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      Functional and clinical studies reveal pathophysiological complexity of CLCN4-related neurodevelopmental condition

      research-article
      1 , 2 , , 3 , , 3 , 1 , 2 , 4 , 5 , 5 , 3 , 6 , 3 , 6 , 7 , 8 , 9 , 10 , 9 , 10 , 9 , 11 , 12 , 11 , 13 , 14 , 13 , 14 , 15 , 16 , 17 , 4 , 18 , 19 , 19 , 20 , 20 , 21 , 21 , 22 , 23 , 24 , 24 , 25 , 26 , 27 , 28 , 29 , 29 , 30 , 31 , 32 , 33 , 34 , 34 , 34 , 35 , 36 , 35 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 43 , 42 , 44 , 45 , 45 , 46 , 47 , 47 , 48 , 49 , 49 , 49 , 50 , 51 , 52 , 53 , 1 , 2 , 1 , 2 , 53 , 53 , 54 , 55 , 56 , 57 , 58 , 58 , 58 , 59 , 35 , 60 , 61 , 61 , 62 , 63 , 62 , 64 , 65 , 66 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 78 , 78 , 79 , 18 , 80 , 81 , 5 ,
      Molecular Psychiatry
      Nature Publishing Group UK
      Autism spectrum disorders, Genetics

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          Abstract

          Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a “shift” of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

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          Most cited references42

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          Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology

          The American College of Medical Genetics and Genomics (ACMG) previously developed guidance for the interpretation of sequence variants. 1 In the past decade, sequencing technology has evolved rapidly with the advent of high-throughput next generation sequencing. By adopting and leveraging next generation sequencing, clinical laboratories are now performing an ever increasing catalogue of genetic testing spanning genotyping, single genes, gene panels, exomes, genomes, transcriptomes and epigenetic assays for genetic disorders. By virtue of increased complexity, this paradigm shift in genetic testing has been accompanied by new challenges in sequence interpretation. In this context, the ACMG convened a workgroup in 2013 comprised of representatives from the ACMG, the Association for Molecular Pathology (AMP) and the College of American Pathologists (CAP) to revisit and revise the standards and guidelines for the interpretation of sequence variants. The group consisted of clinical laboratory directors and clinicians. This report represents expert opinion of the workgroup with input from ACMG, AMP and CAP stakeholders. These recommendations primarily apply to the breadth of genetic tests used in clinical laboratories including genotyping, single genes, panels, exomes and genomes. This report recommends the use of specific standard terminology: ‘pathogenic’, ‘likely pathogenic’, ‘uncertain significance’, ‘likely benign’, and ‘benign’ to describe variants identified in Mendelian disorders. Moreover, this recommendation describes a process for classification of variants into these five categories based on criteria using typical types of variant evidence (e.g. population data, computational data, functional data, segregation data, etc.). Because of the increased complexity of analysis and interpretation of clinical genetic testing described in this report, the ACMG strongly recommends that clinical molecular genetic testing should be performed in a CLIA-approved laboratory with results interpreted by a board-certified clinical molecular geneticist or molecular genetic pathologist or equivalent.
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            DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources.

            Many patients suffering from developmental disorders harbor submicroscopic deletions or duplications that, by affecting the copy number of dosage-sensitive genes or disrupting normal gene expression, lead to disease. However, many aberrations are novel or extremely rare, making clinical interpretation problematic and genotype-phenotype correlations uncertain. Identification of patients sharing a genomic rearrangement and having phenotypic features in common leads to greater certainty in the pathogenic nature of the rearrangement and enables new syndromes to be defined. To facilitate the analysis of these rare events, we have developed an interactive web-based database called DECIPHER (Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources) which incorporates a suite of tools designed to aid the interpretation of submicroscopic chromosomal imbalance, inversions, and translocations. DECIPHER catalogs common copy-number changes in normal populations and thus, by exclusion, enables changes that are novel and potentially pathogenic to be identified. DECIPHER enhances genetic counseling by retrieving relevant information from a variety of bioinformatics resources. Known and predicted genes within an aberration are listed in the DECIPHER patient report, and genes of recognized clinical importance are highlighted and prioritized. DECIPHER enables clinical scientists worldwide to maintain records of phenotype and chromosome rearrangement for their patients and, with informed consent, share this information with the wider clinical research community through display in the genome browser Ensembl. By sharing cases worldwide, clusters of rare cases having phenotype and structural rearrangement in common can be identified, leading to the delineation of new syndromes and furthering understanding of gene function.
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              Rome IV-Functional GI Disorders: Disorders of Gut-Brain Interaction.

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                Author and article information

                Contributors
                elizabeth.palmer@unsw.edu.au
                michael.pusch@ibf.cnr.it
                kalscheu@molgen.mpg.de
                Journal
                Mol Psychiatry
                Mol Psychiatry
                Molecular Psychiatry
                Nature Publishing Group UK (London )
                1359-4184
                1476-5578
                16 November 2022
                16 November 2022
                2023
                : 28
                : 2
                : 668-697
                Affiliations
                [1 ]GRID grid.414009.8, ISNI 0000 0001 1282 788X, Centre for Clinical Genetics, Sydney Children’s Hospital Network, ; Randwick, NSW Australia
                [2 ]GRID grid.1005.4, ISNI 0000 0004 4902 0432, Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, , University of New South Wales, ; Randwick, NSW Australia
                [3 ]GRID grid.419463.d, ISNI 0000 0004 1756 3731, Istituto di Biofisica, CNR, ; Genova, Italy
                [4 ]GRID grid.415994.4, ISNI 0000 0004 0527 9653, Department of Clinical Genetics, , Liverpool Hospital, ; Liverpool, NSW Australia
                [5 ]GRID grid.419538.2, ISNI 0000 0000 9071 0620, Max Planck Institute for Molecular Genetics, Group Development and Disease, ; Berlin, Germany
                [6 ]GRID grid.5640.7, ISNI 0000 0001 2162 9922, Department of Biomedical and Clinical Sciences, , Linköping University, ; Linköping, 581 83 Sweden
                [7 ]GRID grid.411323.6, ISNI 0000 0001 2324 5973, Department of Human Genetics, Gilbert and Rose-Marie Chagoury School of Medicine, , Lebanese American University, ; Byblos, Lebanon
                [8 ]GRID grid.453925.c, Institut Jerome Lejeune, ; Paris, France
                [9 ]GRID grid.4817.a, ISNI 0000 0001 2189 0784, Service de Génétique Médicale, CHU de Nantes, , Nantes Université, ; Nantes, France
                [10 ]GRID grid.462318.a, Nantes Université, CNRS, INSERM, l’Institut du Thorax, ; Nantes, France
                [11 ]GRID grid.415310.2, ISNI 0000 0001 2191 4301, Department of Translational Genomics, Center for Genomic Medicine, , King Faisal Specialist Hospital and Research Center, ; Riyadh, Saudi Arabia
                [12 ]GRID grid.415310.2, ISNI 0000 0001 2191 4301, Department of Neurosciences, , King Faisal Specialist Hospital and Research Center, ; Riyadh, Saudi Arabia
                [13 ]GRID grid.511528.a, Applied and Translational Neurogenomics Group, VIB Center for Molecular Neurology, VIB, ; Antwerp, Belgium
                [14 ]GRID grid.411414.5, ISNI 0000 0004 0626 3418, Neurology Department, Antwerp University Hospital, ; Antwerp, Belgium
                [15 ]GRID grid.5284.b, ISNI 0000 0001 0790 3681, Translational Neurosciences, Faculty of Medicine and Health Science, University of Antwerp, ; Antwerp, Belgium
                [16 ]GRID grid.410566.0, ISNI 0000 0004 0626 3303, Department of Child Neurology & Metabolism, , Ghent University Hospital, ; Ghent, Belgium
                [17 ]GRID grid.5284.b, ISNI 0000 0001 0790 3681, Department of Pediatric Neurology, , Antwerp University Hospital, University of Antwerp, ; Antwerp, Belgium
                [18 ]GRID grid.413973.b, ISNI 0000 0000 9690 854X, Children’s Hospital at Westmead, Sydney Children’s Hospitals Network, ; Sydney, Australia
                [19 ]GRID grid.411167.4, ISNI 0000 0004 1765 1600, Service de Génétique Clinique, Centre Hospitalier Régional Universitaire de Tours, ; Tours, France
                [20 ]GRID grid.413473.6, ISNI 0000 0000 9013 1194, Genetic Center, Akron Children’s Hospital, ; Akron, OH USA
                [21 ]GRID grid.5284.b, ISNI 0000 0001 0790 3681, Center of Medical Genetics, , University Hospital Antwerp/University of Antwerp, ; Edegem, Belgium
                [22 ]GRID grid.5284.b, ISNI 0000 0001 0790 3681, Department of Pediatric Neurology, , University Hospital Antwerp/University of Antwerp, ; Edegem, Belgium
                [23 ]GRID grid.7700.0, ISNI 0000 0001 2190 4373, Institute of Human Genetics, , Heidelberg University, ; Heidelberg, Germany
                [24 ]GRID grid.10392.39, ISNI 0000 0001 2190 1447, Institute of Medical Genetics and Applied Genomics, , University of Tuebingen, ; Tuebingen, Germany
                [25 ]GRID grid.420468.c, Department of Clinical Genetics, , Great Ormond Street Hospital for Children, ; London, UK
                [26 ]GRID grid.83440.3b, ISNI 0000000121901201, Developmental Neurosciences, , UCL Great Ormond Street Institute of Child Health, ; London, UK
                [27 ]GRID grid.420468.c, Department of Neurology, , Great Ormond Street Hospital, ; London, UK
                [28 ]GRID grid.415216.5, ISNI 0000 0004 0641 6277, Wessex Clinical Genetics Service, , Princess Anne Hospital, ; Southampton, UK
                [29 ]GRID grid.415259.e, ISNI 0000 0004 0625 8678, Genetic Services of WA, , King Edward Memorial Hospital, ; Subiaco, WA Australia
                [30 ]GRID grid.4367.6, ISNI 0000 0001 2355 7002, Department of Neuroscience, , Washington University in St Louis School of Medicine, ; St Louis, MI USA
                [31 ]GRID grid.1003.2, ISNI 0000 0000 9320 7537, The University of Queensland, , Queensland Brain Institute, ; St Lucia, QLD Australia
                [32 ]GRID grid.1058.c, ISNI 0000 0000 9442 535X, Centre for Population Genomics, Murdoch Children’s Research Institute, ; Melbourne, Australia
                [33 ]GRID grid.1005.4, ISNI 0000 0004 4902 0432, Garvan Institute of Medical Research, UNSW, ; Sydney, NSW Australia
                [34 ]GRID grid.42327.30, ISNI 0000 0004 0473 9646, Division of Clinical and Metabolic Genetics, , The Hospital for Sick Children, ; Toronto, ON Canada
                [35 ]GRID grid.511220.5, ISNI 0000 0005 0259 3580, Genetics of Learning Disability Service, ; Newcastle, NSW Australia
                [36 ]GRID grid.266842.c, ISNI 0000 0000 8831 109X, University of Newcastle Grow Up Well Priority Research Centre, ; Newcastle, NSW Australia
                [37 ]GRID grid.239559.1, ISNI 0000 0004 0415 5050, Department of Pathology and Laboratory Medicine, , Children’s Mercy Hospital and Clinics, ; MI Kansas City, USA
                [38 ]GRID grid.134936.a, ISNI 0000 0001 2162 3504, Kansas City School of Medicine, , University of Missouri, ; Kansas City, MI USA
                [39 ]GRID grid.239559.1, ISNI 0000 0004 0415 5050, Division of Clinical Genetics, , Children’s Mercy Hospital and Clinics, ; Kansas City, MI USA
                [40 ]GRID grid.412220.7, ISNI 0000 0001 2177 138X, Service de Génétique Médicale, Institut de Génétique Médicale d’Alsace (IGMA), , Hôpitaux Universitaires de Strasbourg, ; Strasbourg, France
                [41 ]GRID grid.11843.3f, ISNI 0000 0001 2157 9291, Laboratoire de Génétique Médicale, UMRS_1112, Institut de Génétique Médicale d’Alsace (IGMA), , Université de Strasbourg et INSERM, ; Strasbourg, France
                [42 ]GRID grid.412201.4, ISNI 0000 0004 0593 6932, Service de Pédiatrie, Hôpital de Hautepierre, Hôpitaux Universitaires de Strasbourg, ; Strasbourg, France
                [43 ]GRID grid.413866.e, ISNI 0000 0000 8928 6711, Laboratoires de Diagnostic Génétique, Institut de Génétique Médicale d’Alsace (IGMA), , Hôpitaux Universitaires de Strasbourg, Nouvel Hôpital Civil, ; Strasbourg, France
                [44 ]GRID grid.412220.7, ISNI 0000 0001 2177 138X, Pediatric Neurology Department, , CHU de Strasbourg, ; Strasbourg, France
                [45 ]Northern Ireland Regional Genetics Service, Belfast, Northern Ireland
                [46 ]GRID grid.412008.f, ISNI 0000 0000 9753 1393, Department of Medical Genetics, , Haukeland University Hospital, ; Bergen, Norway
                [47 ]GRID grid.5379.8, ISNI 0000000121662407, Division of Evolution, Infection and Genomics, School of Biological Sciences, Faculty of Biology, Medicine and Health, , University of Manchester, ; Manchester, UK
                [48 ]GRID grid.416523.7, ISNI 0000 0004 0641 2620, Manchester Centre for Genomic Medicine, , Saint Mary’s Hospital, Manchester University NHS Foundation Trust, ; Manchester, UK
                [49 ]GRID grid.239585.0, ISNI 0000 0001 2285 2675, Department of Neurology, , Division of Child Neurology, Columbia University Irving Medical Center, ; New York, USA
                [50 ]GRID grid.416992.1, ISNI 0000 0001 2179 3554, Texas Tech Health Sciences Center Lubbock and KinderGenome Medical Genetics, ; Dallas, TX USA
                [51 ]Texas Sports Psychiatry and Integrative Health, Austin, TX USA
                [52 ]Hillcrest Internal Medicine, Waco, TX USA
                [53 ]Centre de Génétique Humaine, Institut de Pathologie et de Génétique ASBL, Gosselies, Belgium
                [54 ]GRID grid.413249.9, ISNI 0000 0004 0385 0051, Department of Medical Genomics/Clinical Genetics, , Royal Prince Alfred Hospital, ; Camperdown, Sydney, NSW Australia
                [55 ]GRID grid.414148.c, ISNI 0000 0000 9402 6172, Department of Genetics, , Children’s Hospital of Eastern Ontario, ; Ottawa, ON Canada
                [56 ]GRID grid.416653.3, ISNI 0000 0004 0450 5663, Division of Medical Genetics, Department of Pediatrics, , San Antonio Military Medical Center, ; San Antonio, TX USA
                [57 ]Department of Pediatrics, Long School of Medicine-UT Health San Antonio, San Antonio, TX USA
                [58 ]GRID grid.10417.33, ISNI 0000 0004 0444 9382, Department of Human Genetics, , Radboud University Medical Center, ; Nijmegen, The Netherlands
                [59 ]GRID grid.491357.d, ISNI 0000 0004 0514 1769, Pluryn, Residential Care Setting, ; Groesbeek, The Netherlands
                [60 ]GRID grid.5808.5, ISNI 0000 0001 1503 7226, Centro de Genética Médica Jacinto Magalhães, , Centro Hospitalar Universitário do Porto, ; Porto, Portugal
                [61 ]GRID grid.5808.5, ISNI 0000 0001 1503 7226, Unit for Multidisciplinary Research in Biomedicine, School of Medicine and Biomedical Sciences, , Porto University, ; Porto, Portugal
                [62 ]GRID grid.42399.35, ISNI 0000 0004 0593 7118, Service de Génétique Médicale, CHU Bordeaux, ; Bordeaux, France
                [63 ]GRID grid.412041.2, ISNI 0000 0001 2106 639X, INSERM U1211, Laboratoire Maladies Rares: Génétique et Métabolisme, Bordeaux, Univ., ; Bordeaux, France
                [64 ]GRID grid.414164.2, ISNI 0000 0004 0442 4003, Child Neurology and Neurodevelopmental Medicine Thompson Autism Center, CHOC Hospital, ; Orange County, CA USA
                [65 ]GRID grid.257413.6, ISNI 0000 0001 2287 3919, Indiana University School of Medicine, ; Indianapolis, USA
                [66 ]GRID grid.266100.3, ISNI 0000 0001 2107 4242, University of California, San Diego, Rady Children’s Hospital San Diego, ; San Diego, CA USA
                [67 ]GRID grid.439803.5, North West Thames Regional Genetics Service, , London North West University Healthcare NHS Trust, ; Harrow, London, UK
                [68 ]GRID grid.7445.2, ISNI 0000 0001 2113 8111, Imperial College London, ; London, UK
                [69 ]GRID grid.511123.5, ISNI 0000 0004 5988 7216, West of Scotland Centre for Genomic Medicine, Queen Elizabeth University Hospital, ; Glasgow, UK
                [70 ]GRID grid.411326.3, ISNI 0000 0004 0626 3362, Centrum Medische Genetica, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), ; Brussels, Belgium
                [71 ]GRID grid.14848.31, ISNI 0000 0001 2292 3357, CHU Sainte-Justine Research Center, , University of Montreal, ; Montreal, QC Canada
                [72 ]GRID grid.412408.b, Division Clinical Genetics Texas A&M University Health Science Center, ; College Station, TX USA
                [73 ]GRID grid.10419.3d, ISNI 0000000089452978, Department of Clinical Genetics, , Leiden University Medical Centre, ; Leiden, The Netherlands
                [74 ]GRID grid.39382.33, ISNI 0000 0001 2160 926X, Molecular and Human Genetics, Baylor College of Medicine, ; Houston, TX USA
                [75 ]GRID grid.510928.7, Baylor Genetics Laboratories, ; Houston, TX USA
                [76 ]GRID grid.511058.8, ISNI 0000 0004 0548 4972, Centogene GmbH, ; Rostock, Germany
                [77 ]GRID grid.465138.d, ISNI 0000 0004 0455 211X, Clinical Genomics, Ambry Genetics, ; Aliso Viejo, CA USA
                [78 ]GRID grid.428467.b, ISNI 0000 0004 0409 2707, GeneDx LLC, ; Gaithersburg, MA USA
                [79 ]Neuropediatric Unit, CHU UCL-Namur, Namur, Belgium
                [80 ]GRID grid.413973.b, ISNI 0000 0000 9690 854X, Children’s Hospital at Westmead, Sydney Children’s Hospitals Network, ; Sydney, NSW Australia
                [81 ]GRID grid.120073.7, ISNI 0000 0004 0622 5016, East Anglian Medical Genetics Service, Clinical Genetics, Addenbrooke’s Treatment Centre, Addenbrooke’s Hospital, ; Cambridge, UK
                Author information
                http://orcid.org/0000-0003-1844-215X
                http://orcid.org/0000-0002-8644-8847
                http://orcid.org/0000-0002-1682-523X
                http://orcid.org/0000-0003-4158-341X
                http://orcid.org/0000-0002-0334-2909
                http://orcid.org/0000-0002-6068-8199
                http://orcid.org/0000-0003-1613-6570
                http://orcid.org/0000-0002-8527-2210
                http://orcid.org/0000-0002-6642-8902
                http://orcid.org/0000-0001-7285-1195
                http://orcid.org/0000-0002-3236-0807
                http://orcid.org/0000-0001-8663-6357
                http://orcid.org/0000-0001-7455-934X
                http://orcid.org/0000-0001-7817-9889
                http://orcid.org/0000-0002-5788-392X
                http://orcid.org/0000-0003-4833-3747
                http://orcid.org/0000-0002-1796-2287
                http://orcid.org/0000-0001-9713-7107
                http://orcid.org/0000-0002-1654-9036
                http://orcid.org/0000-0003-1219-4781
                http://orcid.org/0000-0001-6898-3259
                Article
                1852
                10.1038/s41380-022-01852-9
                9908558
                36385166
                b2d01708-72f7-45d7-8590-cbb57033b9fa
                © The Author(s) 2022

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 5 April 2022
                : 10 October 2022
                : 21 October 2022
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100000925, Department of Health | National Health and Medical Research Council (NHMRC);
                Award ID: GNT20081
                Award Recipient :
                Funded by: Fondazione AIRC per la Ricerca sul Cancro (grant # IG 21558) and from the Italian Ministry for University and Research (MIUR) (PRIN 20174TB8KW).
                Funded by: King Salman Center for Disability Research through Research Group no RG-2022-010 and RG-2022-011
                Funded by: FWO grant number 1861419N and the Queen Elisabeth Medical Foundation
                Funded by: FundRef https://doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft (German Research Foundation);
                Award ID: 418081722
                Award ID: 433158657
                Award Recipient :
                Funded by: NIHR GOSH BRC, MRC (MR/T007087/1), GOSH Charity (VS0122) and Rosetrees Trust (CF2\100018)
                Categories
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                © The Author(s), under exclusive licence to Springer Nature Limited 2023

                Molecular medicine
                autism spectrum disorders,genetics
                Molecular medicine
                autism spectrum disorders, genetics

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