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 Ca2+ channel α1A 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 (α1A(A454T)) that does not cause FHM but is associated with the absence of sensorimotor symptoms in a migraine with aura pedigree. α1A(A454T) channels showed weakened regulation of voltage-dependent steady-state inactivation by CaVβ subunits. More interestingy, 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

Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic and functional studies

Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A-p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2-p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing

Anàlisi genètica i funcional de la migranya hemiplègica i la migranya comuna

[cat] Aquesta tesi es centra en la genètica de la migranya. La migranya comuna és un trastorn neurològic caracteritzat per episodis recurrents de mal de cap. Els criteris de la IHS (International Headache Society) subclasifiquen la malaltia en migranya amb aura (MA) i migranya sense aura (MO). L'aura són símptomes neurològics transitoris que poden acompanyar el mal de cap. Les aures més freqüents són les aures visuals, tot i que també existeixen les aures sensorials essent l'aura hemiplègica la seva forma severa. La nostra investigació es va dividir en dues areas d'acord amb la base genètica dels trastorns, d'una banda, s'ha estudiat la genètica complexa de la migranya comuna, d'altra banda s'ha estudiat una forma rara de la migranya que presenta una herència mendeliana anomenada migranya hemiplègica familiar (FHM). Per entendre més la base genètica de la migranya comuna es va utilitzar un estudi d'associació tipus cas-control amb gens candidats. Amb aquesta finalitat es van seleccionar al voltant de 550 pacients amb migranya (MA i MO) i el seu corresponent grup de control. Per tal d'analitzar la seva implicació en la susceptibilitat genètica a la migranya, es van triar gens que codifiquen per als canals de la superfamília heterogeni de potencial receptor transitori (Transient Receptor Potential- TRP) que se sap que estan implicats en les vies nociceptives. Aquesta feina ha donat lloc a una publicació (Carreño et al. SNP variants within the vanilloid TRPV1 and TRPV3 receptor genes are associated with migraine in the Spanish population. Am J Med Genet B Neuropsychiatr Genet. 2012). En el cas particular de les formes monogèniques de FHM es coneixen tres gens involucrats en la malatia (CACNA1A, ATP1A2 i SCN1A), les proteïnes codificades per aquests gens tenen un paper rellevant en la neurotransmissió del glutamat. L'anàlisi funcional de les mutacions que causen FHM han mostrat en última instància un augment de l'alliberament de la neurotransmissió. En el cas de mutacions al CACNA1A s'ha vist un efecte de guany de funció, a diferència de les mutacions al ATP1A2 que presenten un efecte de pèrdua de funció. En aquest treball s'ha fet un screening mutacional per identificar mutacions en pacients per seqüenciació directa. Quan les mutacions eren suficientment interessants s'han generat construccions en vectors d'expressió per subseqüents estudis funcionals en cèl·lules eucariotes. Aquesta feina ha donat lloc a tres publicacions. A la primera (Serra et al. A mutation in the first intracellular loop of CACNA1A prevents P/Q channel modulation by SNARE proteins and lowers exocytosis. Proc Natl Acad Sci. 2010) es va identificar un canvi que modula la funció del canal de CACNA1A. Aquest estudi ajuda a explicar la contribució genètica en la heterogeneïtat clínica d'una família i a entendre millor el mecanisme molecular dels canals de calci tipus P/Q. El segon (Carreño et al. Acute striatal necrosis in hemiplegic migraine with the novo CACNA1A mutation. Headache. 2011) és un informe d'un pacient que presenta una necrosi aguda stratial. Té una rellevància clínica a causa de l'aparició primerenca dels símptomes neurològics previs als atacs hemiplègics. El tercer i últim treball (Carreño et al. Screening of the ATP1A2 and CACNA1A genes in patients with hemiplegic migraine: clinical, genetic and functional studies. [work in progress]) recull l'screening mutacional al gens ATP1A2 i CACNA1A en 19 pacients amb FHM. Es van identificar 5 mutacions prèviament descrites i dues mutacions noves.[eng] This Thesis is focused in migraine genetics, migraine is a prevalent neurological disorder characterized by recurrent episodes of headache. This research was divided in two areas according to the genetic basis of the disorders; on the one hand we studied the common migraine with a complex genetics, on the other hand we studied the rare mendelian forms of familial hemiplegic migraine (FHM). To understand more the genetic basis of the common migraine a case-control association study approach was used with candidate genes. For that purpose, around 550 patients with migraine and their corresponding control group were selected. In order to analyze their involvement in the genetic susceptibility to migraine, we chose genes encoding for channels of the heterogeneous superfamily of Transient Receptor Potential (TRP) which are known to be involved in the nociceptive pathway. In the particular case of FHM, a monogenic form of the disorder, there are three genes known to be involved in the FHM (CACNA1A, ATP1A2 and SCN1A), whose encoded proteins are playing a relevant role in the neurotransmission of the glutamate. Functional analysis of the mutations causing FHM have shown ultimately an increased neurotransmission release. CACNA1A previous studies reveled a gain-of-function effect from FHM mutations, unlike mutations on ATP1A2 that present a loss-of-function effect. Our work consisted on identifying mutations in patients by direct sequencing. If the mutations were interesting enough vector constructions were generated for functional studies in eukaryotic cells. This work gave rise to three publications: First; the identification of a change that modulates the function of the CACNA1A channel. This study contributes to explain the genetic contribution in the clinical heterogeneity of one family and to know more about the molecular mechanism of the P/Q calcium channel. Second; a report of a patient that presents an acute stratial necrosis that had clinical relevance because of the early onset of the neurological symptoms previous to the hemiplegic attacks. Third; a mutational screening of ATP1A2 and CACNA1A genes in 19 patients with FHM. 5 previously described mutations and two new mutations were found. Functional studies were carried out for the newly mutations

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.The funding for this study was provided by the Spanish Ministerio de Economía y Competitividad (SAF2009-13182-C01, SAF2009-13182-C03), AGAUR (2014SGR-0932, 2009SGR-0078) and Fundació La Marató de TV3 (grant 100731). These institutions had no further role in study design, collection, analysis, interpretation of data or in the submission of this paper for publication. CS and OC were supported by Ministerio de Economía y Competitividad (BES-2007-16450 and BES-2010-033895, respectively) and NF-C by Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER, ISCIII). MV-P was supported by a predoctoral grant from VHIR, Barcelona (Spain). CT was supported by the European Union (Marie Curie, PIEF-GA-2009-254930). EC-L is the recipient of the Beatriu de Pinós programme scholarship (BP-DGR 2010)

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

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

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

Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine : clinical, genetic, and functional studies

Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A -p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2 -p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing

Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic, and functional studies

Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A-p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2-p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing.The work was funded by the Spanish Ministry of Science and Innovation, the Spanish Ministry of Economy and Competitiveness,Fondos Europeos de Desarrollo Regional (FEDER) and Plan E (Grants SAF2012 -31089, SAF2012-3814 0, BES-2010-033895, SAF2009-13182-C03-01,/nSAF2009-13182-C03-02, and SAF2009-13182-C03-03), Fondo de Investigación Sanitaria (Cardiovascular Disease Network RD12/0042/0014) and Generalitat de Catalunya (grants 2009SGR0971, 2009SGR0078 and 2009SGR1369). M. A. V. and N. F.-C. are the recipients of an ICREA Academia Award (Generalitat de Catalunya) and a grant from “CIBER-ER,” respectively. C. T. was supported by the European Union (Marie Curie, PIEF-GA-2009-254930)