Mutation Spectrum in the CACNA1A Gene in 49 Patients with Episodic Ataxia

Abstract

Episodic ataxia is an autosomal dominant ion channel disorder characterized by episodes of imbalance and incoordination. The disease is genetically heterogeneous and is classified as episodic ataxia type 2 (EA2) when it is caused by a mutation in the CACNA1A gene, encoding the α_1A subunit of the P/Q-type voltage-gated calcium channel Ca_v2.1. The vast majority of EA2 disease-causing variants are loss-of-function (LoF) point changes leading to decreased channel currents. CACNA1A exonic deletions have also been reported in EA2 using quantitative approaches. We performed a mutational screening of the CACNA1A gene, including the promoter and 3'UTR regions, in 49 unrelated patients diagnosed with episodic ataxia. When pathogenic variants were not found by sequencing, we performed a copy number variant (CNV) analysis to screen for duplications or deletions. Overall, sequencing screening allowed identification of six different point variants (three nonsense and three missense changes) and two coding indels, one of them found in two unrelated patients. Additionally, CNV analysis identified a deletion in a patient spanning exon 35 as a result of a recombination event between flanking intronic Alu sequences. This study allowed identification of potentially pathogenic alterations in our sample, five of them novel, which cover 20% of the patients (10/49). Our data suggest that most of these variants are disease-causing, although functional studies are required.

Figure 1

CACNA1A gene and protein structure with the identified pathogenic variants. CACNA1A gene structure (top figure), with boxes indicating exons. Protein structure of the Ca_v2.1α_1A subunit (bottom figure). The genetic variants reported in this work are indicated by dots. Cyt: cytoplasm; M: cytoplasmic membrane; ES: extracellular space; S: Segment. Reference sequence for cDNA nomenclature: NM_001127221 (nucleotide c.279A corresponding to the initiation codon, ATG). Rererence sequence for protein nomenclature: NP_001120693.

Figure 2

Pedigrees from individuals with identified changes. The code on top of every pedigree corresponds to the proband individual, indicated by a black arrow. Affected individuals are denoted by solid symbols; episodic ataxia is indicated in black and other phenotypes in gray; squares indicate males and circles indicate females. Clinical characteristics are indicated below each individual (EA2: episodic ataxia type 2; FHM: familial hemiplegic migraine; MO: migraine without aura; MA: migraine with aura). Gene variant carrier status is indicated below individuals. A complete screening of the CACNA1A gene was performed by Sanger sequencing and CNV analysis only in the probands (indicated by an arrow), whereas in the other family members where the genotype is indicated, only the variant identified in the proband was tested. In those individuals where the genotype is not shown, DNA was not available for analysis.

Figure 3

CNV studies in the CACNA1A gene of patient 474. (a) Results from the MLPA analysis performed with Coffalyser. The deleted exon 35 is indicated with an arrow. C: control probe. (b) Deletion breakpoint mapping. Sequence chromatogram corresponding to the homologous fragment from AluY and AluSz sequences. The identical shared sequence by the 5′ and 3′ Alu elements located in introns 34 and 35 that mediated the recombination is framed. Location of fragments taken from UCSC Genome Browser on Human Feb. 2009 Assembly (GRCh37/hg19).