Congenital ichthyoses, now renamed epidermal differentiation disorders (EDDs) (syndromic EDD or nonsyndromic EDD), are rare, disabling conditions caused by sequence variations in epidermal barrier genes. However, 5-10% of variants, called "variants of uncertain significance" (VUS), remain uncharacterized, and their pathogenicity is not demonstrated. We developed an approach for classifying VUS in nonsyndromic EDD associated with variant in PNPLA1. We generated PNPLA1-knockout human keratinocytes. PNPLA1, encoded by a missense VUS or by the reference coding sequence, was expressed after lentiviral transduction in the PNPLA1-knockout cells. Transduced cells were used to produce human epidermal equivalents, and the functionality of the normal or VUS-encoded proteins was evaluated. Compared with PNPLA1-knockout human epidermal equivalents re-expressing normal PNPLA1, PNPLA1-knockout human epidermal equivalents showed disrupted synthesis of ω-O-acylceramide, the normal product of PNPLA1, as well as abnormal vesicle-like structures and immature cornified envelopes, characteristic of the epidermis of patients with PNPLA1 variants. Human epidermal equivalents expressing the PNPLA1 VUS showed similar abnormalities, consistent with an impaired PNPLA1 function. This work demonstrated a feasible strategy to help reclassifying missense VUS, which can be extended to other EDD-related genes. Although further efforts are needed to translate this approach into clinical practice and help overcome current diagnostic limitations, such models are valuable tools for pathophysiological and preclinical research on EDDs.

Centre de Microscopie Electronique Appliquée à la Biologie Toulouse University Toulouse France

Faculty of Pharmacy Charles University Hradec Králové Czech Republic

I2MC Inserm Toulouse University Toulouse France

Institute for Advanced Chemistry of Catalonia Barcelona Spain

MetaboHUB MetaToul National Infrastructure of Metabolomics and Fluxomics Toulouse France

PEFAVar and Department of Cell Biology and Cytology Federative Institute of Biology Purpan Hospital University Hospital Toulouse France

Reference Center for Rare Skin Diseases Dermatology Department CHU Toulouse Toulouse University Toulouse France

RESTORE Institute UMR INSERM 1301 CNRS U5070 UT ENVT EFS Toulouse France

Toulouse Institute for Infectious and Inflammatory Diseases Toulouse University CNRS Inserm Toulouse France

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