Episignatures in practice: independent evaluation of published episignatures for the molecular diagnostics of ten neurodevelopmental disorders
Authors: Husson Thomas , Lecoquierre François , Nicolas Gaël , Richard Anne-Claire , Afenjar Alexandra , Audebert-Bellanger Séverine , Badens Catherine , Bilan Frédéric , Bizaoui Varoona , Boland Anne , Bonnet-Dupeyron Marie-Noëlle , Brischoux-Boucher Elise , Bonnet Céline , Bournez Marie , Boute Odile , Brunelle Perrine , Caumes Roseline , Charles Perrine , Chassaing Nicolas , Chatron Nicolas , Cogné Benjamin , Colin Estelle , Cormier-Daire Valérie , Dard Rodolphe , Dauriat Benjamin , Delanne Julian , Deleuze Jean-François , Demurger Florence , Denommé-Pichon Anne-Sophie , Depienne Christel , Dieux Anne , Dubourg Christèle , Edery Patrick , El Chehadeh Salima , Faivre Laurence , Fergelot Patricia , Fradin Mélanie , Garde Aurore , Geneviève David , Gilbert-Dussardier Brigitte , Goizet Cyril , Goldenberg Alice , Gouy Evan , Guerrot Anne-Marie , Guimier Anne , Harzalla Inès , Héron Delphine , Isidor Bertrand , Lacombe Didier , Le Guillou Horn Xavier , Keren Boris , Kuechler Alma , Lacaze Elodie , Lavillaureix Alinoë , Lehalle Daphné , Lesca Gaëtan , Lespinasse James , Levy Jonathan , Lyonnet Stanislas , Morel Godeliève , Jean-Marçais Nolwenn , Marlin Sandrine , Marsili Luisa , Mignot Cyril , Nambot Sophie , Nizon Mathilde , Olaso Robert , Pasquier Laurent , Perrin Laurine , Petit Florence , Pingault Veronique , Piton Amélie , Prieur Fabienne , Putoux Audrey , Planes Marc , Odent Sylvie , Quélin Chloé , Quemener-Redon Sylvia , Rama Mélanie , Rio Marlène , Rossi Massimiliano , Schaefer Elise , Rondeau Sophie , Saugier-Veber Pascale , Smol Thomas , Sigaudy Sabine , Touraine Renaud , Mau-Them Frederic Tran , Trimouille Aurélien , Van Gils Julien , Vanlerberghe Clémence , Vantalon Valérie , Vera Gabriella , Vincent Marie , Ziegler Alban , Guillin Olivier , Campion Dominique , Charbonnier Camille 
Published: October 2023

Variants of uncertain significance (VUS) are a significant issue for the molecular diagnosis of rare diseases. The publication of episignatures as effective biomarkers of certain Mendelian neurodevelopmental disorders has raised hopes to help classify VUS. However, prediction abilities of most published episignatures have not been independently investigated yet, which is a prerequisite for an informed and rigorous use in a diagnostic setting. We generated DNA methylation data from 101 carriers of (likely) pathogenic variants in ten different genes, 57 VUS carriers, and 25 healthy controls. Combining published episignature information and new validation data with a k-nearest-neighbour classifier within a leave-one-out scheme, we provide unbiased specificity and sensitivity estimates for each of the signatures. Our procedure reached 100% specificity, but the sensitivities unexpectedly spanned a very large spectrum. While ATRX, DNMT3A, KMT2D, and NSD1 signatures displayed a 100% sensitivity, CREBBP-RSTS and one of the CHD8 signatures reached <40% sensitivity on our dataset. Remaining Cornelia de Lange syndrome, KMT2A, KDM5C and CHD7 signatures reached 70–100% sensitivity at best with unstable performances, suffering from heterogeneous methylation profiles among cases and rare discordant samples. Our results call for cautiousness and demonstrate that episignatures do not perform equally well. Some signatures are ready for confident use in a diagnostic setting. Yet, it is imperative to characterise the actual validity perimeter and interpretation of each episignature with the help of larger validation sample sizes and in a broader set of episignatures.