The CACNA1B R1389H variant is not associated with myoclonus-dystonia in a large European multicentric cohort

Myoclonus-dystonia (M-D) is a very rare movement disorder, caused in ∼30–50% of cases by mutations in SGCE. The CACNA1B variant c.4166G>A; (p.R1389H) was recently reported as the likely causative mutation in a single 3-generation Dutch pedigree with five subjects affected by a unique dominant M-D syndrome and cardiac arrhythmias. In an attempt to replicate this finding, we assessed by direct sequencing the frequency of CACNA1B c.4166G>A; (p.R1389H) in a cohort of 520 M-D cases, in which SGCE mutations had been previously excluded. A total of 146 cases (28%) had a positive family history of M-D. The frequency of the variant was also assessed in 489 neurologically healthy controls and in publicly available data sets of genetic variation (1000 Genomes, Exome Variant Server and Exome Aggregation Consortium). The variant was detected in a single sporadic case with M-D, but in none of the 146 probands with familial M-D. Overall, the variant was present at comparable frequencies in M-D cases (1 out of 520; 0.19%) and healthy controls (1 out of 489; 0.2%). A similar frequency of the variant was also reported in all publicly available databases. These results do not support a causal association between the CACNA1B c.4166G>A; (p.R1389H) variant and M-D.

Recently, Groen et al. (6) identified the missense variant c.4166G>A; ( p.R1389H) (rs184841813) in CACNA1B [MIM 601012] as the likely causative mutation in a Dutch pedigree with five subjects affected by autosomal dominant M-D lacking mutations in SGCE. Unique features in the pedigree were lower limb orthostatic high-frequency myoclonus, attacks of limb painful cramps and cardiac arrhythmias in three of the affected subjects (7). Sanger sequencing of the CACNA1B exons coding for the protein portion spanning from III-S5 to III-S6 failed to reveal other mutations in a further 47 M-D cases.
The CACNA1B p.(R1389H) substitution represents therefore an excellent candidate as a disease-causing mutation for M-D. However, in the absence of identification of CACNA1B mutations in other unrelated pedigrees, the implication of mutations in this gene as a cause for M-D is not confirmed.
In this study, we assessed the frequency of the CACNA1B c.4166G>A; ( p.R1389H) variant in a large multicentric cohort of M-D cases without mutations in SGCE (both point mutations and copy number variants).

Results
A total of 520 M-D cases (28% were familial) were screened for the presence of the c.4166G>A; ( p.R1389H) variant. Additionally, we assessed the frequency of the variant in whole-exome sequencing data from 489 white healthy controls of UK and US origin and in European cases listed in publicly available data sets of genetic variation (1000 Genomes, Exome Variant Server and Exome Aggregation Consortium). None

Discussion
The advent of next generation sequencing has led to an extraordinary acceleration in the discovery rate of rare genetic variants, the majority of which are of uncertain clinical significance. Hence, a close scrutiny is necessary before causally linking a candidate variant to a disease. To avoid false assignment of pathogenicity, MacArthur et al. (11) have recently proposed guidelines for implicating causality of rare variants in human disease.
In family-based studies, assessment of co-inheritance of a candidate variant with the disease status within family members represents the first requirement to prove causality. , were found to perfectly co-segregate with the disease in the family. De novo mutations in SPTAN1 have been shown to cause a neurological phenotype (West syndrome with severe cerebral hypomyelination, spastic quadriplegia and developmental delay) (12) and more recently a microdeletion encompassing SPTAN1 was detected in a child with epileptic encephalopathy and severe dystonia (13).
Given the clinical presentation pointing towards a possible channelopathy, the authors assumed that the causative variant was the one in CACNA1B (6).
However, co-segregation of a variant with disease in a single pedigree does not establish with certainty its pathogenic role, especially if other co-segregating coding variants and the possibility of a separate undetected pathogenic variant in linkage disequilibrium cannot be convincingly ruled out.
In addition, a candidate variant responsible for a rare disease should be found at a low frequency in population controls, consistent with the proposed model of inheritance and disease prevalence.
M-D is a very rare disorder with a suggested prevalence of around two per million in Europe (14). We would therefore anticipate highly penetrant mutations causing dominant forms of M-D to be absent or extremely rare in the general population. Yet, this is not the case for p.(R1389H), which is present at a considerable frequency in our healthy controls and all publicly available databases (∼0.1-0.3%). According to the Exome Aggregation Consortium database, the carrier frequency of this variant in Europeans is ∼4 times higher than the TOR1A [MIM 605204] c.904_906delGAG deletion (0.026%), which is by far the most common single mutation responsible for dystonia described to date (15). Given this frequency, if c.4166G>A; ( p.R1389H) were a pathogenic variant, we would expect it to be responsible for a large proportion of familial M-D cases. However, in our cohort, not only was the variant not identified in any of the probands with familial M-D, but the overall frequency of the variant did not differ between M-D cases and healthy controls. This does not support a pathogenic effect of the variant even assuming a reduced penetrance.
In conclusion, our study suggests that the role of the CACNA1B variant c.4166G>A; ( p.R1389H) as a cause for M-D is questionable.
Further genetic evidence is needed before designating CACNA1B mutations as a cause for dominant M-D.

Materials and Methods
A total of 520 M-D cases of British, German and Italian origin were recruited in four tertiary movement disorders centers (London, Lübeck, Tübingen and Milan). All selected cases fulfilled the proposed diagnostic criteria for M-D (2). A total of 146 cases (28%) had a positive family history of M-D. All participants provided written informed consent.
M-D cases were screened by direct Sanger sequencing for mutations in exon 28 of CACNA1B (RefSeq NM_000718.3), which contains the c.4166G>A; ( p.R1389H) variant. Each reaction was performed in a 20 µl volume containing 10 µl of FastStart PCR master mix (Roche), 5 µl of water, 2 µl of each primer (5pmol/µl) and 30 ng of genomic DNA. After purification, PCR products were sequenced in both forward and reverse directions using BigDye Terminator v3.1 sequencing chemistry and then were loaded on the ABI3730xl genetic analyser (Applied Biosystems, Foster City, CA, USA). The sequences were analysed with Sequencher software (version 4.9; Gene Codes).
Whole-exome sequencing data from 489 white healthy controls of UK and US origin were provided by the International Parkinson's Disease Genomic Consortium (IPDGC). In short, prior to sequencing, DNA templates were bridge amplified to form clonal clusters inside a flowcell via the cBot cluster generation process. The flowcells were then loaded into the next-generation sequencer Illumina HiSeq 2000. Paired end sequence reads were aligned with Burrows-Wheeler Aligner (BWA) against the reference human genome (UCSC hg19). Duplicate read removal, format conversion and indexing were performed with Picard (http:// picard.sourceforge.net/). The Genome Analysis Toolkit (GATK) was used to recalibrate base quality scores, perform local realignments around possible indels and to call and filter the variants.

Supplementary material
Supplementary Material is available at HMG online.