New CACNA1A deletions are associated to migraine phenotypes

DOI: https://doi.org/10.1186/s10194- 018- 0891- x

Abstract-Summary Familial hemiplegic migraine type 1 (FHM1) is a form of migraine with aura caused by heterozygous mutations in 4 genes: CACNA1A, ATP1A2, SNC1A and PRRT2, but further heterogeneity is expected.

Generation Sequencing by TruSeq Custom Amplicon for CACNA1A and

ATP1A2 gene has been performed.

All genetic variants have been confirmed by Sanger sequencing and all samples were also analyzed with MLPA assay for ATP1A2-CACNA1A genes to detect duplication or deletion.

MLPA analysis showed three deletions in nine sporadic hemiplegic migraine (18%), in three patients with non-hemiplegic migraine (4.1%) and in three patients affected by episodic ataxia (20%).

Two sporadic patients showed a deletion in exons 41–43, while the rest of HM

patients (5) showed a deletion in the terminal part of the CACNA1A gene.

In migraine patients, we have found different subjects affected by different phe-

notypes deleted in exon 47.

Our work demonstrated that deletions in CACNA1A gene may be associated

also to different migraine phenotypes.

Extended: Generation Sequencing by TruSeq Custom Amplicon for CACNA1A, ATP1A2 SCN1A, PRRT2 genes has been performed (Illumina) according to the manufacturers’ instructions.

All genetic variants have been confirmed by Sanger sequencing, PCR-amplified the 47 exons and 80 base pairs of the flanking intronic sequences of CACNA1A (NC_000019.9).

The other reports of deletions in CACNA1A gene are in keeping with these find- ings, suggesting that deletions in the gene are associated with an onset of clinical manifestations before the age of 30 [45, 46].

Background Mutations in the CACNA1A gene have been found to be responsible for three dis- orders with autosomal dominant inheritance: (a) Episodic Ataxia 2 (EA2; MIM: 108500), (b) familial hemiplegic migraine type 1 (FHM1; MIM: 141500), and (c) spinocerebellar ataxia type 6 (SCA6; MIM: 183086).

2.1 Genetics

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Interesting, Pradotto and collaborators [47], described, for the first time, a com- mon mutation in CACNA1A gene in different phenotypes, such as EA2 and SCA6, opening a new prospective about the association between distinct phenotypes caused by the same CACNA1A mutation.

About the last CACNA1A associated disorder, SCA6 disease, CACNA1A caused the pathology by the expansion of CAG repeat in the α1A subunit of the voltage- dependent calcium channel gene [48].

To perform a complete analysis of CACNA1A gene, we have also investigated the expansion of the CAG repeat in the α1A subunit of CACNA1A associated to SCA6 phenotype.

Methods Generation Sequencing by TruSeq Custom Amplicon for CACNA1A, ATP1A2 SCN1A, PRRT2 genes has been performed (Illumina) according to the manufactur- ers’ instructions.

All genetic variants have been confirmed by Sanger sequencing, PCR-amplified the 47 exons and 80 base pairs of the flanking intronic sequences of CACNA1A (NC_000019.9).

Cycle conditions were 40 cycles at 95 °C for 15 s and 60 °C for 1 min according

to the Sybr Green Real Time PCR Protocol (primers are available upon request).

Analysis of relative gene expression data have been performed using real-time quantitative PCR and the 2–ΔΔCT method [49], the starting copy number of the unknown samples was determined in comparison with the known copy number of the calibrator sample, using the 2–(ΔΔCt) method.

To the already published method [50], all samples have been run by sequencer DNA analyser ABI 3031 (microsatellite analysis) and analysed by the Gene Mapper software.

Results The first patient affected by FHM showed the missense mutation E1015K, already described in literature [51] as pathogenetic.

In silico analysis with Polyphen software predicted the I1512T mutation as prob-

ably damaging with a score of 0.999.

Sporadic hemiplegic migraine: two patients showed a deletion in exons 41–43, while the rest of HM patients [52] showed a deletion in the terminal part of the CACNA1A gene (ex 47).

Episodic ataxia: in a female EA patient (and her affected son) we identified a

deletion in exon 12–15, while a second patient showed a deletion in exon 47.

Migraine: the three deleted patients showed the same deletion in exon 47, but

two different phenotypes, as MA [53] and MO [54].

Discussion We report an exhaustive screening of the CACNA1A gene in a large sample of 137 patients with four clinical phenotypes: FHM1, sporadic HM, EA2, MA and MO.

In our cohort of patients, Sanger analysis identified only three point mutations in the CACNA1A gene; the E1015K, previously described associated with different

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2 Mechanisms

migraine phenotypes [51], was identified in a patient with a clinical diagnosis of Hemiplegic migraine without family history.

Patients with EA2 and MA/MO phenotypes resulted negative to sequencing analysis, suggesting that other genes, known to be causative of FHM such as ATP1A2, SNC1A and PRRT2, could be involved and are worth to be screened.

In our cohort of patients negative to Sanger sequencing, we performed MLPA analysis of CACNA1A gene and we found different deletions in the C-terminus region of the gene.

Deletions in the CACNA1A gene have been reported in literature but associated

exclusively to an EA2 phenotype.

Conclusions The four clinical phenotypes we have included in this study (FHM1, sporadic HM, EA2, MA and MO) should be considered part of the same disease spectrum and for this group of diseases it should be kept in mind the genetic and allelic heterogeneity and also the intrafamilial/interfamilial clinical variability.

In patients with migraine phenotypes and episodic ataxia is becoming mandatory the screening of gross rearrangements of CACNA1A gene for a complete genetic workup.

Acknowledgement A machine generated summary based on the work of Grieco, G. S.; Gagliardi, S.; Ricca, I.; Pansarasa, O.; Neri, M.; Gualandi, F.; Nappi, G.; Ferlini, A.; Cereda, C. 2018 in The Journal of Headache and Pain.

Expression of PIAS Genes in Migraine Patients