Functional correlation of ATP1A2 mutations with phenotypic spectrum: from pure hemiplegic migraine to its variant forms

Abstract Background Mutations in ATP1A2, the gene encoding the α2 subunit of Na+/K+-ATPase, are the main cause of familial hemiplegic migraine type 2 (FHM2). The clinical presentation of FHM2 with mutations in the same gene varies from pure FHM to severe forms with epilepsy and intellectual disability, but the correlation of these symptoms with different ATP1A2 mutations is still unclear. Methods Ten ATP1A2 missense mutations were selected according to different phenotypes of FHM patients. They caused pure FHM (FHM: R65W, R202Q, R593W, G762S), FHM with epilepsy (FHME: R548C, E825K, R938P), or FHM with epilepsy and intellectual disability (FHMEI: T378N, G615R, D718N). After ouabain resistance and fluorescence modification, plasmids carrying those mutations were transiently transfected into HEK293T and HeLa cells. The biochemical functions were studied including cell survival assays, membrane protein extraction, western blotting, and Na+/K+-ATPase activity tests. The electrophysiological functions of G762S, R938P, and G615R mutations were investigated in HEK293T cells using whole-cell patch-clamp. Homology modeling was performed to determine the locational distribution of ATP1A2 mutations. Results Compared with wild-type pumps, all mutations showed a similar level of protein expression and decreased cell viability in the presence of 1 µM ouabain, and there was no significant difference among the mutant groups. The changes in Na+/K+-ATPase activity were correlated with the severity of FHM phenotypes. In the presence of 100 µM ouabain, the Na+/K+-ATPase activity was FHM > FHME > FHMEI. The ouabain-sensitive Na+/K+-ATPase activity of each mutant was significantly lower than that of the wild-type protein, and there was no significant difference among all mutant groups. Whole-cell voltage-clamp recordings in HEK293T cells showed that the ouabain-sensitive pump currents of G615R were significantly reduced, while those of G762S and R938P were comparable to those of the wild-type strain. Conclusions ATP1A2 mutations cause phenotypes ranging from pure FHM to FHM with epilepsy and intellectual disability due to varying degrees of deficits in biochemical and electrophysiological properties of Na+/K+-ATPase. Mutations associated with intellectual disability presented with severe impairment of Na+/K+-ATPase. Whether epilepsy is accompanied, or the type of epilepsy did not seem to affect the degree of impairment of pump function.

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RNA expression profiling in brains of familial hemiplegic migraine type 1 knock-in mice

Cephalalgia ◽

10.1177/0333102413502736 ◽

2013 ◽

Vol 34 (3) ◽

pp. 174-182 ◽

Cited By ~ 8

Author(s):

Boukje de Vries ◽

Else Eising ◽

Ludo AM Broos ◽

Stephany C Koelewijn ◽

Boyan Todorov ◽

...

Keyword(s):

Cerebellar Ataxia ◽

Expression Profiles ◽

Quantitative Polymerase Chain Reaction ◽

Brain Regions ◽

Familial Hemiplegic Migraine ◽

Rna Expression ◽

Missense Mutations ◽

Wild Type ◽

Hemiplegic Migraine

Background Various CACNA1A missense mutations cause familial hemiplegic migraine type 1 (FHM1), a rare monogenic subtype of migraine with aura. FHM1 mutation R192Q is associated with pure hemiplegic migraine, whereas the S218L mutation causes hemiplegic migraine, cerebellar ataxia, seizures, and mild head trauma-induced brain edema. Transgenic knock-in (KI) migraine mouse models were generated that carried either the FHM1 R192Q or the S218L mutation and were shown to exhibit increased CaV2.1 channel activity. Here we investigated their cerebellar and caudal cortical transcriptome. Methods Caudal cortical and cerebellar RNA expression profiles from mutant and wild-type mice were studied using microarrays. Respective brain regions were selected based on their relevance to migraine aura and ataxia. Relevant expression changes were further investigated at RNA and protein level by quantitative polymerase chain reaction (qPCR) and/or immunohistochemistry, respectively. Results Expression differences in the cerebellum were most pronounced in S218L mice. Particularly, tyrosine hydroxylase, a marker of delayed cerebellar maturation, appeared strongly upregulated in S218L cerebella. In contrast, only minimal expression differences were observed in the caudal cortex of either mutant mice strain. Conclusion Despite pronounced consequences of migraine gene mutations at the neurobiological level, changes in cortical RNA expression in FHM1 migraine mice compared to wild-type are modest. In contrast, pronounced RNA expression changes are seen in the cerebellum of S218L mice and may explain their cerebellar ataxia phenotype.

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Immunohistochemical characterization of calcitonin gene-related peptide in the trigeminal system of the familial hemiplegic migraine 1 knock-in mouse

Cephalalgia ◽

10.1177/0333102411418847 ◽

2011 ◽

Vol 31 (13) ◽

pp. 1368-1380 ◽

Cited By ~ 26

Author(s):

Rammya Mathew ◽

Anna P Andreou ◽

Linda Chami ◽

Astrid Bergerot ◽

Arn MJM van den Maagdenberg ◽

...

Keyword(s):

Familial Hemiplegic Migraine ◽

Calcitonin Gene Related Peptide ◽

Control Group ◽

Cell Diameter ◽

Trigeminal Ganglia ◽

Wild Type ◽

Hemiplegic Migraine ◽

Related Peptide ◽

Calcitonin Gene ◽

Significant Difference

Introduction: Familial hemiplegic migraine type 1 (FHM-1) is caused by mutations in the CACNA1A gene, with the R192Q mutation being the most common. Elevated calcitonin gene-related peptide (CGRP) levels in acute migraine and clinical trials using CGRP receptor antagonists suggest CGRP-related mechanisms are important in migraine. Methods: Wild-type and R192Q knock-in mice were anaesthetized and perfused. Using immunohistochemical staining, the expression of CGRP in the trigeminocervical complex (TCC) and in the trigeminal and dorsal root ganglia was characterized. Results: There was a 38% reduction in the percentage of CGRP-immunoreactive cells in the trigeminal ganglia ( p < 0.001) of R192Q knock-in mice compared to wild-type animals. The size distribution profile of CGRP-immunoreactive cells within the trigeminal ganglia demonstrated no significant difference in cell diameter between the two groups ( p ≥ 0.56). CGRP expression was also reduced in thoracic ganglia of R192Q knock-in mice (21% vs. 27% in wild-type group; p < 0.05), but not in other ganglia. In addition, decreased CGRP immunoreactivity was observed in the superficial laminae of the TCC in R192Q knock-in mice, when compared to the control group ( p < 0.005). Conclusion: The data demonstrates that the FHM-1 CACNA1A mutation alters CGRP expression in the trigeminal ganglion and TCC. This suggests further study of these animals is warranted to characterize better the role of these mutations in the neurobiology of migraine.

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The familial hemiplegic migraine type 1 mutation K1336E affects direct G protein-mediated regulation of neuronal P/Q-type Ca2+ channels

Cephalalgia ◽

10.1177/0333102412475236 ◽

2013 ◽

Vol 33 (6) ◽

pp. 398-407 ◽

Cited By ~ 8

Author(s):

Edgar Garza-López ◽

Ricardo González-Ramírez ◽

María A Gandini ◽

Alejandro Sandoval ◽

Ricardo Felix

Keyword(s):

G Protein ◽

Familial Hemiplegic Migraine ◽

Inactivation Kinetics ◽

Missense Mutations ◽

Hemiplegic Migraine ◽

Gene Encoding ◽

Dominant Form ◽

Hek 293 ◽

Autosomal Dominant Form

Background Familial hemiplegic migraine type 1 (FHM-1) is an autosomal dominant form of migraine with aura characterized by recurrent migraine, hemiparesis and ataxia. FHM-1 has been linked to missense mutations in the CACNA1A gene encoding the pore-forming subunit of the neuronal voltage-gated P/Q-type Ca2+ channel (CaV2.1α1). Methods Here, we explored the effects of the FHM-1 K1336E mutation on G protein-dependent modulation of the recombinant P/Q-type channel. The mutation was introduced into the human CaV2.1α1 subunit and its functional consequences investigated after heterologous expression in HEK-293 cells using patch-clamp recordings. Results Functional analysis of the K1336E mutation revealed a reduction of Ca2+ current densities, a ∼10 mV left-shift in the current-voltage relationship, and the slowing of current inactivation kinetics. When co-expressed along with the human μ-opioid receptor, application of the agonist DAMGO inhibited whole-cell currents through both the wild-type and the mutant channels. Prepulse facilitation was also reduced by the K1336E mutation. Likewise, the kinetic analysis of the onset and decay of facilitation showed that the mutation affects the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex. Conclusions These results suggest that the extent of G-protein-mediated inhibition is significantly reduced in the K1336E mutant CaV2.1 Ca2+ channels. This alteration would contribute to render the neuronal network hyperexcitable, possibly as a consequence of reduced presynaptic inhibition, and may help to explain some aspects of the FHM-1 pathophysiology.

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Distinct Clinical and Biological Implications of Various DNTMT3A Mutations in Myeloid Neoplasms

Blood ◽

10.1182/blood.v128.22.2872.2872 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2872-2872

Author(s):

Suresh Kumar Balasubramanian ◽

Mai Ali ◽

Taha Bat ◽

Bhumika Patel ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Clinical Features ◽

Bone Marrow Failure ◽

Missense Mutations ◽

Negative Consequences ◽

Wild Type ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

Significant Difference

Abstract DNMT3A, a member of the DNA methyltransferases family along with DNMT1 and DNMT3B, is located on chromosome 2p23. Recurrent somatic mutations in DNMT3A are typically heterozygous and found mostly in non-CBF AML, less frequently in MDS and MPN. DNMT3A mutations are reported with other common myeloid mutations including NPM1, FLT3 and IDH1/2. The most canonical DNMT3A mutations are missense alteration in the R882 codon, accounting for >60% of all DNMT3A mutations and they imply dominant negative consequences. Overall, DNMT3A mutations carry a poor prognosis compared to the AML or MDS with wild type (WT) DNMT3A, although data within different subgroups (e.g., incorporating cytogenetic profiles) are conflicting. We hypothesized that molecular consequence of R882 mutations will differ from those of other somatic alterations of DNMT3A and may also result in distinct clinical features and outcomes. To test this theory, we analyzed a cohort of 1174 patients with myeloid neoplasias including 32% AML, 33% MDS, 13% MDS/MPN, 6% MPN and 16% other bone marrow failure disorders. These cases were subjected to multiamplicon targeted deep NGS including all ORFs of DNMT3A and other recurrently mutated genes. After application of various bioanalytic algorithms, confirmatory sequencing and thus stringent exclusion of all artifacts and germline alterations, we identified 140 somatic mutant cases (12% of the cohort), including 89 missense mutations (53 at R882, 19 at R693 and 17 other non-canonical missense alterations) and 51 truncations/frame shifts (all heterozygous). There was an age-related increase in the incidence of DNMT3A mutations, with the peak occurrence at 35-40 yrs. of age. Mutations in DNMT3A were most common in AML (54% in primary (p) AML, 8% in secondary (s) AML) followed by MDS (28%), MDS/MPN (4%), MPN (3%) and other bone marrow failure disorders (3%). Mutation in the R693 codon and truncating mutations were most commonly associated with MDS (p=.013) and sAML (p=.0013) whereas mutation occurring in codon R882 and other non-canonical missense mutations were frequently associated with pAML (p=.00001). For the whole cohort, DNMT3A mutations were most frequently associated with NPM1 (21% vs 8%, p=.014), FLT3 (24% vs. 2%, p=.0001), and IDH1/2 (26% vs. 8%, p=.001), compared to wild type DNMT3A. However, PRC2 complex mutations were less likely to occur in the context of DNMT3A mutations (6% vs. 24%, p=.0006). Canonical R882 mutation was commonly associated with FLT3 (p=.03) mutations, while truncating mutations were not (p=.03). Analyses of clonal hierarchy by ranking of VAF values demonstrated that 53% of DNMT3A mutations were dominant (mean VAF 39%, range 5-93%) (n=74/140). When DNMT3A mutations were dominant, IDH 1/2 (14%), TET2 (9%), ASXL (5%), PRC2 complex (3%) and BCOR (3%) mutations were common secondary events. In subgroup analyses, 55% of mutations in the R693 codon were dominant compared to 45% in R882 and 47% in truncating mutations. TET2 mutations were the most common associated secondary hits in dominant R693 mutations (n=10) compared to truncating (n=24) and R882 mutations (n=23) (40% vs. 8% vs. none, p=.0001). When DNMT3A mutations are secondary (mean VAF 34%, range 1-60%), as in 47% of our cases (n=66/140), then the common first hits were TET2 (10%), U2AF1 (8%) and cohesin complex (RAD21, SMC3, STAG2) mutations (6%). Dominant DNMT3A mutations correlated with MDS/MPN (60%, p=.007), while secondary DNMT3A mutations correlated with sAML (73%, p=.001). DNMT3A mutant myeloid neoplasms showed worse survival (p<.0001) compared to WT cases. Among different subgroups, there was significant difference in OS between R882, R693, truncating and other non-canonical missense mutations (p=.013). The R882 mutations had worse survival compared to other DNMT3A mutations (p=.003). Non-canonical mutations (truncating and other missense) vs. canonical mutations (R882 and R693) had better survival (p<.04). Survival for mutant R882 DNMT3A was worse compared to truncating mutations (p=.005) while there was no difference between R693 and truncating mutations. Among AML cases, R882 mutations vs. other mutations had worse survival (p=.01) while in MDS and MDS/MPN there was no significant difference in OS. DNTMT3A mutations often occur as founder lesion in AML. Our study shows that different types of mutations other than canonical R882 alterations may have a differential impact on OS and distinct clinical features. Disclosures Carraway: Celgene Corporation: Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

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Whole cell Cl- conductances in mouse choroid plexus epithelial cells do not require CFTR expression

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.6.c1899 ◽

1997 ◽

Vol 272 (6) ◽

pp. C1899-C1907 ◽

Cited By ~ 27

Author(s):

J. D. Kibble ◽

C. Garner ◽

W. H. Colledge ◽

S. Brown ◽

H. Kajita ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Choroid Plexus ◽

Disulfonic Acid ◽

Wild Type ◽

Pipette Solution ◽

Whole Cell ◽

Whole Cell Patch Clamp ◽

Significant Difference ◽

Choroid Plexus Epithelial

Whole cell patch-clamp studies were performed with tissue isolated from the cystic fibrosis (CF) transgenic Cftrm1cam mouse, to determine whether anion currents in choroid plexus epithelial cells require the expression of cystic fibrosis transmembrane conductance regulator (CFTR). Inclusion of 0.25 mM adenosine 3',5'-cyclic monophosphate (cAMP) and 375 nM protein kinase A (PKA) in the pipette solution caused a significant activation of a Cl(-)-selective, inward-rectifying conductance in cells from wild-type and CF mice. The small, outward currents observed in wild-type and CF animals, however, were not activated by cAMP-PKA. There were no significant differences in the size of currents between wild-type, heterozygote, and CF cells in the presence or absence of cAMP-PKA. A second whole cell conductance was activated when cells from wild-type mice were swollen. These volume-activated currents were Cl- selective and exhibited outward rectification. They were Ca2+ independent and ATP dependent and blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The volume-activated channels were also activated in CF mutant cells, and there was no significant difference in the size of the volume-activated currents between wild-type, heterozygote, and CF cells. It is concluded that CFTR neither contributes to the whole cell conductance nor regulates the other anion conductances in choroid plexus epithelial cells.

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The α2β2 isoform combination dominates the astrocytic Na+/K+-ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation

Glia ◽

10.1002/glia.23194 ◽

2017 ◽

Vol 65 (11) ◽

pp. 1777-1793 ◽

Cited By ~ 26

Author(s):

Anca Stoica ◽

Brian Roland Larsen ◽

Mette Assentoft ◽

Rikke Holm ◽

Leanne Melissa Holt ◽

...

Keyword(s):

Atpase Activity ◽

Familial Hemiplegic Migraine ◽

Hemiplegic Migraine

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Mutation analysis of CACNA1A and ATP1A2 genes in Brazilian FHM families

Arquivos de Neuro-Psiquiatria ◽

10.1590/s0004-282x2006000400001 ◽

2006 ◽

Vol 64 (3a) ◽

pp. 549-552

Author(s):

Luciana R. Lopes ◽

Mario Fernando Prieto Peres ◽

Kaate R.J. Vanmolkot ◽

Patrícia R. Tobo ◽

Eliova Zukerman ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Change ◽

Autosomal Dominant ◽

Familial Hemiplegic Migraine ◽

Missense Mutations ◽

Asymptomatic Individual ◽

Hemiplegic Migraine ◽

Dominant Form ◽

The Third ◽

Autosomal Dominant Form

Familial hemiplegic migraine (FHM) is a rare autosomal dominant form of migraine with aura. This disease has been associated with missense mutations in the CACNA1A and ATP1A2 genes. The aim of this study was to identify whether CACNA1A and ATP1A2 are or not related to Brazilian FHM. Here we screened four Brazilian FHM families (total of 26 individuals - 13 affected and 13 asymptomatic or normal) for mutations in both genes. We found an amino acid change in a member of family FHM-D (Arg2206Gly). However since this alteration is not present in all affected individuals and is present in one asymptomatic individual it should be considered a polymorphism. Further studies with additional families will be necessary to reveal the importance of both CACNA1A and ATP1A2 genes on the pathogeneses of FHM in Brazil and to test the third gene (SCN1A) in these FHM families.

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Two novel families with hemiplegic migraine caused by recurrent SCN1A mutation p.F1499L

Cephalalgia ◽

10.1177/0333102417742365 ◽

2017 ◽

Vol 38 (8) ◽

pp. 1503-1508 ◽

Cited By ~ 2

Author(s):

Victoria Schubert ◽

Eva Auffenberg ◽

Saskia Biskup ◽

Karin Jurkat-Rott ◽

Tobias Freilinger

Keyword(s):

Hot Spot ◽

Familial Hemiplegic Migraine ◽

Missense Mutations ◽

Hemiplegic Migraine ◽

Voltage Gated ◽

Channel Gene ◽

Scn1a Mutation ◽

Type 3 ◽

Work Up ◽

Detailed Work

Background Familial hemiplegic migraine type 3 is a monogenic subtype of migraine caused by missense mutations in the neuronal voltage-gated sodium channel gene SCN1A, with 10 different mutations reported so far. In two familial hemiplegic migraine type 3 families, partial cosegregation with a rare eye phenotype (elicited repetitive daily blindness) was previously reported. Methods Two novel familial hemiplegic migraine pedigrees were subjected to genetic analysis and detailed work-up of associated clinical features. Results In both pedigrees, we identified SCN1A mutation p.F1499L, which has been previously associated with familial hemiplegic migraine type 3 and elicited repetitive daily blindness. Both families displayed a pure familial hemiplegic migraine phenotype without evidence of an episodic eye phenotype. Conclusion Like a substantial proportion of other familial hemiplegic migraine type 3 mutations, p.F1499L affects the intracellular linker between domains III and IV of SCN1A, which seems to be a mutational hot-spot. Our new data establish p.F1499L as a recurrent familial hemiplegic migraine type 3 mutation. Elicited repetitive daily blindness seems to be a rare phenomenon in familial hemiplegic migraine type 3, even in carriers of the same mutation.

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Enhanced susceptibility to cortical spreading depression in two types of Na+,K+-ATPase α2 subunit-deficient mice as a model of familial hemiplegic migraine 2

Cephalalgia ◽

10.1177/0333102417738249 ◽

2017 ◽

Vol 38 (9) ◽

pp. 1515-1524 ◽

Cited By ~ 9

Author(s):

Miyuki Unekawa ◽

Keiko Ikeda ◽

Yutaka Tomita ◽

Kiyoshi Kawakami ◽

Norihiro Suzuki

Keyword(s):

Cortical Spreading Depression ◽

Spreading Depression ◽

Propagation Rate ◽

Familial Hemiplegic Migraine ◽

High Dose ◽

High Susceptibility ◽

Hemiplegic Migraine ◽

Gene Encoding ◽

Significant Difference ◽

The Difference

Background Patients with familial hemiplegic migraine type 2 (FHM2) have a mutated ATP1A2 gene (encoding Na+,K+-ATPase α2 subunit) and show prolonged migraine aura. Cortical spreading depression (CSD), which involves mass depolarization of neurons and astrocytes that propagates slowly through the gray matter, is profoundly related to aura. Methods In two types of Atp1a2-defective heterozygous mice, Atp1a2tm1Kwk (C-KO) and Atp1a2tm2Kwk (N-KO), the sensitivity and responsiveness to CSD were examined under urethane anesthesia. Results In both cases, heterozygotes exhibited a low threshold for induction of CSD, faster propagation rate, slower recovery from DC deflection, and profound suppression of the electroencephalogram, compared to wild-type mice. A high dose of KCl elicited repeated CSDs for a longer period, with a tendency for a greater frequency of CSD occurrence in heterozygotes. The difference of every endpoint was slightly greater in N-KO than C-KO. Change of regional cerebral blood flow in response to CSD showed no significant difference. Conclusion Heterozygotes of Atp1a2-defective mice simulating FHM2 demonstrated high susceptibility to CSD rather than cortical vasoreactivity, and these effects may differ depending upon the knockout strategy for the gene disruption. These results suggest that patients with FHM2 may exhibit high susceptibility to CSD, resulting in migraine.

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Presynaptic CaV2.1 calcium channels carrying familial hemiplegic migraine mutation R192Q allow faster recovery from synaptic depression in mouse calyx of Held

Journal of Neurophysiology ◽

10.1152/jn.01183.2011 ◽

2012 ◽

Vol 108 (11) ◽

pp. 2967-2976 ◽

Cited By ~ 15

Author(s):

Carlota González Inchauspe ◽

Francisco J. Urbano ◽

Mariano N. Di Guilmi ◽

Michel D. Ferrari ◽

Arn M. J. M. van den Maagdenberg ◽

...

Keyword(s):

Synaptic Transmission ◽

Close Association ◽

Low Frequency ◽

Familial Hemiplegic Migraine ◽

Missense Mutations ◽

Calyx Of Held ◽

Hemiplegic Migraine ◽

Calcium Sensors ◽

Medial Nucleus ◽

Key Factor

CaV2.1 Ca2+ channels have a dominant and specific role in initiating fast synaptic transmission at central excitatory synapses, through a close association between release sites and calcium sensors. Familial hemiplegic migraine type 1 (FHM-1) is an autosomal-dominant subtype of migraine with aura, caused by missense mutations in the CACNA1A gene that encodes the α1A pore-forming subunit of CaV2.1 channel. We used knock-in (KI) transgenic mice harboring the FHM-1 mutation R192Q to study the consequences of this mutation in neurotransmission at the giant synapse of the auditory system formed by the presynaptic calyx of Held terminal and the postsynaptic neurons of the medial nucleus of the trapezoid body (MNTB). Although synaptic transmission seems unaffected by low-frequency stimulation in physiological Ca2+ concentration, we observed that with low Ca2+ concentrations (<1 mM) excitatory postsynaptic currents (EPSCs) showed increased amplitudes in R192Q KI mice compared with wild type (WT), meaning significant differences in the nonlinear calcium dependence of nerve-evoked transmitter release. In addition, when EPSCs were evoked by broadened presynaptic action potentials (achieved by inhibition of K+ channels) via Cav2.1-triggered exocytosis, R192Q KI mice exhibited further enhancement of EPSC amplitude and charge compared with WT mice. Repetitive stimulation of afferent axons to the MNTB at different frequencies caused short-term depression of EPSCs that recovered significantly faster in R192Q KI mice than in WT mice. Faster recovery in R192Q KI mice was prevented by the calcium chelator EGTA-AM, pointing to enlarged residual calcium as a key factor in accelerating the replenishment of synaptic vesicles.

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