Effect of depletion of PNPLA1 in gene expression during keratinocyte differentiation on reconstructed human epidermis

Congenital ichthyoses are rare and disabling genodermatoses caused by mutations in genes involved in the epidermal barrier. More than 60 genes are known to be involved. However, 5-10% of the genetic variants identified in those genes are rarely or not described and their pathogenicity not yet demonstrated. They are therefore classified as Variants of Uncertain Significance (VUS) that leave number of patients undiagnosed, precluding prognosis, counselling and treatment. We report here, an experimental strategy to reclassify VUS identified in congenital ichthyosis-causing genes tested for PNPLA1 variants. PNPLA is a transacylase essential for the skin barrier formation. It catalyzes the biosynthesis of acylceramides (AcylCer) in the stratum granulosum by esterification of ?-hydroxy ceramides with linoleic acid. We successfully characterized three PNPLA1 VUS identified in three patients with congenital ichthyosis, opening the possibility to extend the approach to other ichthyosis-causing genes. Overall design: We generated immortalized keratinocyte cell lines (N/TERT-2G) CrisprCas9-knockout (KO) for the ichthyosis-causing gene PNPLA1. Three PNPLA1 VUS were ectopically expressed in the corresponding KO cell line by lentiviral transduction and the resulting cells were used to produce reconstructed human epidermis (RHE). To further characterized the KO PNPLA1 icthyosis model, we performed gene expression profiling analysis using data obtained by RNAseq of WT reconstructed epidermis vs 3 PNPLA1 KO clones