CNPY3 variants associated with a recessive developmental and epileptic encephalopathy affect inflammasome and TLR function

BACKGROUND: Canopy FGF signalling regulator 3 (CNPY3) is an endoplasmic reticulum (ER)-resident chaperone essential for toll-like receptor (TLR) folding, trafficking and inflammasome activation. In rare paediatric cases, biallelic CNPY3 variants have been associated with developmental and epileptic encephalopathy (DEE), but available data remain sparse. METHODS: 50 individuals from 38 unrelated families with biallelic CNPY3 variants and DEE were evaluated for exome and genome findings, standardised clinical phenotype descriptions, EEG results, and neuroimaging data. A selection of variants underwent functional analysis focussing on protein stability and localisation, TLR2 function, NLPR3 inflammasome activation, and structural conformation. FINDINGS: Disease onset occurred in early infancy (median age at onset 68 days), with a clinical phenotype comprising global developmental delay (100%) and intractable epilepsy (86%), accompanied by microcephaly (70%), spasticity (77%), growth faltering (63%), stridor (48%), and early mortality (38%). Epileptic spasms and tonic seizures (46% each) were the most frequent seizure types, and EEG findings included suppression-burst (37%) and hypsarrhythmia (28%). Phenotypic severity varied substantially, with most patients being severely affected (median score 16, range 2-20). Most variants occurred in the homozygous state (95%), including 13 missense changes and 7 truncating alleles. Variants were classified as of uncertain significance (71%), followed by likely pathogenic (23%) and pathogenic (6%) according to ACMG/AMP criteria. Genotype-phenotype correlation showed that individuals with truncating variants were, on average, more severely affected than those with non-truncating variants (median score 18 vs 14.5). Protein modelling predicted conformational effects for most missense variants. In cells, frameshift alleles abolished CNPY3 protein expression, whereas nonsense alleles markedly reduced protein levels. ER localisation of CNPY3 remained preserved across tested genotypes; however, capacity to support TLR2 maturation and processing varied substantially. Remarkably, some missense variants permitted TLR2 surface expression but impaired downstream signalling and inflammasome activation, uncoupling trafficking from signal transduction. INTERPRETATION: CNPY3-associated DEE is a severe early-onset disorder with genotype-dependent phenotypic severity, accompanied by allele-specific disruption of innate immune pathways. Our findings reveal separable effects on TLR trafficking, downstream signalling and inflammasome activation, identifying CNPY3 as a key regulator of multiple cellular pathways involved in inflammatory responses and neurodevelopment. FUNDING: Interdisciplinary Center of Clinical Research and Jena School of Molecular Medicine, Jena University Hospital; Friedrich Schiller University Jena; The Wellcome Trust; The Medical Research Council; The MSA Trust; The National Institute for Health Research University College London Hospitals Biomedical Research Centre; The Michael J Fox Foundation; Biotechnology and Biological Sciences Research Council; The Fidelity Trust; Rosetrees Trust; Ataxia UK; Brain Research UK; Sparks GOSH Charity; Alzheimer's Research UK; CureDRPLA; UK Dementia Research Institute; Parkinson's UK; South-Eastern Norway Regional Health Authority, Norway; France Genomic Medicine Plan 2025; Deutsche Forschungsgemeinschaft; European Commission; Federal Government of Germany; State of Thuringia; Pakistan Science Foundation; King Abdullah International Medical Research Center; Science and Technology Development Fund; King Salman Center for Disability Research.

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Journal
EBioMedicine
Published
2026-10-14
DOI
https://doi.org/10.1016/j.ebiom.2026.106506
Primary Topic
Inflammasome and immune disorders
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article
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article

CNPY3 variants associated with a recessive developmental and epileptic encephalopathy affect inflammasome and TLR function

Matthias Waginger, Ralf A. Husain, Sven Stengel, Zafar Iqbal et al.
EBioMedicine
Inflammasome and immune disorders
article

CNPY3 variants associated with a recessive developmental and epileptic encephalopathy affect inflammasome and TLR function

Matthias Waginger, Ralf A. Husain, Sven Stengel, Zafar Iqbal, Caroline Racine, Kimia Kahrizi, Louis Januel, Gulnara Svyatova, Tobias B. Haack, Khadijah Bakur, Constance L. Smith‐Hicks, Margot Grisval, Barbara Oehl‐Jaschkowitz, Agustí Rodríguez‐Palmero, Shazia Maqbool, Rauan B. Kaiyrzhanov, Maha Saad Zaki, Vera Garloff, David Cheillan, Naser Ajami, Vivek Jain, Gabriela E. Oprea, Kamran Salayev, Maureen Mulhern, Peter Huppke, Fatima Rahman, Hamid Galehdari, Stéphanie Efthymiou, Ignacio Rubio, Dolores Bello, Vykuntaraju K. Gowda, Mohammad Reza Abbaszadegan, Lokesh Lingappa, Loreto Ríos‐Pohl, Tristan T. Sands, Andrea Dieckmann, Fawzia Amer, Cécile Acquaviva, Mona Grimmel, Ulviyya Guliyeva, Nour M. Elkhateeb, Erum Afzal, Bader Alhaddad, Iman Sabri Abumansour, Majid Alfadhel, Alexandra Murtazaliyeva, Javeria Raza Alvi, Stefanie Beck‐Wödl, Rola Mohammed Alanazi, Debangana Baruah, Ambrin Fatima, Varunvenkat M. Srinivasan, R. Møller, Michael W Bauer, Hossein Najmabadi, Mathias Toft, Heike de Vries, Abolfazl Rad, Mohammad Miryounesi, Reza Maroofian, Chadi Al Alam, Fowzan Sami Alkuraya, Mohamed Ghait, Hani Akbar Rao, Gholamreza Shariati, Evangeline Wassmer, Eduardo Pérez‐Palma, Anya Revah‐Politi, Niloofar Chamanrou, Tipu Sultan, Carolina Alvarez, Ahmad Farooq, Reza Azizimalamiri, Sohana Nadeem Hashmi, Bhuvandeep Narang, Shahzad Haider, Ehsan Barkhordari, Pardis Nourbakhsh, Christopher Manuel Meudt, Rami Abou Jamra, Henry Houlden, Farhan Bahadar Ali, Muhammad Umair, Julie Vogt, Sidra Kaleem, Ali Mardi, Oliver Ohlenschläger, Sarah Bühler, Anna Chassevent, Hafiz Muhammad Mustafa, Irisa Pjeci, Carolin Tille, Asem Burabayeva, Mina Zamani, Irina Mishina, Vincenzo Salpietro, Seba Nadeef
article en

Abstract

BACKGROUND: Canopy FGF signalling regulator 3 (CNPY3) is an endoplasmic reticulum (ER)-resident chaperone essential for toll-like receptor (TLR) folding, trafficking and inflammasome activation. In rare paediatric cases, biallelic CNPY3 variants have been associated with developmental and epileptic encephalopathy (DEE), but available data remain sparse. METHODS: 50 individuals from 38 unrelated families with biallelic CNPY3 variants and DEE were evaluated for exome and genome findings, standardised clinical phenotype descriptions, EEG results, and neuroimaging data. A selection of variants underwent functional analysis focussing on protein stability and localisation, TLR2 function, NLPR3 inflammasome activation, and structural conformation. FINDINGS: Disease onset occurred in early infancy (median age at onset 68 days), with a clinical phenotype comprising global developmental delay (100%) and intractable epilepsy (86%), accompanied by microcephaly (70%), spasticity (77%), growth faltering (63%), stridor (48%), and early mortality (38%). Epileptic spasms and tonic seizures (46% each) were the most frequent seizure types, and EEG findings included suppression-burst (37%) and hypsarrhythmia (28%). Phenotypic severity varied substantially, with most patients being severely affected (median score 16, range 2-20). Most variants occurred in the homozygous state (95%), including 13 missense changes and 7 truncating alleles. Variants were classified as of uncertain significance (71%), followed by likely pathogenic (23%) and pathogenic (6%) according to ACMG/AMP criteria. Genotype-phenotype correlation showed that individuals with truncating variants were, on average, more severely affected than those with non-truncating variants (median score 18 vs 14.5). Protein modelling predicted conformational effects for most missense variants. In cells, frameshift alleles abolished CNPY3 protein expression, whereas nonsense alleles markedly reduced protein levels. ER localisation of CNPY3 remained preserved across tested genotypes; however, capacity to support TLR2 maturation and processing varied substantially. Remarkably, some missense variants permitted TLR2 surface expression but impaired downstream signalling and inflammasome activation, uncoupling trafficking from signal transduction. INTERPRETATION: CNPY3-associated DEE is a severe early-onset disorder with genotype-dependent phenotypic severity, accompanied by allele-specific disruption of innate immune pathways. Our findings reveal separable effects on TLR trafficking, downstream signalling and inflammasome activation, identifying CNPY3 as a key regulator of multiple cellular pathways involved in inflammatory responses and neurodevelopment. FUNDING: Interdisciplinary Center of Clinical Research and Jena School of Molecular Medicine, Jena University Hospital; Friedrich Schiller University Jena; The Wellcome Trust; The Medical Research Council; The MSA Trust; The National Institute for Health Research University College London Hospitals Biomedical Research Centre; The Michael J Fox Foundation; Biotechnology and Biological Sciences Research Council; The Fidelity Trust; Rosetrees Trust; Ataxia UK; Brain Research UK; Sparks GOSH Charity; Alzheimer's Research UK; CureDRPLA; UK Dementia Research Institute; Parkinson's UK; South-Eastern Norway Regional Health Authority, Norway; France Genomic Medicine Plan 2025; Deutsche Forschungsgemeinschaft; European Commission; Federal Government of Germany; State of Thuringia; Pakistan Science Foundation; King Abdullah International Medical Research Center; Science and Technology Development Fund; King Salman Center for Disability Research.

EBioMedicineVol. 133
Université Claude Bernard Lyon 1 (FR), Aga Khan University (PK), Ahvaz Jundishapur University of Medical Sciences (IR), Oslo University Hospital (NO), Sindh Madressatul Islam University (PK), Boston Children's Hospital (US), Morgan Stanley Children's Hospital (US), Kennedy Krieger Institute (US), Universidad del Desarrollo (CL), Alfaisal University (SA), Johns Hopkins University (US), Cairo University (EG), Inserm (FR), Punjab Institute of Cardiology (PK), Mashhad University of Medical Sciences (IR), University of Southern Denmark (DK), Islamic Azad University, Mashhad (IR), King Saud bin Abdulaziz University for Health Sciences (SA), Shahid Chamran University of Ahvaz (IR), Umm al-Qura University (SA), Columbia University Irving Medical Center (US), Shalamar Hospital (PK), King Faisal Specialist Hospital & Research Centre (SA), Leibniz Institute of Photonic Technology (DE), King Fahad Specialist Hospital (SA), Cambridge University Hospitals NHS Foundation Trust (GB), Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (FR), National Human Genome Research Institute (US), Birmingham Children's Hospital (GB), Hospices Civils de Lyon (FR), King Fahd Armed Forces Hospital (SA), Hospital Luis Calvo Mackenna (CL), CHU Dijon Bourgogne (FR), Klinikum Frankfurt Höchst (DE), South Kazakhstan Medical Academy (KZ), University Hospital Leipzig (DE), Research Centre for Medical Genetics (RU), Laboratoire de recherche en cardiovasculaire, métabolisme, diabétologie et nutrition (FR), Leibniz Institute on Aging - Fritz Lipmann Institute (FLI) (DE), Jena University Hospital (DE), Praxis für Humangenetik (DE), European Brain Research Institute (IT), University of Swabi (PK), Rainbow Children's Hospital (IN), New York Genome Center (US), Hospital Universitari Germans Trias i Pujol (ES), Birmingham Women’s and Children’s NHS Foundation Trust (GB), Indira Gandhi Institute of Child Health (IN), Islamic Azad University of Urmia (IR), University of Social Welfare and Rehabilitation Sciences (IR), UCL Queen Square Institute of Neurology (GB), Center for Sepsis Control and Care (DE), University College London (GB), National Guard Health Affairs (SA), Shahid Beheshti University of Medical Sciences (IR), Columbia University (US), University of Tübingen (DE), Leipzig University (DE)
Michael J. Fox Foundation for Parkinson's Research, Pakistan Science Foundation, Wellcome Trust, Friedrich-Schiller-Universität Jena, Multiple System Atrophy Trust, Brain Research UK, Freistaat Thüringen, Ataxia UK, Rosetrees Trust, Sparks, Deutsche Forschungsgemeinschaft, Alzheimer’s Research UK, Science and Technology Development Fund, Universitätsklinikum Jena, King Abdullah International Medical Research Center, UK Dementia Research Institute, Medical Research Council, Biotechnology and Biological Sciences Research Council, Helse Sør-Øst RHF, University College London Hospitals Biomedical Research Centre
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