Episodic Ataxia

Screening genes using targeted next-generation sequencing methods

Fatima Jaffer1,2, Alice Gardiner1,2, Alan Pittman5, Tracey Graves1,3, Vaneesha Gibbons2, Nicholas Wood2, Michael Hanna1,4 and Henry Houlden1,2,4
MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology 2 Department of Molecular Neuroscience, UCL Institute of Neurology 3 Oxford Radcliffe Hospitals NHS Trust 4 Paroxysmal/Episodic Ataxia Collaborative Group, UCL Institute of Neurology 5 Department of Molecular Neurology, UCL Institute of Neurology
1

Background
Brain channelopathies are a genetically and phenotypically heterogeneous group of disorders caused by mutations in voltage-gated ion channels causing paroxysmal CNS diseases including ataxia, epilepsy, familial hemiplegic migraine, and hyperekplexia. The Episodic Ataxias (EA) are autosomal dominant disorders of channel dysfunction that manifest in the first two decades of life and all characterised by paroxysmal episodes of ataxia. EA types 1 and 2 have been well characterised and are known to cause different clinical features and are due to mutations in KCNA1 gene encoding a potassium channel and CACNA1A which encodes the 1A subunit of the Cav2.1 voltage-gated calcium channel complex (VGCC) respectively (Table 1). Furthermore, EA2 tends to result in a progressive cerebellar syndrome and is allelic with familial hemiplegic migraine (FHM) and spinocerebellar ataxia type 6 (SCA6). Finally EA2 is responsive to acetazolamide therapy in 75% of cases whereas it is usually ineffective in cases of EA1 with a variable response in other EA types. Six other types of EA have been reported in isolated kindreds and not all the causative genes have been identified (Table 1). The UK Genetic Testing Network currently provides tests for EA1 and EA2 only. In addition, the phenotypes of paroxysmal movement disorders including paroxysmal kinesigenic dyskinesias (PKD), paroxysmal non-kinesigenic dyskinesias (PNKD) and FHM are overlapping with EA suggesting that these disorders exist in a spectrum making the genetic cause unclear. Furthermore, it is hypothesised that several of the responsible genes have not yet been identified. The objective of this project is to use next-generation sequencing technology to screen patients with a clinical phenotype suggesting a paroxysmal movement disorder such as EA , PKD, PNKD and FHM to detect novel mutations in known causative genes before moving forward to whole-exome sequencing.

Table 1: EA types clinical features and genetics

EA type Age of onset Duration Other clinical features Gene and/ Channel subunit or Locus

EA 1

10-20 yrs 10-20 yrs

Brief Neuromyotonia, myokymia, (seconds to seizures.Triggers: exertion, minutes) startle, stress Prolonged (hours)

KCNA1; 12p13

Kv1.1 potassium channel

EA 2

Episodic and progressive ataxia, CACNA1A; 1A of Cav2.1 P/ Vertigo, weakness, seizures. 19p13 Q VGCC Triggers: Exertion, alcohol Vertigo, tinnitus, 1q42 headache.Triggers: kinesogenic Vertigo, diplopia, nystagmus, broken smooth pursuit Vertigo, nystgamus, seizures (JME with Arg482X) PATX Unknown Unknown

EA 3 EA 4 EA 5 EA 6

1-40 yrs Brief (minutes) 30-60 yrs 20-60 yrs Prolonged (hours) Prolonged (hours)

CACNB44 4 subunit of ; 2q22 Cav2.1 VGCC EAAT 1 protein of glial glutamate transporter

<10 yrs Prolonged (hours)

Episodic and progressive ataxia, SLC1A3; seizures, migraine, paroxysmal 5p13.2 hemiplegia, cognitive impairment. Triggers: hyperpyrexia Episodic ataxia frequency decreasing with age, vertigo, weakness, dysarthria. Triggers: Exertion Choreoathetosis, limb dystonia, spasticity, paraesthesiae, headache. Triggers: Alcohol, fatigue, exercise 19q13

EA 7

<20 yrs Prolonged (hours)

Unknown

EA 8

2-10 yrs Brief (minutes)

DYT9; 1p31

Probable potassium channel

Methods
We designed a brain channelopathy panel which contains the known coding genes associated with EA, PKD, PNKD and FHM Using the Illumina TruSeq Custom Amplicon, application the patients undergo targeted sequencing of only of the coding regiong these genes. It is quicker and cheaper then Sanger sequencing the same genes. The sequencer used was the Illumina MiSeq (see right) .
Gene KCNA1

Brain channelopathy panel

Associated Disease(s) Episodic Ataxia Type1 No Exons No Amplicons 2 47 3 10 10 4 10 14 10 66 9 16 16 7 13 18

CACNA1A Episodic Ataxia Type2, Hemiplegic Migraine Spinal Cerebellar Ataxia Type6 KCNK18 SLC2A1 SLC1A3 PRRT2 PNKD CACNB4 Hemiplegic Migraine GLUT Deficiency Syndrome Type1 GLUT Deficiency Syndrome Type2 Episodic Ataxia Type6 Paroxysmal Kinesigenic Dyskinesia Paroxysmal Nonkinesigenic Dyskinesia Episodic Ataxia Type 5 Epilepsy

TruSeq Custom Amplicon Design The panel was designed using the Illumina design studio software, specifying either the genes or genomic coordinates of the regions to be sequenced in order to design amplicons covering these regions. Amplicons are 250 base pairs in size. The panel can currently cover up to 650Kb of sequence for up to 95 samples.

Total Number of Amplicons 155 Number of Exons Missed 3

Results and Conclusions

We screened 79 patient samples and 16 positive controls were used for EA, PKD, PNKD and FHM. Of the controls, 10 were identified, and 6 were not. The control mutations that were not seen either because the region in question was not covered well, or the mutation was a large scale deletion. All mutations found were confirmed with Sanger sequencing. The table (below) shows details of the variants identified by the panel in patient samples that were not in databases (1000 Genomes and Exome Variant Server) and are likely to be pathogenic. 10 likely pathogenic variants were found (13% of samples), and of these 50% were in genes not usually tested for the disease.
Patient Diagnosis Gene
EA 2 EA 2 EA 2 PKD PKD PKD Complex EA 2 EA 2 PKD PKD CACNA1A CACNA1A CACNA1A CACNA1A KCNA1 KCNK18 SLC2A1 CACNB4 PRRT2 PRRT2

Mutation
D302N Het R1352X Het G1401E Het K1937X Het S306F Het Y121Lfs*44 Het G18R Het L413Q Het P138A Het 240X Het

Sift

Polyphen

Conserved? Gene Associated with Disease?

Highly Highly Highly Moderately Moderately Moderately Yes Yes Yes No No No No No Yes Yes

Deleterious Probably Damaging Tolerated Tolerated Tolerated Benign Probably Damaging Benign Deleterious Probably Damaging Deleterious Probably Damaging

Problems 3 false positives There is no easy way to tell which exons have not been covered without additional expensive software Copy number variants could not be detected Conclusions Probable pathogenic mutations found in 13% of samples The results also show that the genetic spectrum aspect of ion disorders makes this type of sequencing is especially effective, as it is not always expected gene that is responsible, and it does not let diagnostic bias affect whether a causative mutation is found.

CONTACT DETAILS Brain Channelopathy studies: f.jaffer@ucl.ac.uk