Identification of a novel de novo p.Phe932Ile KCNT1 mutation in a patient with leukoencephalopathy and severe epilepsy

Abstract

We present a male with early infantile epileptic encephalopathy (EIEE) and a leukoencephalopathy in whom whole exome family trio sequencing identified a heterozygous de novo mutation in KCNT1, a sodium gated potassium channel gene. Severely delayed myelination was anecdotally reported in previous cases with KCNT1 mutations. This case reinforces that KCNT1 sequencing should be included in an investigation of patients with severely delayed myelination and epilepsy.

Introduction

Recent advances in MRI analysis have led to significant improvements in the diagnosis of pediatric leukoencephalopathy patients. At least 50% of these patients, however, remain without an ultimate diagnosis¹ – which, in part, may be due to the fact that the clinical and MRI presentation of leukencephalopathies can be highly variable. The advent of whole genome and exome sequencing provides an agnostic platform by which to assess individual leukoencephalopathy cases (ideally within the context of their familial genetics) for known and novel disease-associated mutations.

Case Study

The proband first came to medical attention at one month of age when, after an uneventful gestation and delivery, he was noted to have abnormal movements characterized as myoclonic seizures. Over the ensuing months, seizures evolved to consist of both generalized tonic clonic and myoclonic events, with frequent status epilepticus, which was exacerbated by febrile events, resulting in several dozen hospitalizations. Seizures are now better controlled, but on a regimen of Oxcarbazepine, Topiramate, Levetiracetam and Zonisamide. At approximately two years of age, the patient developed truncal and head titubation and adventitious movements characterized as choreathetoid, which intruded on sleep and residual neurologic function. Upon initial observation and subsequently, the patient showed no evidence of dysmorphic facies, though he was microcephalic (orbitofrontal head circumference >2 standard deviations below the mean). The patient has also suffered from severe developmental stagnation and now, at age 10 years, is able to only minimally communicate with his care providers, is unable to sit or walk and does not have any self-help skills. Neuroimaging, first obtained at several months of age, and repeated serially over the course of his care, showed an initial severe deficit in the development of age appropriate myelination that has never normalized. Indeed, the most recent MRI, performed at 7 years of age, has shown evidence of progression of myelination but at levels that are still severely reduced (Figure 1A-C), consistent with severely delayed myelination.

Figure 1.

Patient MRI, EEG and KCNT1 mutation information. (A-C) Initial studies demonstrated a severe delay in acquisition of normal myelination demonstrated on T2 weighted images (A, T2 axial image obtained at 3 years of age). However over time there was slow gradual progression of myelination (B, T2 axial image obtained at 7 years of age), demonstrated in the deep white matter (white arrows). Sagittal images (C, T1 weighted, 7 years of age) show microcephaly, and a shorted corpus callosum with a thin splenium. (D) EEG showing multifocal spike-wave discharges (bipolar longitudinal montage, epoch length 15 s, sensitivity 7 mcv/mm, tc 0.16 s, HFF 70 Hz). (E) A schematic of KCNT1 and the mutations recently described by Barcia et al. (purple text), Heron et al. (green text), McTague et al. (blue text) or in this study (black text and asterisk). Domain annotation is per Barcia et al. and Interpro. Note that nearly all amino acid changes are found in the cytoplasmic C-terminal protein domain.

Electroencephalogram telemetry done at age two years was characterized by high voltage background slowing (delta range, 0.5-2.5 Hz) and superimposed multifocal interictal sharp discharges with occasional periods of burst-suppression. Habitual clinical events were recorded as myoclonias involving the extremities with associated oral dyskinesias followed by a motionless appearance, without changes in the electrographic background, most consistent with subcortical events. Subsequent EEGs have continued to show an absence of normal background organization with multifocal epileptiform discharges during wakefulness and with activation in sleep, although without ictal events (Figure 1D).

The patient's seizures were refractory to medical management until approximately 7 years of age when the patient was placed on multiple anticonvulsants, at which point they became infrequent. The underlying cause of the seizures and delayed myelination, however, remained enigmatic. Extensive biochemical and genetic testing² revealed no abnormalities other than mildly increased levels of sialic acid in the CSF and urine, and subsequent fibroblast and genetic studies of sialic acid metabolism were unremarkable.

The patient's presentation and severely delayed myelination were consistent with an inherited leukoencephalopathy, and he was therefore enrolled prospectively in a Myelin Disorders Bioregistry Project study on unclassified leukoencephalopathies at Children's National Medical Center (CNMC). To investigate the possible genetic origin of the patient's disease we performed whole exome sequencing on DNA collected from the proband and the unaffected parents. We produced > 19 Gb of sequence for each individual, yielding a mean depth of 57 fold coverage with an average 90.4% of target bases sequenced at least 18 times. Using a custom-developed pipeline that integrates parental variant calls into the analysis of the affected child^3,4, a heterozygous de novo mutation in KCNT1 (MIM 608167; NM_020822.2) was identified in the proband (NM_020822: c.2794T>A, p.F932I; Figure 1E). Sanger sequencing of the trio confirmed that this mutation was specific to the patient, and further analysis showed that it is not present in dbSNP135, the 1000 Genomes Project database or the NHLBI Exome Sequencing Project database. This was the only de novo mutation identified in this screen that passed our filtering criteria. KCNT1 p.F932 is highly conserved (phyloP score of 0.989744), and the p.F932I change is, in principle, the most damaging non-synonymous change found in our screen, with a PolyPhen2 score of 0.998, a SIFT score of 0.99, and MutationTaster score of 0.78.

Discussion

Three recent reports have shown that mutations in KCNT1, a sodium-gated potassium channel,cause at least two forms of severe epilepsy.^5-7 A set of missense KCNT1 mutations were recently described in four families with nocturnal frontal lobe epilepsy (NFLE), including a de novo mutation associated with a sporadic case⁶ (Figure 1E). Additionally, and a total of eight subjects from two studies investigating malignant migrating partial seizures of infancy (MMPSI), one subtype of early infantile epileptic encephalopathy (EIEE), were shown to have five additional KCNT1 mutations, three of which were de novo^5,7 (Figure 1E). Although our patient did not demonstrate the features of MMPSI, he does have features consistent with the broader symptomatic description of EIEE, including refractory myoclonic seizures with onset at 1 month.

KCNT1 encodes the largest potassium channel subunit, and is thought to regulate the hyperpolarization that follows repetitive firing. Functional studies have shown that mutations in the cytoplasmic C-terminal domain lead to constitutive activation of the channel³, which is predicted to disrupt normal neuronal firing and directly lead to epileptogenesis. The novel KCNT1 mutation described here sits immediately adjacent to two of those recently described (Figure 1E), and we therefore conclude that it is the underlying disease-causing change in this case. KCNT1 related epilepsies fall within a broader group of potassium channel related epilepsies that may be amendable to channel specific therapies, and the findings made here will thus direct future epilepsy therapy in this patient.

Previously reported patients with KCNT1 mutations include subjects with malignant migrating partial seizures of infancy who demonstrated delayed myelination. Severely delayed myelination is a non-specific feature of primary disorders of neuronal function, and KCNT1 mutations should be included in an investigation of patients with severely delayed myelination and epilepsy.