CACNA1B gene variants in adult-onset isolated focal dystonia

Isolated dystonia is a heterogeneous group of under-diagnosed movement disorders [1]. The main clinical findings are the dystonic movements and postures, defined by “sustained or intermittent muscle contractions causing abnormal, often repetitive, movements, postures, or both” [1, 2]. Depending on the body distribution, isolated dystonia is classifiable as focal, segmental, multifocal, generalized, and unilateral [2]. In those with adult-onset, the most common form is isolated focal dystonia (IFD), torticollis, and blepharospasm being the typical phenotypes, sometimes with subsequent progression to other regions [1].

Since the discovery that mutations in the TOR1A gene (i.e., DYT1) are responsible for most of the early-onset isolated dystonia cases, a growing number of genes linked to Mendelian forms of dystonia have been identified [3]. The etiopathogenesis of adult-onset dystonia is known to a lesser degree, both genetic predisposition and environmental factors probably playing a role [2, 3].

To date, a total of 26 loci symbolized as DYTs (i.e., DYT1 to DYT25) are known to be involved in dystonia syndromes [4]. Of these, three causative genes have been extensively validated in different populations, namely TOR1A (DYT1), THAP1 (DYT6), and GNAL (DYT25) [1, 4]. Other genes found to be associated with isolated or combined dystonia include ANO3 (DYT24), TUBB4A (DYT4), GCH1 (DYT5a), TH (DYT5b), TAF1 (DYT3), PRKRA (DYT16), ATP1A3 (DYT12), SGCE (DYT11), KCTD17, and CACNA1A [4‐8].

The CACNA1B gene (chromosome 9, q34.3) codes for the pore-forming α1B subunit of the Ca_V2.2 N-type voltage-gated calcium channels, which are essential for neurotransmitter release from cerebral neurons [6, 9, 10]. Common variants in CACNA1B were linked to the risk of cerebral infarction [9], bipolar disorders [10], and schizophrenia [10, 11]. Recently, CACNA1B mutations were also found in association to a particular myoclonus-dystonia syndrome [12] and progressive epilepsy-dyskinesia [13]. The former association was not replicated in a large European cohort, but the study focused only on the previously reported R1389H variant, the CACNA1B gene not being extensively screened [14]. Targeted next-generation sequencing (NGS) can help identify and investigate genes with a great number of exons, such as CACNA1B. Considering its putative role in some primary dystonia syndromes, we screened the CACNA1B gene for disease-causing variants using targeted NGS Ion Torrent PGM in a large cohort of patients with adult-onset IFD. To our knowledge, this is the first study that used targeted NGS to identify potentially predisposing variants in CACNA1B gene.

The research was conducted in accordance with the Helsinki declaration, with approval from the Research Ethics Committee at Colentina Clinical Hospital (Bucharest, Romania), as part of a more extensive project on the genetics of adult-onset IFD (i.e., GENDYS-UEFISCDI, PN-III-P4-ID-PCE-2016-0696). All patients signed a dedicated written informed consent prior to enrolment.

We analyzed samples from 120 adult-onset IFD patients for mutations in CACNA1B gene, using targeted NGS. Amplicons ranged from 120 to 310 pb, with a mean amplicon length of 237 pb and at least 110-fold depth covering 100% of the bases of the targeted gene. The sequence analysis by NGS revealed a novel frameshift mutation c.2291AGG > A (Fig. 1), in exon 19, and a previously reported variant, c.6834T > G (Fig. 2), in exon 47. The mutation/variants are named according to the nomenclature of Human Genome Variation Society. Segregation analyses were not performed for the mutation because paternal DNA was unavailable for testing. The mutation was not found in 120 unrelated control individuals from the same ethnic origin.

For predicting the impact of the novel frameshift mutation, we used three in silico programs: PolyPhene-2, SIFT, and Mutation Taster PolyPhene 2, damaging; SIFT, damaging; Mutation Taster, disease causing. The novel mutation was not reported in the control databases gnomAD, 1000 Genomes, and Exome Variant Server or in any other single-nucleotide polymorphism database. The online software tools predict that c.6834T > G causes the deleterious leucine acid to arginine substitution at position 2215, pLeu2215Arg (PolyPhene 2, unknown; SIFT, tolerated; Mutation Taster, disease causing) in one of two isoforms of CACNA1B. The c.6834T > G variant is predicted as “disease causing” by Mutation Taster, but it can be classified as likely benign, considering that allele frequency is greater than that expected for a disorder. The two variants/mutation were found recurrently; thus, c.6834T > G pLeu2215Arg (rs2278973) was detected in 38 (31.7%) of our 120 IFD patients in homozygous and heterozygous state, and the novel c.2291AGG > A frameshift mutation was present in 6 (5%) unrelated cases in heterozygous state.

The CACNA1B gene codes for the N-type neuronal voltage-gated calcium channels CaV2.2, which may play a role in the development of some movement disorders, including IFD. Prior to our study, genetic analysis of the CACNA1B gene in patients with movement disorder was performed in a limited number of studies, by methods not suited for wide gene screening. Mutations were found only in patients with myoclonus-dystonia, a rare form of combined dystonia [3, 12]. The targeted NGS approach allowed us to quickly screen for disease-causing variants of CACNA1B gene in a cohort of 120 patients with adult-onset IFD. We identified a novel frameshift mutation (i.e., c.2291AGG > A) that may have damaging effects on the CaV2.2 N-type voltage-gated calcium channels. To our knowledge, this is the first study that identifies a disease-causing mutation with loss of function effect in the CACNA1B gene association with adult-onset IFD.

Considering the lack of this mutation in the control group and the putative pathogenicity estimated by the in silico prediction tools, our results support a causal association between c.2291AGG > A variant and IFD. The effect of this frameshift mutation should be further analyzed by functional studies.