# Etiological involvement of KCND1 variants in an X-linked neurodevelopmental disorder with variable expressivity

Tassja Kalm, Claudia Schob, Hanna Völler, Thatjana Gardeitchik, Christian

Gilissen, Rolph Pfundt, Chiara Klöckner, Konrad Platzer, Annick

Klabunde-Cherwon, Markus Ries, et al.

► To cite this version:

Tassja Kalm, Claudia Schob, Hanna Völler, Thatjana Gardeitchik, Christian Gilissen, et al. Etiological involvement of KCND1 variants in an X-linked neurodevelopmental disorder with variable expressivity. American Journal of Human Genetics, 2024, 111 (6), pp.1206-1221. 10.1016/j.ajhg.2024.04.019. hal-04959421

HAL Id: hal-04959421

https://hal.science/hal-04959421v1

Submitted on 20 Feb 2025

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Distributed under a Creative Commons Attribution 4.0 International License
# Etiological involvement of *KCND1* variants in an X-linked neurodevelopmental disorder with variable expressivity

## Authors

Tassja Kalm, Claudia Schob, Hanna Völler, ..., Christian Kubisch, Robert Bähring, Stefan Kindler

## Correspondence

r.baehring@uke.uni-hamburg.de (R.B.),
kindler@uke.de (S.K.)

Hemizygous variants of *KCND1* lead to alterations in the biophysical properties of the encoded ion channel, implicating dysfunction of Kv4.1 voltage-gated potassium channels in the etiology of an X-linked neurodevelopmental disorder with variable expressivity.

<figure><img src="image_2.png" /></figure>
# Etiological involvement of KCND1 variants in an X-linked neurodevelopmental disorder with variable expressivity

Tassja Kalm,<fnref n="1" /> Claudia Schob,² Hanna Völler,² Thatjana Gardeitchik,³ Christian Gilissen,³ Rolph Pfundt,³ Chiara Klöckner,⁴ Konrad Platzer,⁴ Annick Klabunde-Cherwon,⁵ Markus Ries,⁵ Steffen Syrbe,⁵ Francesca Beccaria,⁶ Francesca Madia,⁷ Marcello Scala,⁷,⁸ Federico Zara,⁷,⁸ Floris Hofstede,⁹ Marleen E.H. Simon,¹⁰ Richard H. van Jaarsveld,¹⁰ Renske Oegema,¹⁰ Koen L.I. van Gassen,¹⁰ Sjoerd J.B. Holwerda,¹⁰ Tahsin Stefan Barakat,¹¹,¹²,¹³ Arjan Bouman,¹¹

*(Author list continued on next page)*

## Summary

Utilizing trio whole-exome sequencing and a gene matching approach, we identified a cohort of 18 male individuals from 17 families with hemizygous variants in KCND1, including two *de novo* missense variants, three maternally inherited protein-truncating variants, and 12 maternally inherited missense variants. Affected subjects present with a neurodevelopmental disorder characterized by diverse neurological abnormalities, mostly delays in different developmental domains, but also distinct neuropsychiatric signs and epilepsy. Heterozygous carrier mothers are clinically unaffected. KCND1 encodes the α-subunit of Kv4.1 voltage-gated potassium channels. All variant-associated amino acid substitutions affect either the cytoplasmic N- or C-terminus of the channel protein except for two occurring in transmembrane segments 1 and 4. Kv4.1 channels were functionally characterized in the absence and presence of auxiliary β subunits. Variant-specific alterations of biophysical channel properties were diverse and varied in magnitude. Genetic data analysis in combination with our functional assessment shows that Kv4.1 channel dysfunction is involved in the pathogenesis of an X-linked neurodevelopmental disorder frequently associated with a variable neuropsychiatric clinical phenotype.

## Introduction

Voltage-dependent potassium (Kv) channels represent a diverse group of ion channels encoded by a gene superfamily that includes twelve members (Kv1–12) and about 40 different genes.<fnref n="1" /> The particular physiological role of each Kv channel subtype is determined not only by its cellular abundance and subcellular distribution but also by its specific voltage dependence and gating kinetics.<fnref n="1" /> The three members of the Kv4 subfamily, encoded by the genes KCND1–3, mediate a subthreshold-activating, somatodendritic, rapidly activating and inactivating (A-type) potassium current (I<sub>SA</sub>) in neurons.² I<sub>SA</sub> plays a critical role in distinct aspects of neurophysiology, including the control of low fre-

quency repetitive discharge, regulation of dendritic excitation, and action potential backpropagation, as well as synaptic plasticity.²⁻⁴

Like all members of the Kv channel superfamily, Kv4 α-subunits consist of six transmembrane segments (S1–S6), flanked by cytoplasmic termini. A re-entrant pore loop between S5 and S6 contains the highly conserved potassium channel “signature sequence,” which mediates ion selectivity (Figure 1).⁵ The α-subunits tetramerize to constitute functional channels. Characteristic for Kv1–4 subfamily members, the cytoplasmic N-terminus contains a tetramerization (T1) domain, which mediates subfamily-specific assembly, with a critical involvement of specific Zn²⁺ coordination sites in subfamilies Kv2–4.⁶ Within a

*(Affiliations continued on next page)*

<footnote n="1">¹Institute for Cellular and Integrative Physiology, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; ²Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; ³Department of Human Genetics, Radboud University Medical Center, Nijmegen 6525 GA, the Netherlands; ⁴Institute of Human Genetics, University of Leipzig Medical Center, 04103 Leipzig, Germany; ⁵Division of Pediatric Epileptology, Centre for Paediatric and Adolescent Medicine, University Hospital Heidelberg, 69120 Heidelberg, Germany; ⁶Epilepsy Center, Department of Child Neuropsychiatry, Territorial Social-Health Agency, 46100 Mantova, Italy; ⁷Medical Genetics Unit, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy; ⁸Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, 16145 Genoa, Italy; ⁹Department of General Pediatrics, Wilhelmina Children’s Hospital, University Medical Centre Utrecht, Utrecht, the Netherlands; ¹⁰Department of Clinical Genetics, University Medical Center Utrecht, Utrecht 3584 EA, the Netherlands; ¹¹Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam 3000 CA, the Netherlands; ¹²ENCORE Expertise Center for Neurodevelopmental Disorders, Erasmus MC University Medical Center, Rotterdam 3000 CA, the Netherlands; ¹³Discovery Unit, Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam 3000 CA, the Netherlands; ¹⁴Genomics and Medicine, NIMGenetics, 28108 Madrid, Spain; ¹⁵Pediatric Neurology Department, Quironsalud University Hospital Madrid, School of Medicine, European University of Madrid, 28224 Madrid, Spain; ¹⁶Genomics, Genologica Medica, 29016 Málaga, Spain; ¹⁷Division of Medical Genetics, Department of Specialized Medicine, Montreal Children’s Hospital, McGill University Health Centre, QC H4A 3J1 Montreal, Canada; ¹⁸Department of Human Genetics, McGill University, QC H4A 3J1 Montreal, Canada; ¹⁹Child Neuropsychiatry Unit, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy; ²⁰Pediatric Neurology and Neuromuscular Diseases Unit, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy.</footnote>
Marjon van Slegtenhorst,¹¹ Sara Álvarez,¹⁴ Alberto Fernández-Jaén,¹⁵ Javier Porta,¹⁶ Andrea Accogli,¹⁷,¹⁸
Margherita Maria Mancardi,¹⁹ Pasquale Striano,⁸,²⁰ Michele Iacomino,⁷ Jong-Hee Chae,²¹,²² SeSong Jang,²¹
Soo Y. Kim,²² David Chitayat,²³,²⁴ Saadet Mercimek-Andrews,²⁴,²⁵ Christel Depienne,²⁶ Antje Kampmeier,²⁶
Alma Kuechler,²⁶ Harald Surowy,²⁶ Enrico Silvio Bertini,²⁷ Francesca Clementina Radio,²⁸ Cecilia Mancini,²⁸
Simone Pizzi,²⁸ Marco Tartaglia,²⁸ Lucas Gauthier,²⁹ David Genevieve,³⁰,³¹ Mylène Tharreau,²⁹
Noy Azoulay,³²,³³ Gal Zaks-Hoffer,³³,³⁴ Nesia K. Gilad,³⁴,³⁵ Naama Orenstein,³⁴,³⁵ Geneviève Bernard,¹⁷,³⁶,³⁷
Isabelle Thiffault,³⁸,³⁹,⁴⁰ Jonas Denecke,⁴¹ Theresia Herget,² Fanny Kortüm,² Christian Kubisch,²
Robert Bähring,¹,<fnref n="42" />,* and Stefan Kindler²,<fnref n="42" />,*

tetramer, S5 and S6 surround the central ion conduction pathway with the intertwined and flexible distal S6 segments acting as the cytoplasmic gate, while the peripheral S1-S4 segments (especially the positively charged amino acid residues in S4) act as voltage sensors. Kv channel opening and closing is mediated by direct mechanical coupling of the motile voltage sensors to the cytoplasmic S6 gate via the S4-S5 linkers.⁷

Kv4 channels form complexes with auxiliary β subunits, namely cytosolic Kv channel interacting proteins (KChIPs)¹³ and/or transmembrane dipeptidyl aminopeptidase-like proteins (DPPs).¹⁴ In heterologous cell systems, both KChIPs and DPPs increase Kv4 channel surface levels and modulate channel gating in a β-subunit-specific manner (see Figures S3–S5).¹³⁻¹⁹ Structural and functional analyses revealed that both the cytoplasmic N-terminus, including the T1-domain, and the cytoplasmic C-terminus of Kv4 α-subunits interact with KChIPs, while S1 and S2 are involved in DPP binding.¹⁵,¹⁹⁻²³ It is assumed that native Kv4 channels assemble in a ternary fashion, i.e., with both types of auxiliary β subunits (KChIPs and DPPs, see Figure S1 and Video S1).²¹,²³,²⁴ Since KChIP and DPP interaction sites on Kv4 α-subunits do not overlap, effects of both β subunits on Kv4 channels are expected to be more or less additive.²⁵ These β-subunit-mediated adaptations of channel physiology need to be considered, and the ability to interact with the β subunits needs to be tested when Kv4 channel function is to be evaluated, and a rigorous testing of putative variant pathogenicity is contemplated.

Variants in several Kv channel genes are associated with monogenic types of epilepsy or developmental and epileptic encephalopathy.²⁶ In particular, a number of het-

erozygous missense variants in KCND2 (MIM: 605410) that alter Kv4.2 channel gating have been linked to early-onset global developmental delay of distinct severity, often in combination with seizures, muscular hypotonia, and/or visual impairment.⁸⁻¹⁰,²⁷,²⁸ Also, a hemizygous nonsense variant of X-chromosomal KCND1 (MIM: 300281) has been suggested as a candidate pathogenic variant in a single individual with focal epilepsy, however, with no reference to Kv4.1 channel function.²⁹

Here, we report on a cohort of 18 males with 17 distinct hemizygous KCND1 variants, including two de novo missense variants (DNVs), three maternally inherited protein-truncating variants (PTVs), and 12 maternally inherited missense variants. Our genetic and functional analyses support the etiological involvement of Kv4.1 channel dysfunction in an X-linked neurodevelopmental disorder frequently associated with a variable neuropsychiatric clinical phenotype.

## Material and methods

### Recruitment of probands and genetic analysis

Utilizing GeneMatcher,³⁰ 18 individuals from 17 families were gathered as part of an international collaborative project. In most cases, affected probands were initially examined by their referring physicians, genetic analyses were performed in a diagnostic setting, and genetic findings were subsequently used for secondary research. In all probands, KCND1 variants were independently identified at the referring medical centers as the most-likely disease-causing variant. Biological samples were obtained after written informed consent was given by the affected individuals or their legal guardians. Informed consent for the publication of clinical and genetic data was obtained from all participants. The

Italy; ²¹Department of Pediatrics, Seoul National University College of Medicine, Seoul 110-744, Republic of Korea; ²²Department of Genomic Medicine, Rare Disease Center, Seoul National University Hospital, Seoul 03080, Republic of Korea; ²³The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, Mount Sinai Hospital, University of Toronto ON M5G 1E2 Toronto, Canada; ²⁴Division of Clinical and Metabolic Genetics, Department of Pediatrics, The Hospital for SickKids, University of Toronto, M5G 1X8 Toronto, Canada; ²⁵Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, AB T6G 2H7 Edmonton, Canada; ²⁶Institute of Human Genetics, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany; ²⁷Neuromuscular Disorders, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy; ²⁸Molecular Genetics and Functional Genomics, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy; ²⁹Department of Molecular Genetics and Cytogenomics, Rare and Autoinflammatory Diseases Unit, University Hospital of Montpellier, 34295 Montpellier, France; ³⁰Montpellier University, Inserm U1183, Montpellier, France; ³¹Department of Clinical Genetics, University Hospital of Montpellier, 34295 Montpellier, France; ³²The Genetic Institute of Maccabi Health Services, Rehovot 7610000, Israel; ³³Raphael Recanati Genetics Institute, Beilinson Hospital, Rabin Medical Center, Petach Tikva 49100, Israel; ³⁴Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; ³⁵Pediatric Genetics Unit, Schneider Children's Medical Center of Israel, Petah Tikvah 4920235, Israel; ³⁶Departments of Neurology and Neurosurgery, Pediatrics and Human Genetics, McGill University, Montreal, Canada; ³⁷Child Health and Human Development Program, Research Institute of the McGill University Health Centre, Montreal, Canada; ³⁸Genomic Medicine Center, Department of Pediatrics, Children's Mercy Kansas City, Kansas City, MO, USA; ³⁹UKMC School of Medicine, University of Missouri Kansas City, Kansas City, MO, USA; ⁴⁰Department of Pathology and Laboratory Medicine, Children's Mercy Kansas City, Kansas City, MO, USA; ⁴¹Department of Pediatrics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany

<footnote n="42">⁴²These authors contributed equally</footnote>

<footnote>*Correspondence: r.baehring@uke.uni-hamburg.de (R.B.), kindler@uke.de (S.K.)</footnote>

<footnote>https://doi.org/10.1016/j.ajhg.2024.04.019.</footnote>
A

T57M

Kv4.1 MAAGLATWLPARAAAAGVWLPLAQQLPPAPGVKAS-RGDEVLVNVNSGRTRETWKNTLD
Kv4.2 MAAGVAAALPFARAAAIGWMPVAPGMPAPPROERKRTODALIVNVSGRTFOTWDTLD 59
Kv4.3s MAAGVAAALPFARAAAIGWMPVAPCPAPLAPADKNK-RQDELVIVNVSGRRTFOTWRTTLE 59

T1-domain

R60C

Kv4.1 Y61Cfs*31 GCR0P B92C R99* D107A D115N
Kv4.2 RYPTDLLGSSERDFFFYHPEQTYFFDRDDPIDFRHILNFYRTGKILHYPRHECISAYDEELA 119
Kv4.3s RYPTDLLGSSERDFFFNEDKEYFFDRDEPEVERCVLNFYRTGKILHYPRSECISAYDEELA 119

T1-domain

R146C

Kv4.1 FYGVLPEVGDCLEEYDRRKKENAERLAEDEEAAQADGDPALGPASSLRQLRWAFFENP 179
Kv4.2 FFGLPETIIGDCCYYEKDRRRENAERLDDAADTDTAGESA--LPTMTARQVWRAFFENP 178
Kv4.3s FYGILPEIIGDCCYYEKDRRRENAERLDMDDNDSENN-QES--MPSLSFRQTWMRAFFENP 176

T1-domain

A202T

Kv4.1 HTSTALVFYVTVGFIAVSVIANNVETIPCRGARSRRSREQPOECRGEPAQFAECMDTACV 239
Kv4.2 HTSTMALVFYVTVGFIAVSVIANNVETPCGSS-PGHIKELPCGERYAAVFACFLTACV 237
Kv4.3s HTSTALVFYVTVGFIAVSVIANNVETPCGTV-PGS-KELPCGERYAAVFACFLTACV 234

S1

LIFTGEYLLRFAAPSCRFLSRVSMLIDVDAIVPAYIIGVLVKNDVSGAFVTLRFVRV 299
Kv4.2 MIFTVEYLLRFAAPSRYRFVRSMSIIDVDAIVPAYIIGVLVMDNEDVSGAFVTLRFVRV 297
Kv4.3s MIFTVEYLLRFAAPSRYFVRSMSIIDVDAIVPAYIIGVLVMDNEDVSGAFVTLRFVRV 294

S2

H308Y

Kv4.1 FRIEKFSRHSQLQRILGYTLKSCASELGFLFSLTIMAIIFATVMFYAEKGTNKTNFTSI 359
Kv4.2 FRIEKFSRHSQLQRILGYTLKSCASELGFLFSLTIMAIIFATVMFYAEKGSASSKFTSI 357
Kv4.3s FRIEKFSRHSQLQRILGYTLKSCASELGFLFSLTIMAIIFATVMFYAEKGSASSKFTSI 354

S4 (voltage sensor)

S5

Kv4.1 PAAFWYTIVMTTTLGYDMVPSTIAGKIEFGSICSLSGVLVIAVFVPTVIVSNFSRIYHONQ 419
Kv4.2 PAAFWYTIVMTTTLGYDMVPKTIAGKIEFGSICSLSGVLVIAVFVPTVIVSNFSRIYHONQ 417
Kv4.3s PAFSWYTIVMTTTLGYDMVPKTIAGKIEFGSICSLSGVLVIAVFVPTVIVSNFSRIYHONQ 414

P-loop (selectivity filter)

K450*

Kv4.1 RADKRRAQQVKLARIRALAKSGKTTNAFLQKYKQNGLEDGSG--SQEEGALCVRNRSAFQE 476
Kv4.2 RADKRRAQQKARLARIAAKSGSANAYMOSKRNGLLSNQLOSSE-DEQAFSGKSGSFET 476
Kv4.3s RADKRRAQQKARLARIAKVAKTGSSFEGDPEPDESPESYG--GNVIRVVSAL-630 474

S6

T516S

Kv4.1 QHHHHLLHLECKTTCHEFTDLTFSEALGAVSPGGRT-SRSTSVSSQVPGPSLLSSCCPR 535
Kv4.2 QHHHHLLHLECKTNNHEFVDEQVFEESCMEVATVNRPSSHPSLSQ---GQVHTSCCSR 532
Kv4.3s QHHHHLLHLECKTNNHEFVDEQFOMFQCNQMESSNQYPTRSPSSLSQH---PGHTSCCSR 530

dendritic targeting motif

R536G

Kv4.1 RAKRRAIRLANSTASVSRSGMQEIDELM--AGLRRSHAPQRSRSSLAKPHDSLDNCDSR 593
Kv4.2 RHKKTFRIPNANVSGSHOGSIELGTIOIQCERVTPLSNSRSSLAKMEECVRLKNCQPY 592
Kv4.3s RSKKTHTLPSNLPATRLRSMQELSTHIQGSEQPSLLTSSRSSLNKLADDDGRPNPCTSQ 590

S781

FVAIIISIPTPPANTPDESQ-SSPGGGGRAGSTLRNSLGTCLPFPETVKISSL 647
Kv4.1 FVAIIISIPTPPANTPDESQ-SSPGGGGRAGSTLRNSLGTCLPFPETVKISSL 647
Kv4.2 FVAIIISIPTPPANTPDESQ-SSPGGGGRAGSTLRNSLGTCLPFPETVKISSL 647
Kv4.3s ITTAIIISIPTPPALTPEGESRRPPASPGPNT----------NIPSIASNVVKVVSAL 636

S8

B

<figure><img src="image_3.png" /><figcaption>Figure 1. KCND1 variant-associated amino acid substitutions</figcaption></figure>

(A) Alignment of human Kv4 subfamily members Kv4.1, Kv4.2, and Kv4.3s (short isoform). Kv4.1 amino acid residues affected by the reported *KCND1* variants are shown in color, and respective changes are indicated above using single-letter code (red: initially studied *KCND1* group 1 variants; purple: additional maternally inherited missense variants, group 2). Regions highlighted in gray indicate the tetramerization (T1) domain and transmembrane segments S1–S6, as indicated below. Sequence motifs critical for channel assembly trafficking and function are indicated by bold letters (HX<sub>5</sub>CX<sub>20</sub>CC motif in the T1-domain: Zn<sup>2+</sup> coordination site;<sup>6</sup> positively charged residues in S4: voltage sensor;<sup>7</sup> potassium channel signature sequence GYGD in the pore loop [P]: selectivity filter;<sup>5</sup> critical dynamic coupling residues, glutamate [E] in the S4-S5 linker, and proline-valine [PV] in the distal S6 segment: operation of the cytoplasmic gate;<sup>8,9,10,11</sup> C-terminal di-leucine motif: dendritic targeting<sup>12</sup>). Numbers on the right specify amino acid residue positions. Sequences were aligned using Clustal Omega software (http://www.clustal.org/omega/). Residues that are perfectly conserved (*) or exhibit either strong or weak similarity (: or . for a scoring of >0.5 or ≤0.5, respectively, in the Gonnet

study was performed in accordance with the Declaration of Helsinki protocols and approved by the ethics committees of the respective institutions. Trio whole-exome sequencing (WES) was performed in all subjects, and identified *KCND1* variants were confirmed by Sanger sequencing. The different medical centers involved in this study utilized comparable protocols for DNA isolation, WES, bioinformatic processing, and variant interpretation. Functional impact of identified putative pathogenic variants was predicted via the MetaDome webserver,³¹ the AlphaMissense tool (https://alphamissense.hegelab.org/), the Rare Exome Variant Ensemble Learner (REVEL) algorithm (https://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=revel), the Combined Annotation Dependent Depletion (CADD) tool,³² and the Polymorphism Phenotyping v2 (PolyPhen-2) tool.³³

Plasmids and synthesis of Kv4.1 channels in *Xenopus* oocytes

The human KCND1 cDNA (GenBank: NM_004979.6) encoding full-length Kv4.1 (GenBank: NP_004970.3, UniProtKB Q9NSA2)³⁴ was inserted into pGEM-HE to generate pGEM-OK-hKCND1, which carries an optimized Kozak sequence. Variant-specific mutations were introduced utilizing the Quick-Change II site-directed mutagenesis kit (Agilent), and resulting constructs were verified by Sanger sequencing. Similar to a previous study, pGEM vectors encoding human KChIP2b¹⁵ and DPP6s (a generous gift from Nicole Schmitt, University of Copenhagen), two well-characterized isoforms present in many brain regions, and hereafter referred to as KChIP and DPP, respectively, were used for auxiliary β-subunit synthesis.¹⁰,³⁵ Plasmid linearization, *in vitro* transcription, and cRNA injection in *Xenopus laevis* oocytes were done as previously described.¹⁰ All experimental procedures were in accordance with the national guidelines for the care and use of research animals and were approved by the local authorities. To generate Kv4.1 homotetramers, 5 ng total cRNA were injected per oocyte. Kv4.1 wild type (WT) and variant cRNAs were also co-injected KChIP or DPP cRNAs (5 + 5 ng per oocyte) for binary complex formation (only one type of auxiliary β-subunit) or with both KChIP and DPP cRNAs (2 + 5 + 5 ng per oocyte) for ternary complex formation.

Electrophysiology

Oocytes were used for electrophysiological recordings 1–3 days after cRNA injection. Currents were recorded at room temperature

PAM 250 matrix) are indicated. Note that Kv4.1 amino acid residues in positions 57, 60, 61, 92, 99, 107, 115, 146, 308, 431, 450, 536, and 578 are fully conserved in all three Kv4 subfamily members.

(B) Topology scheme of the Kv4.1 α-subunit (extracellular and cytoplasmic side indicated) with six transmembrane segments (S1–S6, gray boxes) and cytoplasmic N- and C-termini. Positively charged amino acid residues in S4 (+) mediate voltage sensing,<sup>7</sup> and a re-entrant P loop between S5 and S6 harbors the selectivity filter sequence.<sup>5</sup> The T1 domain (gray box) is located in the cytoplasmic N-terminus. Changes in amino acid sequence are indicated in single-letter code. The two amino acid substitutions and three truncation sites, respectively, associated with five KCND1 group 1 variants are depicted as red dots (DNVs R92C and D115N) and red squares (PTVs Y61Cfs*31, R99*, and K450*). All other amino acid substitutions (12 maternally inherited missense variants) are depicted as purple dots. Note that all variant-associated amino acid substitutions, except for p.Ala202Thr (A202T in S1) and p.His308Tyr (H308Y in S4), reside within either the cytoplasmic N- or the cytoplasmic C-terminal domain of the protein and do not affect one of the indicated critical sequence motifs.
(20°C–22°C) under two-electrode voltage clamp as described¹⁰ in a low chloride (15 mM) bath solution containing (in mM) 7.4 NaCl, 88.6 Na-aspartate, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, and 5 HEPES, pH 7.4 with NaOH. Pulse protocols are explained in the figure legends. Data were analyzed using FitMaster (HEKA) and KaleidaGraph (Synergy Software), as described previously.¹⁰,¹¹,³⁶ Current decay kinetics were approximated with a double-exponential function and the kinetics of recovery from inactivation with a single-exponential function. The voltage dependences of activation and steady-state inactivation were analyzed with appropriate Boltzmann functions.¹⁰,¹¹,³⁶ Pooled data are presented as mean ± SD, unless stated otherwise. Statistical analyses for multiple groups are based on ANOVA with Dunnett's post hoc testing and for two groups on Student's t tests.

## Results

### Identification of hemizygous *KCND1* variants associated with variable neurological phenotypes

Utilizing trio WES and GeneMatcher, we identified distinct hemizygous coding variants of *KCND1* in a cohort of 18 male subjects from 17 families. The variant group includes one frameshift, two nonsense, and 12 missense mutations, all of which are maternally inherited, as well as two DNVs (Table 1). Affected individuals display diverse neurological abnormalities, primarily including delays in various developmental domains, distinct neuropsychiatric signs, and seizures (Tables 2 and S1). In each case, trio WES data analysis revealed the respective hemizygous *KCND1* variant as the most-likely genetic cause of the observed clinical phenotype, as no other (likely) pathogenic variants that could explain the observed clinical features were identified. *KCND1* is located on the X chromosome and encodes the Kv4.1 Kv channel α subunit.³⁴ Although Kv4 channels play a crucial role in brain function,² variants in *KCND1* have not been associated with a human disease phenotype in OMIM, though a single individual with focal epilepsy has been reported as hemizygous for a truncating variant in this gene.²⁹ Thus, the two central aims of the present work were to (1) functionally characterize the identified *KCND1* variants, thereby allowing their classification beyond uncertain clinical significance and (2) provide an overview of the phenotypic spectrum associated with hemizygous *KCND1* variants.

Initially, we focused on a set of five variants (group 1) for which the genetic data provide the strongest evidence for pathogenicity, i.e., DNVs and PTVs. The allele frequency in hemizygotes in the gnomAD database (v4.0.0, GRCh38/hg38)³⁷ is very low for the DNV c.274C>T (p.Arg92Cys) (7.54e-6, individual 1) and low for the DNV c.343G>A (p.Asp115Asn) (1.26e-5, individual 2) (Table 1). Both affected amino acid residues in the T1-domain are completely conserved throughout the Kv4 subfamily (Figure 1A). While well-established prediction tools support the pathogenicity of p.Arg92Cys, they provide ambiguous results for p.Asp115Asn (Table 1). We also identified three PTVs, namely c.182_194del (p.Tyr61Cysfs*31) (individual

3), c.295C>T (p.Arg99*) (individual 4), and c.1348A>T (p.Lys450*) (individual 5). While the former is not listed in gnomAD, p.Arg99* and p.Lys450* are both found at very low allele frequencies in hemizygotes (around 5.0e-6). High-scaled C-scores predicted by CADD suggest that the latter two are pathogenic variants. All PTVs are predicted to lead to nonsense-mediated decay (NMD) of the respective transcripts, thus resulting in a complete loss-of-function (LOF) in hemizygous males. However, as NMD is known to exhibit a large variability in its efficiency across mRNAs, cell types, tissues, and individuals, we also considered the possibility of NMD escape.³⁸ In case of NMD escape and synthesis of p.Tyr61Cysfs*31 and p.Arg99*, both truncated proteins are expected to remain cytosolic and be unable to form functional channels due to the lack of any transmembrane domains (Figure 1), thus still leading to complete LOF. In contrast, if synthesized, p.Lys450* contains transmembrane regions S1–S6 (Figure 1) and is hence theoretically able to form functional channels. Thus, from the five group 1 variants, p.Arg92Cys, p.Asp115Asn, and p.Lys450* were subsequently chosen for functional characterization, while cytosolic p.Tyr61Cysfs*31 and p.Arg99*, although patho-physiologically relevant, were not included in these experiments.

### *KCND1* group 1 variants: Functional characterization and associated clinical features

First of all, Kv4.1 WT and variant channels were functionally characterized in a ternary configuration, i.e., in the presence of both types of auxiliary β subunits (KChIP and DPP, Figures 2A and S1 and Video S1), a likely channel configuration in native neuronal tissues²⁴ (see material and methods). For the ternary Kv4.1 channel complexes, the following biophysical parameters were examined under two-electrode voltage clamp in *Xenopus* oocytes (see material and methods): peak current amplitude, macroscopic inactivation kinetics (i.e., kinetics of current decay), kinetics of recovery from inactivation, and voltage dependences of activation and steady-state inactivation.

Both Kv4.1 WT and variant ternary channels, even the truncated protein p.Lys450*, mediated rapidly, activating and inactivating currents with variable amplitudes and decay kinetics in response to depolarizing voltage steps (Figure 2B). Mean peak current amplitudes at a test voltage of +40 mV (19.2 ± 9.52 µA for Kv4.1 WT, *n* = 16) were found to be reduced for all three variants (Figure 2C). Macroscopic inactivation kinetics (τ₁ = 30.7 ± 4.72 ms, relative amplitude = 83 ± 11% and τ₂ = 85.8 ± 56.8 ms for Kv4.1 WT, *n* = 21) were accelerated for p.Asp115Asn, slowed for p.Arg92Cys and p.Lys450*, and the relative amplitude accounted for by τ₁ was significantly decreased for p.Lys450* (Figures 2D and 2F). The kinetics of recovery from inactivation (τrec = 11.2 ± 2.02 ms for Kv4.1 WT, *n* = 16), were unaffected for p.Arg92Cys and p.Asp115Asn but slowed for p.Lys450* (Figures 2E and 2G). Finally, we examined the voltage dependences of activation and steady-state inactivation (Figure 3A). For Kv4.1 WT,
<table><caption>Table 1. Description and in silico analysis of KCND1 variants</caption><thead><tr><th colspan="10">Genetic analysis</th><th colspan="5">Bioinformatic analysis</th></tr><tr><th rowspan="2">Transcript GenBank:<br>Individual NM_004979.6</th><th rowspan="2">Protein GenBank:<br>NP_004970.3</th><th colspan="4">gnomAD v2.1.1</th><th colspan="4">gnomAD v4.0.0</th><th rowspan="2">MetaDome</th><th rowspan="2">REVEL</th><th rowspan="2">CADD</th><th rowspan="2">Alpha- Missense</th><th rowspan="2">Poly- Phen-2</th></tr><tr><th>Allele # of hemi- count zygotes</th><th>Allele frequency</th><th>Allele frequency in hemizygotes</th><th>Allele # of hemi- count zygotes</th><th>Allele frequency</th><th>Allele frequency in hemizygotes</th></tr></thead><tbody><tr><th colspan="14">De novo missense variants</th></tr><tr><td>1</td><td>c.274C>T</td><td>p.Arg92Cys<sup>a</sup></td><td>0</td><td>0</td><td>0</td><td>0</td><td>11</td><td>3</td><td>9.09e-6</td><td>7.54e-6</td><td>1.31</td><td>0.404</td><td>24.7</td><td>0</td><td>1.0</td></tr><tr><td>2</td><td>c.343G>A</td><td>p.Asp115Asn<sup>a</sup></td><td>1</td><td>0</td><td>5.58e-6</td><td>0</td><td>18</td><td>5</td><td>1.49e-5</td><td>1.26e-5</td><td>0.53</td><td>0.165</td><td>23.6</td><td>0.037</td><td>0.388</td></tr><tr><th colspan="14">Maternally inherited truncating variants</th></tr><tr><td>3</td><td>c.182_194del</td><td>p.Tyr61Cysfs*31<sup>a</sup></td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td></tr><tr><td>4</td><td>c.295C>T</td><td>p.Arg99*<sup>a</sup></td><td>2</td><td>0</td><td>1.1e-5</td><td>0</td><td>6</td><td>2</td><td>4.96e-6</td><td>5.03e-6</td><td>N/A</td><td>N/A</td><td>35</td><td>N/A</td><td>N/A</td></tr><tr><td>5</td><td>c.1348A>T</td><td>p.Lys450*<sup>a</sup></td><td>1</td><td>0</td><td>5.51e-6</td><td>0</td><td>5</td><td>1</td><td>8.87e-6</td><td>4.99e-6</td><td>N/A</td><td>N/A</td><td>37</td><td>N/A</td><td>N/A</td></tr><tr><th colspan="14">Maternally inherited missense variants</th></tr><tr><th colspan="14">Variants affecting the N-terminal cytoplasmic domain</th></tr><tr><td>6</td><td>c.170C>T</td><td>p.Thr57Met<sup>a</sup></td><td>3</td><td>0</td><td>1.67e-5</td><td>0</td><td>12</td><td>0</td><td>9.92e-6</td><td>0</td><td>0.14</td><td>0.858</td><td>25.3</td><td>0.909</td><td>0.986</td></tr><tr><td>7</td><td>c.178C>T</td><td>p.Arg60Cys<sup>a</sup></td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>0</td><td>1.82e-6</td><td>0</td><td>0.16</td><td>0.469</td><td>29.0</td><td>0.534</td><td>1.0</td></tr><tr><td>8</td><td>c.238G>C</td><td>p.Gly80Arg</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0.36</td><td>0.101</td><td>18.14</td><td>0.145</td><td>0.001</td></tr><tr><td>9</td><td>c.320G>A</td><td>p.Arg107Gln<sup>a</sup></td><td>0</td><td>0</td><td>0</td><td>0</td><td>6</td><td>2</td><td>4.96e-6</td><td>5.03e-6</td><td>0.55</td><td>0.395</td><td>23.1</td><td>0.225</td><td>0.956</td></tr><tr><td>10</td><td>c.436C>T</td><td>p.Arg146Cys<sup>a</sup></td><td>1</td><td>0</td><td>5.71e-6</td><td>0</td><td>3</td><td>2</td><td>2.74e-6</td><td>8.78e-6</td><td>0.18</td><td>0.807</td><td>26.5</td><td>0.952</td><td>1.0</td></tr><tr><td>11</td><td>c.442G>A</td><td>p.Ala148Thr</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>2.2e-6</td><td>0</td><td>0.23</td><td>0.438</td><td>20.7</td><td>0.112</td><td>0.471</td></tr><tr><th colspan="14">Variants affecting transmembrane domains</th></tr><tr><td>12</td><td>c.604G>A</td><td>p.Ala202Thr</td><td>5</td><td>2</td><td>2.53e-5</td><td>2.83e-5</td><td>130</td><td>35</td><td>1.08e-4</td><td>8.82e-5</td><td>0.74</td><td>0.613</td><td>22.7</td><td>0.207</td><td>0.236</td></tr><tr><td>13</td><td>c.922C>T</td><td>p.His308Tyr<sup>a</sup></td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0.69</td><td>0.774</td><td>25.3</td><td>0.970</td><td>0.786</td></tr><tr><th colspan="14">Variants affecting the C-terminal cytoplasmic domain</th></tr><tr><td>14</td><td>c.1291C>T</td><td>p.Arg431Cys<sup>a</sup></td><td>2</td><td>1</td><td>1.11e-5</td><td>1.56e-5</td><td>5</td><td>3</td><td>4.58e-6</td><td>8.38e-6</td><td>1.79</td><td>0.694</td><td>33</td><td>0.936</td><td>1.0</td></tr><tr><td>15/16</td><td>c.1546A>T</td><td>p.Thr516Ser</td><td>11</td><td>3</td><td>5.81e-5</td><td>4.63e-5</td><td>277</td><td>79</td><td>2.3e-4</td><td>2.01e-4</td><td>0.9</td><td>0.157</td><td>15.87</td><td>0.102</td><td>0.044</td></tr><tr><td>17</td><td>c.1606A>G</td><td>p.Arg536Gly<sup>a</sup></td><td>3</td><td>2</td><td>1.68e-5</td><td>3.11e-5</td><td>32</td><td>14</td><td>2.65e-5</td><td>3.52e-5</td><td>0.46</td><td>0.822</td><td>25.1</td><td>0.194</td><td>0.949</td></tr><tr><td>18</td><td>c.1733A>T</td><td>p.Asn578Ile<sup>a</sup></td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0.55</td><td>0.814</td><td>26.9</td><td>0.756</td><td>1.0</td></tr></tbody></table>

Individuals 15 and 16 are brothers of different age. Allele count, number of identified hemizygotes, and allele frequency of individual variants are derived from the gnomAD database (https://gnomad.broadinstitute.org/, gnomAD v2.1.1, GRCh37/hg19 and gnomAD v4.0.0, GRCh38/hg38; as of January 22, 2024). MetaDome tolerance scores are calculated as described (https://stuart.radboudumc.nl/metadome/). The phred-like scaled C score predicted by CADD (https://wintervar.wglab.org/) ranges from 1 to 99 with higher values indicating more deleterious variants. The AlphaMissense score (https://alphamissense.hegelab.org/) ranges from 0.0 to 1.0 with scores below 0.34 considered likely benign and scores higher than 0.564 being considered likely pathogenic. The REVEL and the PolyPhen-2 scores range from 0.0 (tolerated) to 1.0 (deleterious). CADD, Combined Annotation Dependent Depletion; HGVS, Human Genome Variation Society; N/A, not applicable.

<footnote>ªRespective native amino acids are conserved throughout the Kv4 family.</footnote>
<table><caption>**Table 2.** Clinical features of subjects with hemizygous *de novo* missense or maternally inherited truncating KCND1 group 1 variants</caption><thead><tr><th>Individual #</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th></tr></thead><tbody><tr><td>cDNA variant<br>GenBank:<br>NM_004979.6</td><td>c.274C>T</td><td>c.343G>A</td><td>c.182_194del</td><td>c.295C>T</td><td>c.1348A>T</td></tr><tr><td>Protein variant<br>GenBank:<br>NP_004970.3</td><td>p.Arg92Cys</td><td>p.Asp115Asn</td><td>p.Tyr61Cysfs*31</td><td>p.Arg99*</td><td>p.Lys450*</td></tr><tr><td>Variant origin</td><td>de novo</td><td>de novo</td><td>maternally inherited</td><td>maternally inherited</td><td>maternally inherited</td></tr><tr><td>Age at last<br>examination</td><td>15 years</td><td>9 years 11 months</td><td>16 years</td><td>16 years</td><td>3 years 2 months</td></tr><tr><td>Age at first signs</td><td>10 years</td><td>6 years</td><td>2 years 3 months</td><td>abt 2 years</td><td>neonatal period</td></tr><tr><td>First signs</td><td>anxiety,<br>tics</td><td>speech delay,<br>seizures</td><td>seizures</td><td>speech delay,<br>motor delay,<br>anxiety</td><td>speech delay,<br>motor delay</td></tr><tr><td>Motor development</td><td>normal</td><td>normal</td><td>normal</td><td>delayed</td><td>delayed</td></tr><tr><td>Intellectual<br>development</td><td>average</td><td>low average,<br>TIQ 85</td><td>average</td><td>borderline mental<br>functioning – low<br>average,<br>TIQ 74-86</td><td>extremely low/<br>impaired, TIQ 68</td></tr><tr><td>Speech development</td><td>normal</td><td>delayed</td><td>normal</td><td>delayed</td><td>delayed</td></tr><tr><td>Receptive language</td><td>normal</td><td>below average</td><td>normal</td><td>below average</td><td>below average</td></tr><tr><td>Verbal ability</td><td>normal</td><td>pronunciation problems,<br>grammar problems</td><td>normal</td><td>pronunciation<br>problems<br>at younger age</td><td>pronunciation problems,<br>uses only very short<br>sentences</td></tr><tr><td>Comprehension</td><td>normal</td><td>below average</td><td>normal</td><td>below average</td><td>below average</td></tr><tr><td>Neuropsychiatric<br>signs</td><td>ASD, attention<br>deficit, anxiety,<br>tics</td><td>attention deficit</td><td>none</td><td>ASD, anxiety,<br>emotional<br>problems</td><td>attention deficit</td></tr><tr><td>Seizures/epilepsy</td><td>no</td><td>epilepsy, absences,<br>myoclonus, eyelid<br>myoclonia</td><td>epilepsy, generalized<br>tonic-clonic seizures<br>during sleep until<br>11 years, then seizures<br>remitted, Valproate<br>responsive</td><td>no</td><td>no</td></tr><tr><td>Electro-<br>encephalography</td><td>normal</td><td>irregular polyspikes and<br>spike-waves,<br>photoparoxysmal<br>reaction</td><td>generalized<br>abnormalities<br>at 3 years 8 months,<br>normal<br>at 12 years 6 months</td><td>ND</td><td>ND</td></tr></tbody></table>

abt, about; ASD, autism spectrum disorder; ND, no data; TIQ, total intelligence quotient.

Boltzmann analysis yielded voltages of -6.45 ± 5.42 mV (n = 17) and -57.9 ± 4.30 mV (n = 18) for half-maximal activation and inactivation, respectively. Except for a decreased slope factor (steepening) of the p.Arg92Cys activation curve, the voltage dependence of activation remained unaltered for all variants. The voltage dependence of steady-state inactivation was similar to Kv4.1 WT for p.Arg92Cys but negatively shifted for p.Asp115Asn and p.Lys450* with no significant effects on the steepness of inactivation curves (Figure 3B, results summarized in Table S3). To summarize, functional analyses of group 1 variants p.Arg92Cys, p.Asp115Asn, and p.Lys450* in a ternary channel configuration revealed that (1) all three variants significantly differ from WT, and (2) each of them displays distinct variant-specific alterations of bio-physical channel properties, thus providing evidence for their pathogenicity. Since the two remaining group 1 vari-

ants, p.Tyr61Cysfs*31 and p.Arg99*, which were not functionally studied, are predicted to result in complete LOF, either due to NMD of the respective mRNA or the inability of the encoded protein to form functional channels, the above data collectively suggest that all five identified group 1 KCND1 variants are pathogenic, impairing Kv4.1 channel function and resulting in an X-linked neurodevelopmental disorder with variable expressivity.

From a clinical perspective, group 1 KCND1 variants are associated with a neurodevelopmental disorder variable in severity, with or without seizures (Table 2). First clinical signs were noted between the neonatal period and 10 years of age. Four individuals exhibit neuropsychiatric signs, including autism spectrum disorder (ASD), attention deficit disorder, anxiety, tics, and emotional problems. In addition, three of these subjects present with speech delay, receptive language and comprehension problems, and
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Functional expression and kinetic analysis of inactivation for KCND1 WT and group 1 variants in a ternary configuration</figcaption></figure>

(A) Kv4.1 wild-type channels (WT, gray quadrants) and variant channels with amino acid substitutions (quadrants with red dots) were functionally analyzed in a ternary configuration, i.e., with both KChIP (orange) and DPP (blue, see also Figure S1 and Video S1) in *Xenopus laevis* oocytes (see material and methods).

(B) Macroscopic currents mediated by Kv4.1 WT, p.Arg92Cys, p.Asp115Asn, and p.Lys450* ternary channels. Currents were recorded under two-electrode voltage clamp using depolarizing voltage steps from –100 to +40 mV, as indicated (gray trace: WT; red traces: p.Arg92Cys, p.Asp115Asn, and p.Lys450*).

(C) Bar graph shows mean peak current amplitudes, including individual data points (number of observations indicated). Note that for all functionally tested group 1 variants (red bars) mean peak current amplitudes were smaller than for Kv4.1 WT (gray bar and horizontal dotted line). Water-injected oocytes served as control.

(D) Normalized currents mediated by Kv4.1 WT (gray trace), p.Arg92Cys, p.Asp115Asn, and p.Lys450* (red traces) in a ternary configuration. Currents were elicited by extended voltage pulses, as indicated (2.5 s, +40 mV), to study the kinetics of macroscopic inactivation (i.e., current decay).

(E) Recovery of ternary channels from inactivation was measured using a double-pulse protocol with long control and brief test pulses to +40 mV and variable interpulse durations at –100 mV ($\Delta t$, see inset). The normalized data ($I_{test}/I_{control}$) were plotted against the interpulse duration and described by a single-exponential function (gray symbols: Kv4.1 WT; black symbols: p.Arg92Cys and p.Asp115Asn; red symbols: p.Lys450*; error bars are SEM and smaller than symbols).

(F) Inactivation time constants obtained by fitting the current decay kinetics with a double-exponential function ($\tau_1$: circles, $\tau_2$: triangles) and the relative amplitude of the total decay accounted for by $\tau_1$.

(G) Recovery time constants obtained by fitting the kinetics of recovery from inactivation with a single-exponential function.

(F and G) Gray symbols and horizontal dotted lines: WT; black symbols: values obtained for variant ternary channels with no difference compared to Kv4.1 WT; red symbols: values obtained for variant ternary channels that significantly differ from Kv4.1 WT; number of observations indicated; all statistics based on one way ANOVA with Dunnett's post-hoc testing; significant differences compared to Kv4.1 WT are indicated with \*p < 0.05 or \*\*p < 0.0001.
<figure><img src="image_5.png" /><figcaption>**Figure 3. Voltage dependence of activation and steady-state inactivation**</figcaption></figure>

(A) Voltage dependences of peak conductance activation (circles) and steady-state inactivation (squares) are shown for Kv4.1 wild-type (WT), p.Arg92Cys, p.Asp115Asn, and p.Lys450* ternary channels. For the study of steady-state inactivation, brief control and test pulses to +40 mV were separated by a 10 s conditioning pulse (ΔV between -100 and 0 mV in 10 mV increments, see inset). Normalized data ($I_{test}/I_{control}$) were plotted against the conditioning pulse voltage. For the study of peak conductance activation, test pulses to voltages between -80 and +60 mV (ΔV in 10 mV increments) were applied from -100 mV (see inset). Normalized conductance values were plotted against the test pulse voltage. The data were fitted with appropriate Boltzmann functions.<sup>11,36</sup> Gray symbols: Kv4.1 WT; black symbols: data which do not differ from Kv4.1 WT; red symbols: data that differ from Kv4.1 WT (error bars are SEM).

(B) Voltages of half-maximal inactivation (squares) and half-maximal activation (circles) and corresponding slope factors ($k_{inact}$ and $k_{act}$, respectively); gray symbols and vertical dotted lines: Kv4.1 WT; black symbols: $V_{1/2}$ and $k$ values of variant ternary channels with no difference compared to Kv4.1 WT; red symbols: $V_{1/2}$ and $k$ values of variant ternary channels that significantly differ from Kv4.1 WT; number of observations indicated; all statistics based on one way ANOVA with Dunnett's post-hoc testing; significant differences compared to Kv4.1 WT are indicated with \*p < 0.05 or \*\*p < 0.0001.

impaired intellectual development. Two individuals have different types of epilepsy, while two others present with delayed motor development. There is no apparent genotype-phenotype correlation.

### Maternally inherited KCND1 missense variants (group 2)

In addition to the five group 1 variants described above, we identified 13 male individuals carrying 12 distinct maternally inherited KCND1 missense variants (group 2; Table 1), which were initially considered variants of unknown significance (VUSs). Eleven group 2 variants are private in unrelated males, while p.Thr516Ser was identified in two brothers of different age (individuals 15 and 16; Table 1). The phenotypic spectrum associated with group 2 variants overlaps with the range of phenotypes exhibited by the individuals bearing group 1 variants (Table S1). All 18 individuals present with neurological, albeit rather variable, abnormalities. Frequent clinical signs include impaired intellectual development (14 individuals), delayed speech development (14 individuals), impaired comprehension (13 individuals), neuropsychiatric signs (11 individuals), impaired receptive language (nine individuals), delayed motor development (eight individuals), muscular hypotonia (seven individuals), and different forms of epilepsy (six individuals). Neuropsychiatric features include ASD, attention

deficit, hyperactivity, anxiety, tics, emotional problems, and poor social skills. Of note, the two differently aged brothers carrying the c.1546A>T (p.Thr516Ser) variant (individuals 15 and 16) share a common phenotype with only marginal differences. Both boys present with speech delay, reduced receptive language and comprehension, delayed intellectual development, and abnormalities in cerebral magnetic resonance imaging, while until now only the younger brother has been diagnosed with ASD. Thus, despite a rather large clinical variability within the entire cohort, a particular variant may be associated with a more defined phenotypic pattern. It is also worth noting that the variant p.His308Tyr is the one that deviates the most from Kv4.1 WT in terms of its biophysical parameters (see below). However, since the affected child was only two years old at the time of the most recent medical examination, it is not yet possible to definitively state whether these drastic functional changes correlate with a particularly severe phenotype (individual 13). Subject 14 deceased at the age of eight months due to respiratory insufficiency, probably linked to progressive hypomyelinating encephalopathy. Reportedly, in all cases, the heterozygous carrier mothers are clinically unaffected.

Within group 2, the pathogenicity of six variants is strongly supported by the fact that they are not listed as
<figure><img src="image_6.png" /><figcaption>Figure 4. Functional expression of group 2 variants in a ternary configuration</figcaption></figure>

Mean peak current amplitudes at +40 mV including individual data points (number of observations indicated; gray bar and horizontal dotted line: Kv4.1 WT data from Figure 2C; red bars: variant ternary channel data from Figure 2C; black bars: Peak amplitude values for variant ternary channels with no difference compared to Kv4.1 WT; purple bars: peak amplitude values of variant ternary channels that significantly differ from Kv4.1 WT; all statistics based on one way ANOVA with Dunnett's post-hoc testing; significant differences compared to Kv4.1 WT are indicated with \*p < 0.05 or \*\*p < 0.0001.

hemizygous variants in gnomAD (p.Thr57Met, p.Arg60Cys, p.Gly80Arg, p.Ala148Thr, p.His308Tyr, and p.Asn578Ile). The combined data from four distinct pathogenicity prediction algorithms indicate that six variants (p.Thr57Met, p.Arg146Cys, p.His308Tyr, p.Arg431Cys, p.Arg536Gly, and p.Asn578Ile) are likely pathogenic, while the prediction for the other six variants is rather ambiguous.

On the protein level, nine of the 12 substituted amino acid residues are conserved throughout the Kv4 subfamily (Figure 1A), thus suggesting potential biological relevance. Variant-specific amino acid exchanges primarily reside in the cytoplasmic N- or C-termini of Kv4.1, with only two exceptions affecting S1 or S4 (Figures 1 and S1 and Video S1). While these findings appear to suggest that both intracellular termini of Kv4.1 represent hotspots for disease-associated amino acid substitutions, the exact pathogenic potential of the respective variants in a hemizygous state remains unclear. Thus, after having ensured based on immunocytochemical experiments with epitope-tagged constructs that all variants are synthesized and integrated in the plasma membrane of transfected human cells (data not shown), we aimed at their functional characterization to aid clinical variant classification.

When tested in a ternary configuration (i.e., in the presence of both KChIP and DPP), all 12 maternally inherited missense variants mediated A-type currents and thus allowed further functional characterization. Of note, all group 2 variants as well as the three previously studied group 1 variants were included in the statistical analysis of the functional data. Reduced peak current amplitudes compared to Kv4.1 WT were only seen for p.Arg146Cys and p.His308Tyr (Figure 4). However, macroscopic inactivation kinetics were differentially altered for six out of the 12 variants, with an overall acceleration for p.Arg107Gln and p.Arg146Cys and an overall slowing for p.His308Tyr, p.Thr516Ser, and p.Asn578Ile, while in p.Ala148Thr only the relative amplitude of τ₁ was reduced in comparison to Kv4.1 WT (Figure 5A). Notably, in none of the 12 variants, the recovery from inac-

tivation was affected, except for p.His308Tyr, which showed a remarkable about 100-fold slowing (Figure 5B). The voltage dependences of activation and/or steady-state inactivation were modified for three of the 12 variants. Thus, for p.His308Tyr, both voltage dependences were negatively shifted, whereas for p.Ala202Thr and p.Thr516Ser, both voltage dependences were positively shifted. In addition, increased slope factors (flattening) were obtained for the p.Ala202Thr inactivation curve and the p.His308Tyr activation curve (Figure 5C, results summarized in Table S3). Taken together, when tested in a ternary configuration, significant variant effects on one or more electrophysiological parameters were observed for seven out of 12 group 2 variants. It should be noted that, due to the inclusion of a large number of variants in addition to the initially studied group 1 variants, and particularly due to the drastic effects caused by p.His308Tyr, ANOVA might underestimate the significance of effects associated with some of the variants.

## Variant-associated effects in Kv4.1 homotetramers and binary channel complexes

In the absence of a precise knowledge of the availability of KChIP and DPP subunits at early developmental stages, in specific brain regions, cell types, or subcellular compartments, further electrophysiological experiments were performed with Kv4.1 homotetramers and binary channel complexes (containing only one of the two auxiliary β subunits). First, Kv4.1 WT and variant channels were examined in the absence of auxiliary β subunits to find out whether a variant exerts an intrinsic effect on biophysical properties. Second, channels were synthesized together with either KChIP or DPP to assess possible dysfunctions of binary channel complexes (results summarized in Tables S4–S6). Our experiments revealed for instance that, although inconspicuous in the ternary configuration, p.Gly80Arg, p.Ala202Thr, p.Thr516Ser, and p.Asn578Ile show an increase, whereas p.Arg431Cys shows a decrease in peak current amplitude in the absence of auxiliary β
<figure><img src="image_7.png" /></figure>

subunits. Similarly, macroscopic inactivation kinetics were accelerated for p.Thr57Met and slowed for p.Gly80Arg and p.Arg536Gly, and concerning the voltage dependences, positive shifts were seen for p.Thr57Met and p.Arg92Cys, and negative shifts were seen for p.Asp115Asn, p.Ala148Thr, p.Arg431Cys, and p.Asn578Ile in the absence of auxiliary β subunits but not in the ternary configuration. For other variants, intrinsic functional alterations correspond to those observed in the ternary configuration. Thus, peak current amplitudes were intrinsically lowered for p.Arg92Cys, p.His308Tyr, and p.Lys450*; macroscopic inactivation kinetics were intrinsically slowed for p.Lys450* and p.Asn578Ile; and recovery kinetics were intrinsically slowed for p.His308Tyr. Finally, the voltage dependence of steady-state inactivation was intrinsically shifted positive for p.Ala202Thr and p.Thr516Ser, and both voltage dependences were intrinsically shifted negative for p.His308Tyr. Taken together, our findings show

<caption>**Figure 5.** Biophysical characterization of group 2 variants in a ternary configuration</caption>

(A) Inactivation time constants obtained by fitting the Kv4.1-mediated current decay kinetics with a double-exponential function ($\tau_1$: circles, $\tau_2$: triangles) and the relative amplitude of the total decay accounted for by $\tau_1$ (number of observations indicated).
(B) Recovery time constants obtained by fitting the kinetics of recovery from inactivation with a single-exponential function (number of observations indicated).
(C) Voltages of half-maximal inactivation (squares) and half-maximal activation (circles) and corresponding slope factors ($k_{inact}$ and $k_{act}$, respectively; number of observations indicated).
(A–C) Gray symbols and dotted lines: Kv4.1 WT data from Figures 2F, 2G, and 3B; red symbols: variant ternary channel data from Figures 2F, 2G, and 3B that significantly differ from Kv4.1 WT when testing 15 variants, including p.His308Tyr; black symbols: values obtained for variant ternary channels with no difference compared to Kv4.1 WT (including part of the p.Asp115Asn and p.Lys450* data); purple symbols: values obtained for variant ternary channels (maternally inherited missense variants) that significantly differ from Kv4.1 WT; number of observations indicated; all statistics based on one way ANOVA with Dunnett's post-hoc testing; significant differences compared to Kv4.1 WT are indicated with \*p < 0.05 or \*\*p < 0.0001.

that variant-specific alterations of individual biophysical parameters are often apparent in all four tested channel configurations. On the other hand, the only functional alteration found for p.Arg60Cys was a modest acceleration of macroscopic inactivation

kinetics, reflected by an increase in the relative amplitude of $\tau_1$ in the DPP binary complex.

Of note, the presence of only one β subunit (binary channels) also yielded valuable information on whether a variant channel was still able to functionally interact with KChIP or DPP. As seen for Kv4.1 WT and the majority of variants, the concurrent presence of KChIP slowed the initial phase and accelerated the late phase of macroscopic inactivation, accelerated the recovery from inactivation, and positively shifted the voltage dependence of steady-state inactivation, whereas the presence of DPP accelerated both the initial phase of macroscopic inactivation and recovery from inactivation and negatively shifted the voltage dependences of both activation and steady-state inactivation. These typical β-subunit effects on Kv4.1 channel function were lost for certain variants (see Figures S3–S5) thus representing a further putative pathophysiological mechanism.
## Classification of KCND1 group 2 variants

Finally, an assessment of the pathogenicity probability of group 2 variants should be made, taking into account the above findings. Thus, we relied on the well-established American College of Medical Genetics and Genomics (ACMG) guidelines for clinical variant interpretation.³⁹,⁴⁰ This classification system utilizes the evidence code PS3 based on the results of “well-established” functional assays, which are suitable to discriminate between abnormal and normal gene/protein function. In the past, the highly reproducible and robust two-electrode voltage-clamp technique in Xenopus oocytes has been used successfully to assess the biophysical features of ion channels, including variants of KCND2/Kv4.2⁸,⁹,¹⁰,⁴¹,⁴² and KCND3/Kv4.3,⁴³⁻⁴⁵ as well as other voltage-gated potassium channels.⁴⁶ Therefore, we performed PS3 scoring of KCND1 variants on the basis of the results from this functional assay system. In particular, to rate functional impairment of Kv4.1 variants, we included the five biophysical parameters examined (peak current amplitude, macroscopic inactivation, recovery from inactivation, and the voltage dependences of activation and steady-state inactivation) from all analyzed channel configurations (ternary complexes, Kv4.1 alone, and both binary channel compositions). In addition, we incorporated the functional interaction of Kv4.1 variants with KChIP2 and/or DPP6. Thus, to assess physiological impairment of a given functionally analyzed variant, 29 items were taken into account (Table S7). If four or more of these functional parameters are significantly altered compared to Kv4.1 WT, this is valued as supportive evidence for a pathogenic effect on channel function (ACMG criterion PS3, see discussion). This definition is based on the idea that variants showing normal interaction with auxiliary β subunits meet the requirements to pass the threshold if they differ from Kv4.1 WT in only one of the five biophysical parameters examined but do so consistently in all four channel configurations tested. All but two group 2 variants (p.Arg60Cys and p.Arg536Gly) exceed this threshold value (Table S7), with p.His308Tyr reaching the maximum number of 22 altered parameters, some of which are drastically affected. For comparison, 12 (p.Asp115Asn) and 15 (p.Arg92Cys and p.Lys450*) items are significantly altered in the functionally analyzed pathogenic group 1 variants. According to ACMG classification, three group 2 variants are considered to be likely pathogenic, whereas the nine remaining variants are categorized as VUSs (Table 3).

## Discussion

Here, we describe 17 distinct hemizygous coding variants of KCND1, which were independently identified as the most-likely disease-causing variant in 18 male subjects presenting with a variable neurodevelopmental phenotype. To determine variant pathogenicity, we employed genomic data analyses and comprehensive functional characteriza-

tion of biophysical channel features. Our results provide strong supporting evidence that hemizygous KCND1 variants that differentially alter Kv4.1 channel properties are involved in the pathogenesis of an X-linked neurodevelopmental condition, which is often associated with a variable neuropsychiatric phenotype.

## Clinical phenotypes

The KCND1 variants described herein are associated with a neurodevelopmental disorder characterized by a remarkable variability in the phenotypic expression and severity of the disease. Although the clinically documented onset of the disease varies from neonatal to ten years of age, all but two individuals were younger than five years of age when the first clinical signs were diagnosed. In the two exceptions, it is not known whether an earlier clinical diagnosis would have been possible. The disease phenotype of the entire cohort ranges from rather complex and severe neurological phenotypes to more or less isolated clinical signs. Individual clinical features noticed in isolation were also observed as syndromic attributes. Prevalent clinical signs are intellectual disability, delayed speech development, hindered comprehension, neuropsychiatric signs, impaired receptive language, motor delay, muscular hypotonia, and different types of epilepsy. Neuropsychiatric abnormalities observed in about two-thirds of the subjects include ASD, attention deficit, hyperactivity, anxiety, tics, emotional problems, and poor social skills. There is no obvious genotype-phenotype correlation, e.g., similar genetic alterations or amino acid exchanges affecting identical parts of the channel protein do not appear to result in a common phenotype. Several KCND1 variants, including two predicted LOF variants, are listed as rare hemizygous variants in gnomAD v4.0.0 (Table 1). This finding is consistent with a highly variable disease phenotype that includes adult males with a mild (or late-onset) or even no apparent clinical phenotype, thus potentially broadening the clinical variability described herein. Unfortunately, to our knowledge, there are no suitable published or otherwise available data on a KCND1 LOF animal model to verify this hypothesis. However, in a single individual with childhood-onset focal epilepsy, a hemizygous KCND1 nonsense variant was identified as the prime candidate for a pathogenic variant.²⁹ Moreover, LOF hemizygotes are listed in gnomAD for several well-known X-linked neurodevelopmental genes, including genes that also encode transmembrane proteins with ion channel/transporter function, such as GRIA3⁴⁷ (MIM: 305915) and SLC9A6⁴⁸ (MIM: 300231) for both of which pathogenic LOF variants have been described. It should also be noted that the KCND1 cohort presented in our study is likely to be biased to the more severe end of the phenotypic spectrum, as all subjects were selected based on distinctive clinical abnormalities exhibited during childhood. Taken together, it is tempting to speculate that hemizygous KCND1 variants lead to a wide-ranging continuum of neurodevelopmental abnormalities reaching from clinically largely unremarkable
<table><caption>**Table 3.** Classification of KCND1 group 2 variants</caption><thead><tr><th>Individual</th><th>Transcript GenBank: NM_004979.6</th><th>protein GenBank: NP_004970.3</th><th>Used ACMG criteria</th><th>ACMG classification<sup>a</sup></th></tr></thead><tbody><tr><td colspan="5"><strong>Maternally inherited missense variants</strong></td></tr><tr><td colspan="5"><strong>Variants affecting the N-terminal cytoplasmic domain</strong></td></tr><tr><td>6</td><td>c.170C&gt;T</td><td>p.Thr57Met</td><td>PS3, PM2_sup, PP3</td><td>likely pathogenic</td></tr><tr><td>7</td><td>c.178C&gt;T</td><td>p.Arg60Cys</td><td>PM2_sup, PP3</td><td>VUS</td></tr><tr><td>8</td><td>c.238G&gt;C</td><td>p.Gly80Arg</td><td>PS3, PM2_sup, BP4</td><td>VUS</td></tr><tr><td>9</td><td>c.320G&gt;A</td><td>p.Arg107Gln</td><td>PS3</td><td>VUS</td></tr><tr><td>10</td><td>c.436C&gt;T</td><td>p.Arg146Cys</td><td>PS3, PP3</td><td>VUS</td></tr><tr><td>11</td><td>c.442G&gt;A</td><td>p.Ala148Thr</td><td>PS3, PM2_sup</td><td>VUS</td></tr><tr><td colspan="5"><strong>Variants affecting transmembrane domains</strong></td></tr><tr><td>12</td><td>c.604G&gt;A</td><td>p.Ala202Thr</td><td>PS3, BS2</td><td>VUS</td></tr><tr><td>13</td><td>c.922C&gt;T</td><td>p.His308Tyr</td><td>PS3, PM2_sup, PP3</td><td>likely pathogenic</td></tr><tr><td colspan="5"><strong>Variants affecting the C-terminal cytoplasmic domain</strong></td></tr><tr><td>14</td><td>c.1291C&gt;T</td><td>p.Arg431Cys</td><td>PS3, PP3</td><td>VUS</td></tr><tr><td>15 and 16</td><td>c.1546A&gt;T</td><td>p.Thr516Ser</td><td>PS3, BP4, BS2</td><td>VUS</td></tr><tr><td>17</td><td>c.1606A&gt;G</td><td>p.Arg536Gly</td><td>PP3</td><td>VUS</td></tr><tr><td>18</td><td>c.1733A&gt;T</td><td>p.Asn578Ile</td><td>PS3, PM2_sup, PP3</td><td>likely pathogenic</td></tr></tbody></table>

American College of Medical Genetics and Genomics (ACMG) criteria: PS3, well-established *in vitro* or *in vivo* functional studies supportive of a damaging effect on the gene or gene product; PM2, absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium; PP3, multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.); BP4, multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.); BS2, variant was observed in a hemizygous state in population databases more than expected for disease.

phenotypes to early-onset, rather severe syndromic conditions. While the reasons for this large degree of phenotypic variability are currently unknown, it is important to note that distinct variants of a given gene are unlikely to act in isolation. Instead, other genomic variants may act as genetic modifiers to either alleviate or exacerbate disease severity, thus leading to a variable phenotypic outcome. In the future, it will be interesting to identify genetic modifiers of pathogenic KCND1 variants and determine their mutual interaction. Attractive candidate modifiers are, e.g., genes encoding proteins that interact with Kv4.1, such as members of the KChIP and DPP families.

## ACMG classification

Our genetic and functional data provide strong evidence that all five KCND1 group 1 variants are pathogenic. Based on these findings, the evidence of pathogenicity for all group 2 variants was formally evaluated in accordance with ACMG standards and guidelines (Table 3).<sup>39,40</sup> For functionally tested variants the criterion PS3 (well-established *in vitro* or *in vivo* functional studies supportive of a damaging effect on the gene or gene product) was rated utilizing a functional assessment table based on significance levels with no reference to the direction and amount of change (i.e., gain or LOF; Table S7). In order to meet the PS3 criterion, at least four electrophysiological items need to be significantly altered, accounting for the possibility that a variant may differ from Kv4.1 WT in only

one electrophysiological parameter, but does so in all four tested channel configurations. To keep the scoring system simple, quantitative differences in significant variant effects are not further differentiated. Likewise, all analyzed parameters are assumed to be of equivalent pathogenic significance and are therefore given equal weight. Overall, ACMG classification categorizes three group 2 variants (p.Thr57Met, p.His308Tyr, and p.Asn578Ile) as likely pathogenic, thus providing supporting evidence that these variants are involved in the pathogenesis of a neurodevelopmental disorder. The remaining group 2 missense variants are classified as VUS. Further studies are needed to determine their involvement in pathogenicity.

## Molecular pathophysiology of KCND1 variants

A closer inspection of the structure-function relationships specifically affected by individual variants may help to understand the molecular pathophysiology of the disorder described herein. Strikingly, the vast majority of KCND1 missense variants described herein leads to widely scattered amino acid exchanges within the cytoplasmic N- and C-termini at sites that are presumably not quite essential for Kv4.1 channel function (Figures 1 and S1). Although most amino acid exchanges lead to alterations in the number (mostly a reduction) and nature of polar side chain interactions (Figure S2; Table S2), the resultant biophysical alterations are mostly moderate. In contrast, published heterozygous missense variants in KCND2 associated with a

<footnote><sup>a</sup>To assign variant class, a points-based system to simplify scoring was used.<sup>40</sup> See Table S7 for more details on PS3 scoring.</footnote>
neurodevelopmental disease phenotype alter sites within the S4-S5 linker and the distal S6 segment, core regions of the protein known to play a pivotal role in the operation of the cytoplasmic gate, with a prominent impact on Kv4.2 channel gating.⁸,⁹,¹⁰,¹¹ This obvious difference may reflect natural selection due to the gonosomal and autosomal localization of *KCND1* and *KCND2*, respectively.³⁴ Putative *KCND1* missense variants affecting the S4-S5 linker or the distal S6 segment may be selected against because homo-meric variant channels may lead to embryonic lethality of males harboring these variants. Of note, all *KCND1* missense-variant-conducting mothers are reportedly clini-cally asymptomatic. It will be interesting to see if *KCND1* missense variants affecting the cytoplasmic gate are identi-fied in the future and shown to be associated with a fe-male-specific genetic disease.

In order to define loss- or gain-of-function features for a given variant, the direction of changes in biophysical pa-rameters may be considered. Thus, besides an increase in current amplitude, a slowing of macroscopic inactivation, accelerated recovery kinetics, a positively shifted voltage dependence of steady-state inactivation and/or negatively shifted voltage dependence of activation represent gain-of-function features, and vice versa for LOF. Except for the re-covery kinetics, which, if modified, were always slowed; bio-physical Kv4.1 alterations are observed in either direction. Thus, observed alterations of biophysical parameters are likely to eventually reflect both LOF and gain-of-function. Overall, variant-specific alterations in electrophysiological parameters or a lack of their modulation by auxiliary β sub-units do not appear to correlate with the associated clinical phenotype, neither with individual signs, the sum of phenotypic features, nor the disease severity. Notably, this finding holds true regardless of whether the quantitative and qualitative nature of biophysical changes was taken into account or not (data not shown). Therefore, and for the sake of simplicity, we based the PS3 scoring for ACMG classification of *KCND1* variants exclusively on the sum of significantly altered electrophysiological parameters (Tables S3–S7), thus not accounting for the qualitative and quantitative nature of variant effects.

Cellular mechanism of this neurodevelopmental
disorder

In order to determine cellular mechanisms that may be
critically influenced by Kv4.1 channel dysfunction or com-
plete LOF, it is important to consider the brain regions in
which *KCND1* is expressed and the physiological role of
Kv4.1 channels in specific neurons. While *KCND1* expres-
sion seems to be relatively moderate compared to other ion
channel genes,³⁴,⁴⁹ it is widely expressed throughout the
human brain and has been shown to be involved in the
control of slow repetitive action potential firing in many
different neurons.⁵⁰⁻⁵³ This is reminiscent of the A-type
current function originally identified in primitive ani-
mals,⁵⁴ where these channels appear to already activate
at very negative membrane potentials well below

threshold. Thereby, they cause a delay in firing until
A-type channel inactivation allows for the next spike. It
is obvious that the amount and distribution of A-type
channels on the cell surface as well as their biophysical
properties, especially their macroscopic inactivation ki-
netics, are critical for this function. However, none of the
reported Kv4.1-associated functional roles, including hip-
pocampal pattern separation,⁵² clock gene expression,⁵¹
control of striatal circuitry,⁵³ or nociception,⁵⁰ appear to
directly relate to the neurodevelopmental disease pheno-
type described herein. Intriguingly, Kv4.1 has also been
implicated in the control of cell proliferation, not only in
tumor cells,⁵⁵,⁵⁶ but also in neural stem and progenitor
cells.⁵⁷ Thus, at certain developmental stages, even minor
disturbances in Kv4.1 channel levels and/or biophysical
properties, representing either gain of function or LOF,
may significantly affect neuronal cell proliferation and
migration, thus impairing neuronal development. In addi-
tion, clock gene expression reportedly under the control of
Kv4.1 channel activity⁵¹ is known to regulate cortical
development,⁵⁸ thus representing a further possible expla-
nation for the high phenotypic variability of the described
disorder to be explored in the future.

Data and code availability

Additional methodological, genetic, and clinical data sup-
porting the findings of this study are available from the
corresponding authors upon reasonable request. In some
cases, specific privacy regulations and the consent under
which specific data were obtained may not permit the
sharing of additional findings, e.g., raw next-generation
sequencing files.

Supplemental information

Supplemental information can be found online at https://doi.org/
10.1016/j.ajhg.2024.04.019.

Acknowledgments

We thank the family members for their participation and collabo-
ration. This study makes use of data generated by the DECIPHER
community.⁵⁹ A full list of centers that contributed to the genera-
tion of the data is available from http://decipher.sanger.ac.uk and
via email from decipher@sanger.ac.uk. This work was supported by
the Deutsche Forschungsgemeinschaft (BA2055/1-3 and BA2055/
6-1 to R.B. and SCHO 1921/2-1 to C.S.), the Netherlands Organisa-
tion for Scientific Research (ZonMw Vidi, grant 09150172110002
to T.S.B.), an Erasmus MC Fellowship 2017 (to T.S.B.), the Erasmus
MC Human Disease Model Award 2018 (to T.S.B.), PNRR-MUR-
M4C2 PE0000006 Research Program “MNESYS” - A multiscale in-
tegrated approach to the study of the nervous system in health
and disease (to P.S.), the Italian Ministry of Health (CCR-2017-
23669081 and RF-2021-12374963 to M.T.). IRCCS “G. Gaslini” is
a member of ERN-Epicare. Funding bodies did not have any influ-
ence on study design, results and data interpretation, or final
manuscript.
**Declaration of interests**

The authors declare no competing interests.

Received: November 9, 2023

Accepted: April 25, 2024

Published: May 20, 2024

**Web resources**

CADD, https://wintervar.wglab.org/

GeneMatcher, https://genematcher.org/

gnomAD database, https://gnomad.broadinstitute.org/

MetaDome, https://stuart.radboudumc.nl/metadome/

PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/

REVEL, https://sites.google.com/site/revelgenomics/

SIFT, https://sift.bii.a-star.edu.sg/

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