Mena Abdelsayed

Exome Sequencing and the Management of Neurometabolic Disorders

BACKGROUND Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patients clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Childrens Hospital Foundation and others.).

Loss-of-function mutations in SCN4A cause severe foetal hypokinesia or 'classical' congenital myopathy

See Cannon (doi: 10.1093/brain/awv400 ) for a scientific commentary on this article. Dominant gain-of-function mutations in SCN4A , which encodes the α-subunit of the voltage-gated sodium channel, are a common cause of myotonia and periodic paralysis. Zaharieva et al. now report recessive loss-of-function SCN4A mutations in 11 patents with congenital myopathy. The mutations cause fully non-functional channels or result in reduced channel activity.

Triggers for arrhythmogenesis in the Brugada and long QT 3 syndromes.

Cardiac arrhythmias are a prevalent cause of morbidity and mortality. In many cases, inheritable mutations in the genes encoding cardiac ion channels are the underlying cause of arrhythmias. Relative to other arrhythmogenic disorders, Brugada syndrome (BrS) is recently identified and not well-understood. Although most often referred to as a disease of cardiac sodium channels, familial BrS is now associated with 9 different genes. Of these genes, 4 alter sodium currents, and the most common known genetic cause remains loss-of-function mutants in the cardiac sodium channel gene SCN5A. Long QT syndrome (LQTs) has a much longer history and is associated with at least 17 genes. LQT3, which is the third most common LQTs, is due to gain-of-function mutations in SCN5A. The first sign for BrS and LQTs patients may be sudden death. The triggers for these sudden deaths include exercise, fever, ischemia, and drug use. In this paper we review the effects of acidosis and fever on BrS and LQTs, discuss Brugada phenocopy syndrome drawing from published literature, and present our own patch-clamp data from mutant channels at low pH. We show that, at low pH, there is a preferential block of peak currents and preferential increase of persistent current in a common BrS/LQTs mutant compared to wild type sodium channels. Our data complements the existing literature on the importance of environmental triggers to arrhythmias.

Voltage-gated sodium channels: pharmaceutical targets via anticonvulsants to treat epileptic syndromes.

Epilepsy is a brain disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels, Nav1.1 and Nav1.2. The effects of antiepileptic drugs (AEDs) as effective therapies for epilepsy have been characterized by extensive research. Most of the classic AEDs targeting Nav share a common mechanism of action by stabilizing the channel’s fast-inactivated state. In contrast, novel AEDs, such as lacosamide, stabilize the slow-inactivated state in neuronal Nav1.1 and Nav1.7 isoforms. This paper reviews the different mechanisms by which this stabilization occurs to determine new methods for treatment.

Differential thermosensitivity in mixed syndrome cardiac sodium channel mutants

The E1784K mixed syndrome mutant of the cardiac voltage‐gated sodium channel, NaV1.5, responds differently to temperature changes compared to the R1193Q mutant and wild‐type (WT) NaV1.5. In E1784K, elevated temperature causes a larger increase in persistent current; there is also an increase in use‐dependent inactivation at 1 Hz, which is not apparent at 3 Hz. WT NaV1.5 and R1193Q channels respond similarly to temperature changes. Action potential modelling (from extrapolated temperature coefficient (Q10)values) predicts the effects of differential temperature sensitivity on the cardiac action potential: greater attenuation of the epicardial action potential occurs in E1784K as temperature shifts from hypothermic to hyperthermic conditions, and when transient outward potassium currents are increased. The results from the action potential model predict that, at febrile temperatures, E1784K channels results in a larger transmural voltage gradient. Hyperthermia exacerbates the Brugada syndrome 1 (BrS1) phenotype, which may be arrhythmogenic in E1784K mutants.

Effects of Amiodarone and N-desethylamiodarone on Cardiac Voltage-Gated Sodium Channels

Amiodarone (AMD) is a potent antiarrhythmic drug with high efficacy for treating atrial fibrillation and tachycardia. The pharmacologic profile of AMD is complex. AMD possesses biophysical characteristics of all of class I, II, III, and IV agents. Despite its adverse side effects, AMD remains the most commonly prescribed antiarrhythmic drug. AMD was described to prolong the QT interval and can lead to torsades de pointes. Our goal was to study the effects of AMD on peak and late sodium currents (INa,P and INa,L) and determine whether these effects change as AMD is metabolized into N-desethylamiodarone (DES). We hypothesized that AMD and DES block both INa,P and INa,L with similar profiles due to structural similarities. Given the inherent small amounts of INa,L in NaV1.5, we screened AMD and DES against the Long QT-3-causing mutation, ΔKPQ, to better detect any drug-mediated effect on INa,L. Our results show that AMD and DES do not affect WT or ΔKPQ activation; however, both drugs altered the apparent valence of steady-state fast-inactivation. In addition, AMD and DES preferentially block ΔKPQ peak conductance compared to WT. Both compounds significantly increase INa,L and window currents. We conclude that both compounds have pro-arrhythmic effects on NaV1.5, especially ΔKPQ; however, DES seems to have a greater pro-arrhythmic effect than AMD.

A thermosensitive mutation alters the effects of lacosamide on slow inactivation in neuronal voltage-gated sodium channels, NaV1.2.

Epilepsy is a disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels (NaVs). The C121W mutation of the β1 subunit, in particular, gives rise to the thermosensitive generalized epilepsy with febrile seizures plus (GEFS+) phenotype. Lacosamide is used to treat epileptic seizures and is distinct from other anti-seizure drugs by targeting NaV slow-inactivation. We studied the effects of a physiologically relevant concentration of lacosamide on the biophysical properties of NaV1.2 channels associated with either WT-β1 or the mutant C121W-β1 subunit. Biophysical parameters were measured at both normal (22°C) and elevated (34°C) temperatures to elicit the differential temperature-sensitivity of C121W. Lacosamide was more effective in NaV1.2 associated with the WT-β1 than with C121W-β1 at either temperature. There is also a more potent effect by lacosamide on slow inactivation at elevated temperatures. Our data suggest a modulatory role is imparted by the β1 subunit in the interaction between the drug and the channel.

A Mixed Periodic Paralysis & Myotonia Mutant, P1158S, Imparts pH-Sensitivity in Skeletal Muscle Voltage-gated Sodium Channels

Skeletal muscle channelopathies, many of which are inherited as autosomal dominant mutations, include myotonia and periodic paralysis. Myotonia is defined by a delayed relaxation after muscular contraction, whereas periodic paralysis is defined by episodic attacks of weakness. One sub-type of periodic paralysis, known as hypokalemic periodic paralysis (hypoPP), is associated with low potassium levels. Interestingly, the P1158S missense mutant, located in the third domain S4-S5 linker of the “skeletal muscle”, Nav1.4, has been implicated in causing both myotonia and hypoPP. A common trigger for these conditions is physical activity. We previously reported that Nav1.4 is relatively insensitive to changes in extracellular pH compared to Nav1.2 and Nav1.5. Given that intense exercise is often accompanied by blood acidosis, we decided to test whether changes in pH would push gating in P1158S towards either phenotype. Our results suggest that, unlike in WT-Nav1.4, low pH depolarizes the voltage-dependence of activation and steady-state fast inactivation, decreases current density, and increases late currents in P1185S. Thus, P1185S turns the normally pH-insensitive Nav1.4 into a proton-sensitive channel. Using action potential modeling we predict a pH-to-phenotype correlation in patients with P1158S. We conclude that activities which alter blood pH may trigger the noted phenotypes in P1158S patients.

The efficacy of Ranolazine on E1784K is altered by temperature and calcium

E1784K is the most common mixed syndrome SCN5a mutation underpinning both Brugada syndrome type 1 (BrS1) and Long-QT syndrome type 3 (LQT3). The charge reversal mutant enhances the late sodium current (INa) passed by the cardiac voltage-gated sodium channel (NaV1.5), delaying cardiac repolarization. Exercise-induced triggers, like elevated temperature and cytosolic calcium, exacerbate E1784K late INa. In this study, we tested the effects of Ranolazine, the late INa blocker, on voltage-dependent and kinetic properties of E1784K at elevated temperature and cytosolic calcium. We used whole-cell patch clamp to measure INa from wild type and E1784K channels expressed in HEK293 cells. At elevated temperature, Ranolazine attenuated gain-of-function in E1784K by decreasing late INa, hyperpolarizing steady-state fast inactivation, and increasing use-dependent inactivation. Both elevated temperature and cytosolic calcium hampered the capacity of Ranolazine to suppress E1784K late INa. In-silico action potential (AP) simulations were done using a modified O’Hara Rudy (ORd) cardiac model. Simulations showed that Ranolazine failed to shorten AP duration, an effect augmented at febrile temperatures. The drug-channel interaction is clearly affected by external triggers, as reported previously with ischemia. Determining drug efficacy under various physiological states in SCN5a cohorts is crucial for accurate management of arrhythmias.

SCN5A mutations in 442 neonates and children: genotype–phenotype correlation and identification of higher-risk subgroups

Aims To clarify the clinical characteristics and outcomes of children with SCN5A-mediated disease and to improve their risk stratification. Methods and results A multicentre, international, retrospective cohort study was conducted in 25 tertiary hospitals in 13 countries between 1990 and 2015. All patients ≤16 years of age diagnosed with a genetically confirmed SCN5A mutation were included in the analysis. There was no restriction made based on their clinical diagnosis. A total of 442 children {55.7% boys, 40.3% probands, median age: 8.0 [interquartile range (IQR) 9.5] years} from 350 families were included; 67.9% were asymptomatic at diagnosis. Four main phenotypes were identified: isolated progressive cardiac conduction disorders (25.6%), overlap phenotype (15.6%), isolated long QT syndrome type 3 (10.6%), and isolated Brugada syndrome type 1 (1.8%); 44.3% had a negative electrocardiogram phenotype. During a median follow-up of 5.9 (IQR 5.9) years, 272 cardiac events (CEs) occurred in 139 (31.5%) patients. Patients whose mutation localized in the C-terminus had a lower risk. Compound genotype, both gain- and loss-of-function SCN5A mutation, age ≤1 year at diagnosis in probands and age ≤1 year at diagnosis in non-probands were independent predictors of CE. Conclusion In this large paediatric cohort of SCN5A mutation-positive subjects, cardiac conduction disorders were the most prevalent phenotype; CEs occurred in about one-third of genotype-positive children, and several independent risk factors were identified, including age ≤1 year at diagnosis, compound mutation, and mutation with both gain- and loss-of-function.