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

ADN forense: problemas éticos y jurídicos

Coordinació: María Casado y Margarita GuillénLos análisis genéticos afectan a derechos fundamentales, por lo que el uso de esta información debe estar supeditado a vías de control democrático. En la actualidad, la lucha contra grandes delitos, como por ejemplo el terrorismo, aparentemente legitima a invadir derechos antes considerados intangibles. La presente obra, fruto del trabajo multidisciplinar llevado a cabo por juristas, filósofos, biólogos, técnicos y médicos, tiene por objetivo poner de manifiesto cuáles son los problemas ético-jurídicos derivados de la obtención, el análisis y el almacenamiento del ADN, así como sus usos judiciales y extrajudiciales. Ante el difícil equilibrio entre libertad individual y seguridad colectiva, este libro ayuda a comprender los conflictos que subyacen en el manejo de una herramienta informativa tan poderosa como son las muestras y los perfiles del ADN

Comportament funcional heterogeni de les mutacions "missense" a l'extrem N-terminal de CFTR

[cat] La Fibrosi Quística (FQ; MIM# 219700) és una de les malalties letals més comuns en les poblacions d'origen caucàsic, amb herència autosòmica recessiva. El seu fenotip clínic es caracteritza per l'acumulació de moc viscós en òrgans exocrins, amb conseqüències patològiques en el tracte respiratori, gastrointestinal i urogenital. Mutacions al gen CFTR (cystic fibrosis transmembrane conductance regulator ABCC7; MIM# 602421) són responsables de la malaltia. Fins al moment, ja s'han descrit més de 1600 mutacions en el gen CFTR. La gran majoria són mutacions puntuals de les quals: 42% són mutacions de sentit erroni (missense). El gen CFTR codifica per una proteïna de membrana de 1480 aminoàcids, que pertany a la gran família dels transportadors ABC. La proteïna CFTR presenta una estructura simètrica, formada per dos motius repetits, cadascun d'ells format per una regió transmembrana (TMD), composada per sis α-hèlix, i una regió citosòlica hidrofílica d'unió a ATP (NBD). Aquests dos motius queden units mitjançant un domini regulador (R), citosòlic i hidrofílic, que és fosforila per PKA i PKC. El paper principal que desenvolupa el canal CFTR en la part apical de les cèl·lules epitelials és el transport de fluid transepitelial i electròlits a través de la membrana, activitat que ve regulada directament per la fosforilació de PKA. Aquest canal permet el trànsit d'ions de clorur a través d'ell en qualsevol direcció, i es caracteritza per tenir una baixa conductància unitària (la seva conductància és de 7-10 pS). Existeixen moltes evidències en la literatura on les mutacions de sentit erroni, independentment de la seva localització en el gen CFTR, poden afectar a la funció del canal CFTR a diferents nivells: biogènesi de la proteïna, transport i localització, propietats electrofisiològiques del canal. Així que l'objectiu general de la tesi va ser l'anàlisi molecular i funcional de vuit mutacions amb sentit erroni (p.P5L, p.S50P, p.E60K, p.R75Q, p.G85E, p.G85V, p.Y89C, p.E92K) localitzades en la regió N-terminal de CFTR, i identificades majoritàriament a la població espanyola. Per portar a terme els objectius de la tesi, es van generar les vuit mutacions per mutagènesi dirigida en el vector pCMVCFTRNot6.2wt, i es van expressar en cèl·lules epitelials HEK293. Es va analitzar el processament i patró de maduració de les proteïnes mutants per Western blot i la seva localització cel·lular per immunocitoquímica. I posteriorment, es van caracteritzar funcionalment els canals mutants mitjançant la tècnica de Patch clamp en configuració whole cell i excised inside-out. Vam observar que cinc de les mutacions analitzades, p.S50P, p.E60K, p.G85E, p.G85V i p.E92K, provoquen un processament i una maduració incorrecte de la proteïna. Totes cinc presenten un comportament molt similar a la mutació p.F508del, podent-se classificar aquestes mutacions dins la classe II. La mutació p.P5L afecta a la funció de CFTR a dos nivells: a nivell de la biogènesi i a nivell de l'activitat del canal. Aquesta mutació provoca una disminució de la densitat de canals a la membrana citoplasmàtica. La conductància i la cinètica del canal també s'alteren. La proteïna mutant p.Y89C afecta a l'activitat del canal. Aquesta mutació altera específicament la cinètica del canal. El canal CFTR corresponent a la mutació p.R75Q presenta un patró de maduració i un comportament funcional del canal anàleg a la proteïna CFTR no mutada. Els nostres estudis corroboren la classificació de p.R75Q com a mutació sense expressió clínica. El comportament sever de la majoria de les mutacions avaluades, afectant la biogènesi i/o l'activitat del canal, ressalta la importància funcional de la cua N-terminal i del segment TM1 en la fisiologia de CFTR.[eng] Over 1,600 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure-function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology

Cross talk between β subunits, intracellular Ca2+ signaling, and SNAREs in the modulation of CaV 2.1 channel steady-state inactivation

Modulation of CaV 2.1 channel activity plays a key role in interneuronal communication and synaptic plasticity. SNAREs interact with a specific synprint site at the second intracellular loop (LII-III) of the CaV 2.1 pore-forming α1A subunit to optimize neurotransmitter release from presynaptic terminals by allowing secretory vesicles docking near the Ca2+ entry pathway, and by modulating the voltage dependence of channel steady-state inactivation. Ca2+ influx through CaV 2.1 also promotes channel inactivation. This process seems to involve Ca2+ -calmodulin interaction with two adjacent sites in the α1A carboxyl tail (C-tail) (the IQ-like motif and the Calmodulin-Binding Domain (CBD) site), and contributes to long-term potentiation and spatial learning and memory. Besides, binding of regulatory β subunits to the α interaction domain (AID) at the first intracellular loop (LI-II) of α1A determines the degree of channel inactivation by both voltage and Ca2+ . Here, we explore the cross talk between β subunits, Ca2+ , and syntaxin-1A-modulated CaV 2.1 inactivation, highlighting the α1A domains involved in such process. β3 -containing CaV 2.1 channels show syntaxin-1A-modulated but no Ca2+ -dependent steady-state inactivation. Conversely, β2a -containing CaV 2.1 channels show Ca2+ -dependent but not syntaxin-1A-modulated steady-state inactivation. A LI-II deletion confers Ca2+ -dependent inactivation and prevents modulation by syntaxin-1A in β3 -containing CaV 2.1 channels. Mutation of the IQ-like motif, unlike CBD deletion, abolishes Ca2+ -dependent inactivation and confers modulation by syntaxin-1A in β2a -containing CaV 2.1 channels. Altogether, these results suggest that LI-II structural modifications determine the regulation of CaV 2.1 steady-state inactivation either by Ca2+ or by SNAREs but not by both.This work was supported by the Spanish Ministry of Economy and Competitiveness (Grants SAF2012‐31089 and SAF2015‐69762‐R to JMF‐F, and Grant MDM‐2014‐0370 through the “María de Maeztu” Programme for Units of Excellence in R&D to “Departament de Ciències Experimentals i de la Salut”), and FEDER Funds

Rare variants in calcium homeostasis modulator 1 (CALHM1) found in early onset alzheimer's disease patients alter calcium homeostasis

Calcium signaling in the brain is fundamental to the learning and memory process and there is evidence to suggest that its dysfunction is involved in the pathological pathways underlying Alzheimer’s disease (AD). Recently, the calcium hypothesis of AD has received support with the identification of the non-selective Ca2+-permeable channel CALHM1. A genetic polymorphism (p. P86L) in CALHM1 reduces plasma membrane Ca2+ permeability and is associated with an earlier age-at-onset of AD. To investigate the role of CALHM1 variants in early-onset AD (EOAD), we sequenced all CALHM1 coding regions in three independent series comprising 284 EOAD patients and 326 controls. Two missense mutations in patients (p.G330D and p.R154H) and one (p.A213T) in a control individual were identified. Calcium imaging analyses revealed that while the mutation found in a control (p.A213T) behaved as wild-type CALHM1 (CALHM1-WT), a complete abolishment of the Ca2+ influx was associated with the mutations found in EOAD patients (p.G330D and p.R154H). Notably, the previously reported p. P86L mutation was associated with an intermediate Ca2+ influx between the CALHM1-WT and the p.G330D and p.R154H mutations. Since neither expression of wild-type nor mutant CALHM1 affected amyloid ß-peptide (Aß) production or Aß-mediated cellular toxicity, we conclude that rare genetic variants in CALHM1 lead to Ca2+ dysregulation and may contribute to the risk of EOAD through a mechanism independent from the classical Aß cascade.This study was supported by grants from Instituto de Salud Carlos III (PI12/01311, PI10/000587, Red HERACLES RD12/0042/0014), Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, Spain), Spanish Ministry of Economy and Competiveness (SAF2012-38140), FEDER Funds, and Generalitat de Catalunya (SGR05-266). Council of the Academy of Finland, EVO grant 5772708 of Kuopio University Hospital, the Strategic Funding of the University on Eastern Finland (UEF-Brain) (to M.H and H.S). M.A.V. is the recipient of an ICREA Academia Awar

Défis contemporains de la parenté

Pourquoi la notion de parenté que l'on croyait aussi intangible qu'intemporelle est-elle aujourd'hui soumise aux plus profonds bouleversements ? Quels changements ont affecté la parenté ces dernières décennies ? Les auteurs, issus d'horizons géographiques et disciplinaires variés, confrontent positions et analyses autour de trois thématiques : les cadres et enjeux juridiques des nouvelles parentés, la parenté à l'âge des biotechnologies et des adoptions internationales, les changements de perception du corps et de la personne. Ce livre nous entraîne au cœur des controverses soulevées par les nouvelles techniques de procréation (familles mono- et homo-parentales, fécondation in vitro, dons de gamètes, etc.). Il témoigne de l'évolution profonde de la parenté, tant au niveau des savoirs et des techniques que des représentations et des valeurs communes. La filiation ne va plus de soi... La diversité et la profondeur de l'étude, marquée par une approche anthropologique, rendent cet ouvrage incontournable pour la compréhension des nouveaux défis de la parenté, en pleine (r-)évolution

ADN forense: problemas éticos y jurídicos

Los análisis genéticos afectan a derechos fundamentales, por lo que el uso de esta información debe estar supeditado a vías de control democrático. En la actualidad, la lucha contra grandes delitos, como por ejemplo el terrorismo, aparentemente legitima a invadir derechos antes considerados intangibles. La presente obra, fruto del trabajo multidisciplinar llevado a cabo por juristas, filósofos, biólogos, técnicos y médicos, tiene por objetivo poner de manifiesto cuáles son los problemas ético-jurídicos derivados de la obtención, el análisis y el almacenamiento del ADN, así como sus usos judiciales y extrajudiciales. Ante el difícil equilibrio entre libertad individual y seguridad colectiva, este libro ayuda a comprender los conflictos que subyacen en el manejo de una herramienta informativa tan poderosa como son las muestras y los perfiles del ADN

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)

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