Sanjeev Rajakulendran

Neuronal P/Q-type calcium channel dysfunction in inherited disorders of the CNS

The past two decades have witnessed the emergence of a new and expanding field of neurological diseases—the genetic ion channelopathies. These disorders arise from mutations in genes that encode ion channel subunits, and manifest as paroxysmal attacks involving the brain or spinal cord, and/or muscle. The voltage-gated P/Q-type calcium channel (P/Q channel) is highly expressed in the cerebellum, hippocampus and cortex of the mammalian brain. The P/Q channel has a fundamental role in mediating fast synaptic transmission at central and peripheral nerve terminals. Autosomal dominant mutations in the CACNA1A gene, which encodes voltage-gated P/Q-type calcium channel subunit α1 (the principal pore-forming subunit of the P/Q channel) are associated with episodic and progressive forms of cerebellar ataxia, familial hemiplegic migraine, vertigo and epilepsy. This Review considers, from both a clinical and genetic perspective, the various neurological phenotypes arising from inherited P/Q channel dysfunction, with a focus on recent advances in the understanding of the pathogenetic mechanisms underlying these disorders.

Episodic Ataxia Type 1: A Neuronal Potassium Channelopathy

SummaryEpisodic ataxia type 1 is a paroxysmal neurological disorder characterized by short-lived attacks of recurrent midline cerebellar dysfunction and continuous motor activity. Mutations in KCN1A, the gene encoding Kv1.1, a voltage-gated neuronal potassium channel, are associated with the disorder. Although rare, the syndrome highlights the fundamental features of genetic ion-channel diseases and serves as a useful model for understanding more common paroxysmal disorders, such as epilepsy and migraine. This review examines our current understanding of episodic ataxia type 1, focusing on its clinical and genetic features, pathophysiology, and treatment.

Refined exercise testing can aid dna‐based diagnosis in muscle channelopathies

To improve the accuracy of genotype prediction and guide genetic testing in patients with muscle channelopathies we applied and refined specialized electrophysiological exercise test parameters.

Genetic and functional characterisation of the P/Q calcium channel in episodic ataxia with epilepsy

Mutations in CACNA1A, which encodes the principal subunit of the P/Q calcium channel, underlie episodic ataxia type 2 (EA2). In addition, some patients with episodic ataxia complicated by epilepsy have been shown to harbour CACNA1A mutations, raising the possibility that P/Q channel dysfunction may be linked to human epilepsy. We undertook a review of all published CACNA1A EA2 cases and this showed that 7% have epilepsy – representing a sevenfold increased epilepsy risk compared to the background population risk (P < 0.001). We also studied a series of 17 individuals with episodic ataxia accompanied by epilepsy and/or clearly epileptiform electroencephalograms (EEGs). We screened the entire coding region of CACNA1A for point mutations and rearrangements to determine if genetic variation in the gene is associated with the epilepsy phenotype, and measured the functional impact of all missense variations on heterologously expressed P/Q channels. We identified two large scale deletions and two new missense mutations in CACNA1A. When expressed, L621R had little detectable effect on P/Q channel function, while the other missense change, G540R, caused an approximately 30% reduction in current density. In nine patients we also identified the previously reported non‐synonymous coding variants (E921D and E993V) which also resulted in impairment of P/Q channel function. Taken together, 12 of the 17 patients have genetic changes which decrease P/Q channel function. We conclude that variants in the coding region of CACNA1A that confer a loss of P/Q‐type channel function are associated with episodic ataxia and epilepsy. Our data suggest that functional stratification of all variants, including common polymorphisms, rare variants and novel mutations, may provide new insights into the mechanisms of channelopathies.

Large scale calcium channel gene rearrangements in episodic ataxia and hemiplegic migraine: implications for diagnostic testing

Background: Episodic ataxia type 2 (EA2) and familial hemiplegic migraine type 1 (FHM1) are autosomal dominant disorders characterised by paroxysmal ataxia and migraine, respectively. Point mutations in CACNA1A, which encodes the neuronal P/Q-type calcium channel, have been detected in many cases of EA2 and FHM1. The genetic basis of typical cases without CACNA1A point mutations is not fully known. Standard DNA sequencing methods may miss large scale genetic rearrangements such as deletions and duplications. The authors investigated whether large scale genetic rearrangements in CACNA1A can cause EA2 and FHM1. Methods: The authors used multiplex ligation dependent probe amplification (MLPA) to screen for intragenic CACNA1A rearrangements. Results: The authors identified five previously unreported large scale deletions in CACNA1A in seven families with episodic ataxia and in one case with hemiplegic migraine. One of the deletions (exon 6 of CACNA1A) segregated with episodic ataxia in a four generation family with eight affected individuals previously mapped to 19p13. In addition, the authors identified the first pathogenic duplication in CACNA1A in an index case with isolated episodic diplopia without ataxia and in a first degree relative with episodic ataxia. Conclusions: Large scale deletions and duplications can cause CACNA1A associated channelopathies. Direct DNA sequencing alone is not sufficient as a diagnostic screening test.

Clinical, genetic, neurophysiological and functional study of new mutations in episodic ataxia type 1

Background and objective Heterozygous mutations in KCNA1 cause episodic ataxia type 1 (EA1), an ion channel disorder characterised by brief paroxysms of cerebellar dysfunction and persistent neuromyotonia. This paper describes four previously unreported families with EA1, with the aim of understanding the phenotypic spectrum associated with different mutations. Methods 15 affected individuals from four families underwent clinical, genetic and neurophysiological evaluation. The functional impact of new mutations identified in the KCNA1 gene was investigated with in vitro electrophysiology and immunocytochemistry. Results Detailed clinical documentation, dating back to 1928 in one family, indicates that all patients manifested episodic ataxia of varying severity. Four subjects from three families reported hearing impairment, which has not previously been reported in association with EA1. New mutations (R167M, C185W and I407M) were identified in three out of the four families. When expressed in human embryonic kidney cells, all three new mutations resulted in a loss of Kv1.1 channel function. The fourth family harboured a previously reported A242P mutation, which has not been previously described in association with ataxia. Conclusions The genetic basis of EA1 in four families is established and this report presents the earliest documented case from 1928. All three new mutations caused a loss of Kv1.1 channel function. The finding of deafness in four individuals raises the possibility of a link between Kv1.1 dysfunction and hearing impairment. Our findings broaden the phenotypic range associated with mutations in KCNA1.

Nongenetic factors influence severity of episodic ataxia type 1 in monozygotic twins

Objective: Episodic ataxia type 1 (EA1) is a monogenic channelopathy caused by mutations of the potassium channel gene KCNA1. Affected individuals carrying the same mutation can exhibit considerable variability in the severity of ataxia, neuromyotonia, and other associated features. We investigated the phenotypic heterogeneity of EA1 in 2 sets of identical twins to determine the contribution of environmental factors to disease severity. One of the mutations was also found in a distantly related family, providing evidence of the influence of genetic background on the EA1 phenotype. Methods: We evaluated 3 families with an EA1 phenotype, 2 of which included monozygotic twins. We sequenced the KCNA1 gene and studied the biophysical consequences of the mutations in HEK cells. Results: We identified a new KCNA1 mutation in each pair of twins. Both pairs reported striking differences in the clinical severity of symptoms. The F414S mutation identified in one set of twins also occurred in a distantly related family in which seizures complicated the EA1 phenotype. The other twins had an R307C mutation, the first EA1 mutation to affect an arginine residue in the voltage-sensor domain. Both mutants when expressed exerted a dominant-negative effect on wild-type channels. Conclusion: These results broaden the range of KCNA1 mutations and reveal an unexpectedly large contribution of nongenetic factors to phenotypic variability in EA1. The occurrence of epilepsy in 1 of 2 families with the F414S mutation suggests an interplay of KCNA1 with other genetic factors.

Candidate Screening of the TRPC3 Gene in Cerebellar Ataxia

The hereditary cerebellar ataxias are a diverse group of neurodegenerative disorders primarily characterised by loss of balance and coordination due to dysfunction of the cerebellum and its associated pathways. Although many genetic mutations causing inherited cerebellar ataxia have been identified, a significant percentage of patients remain whose cause is unknown. The transient receptor potential (TRP) family member TRPC3 is a non-selective cation channel linked to key signalling pathways that are affected in cerebellar ataxia. Furthermore, genetic mouse models of TRPC3 dysfunction display cerebellar ataxia, making the TRPC3 gene an excellent candidate for screening ataxic patients with unknown genetic aetiology. Here, we report a genetic screen for TRPC3 mutations in a cohort of 98 patients with genetically undefined late-onset cerebellar ataxia and further ten patients with undefined episodic ataxia. We identified a number of variants but no causative mutations in TRPC3. Our findings suggest that mutations in TRPC3 do not significantly contribute to the cause of late-onset and episodic human cerebellar ataxias.

A Clinical, Neuropathological and Genetic Study of Homozygous A467T POLG-Related Mitochondrial Disease

Mutations in the nuclear gene POLG (encoding the catalytic subunit of DNA polymerase gamma) are an important cause of mitochondrial disease. The most common POLG mutation, A467T, appears to exhibit considerable phenotypic heterogeneity. The mechanism by which this single genetic defect results in such clinical diversity remains unclear. In this study we evaluate the clinical, neuropathological and mitochondrial genetic features of four unrelated patients with homozygous A467T mutations. One patient presented with the severe and lethal Alpers-Huttenlocher syndrome, which was confirmed on neuropathology, and was found to have a depletion of mitochondrial DNA (mtDNA). Of the remaining three patients, one presented with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), one with a phenotype in the Myoclonic Epilepsy, Myopathy and Sensory Ataxia (MEMSA) spectrum and one with Sensory Ataxic Neuropathy, Dysarthria and Ophthalmoplegia (SANDO). All three had secondary accumulation of multiple mtDNA deletions. Complete sequence analysis of muscle mtDNA using the MitoChip resequencing chip in all four cases demonstrated significant variation in mtDNA, including a pathogenic MT-ND5 mutation in one patient. These data highlight the variable and overlapping clinical and neuropathological phenotypes and downstream molecular defects caused by the A467T mutation, which may result from factors such as the mtDNA genetic background, nuclear genetic modifiers and environmental stressors.

Episodic ataxia type 1 mutations differentially affect neuronal excitability and transmitter release.

SUMMARY Heterozygous mutations of KCNA1, the gene encoding potassium channel Kv1.1 subunits, cause episodic ataxia type 1 (EA1), which is characterized by paroxysmal cerebellar incoordination and interictal myokymia. Some mutations are also associated with epilepsy. Although Kv1.1-containing potassium channels play important roles in neuronal excitability and neurotransmitter release, it is not known how mutations associated with different clinical features affect the input-output relationships of individual neurons. We transduced rat hippocampal neurons, which were cultured on glial micro-islands, with lentiviruses expressing wild-type or mutant human KCNA1, and injected either depolarizing currents to evoke action potentials or depolarizing voltage commands to evoke autaptic currents. α-Dendrotoxin and tetraethylammonium allowed a pharmacological dissection of potassium currents underlying excitability and neurotransmission. Overexpression of wild-type Kv1.1 decreased both neuronal excitability and neurotransmitter release. By contrast, the C-terminus-truncated R417stop mutant, which is associated with severe drug-resistant EA1, had the opposite effect: increased excitability and release probability. Another mutant, T226R, which is associated with EA1 that is complicated by contractures and epilepsy, had no detectable effect on neuronal excitability; however, in common with R417stop, it markedly enhanced neurotransmitter release. The results provide direct evidence that EA1 mutations increase neurotransmitter release, and provide an insight into mechanisms underlying the phenotypic differences that are associated with different mutations.