De novo KCNA6 variants with attenuated KV 1.6 channel deactivation in patients with epilepsy

Abstract Objective Mutations in the genes encoding neuronal ion channels are a common cause of Mendelian neurological diseases. We sought to identify novel de novo sequence variants in cases with early infantile epileptic phenotypes and neurodevelopmental anomalies. Methods Following clinical diagnosis, we performed whole exome sequencing of the index cases and their parents. Identified channel variants were expressed in Xenopus oocytes and their functional properties assessed using two‐electrode voltage clamp. Results We identified novel de novo variants in KCNA6 in four unrelated individuals variably affected with neurodevelopmental disorders and seizures with onset in the first year of life. Three of the four identified mutations affect the pore‐lining S6 α‐helix of KV1.6. A prominent finding of functional characterization in Xenopus oocytes was that the channel variants showed only minor effects on channel activation but slowed channel closure and shifted the voltage dependence of deactivation in a hyperpolarizing direction. Channels with a mutation affecting the S6 helix display dominant effects on channel deactivation when co‐expressed with wild‐type KV1.6 or KV1.1 subunits. Significance This is the first report of de novo nonsynonymous variants in KCNA6 associated with neurological or any clinical features. Channel variants showed a consistent effect on channel deactivation, slowing the rate of channel closure following normal activation. This specific gain‐of‐function feature is likely to underlie the neurological phenotype in our patients. Our data highlight KCNA6 as a novel channelopathy gene associated with early infantile epileptic phenotypes and neurodevelopmental anomalies.


Subject recruitment and diagnosis
The 4 individuals carrying de novo KCNA6 intragenic variants reported in the present study were recruited from different research groups internationally. Individual 1 was followed up at Alberta Children's Hospital (Calgary, Canada) and genetically investigated at BluePrint Genetics. Individual 2 was followed up at University Hospital "Vall d'Hebron" (Barcelona, Spain) and was genetically investigated by trio-whole exome sequencing (WES) as part of the SYNAPS Study Group (http://neurogenetics.co.uk/synaptopathies-synaps/) initiative. Individual 3 was followed up and genetically investigated at the University Hospital of Nantes (Nantes, France). Individual 4 was followed up at the Benioff University Children's Hospital (San Francisco, USA) and genetically investigated at GeneDx. Routine clinical genetic and metabolic screenings performed during initial workup was negative in each case, which warranted further investigation on a research basis. The study was approved by the ethics committee of the University College London (07/Q0512/26) and additional local ethics committees of the participating centres.

Supplemental Case reports Individual 1
Patient 1 is a 7-year-old Canadian boy born at term with caesarean section after uneventful pregnancy.
He was small for gestational age and required to be admitted to NICU due to poor weight gain. At birth, his occipitofrontal circumference (OFC) was within normal range (50 th ) and birth weight was 2000 gr (>3 rd centile). He has an history of developmental delay since the first months of life. He acquired the ability of sitting autonomously at the age of 12 months and started to walk since the age of 18 months. Abnormal social communication was noticed in the first years of life with late social smiling. Speech delay was present with first meaningful words pronounced at the age of 3 years, but he lately improved with speech therapy. In the first years of life macrocytic anaemia was also noted.
He has some distinctive facial features, including pointed chin, mildly flat midface and thin upper lip.
At his last follow-up appointment at the age of 8 years he was diagnosed with short stature (3-10 th centile); on neurological examination muscle weakness and abnormal motor coordination and clumsiness were noted (e.g., difficulties with running, jumping, etc.) on examination. During his childhood, he displayed some behavioural disturbances and signs of sensory sensitivities and was diagnosed with autism spectrum disorder. There was no history of focal neurological deficits and he never had seizures. He has not showed developmental regression. Trio WES done at BluePrint Genetics did not identify any copy number or single nucleotide variants in disease-causing genes. A de novo non-synonymous variant in KCNA6 [NM_002235.3:c.783C>G; (p.Asp261Glu)] was found in the trios WES data.

Individual 2
Individual 2 is a 22-years-old Spanish female. She was born to healthy, unrelated parents. The rest of her family history is non-contributory. Gestation and term delivery were unremarkable. At birth, her weight was 2900gr (25th centile). Neonatal period was uneventful. At the age 3 of months, she started to present seizures. The episodes occurred on a daily basis and featured brief upward gaze deviation, uni-or bilateral upper limb flexion with altered level of consciousness. The episodes usually lasted a few seconds (less than 10-20 sec) and occurred upon awakening. Between the ages of 6 months and 5 years the patient suffered multiple episodes (up to ten per day) of tonic or tonic-clonic seizures, which were refractory to several drugs, including sodium valproate, vigabatrin and clobazam. Lamotrigine and carbamazepine did not improve her condition. At around 5 years of age, treatment with ethosuximide brought a clear improvement and she has subsequently had good seizure control on this drug. In fact, seizures recurred every time dosing has been reduced in an attempt to taper the medication. Over the years, EEG did show focal or generalized epileptiform abnormalities (not shown), but in recent years have been remarkably normal. Brain CT scan and MRI did not disclose any abnormality. The patient was delayed from early on and she attained independent gait and first meaningful words by age 2. She currently displays mild intellectual disability, is able to read and write simple sentences and attends special school with occupational therapy. She has some distinctive facial features including mild retrognathia and gingival hyperplasia. At her current age, she has poor language syntax and vocabulary and prominent echolalia. She has no major gross or fine motor disabilities, or motor or sensory deficits and she is independent for most basic activities of daily living. She does not show impaired social communication. On neurological examination, she has no motor or sensory deficits, but she displays postural and kinetic high-frequency bimanual tremor, but there is no rigidity, or other extrapyramidal, or pyramidal signs and gait and stance are normal

Individual 3
Individual 3 is a 6-year-old French boy born at term via caesarean section after an uneventful pregnancy. Familial history was negative for neurological or genetic disorders. His growth parameters, including OFC at birth were within normal range. He has a history of mild developmental delay, became able to sit without support at 9 months of age and started to walk autonomously at the age of 17 months. Since the age of 5 months, he presented with multiple focal seizures triggered by fever and characterized by clonic movements at upper limbs lasting few seconds and followed by hypotonia and loss of consciousness. Supplementary Video 2 shows a focal seizure starting with left eye and head deviation and gaze staring followed by tonic posturing of left side (1' 10'') and ending with clonic movements of the upper limbs; the ictal EEG reveals an epileptic activity starting from the right posterior cortical areas and then involving the whole hemisphere. In addition, since the age of 9 months she did also manifest absence seizures (Supplementary Video 2, 1' 28'') associated with mild eye rolling and subtle eyelid myoclonia. EEG showed interictal bilateral slow spike and wave discharges. Brain MRI was normal. Valproic acid was started but he continued to experience feverrelated convulsive seizures; clobazam and stiripentol add-on did result in good control for 10 months.
He was suspected for Dravet syndrome given the history of fever-related epilepsy but SCN1A testing was negative. At the age of 22 months, he experienced focal (clonic) seizures recurred and he also experienced tonic-clonic seizures with fever. Increased valproic acid and stiripentol dosages were effective. His language and cognitive skills are impaired, he started to pronounce first words at the age of 3 years and first sentences at the age of 5 years. He currently attends speech therapy sessions.
At school, learning difficulties and impaired concentration were noticed and he is supported by a psychomotor therapist. At the last follow up (6 years

Individual 4
Individual 4 is a 2-year-old American girl born via vaginal birth after induced labor at 41 weeks of gestation. Pregnancy was uncomplicated and at birth her growth parameters were all within normal range. Since the age of 3 months, she developed febrile and afebrile focal seizures with eye deviation, perioral cyanosis and asymmetric tonic extension of the upper extremities followed by bilateral asynchronous/synchronous clonic movements and sometimes associated with facial twitching, perioral clonias and oral automatisms ( Supplementary Videos 3 and 4). These episodes, usually lasted seconds to minutes and occurred from drowsiness/sleep. After each episode, the girl is frequently lethargic and drowsy. At the age of 5 months, ictal EEG recorded a focal seizure, starting from the temporal anterior areas ( Figure S2A) and rapidly involving the motor cortex (Supplementary Figure   2B); 20 seconds later the seizure involves both hemispheres ( Figure S2C) and ends with a diffuse slowing of cortical activity ( Figure S2D). Therapy with levetiracetam was ineffective and then switched to carbamazepine with benefit. Her developmental history is so far normal and ageappropriate. At her last follow-up appointment at the age of 2 years, neurological examination was normal. Brain MRI studies and metabolic screening were unrevealing. She underwent trio-WES at Protocol consists of 30 5 ms pulses to +40 mV from holding voltage of -80 mV applied every 15 ms.
Capacitive currents at the beginning of the test pulse can be seen as -P/4 subtraction protocol was not applied. Note that while the current responses of all the pulses are overlapping for channels containing wild-type KV1.6 channel, there is a clear increase in current amplitude for KV1.1+T449I channel following the first pulse. Scale bars: x: 1 ms, y: 5 µA. B) Data from (A) but the current of the first trace is subtracted from the following pulses. Note large increase in KV1.1+T449I currents before and during the test pulses. Scale bars: x: 1 ms, y: 2 µA. C) Current increase following the first pulse for each KV1.6 variant co-expressed with KV1.1 wild-type channel (WT KV1.6 (grey), D261E (blue), V447F (green), T449I (orange), V456L (red)). KV1.1 WT is shown in black) at indicated frequencies.
For each cell the mean current increase of pulses 2-30 compared to the first pulse is measured and normalised to the peak current amplitude at the end of the voltage pulse to +40 mV. Data is mean ± SEM of 4-9 cells for each KV1.6 variant and homomeric KV1.1 channel.