Eunson, Louise Helen; (2001) Clinical, genetic and expression studies of human CNS channelopathies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes clinical, molecular genetic and electrophysiological studies in two dominantly inherited CNS disorders; episodic ataxia type 1 (EA1) and episodic ataxia type 2 (EA2). Shortly before this work was initiated, evidence had been published that EA1 associated with mutations in the voltage gated potassium channel gene (KCNA1) and EA2 associated with mutations in the P/Q-type voltage gated calcium channel gene (CACNA1A). However, the frequency of mutations in these genes in patients with EA1 and EA2 was unknown. Furthermore, the range of phenotypes associated with mutations in these channels and their molecular mechanisms had not been defined. Through National and International collaboration, 42 individuals were identified from 20 families with a phenotype compatible with Kvl.l dysfunction. In addition, 29 individuals were identified from 20 families with a phenotype compatible with P/Q-type calcium channel dysfunction. Five previously unreported pathogenic heterozygous mutations were identified (A242P, P244FI, T226R, V404I and R417X) in the KCNAl gene. Although two of these mutations associated with typical EA1, three mutations associated with phenotypes which had not been reported previously. These included EA1 with epilepsy, epilepsy with myokymia and isolated myokymia. In addition to observing this new phenotypic variation between families, marked phenotypic variation within one family was observed. In family F, with the T226R mutation, the index case exhibited unusually severe disabling neuromyotonia without episodic ataxia, while his mother exhibited typical EA1 with mild neuromyotonia. This study shows the mechanism for this variability was not due to polymorphisms in a related potassium channel Kv1.2, which is known to co-assemble with Kvl.l. Using site directed mutagenesis, cDNA constructs harbouring each of the novel mutations were produced and the electrophysiological consequences of these mutations on the function of the potassium channel in a Xenopus laevis Oocyte system was studied. This revealed that all mutations reduced the delayed rectifying function of this channel to different degrees. This is predicted to result in increased neuronal excitability, which is likely to be the basis of the clinical phenotypes. There was some correlation between the electrophysiological data and the severity of the clinical phenotype. Five previously unreported heterozygous mutations were identified in CACNAlA (R1820X, Y1854X, 3404 ins ATCCAATCC, 6056 + 4 del AGTG and 3698 + 1 G>A) which are likely to be pathogenic. Although three of these new mutations (Y1854X, 6056 + 4 del AGTG and 3698 + 1 G>A) associated with typical EA2 phenotypes, one of them represented a previously undescribed mutational mechanism of intronic deletion (6056 + 4 del AGTG). RT-PCR analysis of this mutation provided evidence that the RNA was unstable. Two of the new mutations associated with previously unrecognised phenotypes. The first patient (R1820X) exhibited absence epilepsy and mental retardation in addition to EA2. This is the first evidence that dysfunction of this calcium channel may associate with epilepsy in humans and provides a link with a large body of data in mouse models of absence epilepsy. The second patient (3404 ins ATCCAATCC) exhibited a late onset, pure, non-episodic ataxia, similar to the phenotype associated with an abnormal CAG repeat expansion in the C-terminal region of CACNAlA (resulting in spinocerebellar ataxia type 6). One of the EA2 families without a mutation in CACNAlA was large enough to confirm linkage to CACNAlA. This suggests that mutations in non-coding regions of CACNAlA may also cause EA2. A cDNA construct harbouring the R1820X mutation was produced, allowing detailed molecular expression studies in the Xenopus laevis Oocyte system. This showed that the R1820X mutation resulted in a loss of function of the calcium channel. This is likely to be the basis of the disease, including the epilepsy, in this patient. These studies have identified novel pathogenic mutations in KCNAl and CACNAlA and have extended our knowledge about the clinical phenotypes associated with dysfunction of these ion channels. In addition, it has provided insights into the molecular mechanisms that underlie these CNS channelopathies.

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