The handling editor was Renee Shellhaas, MD, MS.
Appendix Authors

<table><thead><tr><td>Name</td><td>Location</td><td>Contribution</td></tr></thead><tbody><tr><td>Hannah C. Happ, BA</td><td>Ken and Ruth Davee<br>Department of Neurology,<br>Northwestern University<br>Feinberg School of Medicine,<br>Chicago, IL</td><td>Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; and analysis or interpretation of data</td></tr><tr><td>Lynette G. Sadleir, MBChB, MD</td><td>University of Otago,<br>Wellington, New Zealand</td><td>Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; and analysis or interpretation of data</td></tr><tr><td>Matthew Zemel, DO</td><td>University of Washington,<br>Seattle, WA</td><td>Major role in the acquisition of data and analysis or interpretation of data</td></tr><tr><td>Guillem de Valles-Ibañez, PhD</td><td>Epilepsy Research Centre,<br>Department of Medicine,<br>The University of<br>Melbourne, Austin<br>Health, Heidelberg, VIC,<br>Australia</td><td>Major role in the acquisition of data and analysis or interpretation of data</td></tr><tr><td>Michael S. Hildebrand, PhD</td><td>Duke University Medical<br>Center, Durham, NC</td><td>Major role in the acquisition of data and analysis or interpretation of data</td></tr><tr><td>Allyn McConkie Rosell, PhD</td><td>Duke University Medical<br>Center, Durham, NC</td><td>Major role in the acquisition of data and analysis or interpretation of data</td></tr><tr><td>Marie McDonald, MD</td><td>Duke University Medical<br>Center, Durham, NC</td><td>Major role in the acquisition of data</td></tr><tr><td>Halie May, CGC</td><td>Institute for Genomic<br>Medicine, Columbia<br>University Irving<br>Medical Center, New<br>York, NY</td><td>Major role in the acquisition of data</td></tr><tr><td>Tristan Sands, MD</td><td>Institute for Genomic<br>Medicine, Columbia<br>University Irving Medical<br>Center, New York, NY</td><td>Major role in the acquisition of data</td></tr><tr><td>Vimla Aggarwal, MBBS</td><td>Department of Pathology<br>and Cell Institute for<br>Genomic Medicine,<br>Columbia University<br>Irving Medical Center,<br>New York, NYBiology,<br>Columbia University<br>Irving Medical Center,<br>New York, NY</td><td>Major role in the acquisition of data</td></tr><tr><td>Christopher Elder, MD</td><td>Department of Neurology,<br>Columbia University Irving<br>Medical Center, New York, NY</td><td>Major role in the acquisition of data</td></tr><tr><td>Timothy Feyma, MD</td><td>Gillette Children's<br>Specialty Healthcare, St.<br>Paul, MN</td><td>Major role in the acquisition of data</td></tr><tr><td>Allan Bayat, MD</td><td>Department of Epilepsy<br>Genetics and Personalized<br>Medicine, Danish Epilepsy<br>Center, Dianalund, Denmark;<br>Institute of Regional Health<br>Research, University of<br>Southern Denmark,<br>Denmark</td><td>Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; and analysis or interpretation of data</td></tr></tbody></table>

Appendix (continued)

<table><thead><tr><td>Name</td><td>Location</td><td>Contribution</td></tr></thead><tbody><tr><td>Rikke S. Møller, PhD</td><td>Department of Epilepsy<br>Genetics and Personalized<br>Medicine, Danish Epilepsy<br>Center, Dianalund,<br>Denmark; Institute of<br>Regional Health Research,<br>University of Southern<br>Denmark, Denmark</td><td>Drafting/revision of the manuscript for content, including medical writing for content, and major role in the acquisition of data</td></tr><tr><td>Christina D. Fenger, PhD</td><td>Department of Epilepsy<br>Genetics and Personalized<br>Medicine, Danish Epilepsy<br>Center, Dianalund, Denmark;<br>Amplexa Genetics, Odense,<br>Denmark</td><td>Drafting/revision of the manuscript for content, including medical writing for content, and major role in the acquisition of data</td></tr><tr><td>Jens Erik Klint Nielsen, MD</td><td>Department of Clinical<br>Medicine, Zealand<br>University Hospital,<br>Roskilde, Denmark</td><td>Major role in the acquisition of data</td></tr><tr><td>Anita N. Datta, MD</td><td>University of British<br>Columbia, Vancouver,<br>Canada</td><td>Major role in the acquisition of data</td></tr><tr><td>Kathleen M. Gorman, MD</td><td>The Department of<br>Neurology and Clinical<br>Neurophysiology, Children's<br>Health Ireland at Temple St.,<br>Temple Street, Dublin 1,<br>Ireland; School of Medicine<br>and Medical Science,<br>University College Dublin,<br>Dublin 4, Ireland</td><td>Drafting/revision of the manuscript for content, including medical writing for content, and major role in the acquisition of data</td></tr><tr><td>Mary D. King, MD</td><td>The Department of<br>Neurology and Clinical<br>Neurophysiology, Children's<br>Health Ireland at Temple St.,<br>Temple Street, Dublin 1,<br>Ireland; School of Medicine<br>and Medical Science,<br>University College Dublin,<br>Dublin 4, Ireland</td><td>Major role in the acquisition of data</td></tr><tr><td>Natalia D. Linhares, PhD</td><td>Genuity Science, Dublin,<br>Ireland</td><td>Major role in the acquisition of data</td></tr><tr><td>Barbara K. Burton, MD</td><td>Ann & Robert H. Lurie<br>Children's Hospital of<br>Chicago, Chicago, IL;<br>Department of Pediatrics,<br>Northwestern<br>University Feinberg<br>School of Medicine,<br>Chicago, IL</td><td>Major role in the acquisition of data</td></tr><tr><td>Andrea Paras, MD</td><td>Ann & Robert H. Lurie<br>Children's Hospital of<br>Chicago, Chicago, IL;<br>Department of Pediatrics,<br>Northwestern University<br>Feinberg School of Medicine,<br>Chicago, IL</td><td>Major role in the acquisition of data</td></tr><tr><td>Sian Ellard, PhD</td><td>Exeter Genomics<br>Laboratory, Royal<br>Devon University<br>Healthcare NHS<br>Foundation Trust,<br>Exeter, UK; Institute of<br>Clinical and Biomedical<br>Science, University of<br>Exeter, UK</td><td>Major role in the acquisition of data and analysis or interpretation of data</td></tr></tbody></table>
Appendix (continued)

<table><thead><tr><td>Name</td><td>Location</td><td>Contribution</td></tr></thead><tbody><tr><td>Julia Rankin, MD</td><td>Dept Clinical Genetics, Royal Devon University Healthcare NHS Foundation Trust, Exeter, UK</td><td>Major role in the acquisition of data</td></tr><tr><td>Anju Shukla, MBBS</td><td>Department of Medical Genetics, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, India</td><td>Major role in the acquisition of data</td></tr><tr><td>Purvi Majethia</td><td>Department of Medical Genetics, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, India</td><td>Major role in the acquisition of data</td></tr><tr><td>Rory J. Olson, MD</td><td>Center for Individualized Medicine, Mayo Clinic, Rochester, MN</td><td>Major role in the acquisition of data</td></tr><tr><td>Karthik Muthusamy, MBBS</td><td>Center for Individualized Medicine, Mayo Clinic, Rochester, MN; Department of Clinical Genomics, Mayo Clinic, Rochester, MN</td><td>Major role in the acquisition of data</td></tr><tr><td>Lisa A. Schimmenti, MD</td><td>Center for Individualized Medicine, Mayo Clinic, Rochester, MN; Department of Clinical Genomics, Mayo Clinic, Rochester, MN</td><td>Major role in the acquisition of data</td></tr><tr><td>Keith Starnes, MD</td><td>Department of Neurology, Mayo Clinic, Rochester, MN</td><td>Major role in the acquisition of data</td></tr><tr><td>Lucie Sedláčková PhD</td><td>Neurogenetic Laboratory, Department of Pediatric Neurology, Second Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, Czech Republic; Epilepsy Research Centre Prague—EpiReC consortium, Motol University Hospital is a full member of the ERN EpiCARE</td><td>Major role in the acquisition of data</td></tr><tr><td>Katalin Šterbová MD</td><td>Epilepsy Research Centre Prague—EpiReC consortium, Motol University Hospital is a full member of the ERN EpiCARE; Department of Pediatric Neurology, Second Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, Czech Republic</td><td>Major role in the acquisition of data</td></tr><tr><td>Markéta Vičková PhD</td><td>Epilepsy Research Centre Prague—EpiReC consortium, Motol University Hospital is a full member of the ERN EpiCARE; Biology and Medical Genetics, Second Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, Czech Republic</td><td>Major role in the acquisition of data</td></tr></tbody></table>

Appendix (continued)

<table><thead><tr><td>Name</td><td>Location</td><td>Contribution</td></tr></thead><tbody><tr><td>Petra Lašuthová PhD</td><td>Neurogenetic Laboratory, Department of Pediatric Neurology, Second Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, Czech Republic; Epilepsy Research Centre Prague—EpiReC consortium, Motol University Hospital is a full member of the ERN EpiCARE</td><td>Major role in the acquisition of data</td></tr><tr><td>Alena Jahodová PhD</td><td>Epilepsy Research Centre Prague—EpiReC consortium, Motol University Hospital is a full member of the ERN EpiCARE; Department of Pediatric Neurology, Second Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, Czech Republic</td><td>Major role in the acquisition of data</td></tr><tr><td>Brenda E. Porter, MD, PhD</td><td>Stanford University School of Medicine, Palo Alto, CA</td><td>Major role in the acquisition of data</td></tr><tr><td>Nathalie Couque, MD</td><td>Laboratoire de biologie médicale multisites Seqoia-FMG2025, Laboratoire Génétique Moléculaire Robert-Debré, Paris, France</td><td>Major role in the acquisition of data</td></tr><tr><td>Estelle Colin</td><td>Service de Génétique, CHU d'Angers, Angers, France</td><td>Major role in the acquisition of data</td></tr><tr><td>Clément Prouteau, MD</td><td>Service de Génétique, CHU d'Angers, Angers, France</td><td>Major role in the acquisition of data</td></tr><tr><td>Corinne Collet</td><td>Laboratoire de biologie médicale multisites Seqoia-FMG2025, Laboratoire Génétique Moléculaire Robert-Debré, Paris, France</td><td>Major role in the acquisition of data</td></tr><tr><td>Thomas Smol, MD</td><td>Univ. Lille, CHU Lille, ULR7364—RADEME, Institut de Genetique Medicale, F-59000 Lille, France</td><td>Major role in the acquisition of data</td></tr><tr><td>Roseline Caumes, MD</td><td>Univ. Lille, CHU Lille, ULR7364—RADEME, Clinique de Genetique, F-59000 Lille, France</td><td>Major role in the acquisition of data</td></tr><tr><td>Fleur Vansenne, MD</td><td>Univeristy Medical Center Groningen, Groningen, the Netherlands</td><td>Major role in the acquisition of data</td></tr><tr><td>Francesca Bisulli, MD, PhD</td><td>Department of Biomedical and NeuroMotor Sciences, University of Bologna, Bologna, Italy; IRCCS Istituto delle Scienze Neurologiche di Bologna, Full Member of the ERN EpiCARE Bologna, Italy</td><td>Major role in the acquisition of data</td></tr><tr><td>Laura Licchetta, MD, PhD</td><td>IRCCS Istituto delle Scienze Neurologiche di Bologna, Full Member of the ERN EpiCARE Bologna, Italy</td><td>Major role in the acquisition of data</td></tr></tbody></table>

Continued
Appendix (continued)

<table><thead><tr><td>Name</td><td>Location</td><td>Contribution</td></tr></thead><tbody><tr><td>Richard Person, PhD</td><td>GeneDx, Gaithersburg, MD</td><td>Major role in the acquisition of data and analysis or interpretation of data</td></tr><tr><td>Erin Torti, MS</td><td>GeneDx, Gaithersburg, MD</td><td>Major role in the acquisition of data and analysis or interpretation of data</td></tr><tr><td>Kirsty McWalter, CGC</td><td>GeneDx, Gaithersburg, MD</td><td>Major role in the acquisition of data and analysis or interpretation of data</td></tr><tr><td>Richard Webster, MD</td><td>T.Y. Nelson Department of Neurology and Neurosurgery, Children's Hospital at Westmead, Westmead, New South Wales, Australia</td><td>Major role in the acquisition of data</td></tr><tr><td>Elizabeth E. Gerard, MD</td><td>Ken and Ruth Davee Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL</td><td>Analysis or interpretation of data</td></tr><tr><td>Gaetan Lesca, MD, PhD</td><td>Department of Medical Genetics, University Hospital of Lyon, Claude Bernard Lyon 1 University, Lyon, France</td><td>Drafting/revision of the manuscript for content, including medical writing for content, and major role in the acquisition of data</td></tr><tr><td>Pierre Szepetowski, MD, PhD</td><td>INSERM, Aix-Marseille University, INMED, Marseille, France</td><td>Drafting/revision of the manuscript for content, including medical writing for content, and major role in the acquisition of data</td></tr><tr><td>Ingrid E. Scheffer, PhD</td><td>Department of Neurology, Royal Children's Hospital, Department of Paediatrics, The University of Melbourne, and Murdoch Children's Research Institute, Parkville, Victoria, Australia; The Florey Institute of Neuroscience and Mental Health, Victoria, Australia</td><td>Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; and analysis or interpretation of data</td></tr><tr><td>Heather C. Mefford, MD, PhD</td><td>Center for Pediatric Neurologic Disease Research, St. Jude Children's Research Hospital, Memphis, TN</td><td>Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; study concept or design; and analysis or interpretation of data</td></tr><tr><td>Gemma L. Carvill, PhD</td><td>Ken and Ruth Davee Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL; Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL; Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL</td><td>Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; study concept or design; and analysis or interpretation of data</td></tr></tbody></table>

References

1. Bauer CK, Schwarz JR. Ether-a-go-go K(+) channels: effective modulators of neuronal excitability. *J Physiol.* 2018;596(5):769-783. doi: 10.1113/jp275477

2. Simons C, Rash LD, Crawford J, et al. Mutations in the voltage-gated potassium channel gene KCNH1 cause Temple-Baraitser syndrome and epilepsy. *Nat Genet.* 2015;47(1):73-77. doi: 10.1038/ng.3153

3. Kortum F, Caputo V, Bauer CK, et al. Mutations in KCNH1 and ATP6V1B2 cause Zimmermann-Laband syndrome. *Nat Genet.* 2015;47(6):661-667. doi: 10.1038/ng.3282

4. Gripp KW, Smithson SF, Scurr IJ, et al. Syndromic disorders caused by gain-of-function variants in KCNH1, KCNK4, and KCNN3-a subgroup of K(+) channelopathies. *Eur J Hum Genet.* 2021;29(9):1384-1395. doi: 10.1038/s41431-021-00818-9

5. Mastrangelo M, Scheffer IE, Bramswig NC, et al. Epilepsy in KCNH1-related syndromes. *Epileptic Disord.* 2016;18(2):123-136. doi: 10.1684/epd.2016.0830

6. Veeramah KR, Johnstone L, Karafet TM, et al. Exome sequencing reveals new causal mutations in children with epileptic encephalopathies. *Epilepsia.* 2013;54(7):1270-1281. doi: 10.1111/epi.12201

7. Minardi R, Licchetta L, Baroni MC, et al. Whole-exome sequencing in adult patients with developmental and epileptic encephalopathy: it is never too late. *Clin Genet.* 2020;98(5):477-485. doi: 10.1111/cge.13823

8. Imafidon ME, Sikkema-Raddatz B, Abbott KM, et al. Strategies in rapid genetic diagnostics of critically ill children: experiences from a Dutch university hospital. *Front Pediatr.* 2021;9:600556. doi: 10.3389/fped.2021.600556

9. Saganich MJ, de Miera EVS, Nadal MS, Baker H, Coetzee WA, Rudy B. Cloning of components of a novel subthreshold-activating K(+) channel with a unique pattern of expression in the cerebral cortex. *J Neurosci.* 1999;19(24):10789-10802. doi: 10.1523/jneurosci.19-24-10789.1999

10. Jeng CJ, Chang CC, Tang CY. Differential localization of rat Eag1 and Eag2 K+ channels in hippocampal neurons. *Neuroreport* 2005;16(3):229-233. doi: 10.1097/00001756-200502280-00005

11. Yang Y, Vasylyev DV, Dib-Hajj F, et al. Multistate structural modeling and voltage-clamp analysis of epilepsy/autism mutation Kv10.2-R327H demonstrate the role of this residue in stabilizing the channel closed state. *J Neurosci.* 33(42):2013:16586-16593. doi: 10.1523/jneurosci.2307-13.2013

12. Scheffer IE, Berkovic S, Capovilla G, et al. ILAE classification of the epilepsies: position paper of the ILAE commission for classification and terminology. *Epilepsia.* 2017;58(4):512-521. doi: 10.1111/epi.13709

13. Fisher RS, Cross JH, French JA, et al. Operational classification of seizure types by the international league against epilepsy: position paper of the ILAE commission for classification and terminology. *Epilepsia.* 2017;58(4):522-530. doi: 10.1111/epi.13670

14. Carvill GL, Heavin SB, Yendle SC, et al. Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1. *Nat Genet.* 2013;45(7):825-830. doi: 10.1038/ng.2646

15. Sobreira N, Schiettecatte F, Valle D, Hamosh A. GeneMatcher: a matching tool for connecting investigators with an interest in the same gene. *Hum Mutat.* 2015;36(10):928-930. doi: 10.1002/humu.22844

16. Epi25, Collaborative Electronic address sberkovic@unimelbeduau, Epi25 Collaborative. Ultra-rare genetic variation in the epilepsies: a whole-exome sequencing study of 17,606 individuals. *Am J Hum Genet.* 2019;105(2):267-282. doi: 10.1016/j.ajhg.2019.05.020

17. gnomAD [online]. Available at: gnomad.broadinstitute.org

18. TopMED [online]. Available at: bravo.sph.umich.edu/freeze3a/hg19/

19. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of medical genetics and genomics and the association for molecular pathology. *Genet Med.* 2015;17(5):405-424. doi: 10.1038/gim.2015.30

20. Pettersen EF, Goddard TD, Huang CC, et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. *Protein Sci.* 2021;30(1):70-82. doi: 10.1002/pro.3943

21. Rentzsch P, Schubach M, Shendure J, Kircher M. CADD-Splice-improving genome-wide variant effect prediction using deep learning-derived splice scores. *Genome Med.* 2021;13(1):31. doi: 10.1186/s13073-021-00835-9

22. McTague A, Howell KB, Cross JH, Kurian MA, Scheffer IE. The genetic landscape of the epileptic encephalopathies of infancy and childhood. *Lancet Neurol.* 2016;15(3):304-316. doi: 10.1016/s1474-4422(15)00250-1

23. Barcia G, Fleming MR, Deligniere A, et al. De novo gain-of-function KCNT1 channel mutations cause malignant migrating partial seizures of infancy. *Nat Genet.* 2012;44(11):1255-1259. doi: 10.1038/ng.2441

24. Ambrosino P, Soldovieri MV, Bast T, et al. De novo gain-of-function variants in KCNT2 as a novel cause of developmental and epileptic encephalopathy. *Ann Neurol.* 2018;83(6):1198-1204. doi: 10.1002/ana.25248

25. Miceli F, Soldovieri MV, Ambrosino P, et al. Early-onset epileptic encephalopathy caused by gain-of-function mutations in the voltage sensor of Kv7.2 and Kv7.3 potassium channel subunits. *J Neurosci.* 2015;35(9):3782-3793. doi: 10.1523/jneurosci.4423-14.2015

26. Torkamani A, Bersell K, Jorge BS, et al. De novo KCNB1 mutations in epileptic encephalopathy. *Ann Neurol.* 2014;76(4):529-540. doi: 10.1002/ana.24263

27. Syrbe S, Hedrich UBS, Riesch E, et al. De novo loss- or gain-of-function mutations in KCNA2 cause epileptic encephalopathy. *Nat Genet.* 2015;47(4):393-399. doi: 10.1038/ng.3239

28. Niday Z, Tzingounis AV. Potassium Channel gain of function in epilepsy: an unresolved paradox. *Neuroscientist.* 2018;24(4):368-380. doi: 10.1177/1073858418763752

29. Karczewski KJ, Francioli LC, Tiao G, et al. The mutational constraint spectrum quantified from variation in 141, 456 humans. *Nature.* 2020;581(7809):434-443. doi: 10.1038/s41586-020-2308-7
30. Hamilton MJ, Suri M. "Electrifying dysmorphology": potassium channelopathies causing dysmorphic syndromes. *Adv Genet.* 2020;105:137-174. doi: 10.1016/bs.adgen.2020.03.002

31. Aubert Mucca M, Patat O, Whalen S, et al. Patients with KCNH1-related intellectual disability without distinctive features of Zimmermann-Laband/Temple-Baraitser syndrome. *J Med Genet.* 2021;59(S):505-510. doi: 10.1136/jmedgenet-2020-107511

32. Fukai R, Saitsu H, Tsurusaki Y, et al. De novo KCNH1 mutations in four patients with syndromic developmental delay, hypotonia and seizures. *J Hum Genet.* 2016;61(S): 381-387. doi: 10.1038/jhg.2016.1

33. *The Human Protein Atlas*. Updated December 7, 2022. proteinatlas.org/

34. GTEx Portal. gtexportal.org/home/

35. Omasits U, Ahrens CH, Muller S, Wollscheid B. Protter: interactive protein feature visualization and integration with experimental proteomic data. *Bioinformatics*. 2014; 30(6):884-886. doi: 10.1093/bioinformatics/btt607

36. Silk M, Petrovski S, Ascher DB. MTR-Viewer: identifying regions within genes under purifying selection. *Nucleic Acids Res.* 2019;47(W1):W121–W126. doi: 10.1093/nar/gkz457