DNA methylation classifiers ("episignatures") help to determine the pathogenicity of variants of uncertain significance (VUS). However, their sensitivity is limited due to their training on unambiguous cases with strong-effect variants so that the classification of variants with reduced effect size or in mosaic state may fail. Moreover, episignature evaluation of mosaics as a function of their degree of mosaicism has not been developed so far. We improved episignatures with respect to three categories. Applying (i) minimum-redundancy-maximum-relevance feature selection we reduced their length by up to one order of magnitude without loss of accuracy. Performing (ii) repeated re-training of a support vector machine classifier by step-wise inclusion of cases in the training set that reached probability scores larger than 0.5, we increased the sensitivity of the episignature-classifiers by 30%. In the newly diagnosed patients we confirmed the association between DNA methylation aberration and age at onset of KMT2B-deficient dystonia. Moreover, we found evidence for allelic series, including KMT2B-variants with moderate effects and comparatively mild phenotypes such as late-onset focal dystonia. Retrained classifiers also can detect mosaics that previously remained below the 0.5-threshold, as we showed for KMT2D-associated Kabuki syndrome. Conversely, episignature-classifiers are able to revoke erroneous exome calls of mosaicism, as we demonstrated by (iii) comparing presumed mosaic cases with a distribution of artificial in silico-mosaics that represented all the possible variation in degree of mosaicism, variant read sampling and methylation analysis.

Centre de Génétique Humaine Institut de Pathologie et de Génétique ASBL 6041 Gosselies Belgium

Centre for Rare Diseases University of Tuebingen 72076 Tuebingen Germany

Chair of Neurogenetics Technical University of Munich School of Medicine 81675 Munich Germany

Department of Neurology Charles University 1st Faculty of Medicine and General University Hospital Prague 12108 Prague Czech Republic

Department of Neurology Medizinische Universität 6020 Insbruck Austria

Department of Neurology Technical University of Munich School of Medicine 81675 Munich Germany

Department of Pediatric and Adolescent Medicine Medical University of Vienna 1090 Wien Austria

Fondazione IRCCS Istituto Neurologico Carlo Besta 20133 Milano Italy

Institute for Genomic Statistics and Bioinformatics Universität Bonn 53127 Bonn Germany

Institute of Human Genetics School of Medicine University Hospital Bonn 53127 Bonn Germany

Institute of Human Genetics Technical University of Munich School of Medicine 81675 Munich Germany

Institute of Human Genetics Universitätsklinikum Essen 45122 Essen Germany

Institute of Human Genetics Universitätsklinikum Schleswig Holstein 23538 Lübeck Germany

Institute of Medical Genetics and Applied Genomics University of Tuebingen 72076 Tübingen Germany

Institute of Neurogenomics Helmholtz Munich 85764 Neuherberg Germany

Munich Cluster for Systems Neurology 81377 Munich Germany

Neurogenetic Systems Analysis Group Institute of Neurogenomics Helmholtz Munich 85764 Neuherberg Germany

Neuromuscular Research Department Center for Anatomy and Cell Biology Medical University of Vienna 1090 Vienna Austria

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