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      Clinically severe CACNA1A alleles affect synaptic function and neurodegeneration differentially

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          Abstract

          Dominant mutations in CACNA1A, encoding the α-1A subunit of the neuronal P/Q type voltage-dependent Ca 2+ channel, can cause diverse neurological phenotypes. Rare cases of markedly severe early onset developmental delay and congenital ataxia can be due to de novo CACNA1A missense alleles, with variants affecting the S4 transmembrane segments of the channel, some of which are reported to be loss-of-function. Exome sequencing in five individuals with severe early onset ataxia identified one novel variant (p.R1673P), in a girl with global developmental delay and progressive cerebellar atrophy, and a recurrent, de novo p.R1664Q variant, in four individuals with global developmental delay, hypotonia, and ophthalmologic abnormalities. Given the severity of these phenotypes we explored their functional impact in Drosophila. We previously generated null and partial loss-of-function alleles of cac, the homolog of CACNA1A in Drosophila. Here, we created transgenic wild type and mutant genomic rescue constructs with the two noted conserved point mutations. The p.R1673P mutant failed to rescue cac lethality, displayed a gain-of-function phenotype in electroretinograms (ERG) recorded from mutant clones, and evolved a neurodegenerative phenotype in aging flies, based on ERGs and transmission electron microscopy. In contrast, the p.R1664Q variant exhibited loss of function and failed to develop a neurodegenerative phenotype. Hence, the novel R1673P allele produces neurodegenerative phenotypes in flies and human, likely due to a toxic gain of function.

          Author summary

          Calcium channels control the levels of calcium within cells and are important in human health. Indeed, groups of patients with disorders of balance known as ataxia have been found to have mutations in a calcium channel gene in the human genome called CACNA1A. CACNA1A mutations have also been observed in patients with particular forms of migraine leading to temporary paralysis on one side of the body (hemiplegia). Mutations in CACNA1A are increasingly found in even more severe brain phenotypes in childhood. This research focused on a group of 5 patients with that particularly severe CACNA1A-related disease. One of the patients had a particular genetic misspelling in CACNA1A while the other four had nearby misspellings. We used the fruitfly, Drosophila melanogaster, to generate flies with these same misspellings in a genetic background that lacked the fly version of the calcium channel. Interestingly, by studying these flies we saw differences between the mutation in Patient 1 and the other four patients. These differences suggest one of the mutations produces more neurodegeneration, and indeed we see more degeneration in that patient. The fly studies allowed us to understand the function of the mutations in these patients, and were helpful in guiding treatment decisions.

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          Most cited references29

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          Voltage-gated calcium channels.

          Voltage-gated calcium (Ca(2+)) channels are key transducers of membrane potential changes into intracellular Ca(2+) transients that initiate many physiological events. There are ten members of the voltage-gated Ca(2+) channel family in mammals, and they serve distinct roles in cellular signal transduction. The Ca(V)1 subfamily initiates contraction, secretion, regulation of gene expression, integration of synaptic input in neurons, and synaptic transmission at ribbon synapses in specialized sensory cells. The Ca(V)2 subfamily is primarily responsible for initiation of synaptic transmission at fast synapses. The Ca(V)3 subfamily is important for repetitive firing of action potentials in rhythmically firing cells such as cardiac myocytes and thalamic neurons. This article presents the molecular relationships and physiological functions of these Ca(2+) channel proteins and provides information on their molecular, genetic, physiological, and pharmacological properties.
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            Structure of the voltage-gated calcium channel Cav1.1 at 3.6 Å resolution.

            The voltage-gated calcium (Cav) channels convert membrane electrical signals to intracellular Ca(2+)-mediated events. Among the ten subtypes of Cav channel in mammals, Cav1.1 is specified for the excitation-contraction coupling of skeletal muscles. Here we present the cryo-electron microscopy structure of the rabbit Cav1.1 complex at a nominal resolution of 3.6 Å. The inner gate of the ion-conducting α1-subunit is closed and all four voltage-sensing domains adopt an 'up' conformation, suggesting a potentially inactivated state. The extended extracellular loops of the pore domain, which are stabilized by multiple disulfide bonds, form a windowed dome above the selectivity filter. One side of the dome provides the docking site for the α2δ-1-subunit, while the other side may attract cations through its negative surface potential. The intracellular I-II and III-IV linker helices interact with the β1a-subunit and the carboxy-terminal domain of α1, respectively. Classification of the particles yielded two additional reconstructions that reveal pronounced displacement of β1a and adjacent elements in α1. The atomic model of the Cav1.1 complex establishes a foundation for mechanistic understanding of excitation-contraction coupling and provides a three-dimensional template for molecular interpretations of the functions and disease mechanisms of Cav and Nav channels.
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              Genome-wide patterns and properties of de novo mutations in humans

              Mutations create variation in the population, fuel evolution, and cause genetic diseases. Current knowledge about de novo mutations is incomplete and mostly indirect 1–10 . Here, we analyze 11,020 de novo mutations from whole-genomes of 250 families. We show that de novo mutations in offspring of older fathers are not only more numerous 11–13 but also occur more frequently in early-replicating, genic regions. Functional regions exhibit higher mutation rates due to CpG dinucleotides and reveal signatures of transcription-coupled repair, while mutation clusters with a unique signature point to a novel mutational mechanism. Mutation and recombination rates independently associate with nucleotide diversity, and regional variation in human-chimpanzee divergence is only partly explained by mutation rate heterogeneity. Finally, we provide a genome-wide mutation rate map for medical and population genetics applications. Our results reveal novel insights and refine long-standing hypotheses about human mutagenesis.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: ResourcesRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: ResourcesRole: Writing – review & editing
                Role: InvestigationRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                24 July 2017
                July 2017
                : 13
                : 7
                : e1006905
                Affiliations
                [1 ] Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States of America
                [2 ] Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, United States of America
                [3 ] Baylor-Hopkins Center for Mendelian Genomics, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States of America
                [4 ] University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States of America
                [5 ] Nationwide Children’s Hospital & The Ohio State University, Columbus, OH, United States of America
                [6 ] Department of Neurology and Neurotherapeutics, UT Southwestern Medical Center andTexas Scottish Rite Hospital, Dallas, TX, United States of America
                [7 ] Houston Specialty Clinic, Houston, TX, United States of America
                [8 ] Department of Pediatrics, Baylor College of Medicine, Houston, TX, United States of America
                [9 ] Texas Children’s Hospital, Houston, TX, United States of America
                [10 ] Howard Hughes Medical Institute, Houston TX, United States of America
                Stanford University School of Medicine, UNITED STATES
                Author notes

                The Department of Molecular and Human Genetics at Baylor College of Medicine derives revenue from molecular testing offered at the Baylor Genetics Laboratories. JRL has stock ownership in 23 and Me, is a paid consultant for Regeneron Pharmaceuticals, has stock options in Lasergen, Inc., and is a co-inventor on multiple United States and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, and bacterial genomic fingerprinting.

                ¶ Membership of the UDN is provided in the supporting information.

                Author information
                http://orcid.org/0000-0001-5664-7987
                http://orcid.org/0000-0003-2172-8036
                http://orcid.org/0000-0001-9888-0779
                http://orcid.org/0000-0002-6644-6385
                http://orcid.org/0000-0001-9928-9032
                http://orcid.org/0000-0001-8573-4211
                http://orcid.org/0000-0001-5245-5910
                Article
                PGENETICS-D-17-00669
                10.1371/journal.pgen.1006905
                5557584
                28742085
                8d626f55-ea55-498e-b4da-92e02a78ac99
                © 2017 Luo et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 3 April 2017
                : 3 July 2017
                Page count
                Figures: 5, Tables: 1, Pages: 20
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000065, National Institute of Neurological Disorders and Stroke;
                Award ID: U54NS093793
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R24OD022005
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: R01GM067858
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: U01HG007709
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000051, National Human Genome Research Institute;
                Award ID: UM1 HG006542
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000011, Howard Hughes Medical Institute;
                Award ID: N/A
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000893, Simons Foundation;
                Award ID: 368479
                Award Recipient :
                Funded by: Michael Fund International Foundation for Genetics Research (US)
                Award Recipient :
                This work was supported in part by NIH grants U54NS093793, R24OD022005, and R01GM067858 to HJB, by U01HG007709 to BL, by UM1 HG006542 to JRL, and by Simons Foundation Functional Screen (368479 to MFW). RAL is supported in part by the Genetic Resource Association of Texas (GReAT). HJB is an investigator of the Howard Hughes Medical Institute (HHMI). In addition, funding was received by NIH/NIGMS T32 GM007526 to BL and NIH/NICHD U54 HD083092 for Intellectual and Developmental Disabilities Research Center (IDDRC). The sponsors and funding agencies had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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                All relevant data are within the paper and its Supporting Information files.

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