Recurrent KCNT2 missense variants affecting p.Arg190 result in a recognizable phenotype

KCNT2 variants resulting in substitutions affecting the Arg190 residue have been shown to cause epileptic encephalopathy and a recognizable facial gestalt. We report two additional individuals with intellectual disability, dysmorphic features, hypertrichosis, macrocephaly and the same de novo KCNT2 missense variants affecting the Arg190 residue as previously described. Notably, neither patient has epilepsy. Homology modeling of these missense variants revealed that they are likely to disrupt the stabilization of a closed channel conformation of KCNT2 resulting in a constitutively open state. This is the first report of pathogenic variants in KCNT2 causing a developmental phenotype without epilepsy.

efflux from the cell, resulting in the membrane potential becoming more negative and hence repolarization. The ability of K + channels to repolarize and hyperpolarize nerve and muscle cells helps to control action potential frequency and duration (Humphries & Dart, 2015). K + channels can be categorized by the stimulus to which they are activated and include voltage-gated (K V ), calcium-activated (K Ca ), inwardly rectifying (K IR ), ATP-sensitive (K ATP ), and sodium-activated (K Na ) channels. A recent systematic review identified 19 potassium channelopathies implicated in a variety of neurodevelopmental disorders (Kessi et al., 2020).
Humans have two K (Na) channel subunits, Slack and Slick, encoded by KCNT1 (OMIM 608167) and KCNT2 (OMIM 610044), respectively, that rectify outwardly. KCNT1 and KCNT2 share $74% sequence identity (Bhattacharjee et al., 2003), show similar singlechannel conductance, modulate the hyperpolarization that occurs following repetitive firing and form hetero-tetrameric channels in several brain regions such as the oculomotor nucleus and the medial nucleus of the trapezoid body (Chen, Kronengold, et al., 2009). A key difference between KCNT1 and KCNT2 is that KCNT1 channels have an absolute requirement for Na + for channel opening, whereas KCNT2 channels maintain a basal level of activity in the absence of Na + (Bhattacharjee et al., 2003).
Pathogenic variants in KCNT1 have been recently identified to cause autosomal dominant nocturnal frontal lobe epilepsy (OMIM #615005) and epilepsy of infancy with migrating focal seizures (Barcia et al., 2019), as well as, early infantile epileptic encephalopathy with severe dystonia (OMIM #614959) (Gertler et al., 2019;Martin et al., 2014). The majority of cases are caused by gain-of-function variants, however, a single missense variant resulting in loss-offunction (p.Phe932Ile) has been described in a patient with epilepsy and leukoencephalopathy (Evely et al., 2017).
KCNT2 has recently been described as a human disorder gene (OMIM #617771) with only eight patients in total reported so far. Mao et al. reported two patients with epilepsy of infancy with migrating focal seizures and de novo truncating variants in KCNT2 (p.Lys564* and p.Leu48Glufs*43) (Mao et al., 2020). Gururaj et al. reported a patient with a de novo p.Phe240Leu missense variant and epileptic encephalopathy with no dysmorphic features (Gururaj et al., 2017). The p.Phe240 residue is situated in the channel pore helix and the authors concluded that this particular variant causes a "change-in-function" by altering a K + channel that is usually upregulated by Cl À to become a Na + channel down-regulated by Cl À . Inuzuka et al. (2020) reported another patient with a de novo p.Thr242Asn variant, which lies in the same transmembrane domain, with a non-dysmorphic epileptic encephalopathy phenotype (Inuzuka et al., 2020). Alagoz et al. (2020)  The probands had epilepsy, intellectual disability, hypertrichosis, and coarse facial features. Electrophysiological studies revealed a gain-offunction and constitutive activation for both variants. The gain-offunction effect was more pronounced with the substitution of positively charged arginine with neutral proline than with histidine. This suggested that substitution with partially protonated histidine may allow for the maintenance of some, but not all, charge interactions necessary for channel function. Prior to reports of pathogenic variants in KCNT2 in humans, the mechanism of closure of the KCNT2 channel had been investigated by Dai et al. (2010) who performed electrophysiological studies on cRNA-transduced Xenopus oocytes.
p.Arg190 was identified as an important candidate residue for channel gating due to its location in a transmembrane linker region. Mutation of p.Arg190 to Glu, Gln, and Ala showed that charge reversal or neutralization led to constitutive activation of the channel (Dai et al., 2010). Of note, substitution of p.Arg190 for another positively charged amino acid, lysine, resulted in channels with wild-type properties (i.e., normal function).
Given so few cases are reported, the mutational spectrum, clinical features, and the genotype-phenotype relations in KCNT2-related disorders remain undefined. We report two new individuals with a recognizable neurodevelopmental disorder without epilepsy and recurrent de novo KCNT2 variants affecting the Arg190 residue. Using

| Case ascertainment
The index case was identified through a local re-analysis project of genome data from the 100,000 Genomes project via a previously described pipeline (Faundes et al., 2018;Vaz et al., 2019 The proband is the second child born to non-consanguineous Caucasian parents. Pregnancy was uncomplicated and she was born at term by normal vaginal delivery weighing 3.3 kg (+0.15 SD). Concerns regarding hypotonia and delay in her general development were raised during early infancy. She sat unsupported at 1 year of age and was walking unsteadily at the age of 2 years. She started using single words at the age of 2 years and three-word phrases at the age of 4 years 2 months. She attended school with a statement of educational needs due to severe learning difficulties. Currently, at the age of 32 years, she lives in residential care. She has never suffered from seizures.
She developed pubic and axillary hair at the age of 8 years before menarche aged 14 years. Her periods were irregular and a pelvic ultrasound scan which revealed polycystic ovaries. She had impaired fasting glucose at the age of 18 years and was treated with metformin until 21 years, when her fasting glucose had normalized. At 14 years, her height was 170 cm (+1.46 SD), her weight was 50.6 kg (+0.13 SD), and occipitofrontal circumference (OFC) was 58 cm (+3.35 SD).
A CT brain aged 2 years was reported as normal. Her urine mucopolysaccharides, amino acids and organic acids profile, and plasma very long chain fatty acids were all within normal limits. Her thyroid function was also normal. Fragile X testing, karyotyping, and chromosomal microarray showed normal results.
The proband and her parents were recruited to the 100,000 Genomes Project (Caulfield et al., 2019) (Richards et al., 2015). No other plausible candidates were identified during this re-analysis.

| Individual 2
The proband was born to non-consanguineous Caucasian parents.
She has an older maternal half-sister and a younger brother. Pregnancy had been unremarkable except for an iron transfusion at 32 weeks for anemia. She was born at 41 weeks, weighing 4.88 kg (+3.15 SD) with birth OFC 38 cm (+3.48 SD).
She first sat unsupported aged 6 months but delay in motor development was noted at 17 months as she was falling frequently and appeared to have poor leg coordination. She was walking independently by age 22 months. Her speech and language development was significantly delayed. Currently, aged 5 years 7 months, she speaks a few single words but also uses non-verbal gestures. She has moderate intellectual disability and with autistic traits. Her behavior has been described as hyperactive with a tendency for aggressive outbursts and she has difficulties with sleep requiring melatonin. She has never had seizures. She has slightly reduced tone in her legs but otherwise normal neurological examination. She suffers with constipation which has required regular Movicol and is also prone to wheezing episodes, for which she takes preventative inhalers.
At 4 years 8 months, her height was 108 cm (+0.57 SD) and OFC 54.4 cm (+3.00 SD). She has prominent eyebrows and long lashes, big ears, small square teeth, widely spaced teeth and hypertrichosis, mainly affecting her arms and back (Figure 1b). Her brain MRI scan at 18 months of age was reported to be normal. Gene-agnostic trio exome sequencing in the clinical setting revealed a de novo NM_198503.3:c.569G > A (p.Arg190His) missense variant. This variant was also classified as Pathogenic according to the American College of Medical Genetics (ACMG) guidelines (PS2, PM2, PM5, PP3, PP2) (Richards et al., 2015). No other likely causative rare de novo or biallelic variants were identified through the exome sequencing analysis.
3.2 | Homology modeling K Na channels resemble K v channels in topography with six hydrophobic, transmembrane segments (S1-S6) along with a pore-lining loop found between S5 and S6 (Kaczmarek, 2013). Subunits assemble as tetramers to form a functional channel. The KCNT2 p.Arg190 residue is located within the S4 and S5 linker region and is evolutionary conserved among species (Ambrosino et al., 2018). p.Arg190 creates a constriction between the cytoplasmic domains immediately before Asp36,9 and p.Arg372 (Figure 1c) and the distances between these residues appear to be closer to the wild-type closed conformation.

| DISCUSSION
We describe two individuals with de novo missense KCNT2 variants affecting the same residue at p.Arg190, which has been implicated in epileptic encephalopathy previously. Individual #1 presented with all of which were also seen in the two previously reported p.Arg190 cases (Ambrosino et al., 2018). Their previous occurrence in affected individuals and absence of these variants from population databases lends weight to their pathogenicity.
Comparison of the clinical features of the two individuals described here with the eight previously described cases showed that all individuals were affected by intellectual disability. Epilepsy was seen in all mutation types although our two p. Arg190 causing a syndromic disorder with a recognizable facial gestalt. In these two cases, however, epilepsy was absent and we describe a new association with macrocephaly.
The occurrence of macrocephaly in these two individuals is noteworthy as constitutive activity of other ion channels, such as KCNB1, leading to cytoplasmic K+ loss have been linked with excessive neuronal apoptosis (Kondratskyi et al., 2015), although a decrease in cytoplasmic K+ is not obligatory for apoptosis (Börjesson et al., 2011).
Interestingly, KCNT2 expression, unlike that of KCNT1, has been found to be predominantly under control of NF-κβ, which is released during stressful stimuli such as hypoxia and injury (Tomasello et al., 2015). As a putative neuroprotective channel, constitutive activation of KCNT2 may not have the same effect on neuronal apoptosis as other previously studied ion channels.
The basis of the phenotypic differences between KCNT2 p. Given that KCNT2 appears to be tolerant of loss-of-function variants (pLi 0.04, pLEOUF 0.37), haploinsufficiency would appear unlikely to be the mechanism. mRNA or protein studies were not performed to prove these variants were truly loss-of-function and indeed, cells cotransfected with wild-type KCNT1, wild-type KCNT2 and also KCNT2 p.Leu48Glufs*43 showed currents similar to cells expressing KCNT1 alone, which may indicate a dominant negative effect for this variant.
Using homology modeling, we have shown that p.Arg190 participates in key interactions with neighboring charged residues in order to stabilize the closed channel state. Substitution of the charged arginine residue with uncharged amino acids results in a constitutive open state and recapitulates the gain-of-function effects seen in previous electrophysiological studies.
KCNT2 is highly expressed in the hippocampus and amygdala, where KCNT1 is relatively less highly expressed (Human Protein Atlas available from http://www.proteinatlas.org) (Pontén et al., 2008;Uhlen et al., 2017). This difference in expression may suggest then KCNT2 functions independently from KCNT1 in these regions (Bhattacharjee et al., 2005). KCNT2 also has a consensus ATP binding site (amino acids 1032-1038), which, when occupied by ATP, inhibits activity of the channel. A unique attribute of KCNT2 is the requirement for ATP to dissociate from a site near the C-terminus, in the presence of elevated intracellular Na + , to allow channel activation.
During times of metabolic stress, such as hypoxia or even epileptiform activity, which both cause a reduction in ATP and elevation in intracellular Na + , it is postulated that KCNT2 channels play a neuroprotective role by limiting excitability and maintaining a hyperpolarized membrane potential (Bhattacharjee et al., 2003). How a gain-of-function variant could bring about the phenotype observed in our cases is not currently understood. The occurrence of both loss-of-function and gain-of-function variants in the spectrum of KCNT2-associated developmental disorders indicates a delicate balance in maintaining membrane potential, which is disturbed in these conditions. The occurrence of hypertrichosis in several K + channelopathies is also notable. The opening of intracellular K + channels has been suggested as a mechanism regulating hair growth. Several antihypertensive compounds, most notably minoxidil and diazoxide, have the known side effect of excessive hair growth (Suchonwanit et al., 2019;Uno et al., 1990). These have been found to induce hypertrichosis by enhancing the flux of potassium ions (Buhl et al., 1992). It is interesting that in the cases described here, and by  (Tomasello et al., 2017), which is interesting, given that CGRP, along with other neuropeptides such as substance P, is known to regulate hair growth (Samuelov et al., 2012).
In a similar manner, gain-of-function variants in ABCC9, which contributes a subunit to SUR2, a K ATP channel, cause Cantu syndrome (OMIM #239850). Cantu syndrome is another developmental disorder sharing phenotypic overlap, particularly hypertrichosis, with the KCNT2 p.Arg190Arg/His phenotype. SUR2 is also a known pharmacological target of minoxidil (Ohko et al., 2020).  would be pathogenic. Our homology modeling of p.Arg190Cys seen in gnomAD would predict this to result in the same gain-of-function seen with the other charge-neutralizing substitutions, however, the distance between the charged residues is less affected than the other variants. In this case, most distances are shorter than those seen in the p.Arg190Ala substitution, which has the shortest distances for all experimentally proven gain-of-function variants (Supplementary Table 1). Of note, this variant does not occur in the gnomAD control group but rather the "non-cancer" group and hence some variants at this position may be either benign or have reduced penetrance.
In conclusion, we report two new cases of variants affecting the p.
Arg190 residue in KCNT2 causing a recognizable neurodevelopmental disorder. In our cases, epilepsy was not a feature and we also expand this phenotype to include macrocephaly. Differential diagnosis included metabolic storage disorders. We show that p.Arg190 is a critical charged residue and reversal or neutralization of this charge is predicted to result in constitutive channel activation.

ACKNOWLEDGMENTS
We thank all family members for their participation and collaboration.
We would also like to thank Exeter Genomics Laboratory who under-

CONFLICT OF INTERESTS
All authors have no conflicts of interest to declare for this work.  Abbreviations: GoF, gain-of-function; LoF, loss-of-function; NS, not stated; OFC, occipitofrontal circumference.
Siddharth Banka, Jill Clayton-Smith and Simon Lovell supervised the findings of this work. All authors discussed the results and contributed to the final manuscript.

DATA AVAILABILITY STATEMENT
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.