WHOLE EXOME SEQUENCING OF HEMIPLEGIC MIGRAINE PATIENTS SHOWS AN INCREASED BURDEN OF MISSENSE VARIANTS IN CACNA1H AND CACNA1I GENES 195 9 The expression of CACNA1x genes varies considerably and, with the exception of CACNA1S, all are expressed in the brain.25 In addition to CACNA1A being a well-known HM gene, there have been rare reports on other CACNA1x genes possibly involved in HM-relevant phenotypes. For instance, a link between hemiplegic migraine and brain stem aura migraine has been suggested for CACNA1E 26, and headache with neurological deficits and cerebrospinal fluid lymphocytosis (HaNDL), a headache syndrome with much phenotypic resemblance to HM, has been linked to the occurrence of antibodies against CACNA1H,27 a gene implicated in childhood epilepsy although this has recently been debated.28 Furthermore, using a systems genetics approach Rasmussen et al.29 identified CACNA1B as one of the genes commonly mutated in migraine families. Finally, CACNA1A was identified as a risk locus for common migraine, as well as being one of the three genes specific for migraine with aura.30 Although no definite proof for a causal link was provided in any of these cases, the existing data can be regarded as supportive evidence for a spectrum ranging from rare to common variants contributing to certain extent to the risk for both common and hemiplegic migraine. This variety of observed variants makes the family of CACNA1x genes an interesting candidate for burden testing in HM, with relevance, foremost, to patients with a complex genetic basis. Whole exome sequencing (WES) enables comprehensive exploration of missense variants and investigation of their role in complex traits.When considering that these missense variants are unlikely to be causing HM as monogenic factors as occurs for patients with specific CACNA1A, ATP1A2 and SCN1A mutations,5-7 burden testing is a potential way to explore their potential synergistic effect on increasing HM disease risk. Burden testing typically requires a set of qualifying variants, often rare, protein-altering variants in the case of a monogenic condition. However, following on from the hypothesis that HM seems not a monogenic disorder in all patients, the accumulation of both rare and common protein-altering variants may be relevant in terms of disease susceptibility. The use of large publicly available WES datasets from general population controls can be incorporated in burden testing to gain more reliable estimates of gene-wide susceptibility. We hypothesize that the burden of multiple missense variants in CACNA1x genes increases the risk for HM. Burden being the aggregation of both rare and common variants as well as the increased presence of a variant in cases compared to controls. To this end, we here used WES data from a large Australian HM patient cohort to identify missense variants in eight calcium channel genes (CACNA1A, -B, -C, -D, -E, -G, -H, and -I) and determined whether the aggregated effect of the variants across the genes was higher than observed in general population controls. Results were validated in an independent Dutch clinical HM cohort. Methods Study cohorts The study consisted of two cohorts of HM patients: an Australian cohort of 184 patients (discovery cohort) and a Dutch cohort of 32 patients (replication cohort). Importantly, patients were a priori
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