Exploring transcriptomic and genomic latent variable correction approaches in differential expression analysis
Abstract
Abstract Background Differential expression analysis is a central tool for studying the biological processes altered in human diseases via transcriptomic signatures. However, transcriptomic datasets are systematically confounded by latent variables from two distinct sources: unmeasured technical and biological heterogeneity within the expression data, and expression differences driven by population stratification. Correction using expression-based surrogate variables (SVs) and genotype-based principal components (PCs) addresses these sources independently, yet no study has directly evaluated their combined use against either method alone within a differential expression framework. In this study we hypothesised that simultaneously including both correction layers would produce more biologically valid and reproducible results than either approach alone, and tested this in two independent RNA-seq datasets of amyotrophic lateral sclerosis (ALS) cases and controls with matching genotype data. Results Four nested differential expression models (corrected for PC-only, SV-only, both SV and PC, and neither PCs nor SVs) were evaluated across the KCLBB (96 cases and 52 controls) and ALS Consortium (272 cases and 35 controls) datasets. Models were evaluated on: cross-dataset effect size concordance, cross-dataset replicability quantified by the Jaccard Similarity Index, and biological recall against a curated reference set of 66 known ALS genes. The combined SV+PC framework consistently outperformed simpler models across all metrics. Replicability improved nearly ten-fold compared to the non-corrected model, (Jaccard index: 2.28% to 19.5%), and the combined framework exhibited a statistically significant 2.1% gain over the SV-only model. The biological recall ALS genes recovered doubled comparing to the SV correction alone. Crucially, effect size stability was preserved, with the combined model expanding the shared transcriptomic signal without sacrificing consistency. These findings remained generally robust to PC number in sensitivity analyses. Conclusions This study found that SVs and genotype PCs address non-redundant sources of confounding, and we recommend their combined use as standard practice in differential expression analysis where matched genotype data are available. Notably PCs capturing population structure can also be derived directly from RNA-seq data, extending the applicability of this framework to studies lacking matched genotype data. Although this analysis was restricted to ALS datasets, we expect these findings to generalise to other traits.
Authors 91
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Affiliation as printed
Department of Biostatistics & Health Informatics, Institute of Psychiatry Psychology & Neuroscience, King’s College London, London, UK
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Affiliation as printed
Department of Biostatistics & Health Informatics, Institute of Psychiatry Psychology & Neuroscience, King’s College London, London, UK
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Affiliation as printed
Department of Biostatistics & Health Informatics, Institute of Psychiatry Psychology & Neuroscience, King’s College London, London, UK
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Affiliation as printed
Department of Biostatistics & Health Informatics, Institute of Psychiatry Psychology & Neuroscience, King’s College London, London, UK
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Affiliation as printed
Center for Genomics of Neurodegenerative Disease (CGND), New York Genome Center, New York, NY, USA
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University of Maryland, Baltimore · Temple University
Affiliation as printed
Department of Neurology, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, USA
University of Maryland Brain and Tissue Bank and NIH NeuroBioBank, Baltimore, USA
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Affiliation as printed
Department of Biomedical Sciences, Cedars-Sinai Biomanufacturing Center, Board of Governors Regenerative Medicine Institute and Brain Program, Cedars-Sinai Medical Center, Los Angeles, USA
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Affiliation as printed
Department of Biochemistry and Molecular Biology, Penn State Institute for Personalized Medicine, The Pennsylvania State University, Hershey, PA, USA
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Department of Neurology, The Pennsylvania State University, Hershey, PA, USA
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Affiliation as printed
Department of Neurology, Henry Ford Health System, Detroit, MI, USA
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Affiliation as printed
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
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Affiliation as printed
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
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Columbia University Irving Medical Center · Columbia University
Affiliation as printed
Department of Neurology, Center for Motor Neuron Biology and Disease, Institute for Genomic Medicine, Columbia University, New York, NY, USA
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Massachusetts Institute of Technology
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Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
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Johns Hopkins University · Johns Hopkins Medicine · Johns Hopkins Hospital
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Department of Neurology, Johns Hopkins School of Medicine, Baltimore, MD, USA
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Leiden University Medical Center · Amsterdam UMC Location University of Amsterdam
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Department of Neurogenetics, Academic Medical Centre, Amsterdam and Leiden University Medical Center, Leiden, The Netherlands
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University of California, San Francisco
Affiliation as printed
Department of Medicine, Lung Biology Center, University of California, San Francisco, CA, USA
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Massachusetts General Hospital
Affiliation as printed
Neuromuscular Division, Department of Neurology, ALS Multidisciplinary Clinic, Harvard Medical School, and Neurological Clinical Research Institute, Massachusetts General Hospital, Boston, MA, USA
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Queen Mary University of London · Basildon and Thurrock University Hospitals NHS Foundation Trust · Basildon Hospital · Blizard Institute
Affiliation as printed
Centre for Neuroscience and Trauma, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK
Department of Neurology, Basildon University Hospital, Basildon, UK
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National Hospital for Neurology and Neurosurgery · UCL Queen Square Institute of Neurology · University College London
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Institute of Neurology, National Hospital for Neurology and Neurosurgery, University College London, London, UK
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The Jackson Laboratory, Bar Harbor, ME, USA
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University of California, Irvine
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Department of Psychiatry & Human Behavior, Department of Biological Chemistry, School of Medicine, and Department of Neurobiology and Behavior, School of Biological Sciences, University California, Irvine, CA, USA
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Gladstone Institutes · Taube Koret Center
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Taube/Koret Center for Neurodegenerative Disease Research, Roddenberry Center for Stem Cell Biology and Medicine, Gladstone Institute, San Francisco, USA
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Affiliation as printed
Department of Neurology & Sensory Organs, University of Thessaly, Thessaly, Greece
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Washington University in St. Louis
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Department of Neurology, Washington University in St. Louis, St. Louis, MO, USA
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MRC Centre for Regenerative Medicine
Affiliation as printed
Centre for Clinical Brain Sciences, Euan MacDonald Centre for Motor Neurone Disease Research, Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, Edinburgh, UK
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MRC Centre for Regenerative Medicine
Affiliation as printed
Centre for Clinical Brain Sciences, Euan MacDonald Centre for Motor Neurone Disease Research, Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, Edinburgh, UK
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Affiliation as printed
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
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Icahn School of Medicine at Mount Sinai
Affiliation as printed
Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
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Affiliation as printed
Center for Neurodegenerative Disorders, Department of Neurology, The Lewis Katz School of Medicine, Temple University, Philadelphia, PA, USA
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Affiliation as printed
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
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Broad Institute · Brigham and Women's Hospital · Harvard University
Affiliation as printed
Computer Science and Systems Biology Program, Ann Romney Center for Neurological Diseases, Department of Neurology and Division of Genetics in Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA
Harvard Medical School, Boston, MA, USA
Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA
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Brigham and Women's Hospital · Harvard University
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Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
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Affiliation as printed
Department of Anesthesiology, Stony Brook University, Stony Brook, NY, USA
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National Institutes of Health · National Institute of Neurological Disorders and Stroke
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Section of Infections of the Nervous System, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA
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Barrow Neurological Institute · St. Joseph's Hospital and Medical Center
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Department of Neurobiology, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix, AZ, USA
Department of Neurology, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix, AZ, USA
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Affiliation as printed
Department of Neurology, Division of Neuromuscular Medicine, Columbia University, New York, NY, USA
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Amsterdam UMC Location University of Amsterdam · University of Amsterdam
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Department of Neuropathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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Department of Biology and Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA, USA
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Affiliation as printed
Department of Bioengineering, School of Engineering and Applied Sciences, New York Stem Cell Foundation, University of Pennsylvania, Philadelphia, PA, USA
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Allen Institute for Brain Science
Affiliation as printed
Department of Pathology, Fishberg Department of Neuroscience, Ronald M. Loeb Center for Alzheimer’s Disease, Icahn School of Medicine at Mount Sinai, Friedman Brain Institute, New York, NY, USA
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Harvard University · Massachusetts General Hospital
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Department of Neurology, Harvard Medical School, Neurological Clinical Research Institute, Massachusetts General Hospital, Boston, MA, USA
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Hospital for Special Surgery · Weill Cornell Medical Center
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Department of Neurology, Hospital for Special Surgery and Weill Cornell Medical Center, New York, NY, USA
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Overlook Medical Center · Morristown Medical Center · Atlantic Health System
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Medical Genetics, Atlantic Health System, Morristown Medical Center, Morristown, NJ, USA
Overlook Medical Center, Summit, NJ, USA
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National and Kapodistrian University of Athens · Biomedical Research Foundation of the Academy of Athens · Eginition Hospital
Affiliation as printed
1st Department of Neurology, Medical School, Eginition Hospital, National and Kapodistrian University of Athens, Athens, Greece
Center of Clinical Research, Experimental Surgery and Translational Research, Biomedical Research Foundation of the Academy of Athens (BRFAA), 4 Soranou Efesiou Street, 11527, Athens, Greece
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Hebrew University of Jerusalem · Hadassah Medical Center
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Neuromuscular/EMG Service and ALS/Motor Neuron Disease Clinic, Hebrew University-Hadassah Medical Center, Jerusalem, Israel
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Affiliation as printed
Board of Governors Regenerative Medicine Institute, Los Angeles, CA, USA
Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, CA, USA
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Massachusetts General Hospital
Affiliation as printed
Neurological Clinical Research Institute, Massachusetts General Hospital, Boston, MA, USA
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Icahn School of Medicine at Mount Sinai
Affiliation as printed
Departments of Neuroscience, and Genetics and Genomic Sciences, Ronald M. Loeb Center for Alzheimer’s Disease, Icahn School of Medicine at Mount Sinai, New York, NY, USA
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Harvard University · Spaulding Rehabilitation Hospital
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Department of Physical Medicine & Rehabilitation, Harvard Medical School, Spaulding Rehabilitation Hospital, Boston, MA, USA
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Children's Hospital of Philadelphia · University of Pennsylvania
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Center for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA
Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
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Euan MacDonald Centre for Motor Neuron Disease Research · University of Edinburgh
Affiliation as printed
Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK
Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, UK
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Icahn School of Medicine at Mount Sinai
Affiliation as printed
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, Icahn Institute of Data Science and Genomic Technology, New York, NY, USA
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University of Maryland, Baltimore
Affiliation as printed
University of Maryland Brain and Tissue Bank and NIH NeuroBioBank, Baltimore, USA
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Georgetown University Medical Center · Georgetown Lombardi Comprehensive Cancer Center
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Department of Neuropathology, Georgetown Brain Bank, Georgetown Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC, USA
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University of California, San Francisco
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Neuroradiology Section, Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA, USA
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Tel Aviv Sourasky Medical Center
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Neuromuscular Diseases Unit, Department of Neurology, Sackler Faculty of Medicine, Tel Aviv Sourasky Medical Center, Tel-Aviv University, Tel-Aviv, Israel
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University of California San Diego
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Department of Neuroscience, University of California San Diego, La Jolla, CA, USA
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Affiliation as printed
Department of Neurology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
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Columbia University Irving Medical Center · Columbia University
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Department of Neurology, Columbia University Medical Center, New York, NY, USA
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University of California, San Francisco
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Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA
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University of California, San Francisco
Affiliation as printed
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA
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Hebrew University of Jerusalem
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Hadassah Hebrew University, Jerusalem, Israel
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Affiliation as printed
Department of Translational Neuroscience, Barrow Neurological Institute, Phoenix, AZ, USA
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Translational Genomics Research Institute
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The Translational Genomics Research Institute (TGen), Phoenix, AZ, USA
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Broad Institute, Cambridge, MA, USA
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Affiliation as printed
Broad Institute, Cambridge, MA, USA
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Massachusetts General Hospital
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Massachusetts General Hospital, Boston, MA, USA
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Northwell Health · Feinstein Institute for Medical Research
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Institute of Molecular Medicine, Feinstein Institutes for Medical Research, Northwell Health, Manhasset, NY, USA
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Korea Advanced Institute of Science and Technology
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Korea Advanced Institute of Science and Technology (KAIST), Daejeon, South Korea
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Maze Therapeutics, South San Francisco, USA
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Maze Therapeutics, South San Francisco, USA
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Bristol-Myers Squibb (United States)
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Bristol-Myers Squibb, New York, USA
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Bristol-Myers Squibb (United States)
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Bristol-Myers Squibb, New York, USA
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Perron Institute for Neurological and Translational Science
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Perron Institute for Neurological and Translational Science, Nedlands, Australia
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National Institute of Allergy and Infectious Diseases
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Integrative Immunobiology Section, National Institute of Allergy and Infectious Disease, NIH, Bethesda, USA
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Institute on Aging · National Institute on Aging
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Neuromuscular Disease Research Section, National Institute of Aging, Baltimore, USA
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Affiliation as printed
Pfizer, New York, USA
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Pfizer, New York, USA
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Regeneron, Tarrytown, USA
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Affiliation as printed
Regeneron, Tarrytown, USA
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Affiliation as printed
Regeneron, Tarrytown, USA
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Affiliation as printed
Regeneron, Tarrytown, USA
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Affiliation as printed
Regeneron, Tarrytown, USA
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Affiliation as printed
Regeneron, Tarrytown, USA
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Affiliation as printed
Regeneron, Tarrytown, USA
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Affiliation as printed
Regeneron, Tarrytown, USA
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Affiliation as printed
Insitro, South San Francisco, USA
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Affiliation as printed
Insitro, South San Francisco, USA
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Alfredo Iacoangeli corresponding
The University of Western Australia · King's College London · South London and Maudsley NHS Foundation Trust · Perron Institute for Neurological and Translational Science
Affiliation as printed
Department of Biostatistics & Health Informatics, Institute of Psychiatry Psychology & Neuroscience, King’s College London, London, UK
NIHR Maudsley Biomedical Research Centre (BRC), South London and Maudsley NHS Foundation Trust, 16 De Crespigny Park, London, SE5 8AF, UK
Perron Institute for Neurological and Translational Science, University of Western Australia Medical School, Perth, Australia
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Oliver Pain corresponding
Affiliation as printed
Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology & Neuroscience, Maurice Wohl Clinical Neuroscience Institute, King’s College London, London, UK
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