Genome-wide DNA methylation analysis in an antimigraine-treated preclinical model of cortical spreading depolarization.

BACKGROUND: Cortical spreading depolarization, the cause of migraine aura, is a short-lasting depolarization wave that moves across the brain cortex, transiently suppressing neuronal activity. Prophylactic treatments for migraine, such as topiramate or valproate, reduce the number of cortical spreading depression events in rodents. OBJECTIVE: To investigate whether cortical spreading depolarization with and without chronic treatment with topiramate or valproate affect the DNA methylation of the cortex. METHODS: Sprague-Dawley rats were intraperitoneally injected with saline, topiramate or valproate for four weeks when cortical spreading depolarization were induced and genome-wide DNA methylation was performed in the cortex of six rats per group. RESULTS: The DNA methylation profile of the cortex was significantly modified after cortical spreading depolarization, with and without topiramate or valproate. Interestingly, topiramate reduced by almost 50% the number of differentially methylated regions, whereas valproate increased them by 17%, when comparing to the non-treated group after cortical spreading depolarization induction. The majority of the differentially methylated regions lay within intragenic regions, and the analyses of functional group over-representation retrieved several enriched functions, including functions related to protein processing in the cortical spreading depolarization without treatment group; functions related to metabolic processes in the cortical spreading depolarization with topiramate group; and functions related to synapse and ErbB, MAPK or retrograde endocannabinoid signaling in the cortical spreading depolarization with valproate group. CONCLUSIONS: Our results may provide insights into the underlying physiological mechanisms of migraine with aura and emphasize the role of epigenetics in migraine susceptibility.

The contribution of CACNA1A, ATP1A2 and SCN1A mutations in hemiplegic migraine: A clinical and genetic study in Finnish migraine families.

Objective To study the position of hemiplegic migraine in the clinical spectrum of migraine with aura and to reveal the importance of CACNA1A, ATP1A2 and SCN1A in the development of hemiplegic migraine in Finnish migraine families. Methods The International Classification of Headache Disorders 3rd edition criteria were used to determine clinical characteristics and occurrence of hemiplegic migraine, based on detailed questionnaires, in a Finnish migraine family collection consisting of 9087 subjects. Involvement of CACNA1A, ATP1A2 and SCN1A was studied using whole exome sequencing data from 293 patients with hemiplegic migraine. Results Overall, hemiplegic migraine patients reported clinically more severe headache and aura episodes than non-hemiplegic migraine with aura patients. We identified two mutations, c.1816G>A (p.Ala606Thr) and c.1148G>A (p.Arg383His), in ATP1A2 and one mutation, c.1994C>T (p.Thr665Met) in CACNA1A. Conclusions The results highlight hemiplegic migraine as a clinically and genetically heterogeneous disease. Hemiplegic migraine patients do not form a clearly separate group with distinct symptoms, but rather have an extreme phenotype in the migraine with aura continuum. We have shown that mutations in CACNA1A, ATP1A2 and SCN1A are not the major cause of the disease in Finnish hemiplegic migraine patients, suggesting that there are additional genetic factors contributing to the phenotype.

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

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 α1A subunit of the P/Q-type voltage-gated calcium channel Cav2.1. 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.

Meta-analysis of 375,000 individuals identifies 38 susceptibility loci for migraine.

Migraine is a debilitating neurological disorder affecting around one in seven people worldwide, but its molecular mechanisms remain poorly understood. There is some debate about whether migraine is a disease of vascular dysfunction or a result of neuronal dysfunction with secondary vascular changes. Genome-wide association (GWA) studies have thus far identified 13 independent loci associated with migraine. To identify new susceptibility loci, we carried out a genetic study of migraine on 59,674 affected subjects and 316,078 controls from 22 GWA studies. We identified 44 independent single-nucleotide polymorphisms (SNPs) significantly associated with migraine risk (P < 5 × 10(-8)) that mapped to 38 distinct genomic loci, including 28 loci not previously reported and a locus that to our knowledge is the first to be identified on chromosome X. In subsequent computational analyses, the identified loci showed enrichment for genes expressed in vascular and smooth muscle tissues, consistent with a predominant theory of migraine that highlights vascular etiologies.

GNAO1 encephalopathy: further delineation of a severe neurodevelopmental syndrome affecting females.

BACKGROUND: De novo heterozygous mutations in the GNAO1 gene, encoding the Gα o subunit of G-proteins, are the cause of a severe neurodevelopmental disorder, featuring early infantile seizures, profound cognitive dysfunction and, occasionally, movement disorder (early infantile epileptic encephalopathy-17). METHODS: We report a further case of this association in a 20 month-old Spanish girl with neonatal-onset refractory seizures, progressive microcephaly, oral-lingual dyskinesia and nearly absent psychomotor development. We performed whole-exome sequencing, a computational structural analysis of the novel gene variant identified and reviewed the previously reported cases. RESULTS: Trio whole-exome-sequencing uncovered a de novo p.Leu199Pro GNAO1 mutation. Computational structural analysis indicates this novel variant adversely affects the stability of the G-protein heterotrimeric complex as a whole. Of note, our patient showed a sustained seizure reduction while on a ketogenic diet. CONCLUSIONS: With this observation, a total of twelve patients with GNAO1 encephalopathy have been reported. Oral-lingual dyskinesia and responsiveness of seizures to ketogenic diet are novel features. The distorted sex ratio (12/12 females) of the condition remains unexplained; a differential gender effect of the disruption of G-protein- mediated signal transduction on the developing brain can be hypothesized.

Chiari malformation type I: a case-control association study of 58 developmental genes.

Chiari malformation type I (CMI) is a disorder characterized by hindbrain overcrowding into an underdeveloped posterior cranial fossa (PCF), often causing progressive neurological symptoms. The etiology of CMI remains unclear and is most likely multifactorial. A putative genetic contribution to CMI is suggested by familial aggregation and twin studies. Experimental models and human morphometric studies have suggested an underlying paraxial mesoderm insufficiency. We performed a case-control association study of 303 tag single nucleotide polymorphisms (SNP) across 58 candidate genes involved in early paraxial mesoderm development in a sample of 415 CMI patients and 524 sex-matched controls. A subgroup of patients diagnosed with classical, small-PCF CMI by means of MRI-based PCF morphometry (n = 186), underwent additional analysis. The genes selected are involved in signalling gradients occurring during segmental patterning of the occipital somites (FGF8, Wnt, and retinoic acid pathways and from bone morphogenetic proteins or BMP, Notch, Cdx and Hox pathways) or in placental angiogenesis, sclerotome development or CMI-associated syndromes. Single-marker analysis identified nominal associations with 18 SNPs in 14 genes (CDX1, FLT1, RARG, NKD2, MSGN1, RBPJ1, FGFR1, RDH10, NOG, RARA, LFNG, KDR, ALDH1A2, BMPR1A) considering the whole CMI sample. None of these overcame corrections for multiple comparisons, in contrast with four SNPs in CDX1, FLT1 and ALDH1A2 in the classical CMI group. Multiple marker analysis identified a risk haplotype for classical CMI in ALDH1A2 and CDX1. Furthermore, we analyzed the possible contributions of the most significantly associated SNPs to different PCF morphometric traits. These findings suggest that common variants in genes involved in somitogenesis and fetal vascular development may confer susceptibility to CMI.

Paroxysmal non-kinesigenic dyskinesia due to a PNKD recurrent mutation: report of two Southern European families.

Paroxysmal non-kinesigenic dyskinesia (PNKD) is an autosomal dominant disorder characterized by attacks of dystonic or choreathetotic movements precipitated by stress, fatigue, coffee, alcohol or menstruation. In this report we present two families with PNKD of Southern European origin carrying a PNKD recurrent mutation. Incomplete penetrance and intrafamilial variability was detected in both families. Treatment with valproic acid and levetiracetam provided favorable response.

Paroxysmal exercise-induced dyskinesia, writer's cramp, migraine with aura and absence epilepsy in twin brothers with a novel SLC2A1 missense mutation.

We report two monochorionic twins that progressively developed, between ages 5 and 10, a combination of episodic neurological disorders including paroxysmal exercise-induced dyskinesia, migraine without or with aura, absence seizures and writer's cramp. CSF/serum glucose ratio was moderately decreased in both patients. Mutational analysis of SLC2A1 gene identified a de novo heterozygous missense mutation in exon 4. This novel mutation has been previously showed to disrupt glucose transport in vitro. Both patients showed immediate and near-complete response to ketogenic diet. This clinical observation suggests that a high index of suspicion for GLUT1 deficiency syndrome is warranted in evaluating patients with multiple neurological paroxysmal events.

A mutation in the first intracellular loop of CACNA1A prevents P/Q channel modulation by SNARE proteins and lowers exocytosis.

Familial hemiplegic migraine (FHM)-causing mutations in the gene encoding the P/Q Ca(2+) channel alpha(1A) subunit (CACNA1A) locate to the pore and voltage sensor regions and normally involve gain-of-channel function. We now report on a mutation identified in the first intracellular loop of CACNA1A (alpha(1A(A454T))) that does not cause FHM but is associated with the absence of sensorimotor symptoms in a migraine with aura pedigree. Alpha(1A(A454T)) channels showed weakened regulation of voltage-dependent steady-state inactivation by Ca(V)beta subunits. More interestingly, A454T mutation suppressed P/Q channel modulation by syntaxin 1A or SNAP-25 and decreased exocytosis. Our findings reveal the importance of I-II loop structural integrity in the functional interaction between P/Q channel and proteins of the vesicle-docking/fusion machinery, and that genetic variation in CACNA1A may be not only a cause but also a modifier of migraine phenotype.

Two-stage case-control association study of dopamine-related genes and migraine.

BACKGROUND: We previously reported risk haplotypes for two genes related with serotonin and dopamine metabolism: MAOA in migraine without aura and DDC in migraine with aura. Herein we investigate the contribution to migraine susceptibility of eight additional genes involved in dopamine neurotransmission. METHODS: We performed a two-stage case-control association study of 50 tag single nucleotide polymorphisms (SNPs), selected according to genetic coverage parameters. The first analysis consisted of 263 patients and 274 controls and the replication study was composed by 259 cases and 287 controls. All cases were diagnosed according to ICHD-II criteria, were Spanish Caucasian, and were sex-matched with control subjects. RESULTS: Single-marker analysis of the first population identified nominal associations of five genes with migraine. After applying a false discovery rate correction of 10%, the differences remained significant only for DRD2 (rs2283265) and TH (rs2070762). Multiple-marker analysis identified a five-marker T-C-G-C-G (rs12363125-rs2283265-rs2242592-rs1554929-rs2234689) risk haplotype in DRD2 and a two-marker A-C (rs6356-rs2070762) risk haplotype in TH that remained significant after correction by permutations. These results, however, were not replicated in the second independent cohort. CONCLUSION: The present study does not support the involvement of the DRD1, DRD2, DRD3, DRD5, DBH, COMT, SLC6A3 and TH genes in the genetic predisposition to migraine in the Spanish population.

Contribution of syntaxin 1A to the genetic susceptibility to migraine: a case-control association study in the Spanish population.

Migraine is a common neurological disorder with a complex inheritance pattern. Mutations in genes encoding proteins that are involved in ion transport across the neuronal membrane have been linked to rare monogenic variants of migraine. These or other related genes and proteins are also candidates to be involved in the inherited predisposition to the more common forms of migraine without aura (MO) or migraine with aura (MA). One of these proteins, syntaxin 1A, encoded by the STX1A gene, is a key molecule in ion channel regulation and synaptic exocytosis. We assessed the contribution of STX1A to migraine by analyzing three SNPs that cover the entire gene (rs6951030-rs941298-rs4363087), in a case-control association study in 210 migraine patients (102 MO, 86 MA, 22 hemiplegic migraine) and 210 sex-matched unrelated controls. The single-marker analysis revealed significant differences in both allele frequencies (P=0.0087, OR=1.48) and genotype distributions (P=0.0133) of the rs941298 SNP between migraineurs and controls, with an overrepresentation of T-allele carriers in the migraine sample (OR=1.78). We subsequently performed a haplotype-based analysis and observed evidence of an overrepresentation of the A-T-G (rs6951030-rs941298-rs4363087) allelic combination in migraine patients and an increased frequency of carriers of this risk haplotype (P=0.008, OR=1.71). These differences remained significant when patients were subdivided into MO and MA. When the control series was enlarged for rs941298, we confirmed the association only with the whole migraine group.

Late-onset episodic ataxia type 2 associated with a novel loss-of-function mutation in the CACNA1A gene.

We report a patient with typical features of episodic ataxia type 2 (EA2) but with onset in the sixth decade and associated interictal hand dystonia. He was found to bear the novel heterozygous missense mutation p.Gly638Asp (c.1913G>A) in the CACNA1A gene. Functional analysis of the mutation on P/Q channels expressed in HEK 293 cells revealed a reduction of Ca(2+) current densities, a left-shift in the apparent reversal potential, the slowing of inactivation kinetics and the increase in the rate of current recovery from inactivation. These results are consistent with a decrease in Ca(2+) permeability through mutant P/Q channels. To our knowledge, this is just the second patient with late onset EA2 linked to a CACNA1A mutation and the first to carry a loss-of-function missense mutation.

Familial hemiplegic migraine: linkage to chromosome 14q32 in a Spanish kindred.

We sought to map the disease-causing gene in a large Spanish kindred with familial hemiplegic migraine (FHM). Patients were classified according to the ICHD-II criteria. After ruling out linkage to known migraine genetic loci, a single nucleotide polymorphism-based, 0.62-cM density genome-wide scan was performed. Among 13 affected subjects, FHM was the prevailing migraine phenotype in six, migraine with aura in four and migraine without aura in three. Linkage analysis revealed a disease locus in a 4.15-Mb region on 14q32 with a maximum two-point logarithm of odds (LOD) score of 3.1 and a multipoint parametric LOD score of 3.8. This genomic region does not overlap with the reported migraine loci on 14q21-22. Sequence analysis of three candidate genes in the region, SLC24A4, ATXN3 and ITPK1, failed to show disease-causing mutations in our patients. Genetic heterogeneity in FHM may be greater than previously suspected.