Episignature analysis of moderate effects and mosaics

Abstract

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.

Nanopore sequencing-based episignature detection

“…Interestingly, our classifier did detect the episignature in a mosaic heterozygous patient (EPI_19), albeit with a lower confidence score, reflecting the low-grade mosaicism for the pathogenic variant. Mosaicism detection can be improved by including mosaic samples in the training set and lowering the positive confidence score threshold [33]. Episignature evaluation was important in this boy as it contributed to classifying the X-linked ATRX variant as benign and reassuring the parents of low recurrence risk for future pregnancies.…”

Chromatinopathies: insight in clinical aspects and underlying epigenetic changes

“…It also facilitates the functional evaluation of variants of unknown significance and the characterization of newly recognized syndromes, such as Pilarowski-Bjornsonn syndrome and Beck-Fahrner syndrome (Levy et al 2021;Beck et al 2020;Pilarowski et al 2018;Luperchio et al 2021). Episignature profiling has also been applied in mosaic variants evaluation, including low-level mosaicism, in such epigenes as KMT2C and KMT2D, resulting in Kleefstra 2 syndrome and Kabuki syndrome 1, respectively (Montano et al 2022;Oexle et al 2023). Moreover, DNA methylation biomarkers can also be used for disease monitoring when precision-targeted treatments are applied to reverse the DNA methylation episignature.…”

2023 in the European Journal of Human Genetics

“…DNA episignatures are well established to define variant pathogenicity for an increasing range of genes. Oexle report epigenetic effects of mosaic variants and hypomorphic variants [35]. EJHG reported emerging DNA methylation signatures for HNRNPU variants [36] and Renpenning syndrome [37].…”

“…This study provides this independent evaluation, with appropriate methods and interesting results that are extremely useful for clinical practice." Dr Orsetta Zuffardi selected Oxele et al [35]: "The authors were able to increase the sensitivity of the episignature-classifiers by 30%, giving novel insights. Indeed, they demonstrate that both hypomorphic variants and mosaics can lead to minimal alterations of genome methylation which are associated with relatively mild phenotypes or late-onset focal diseases such as late-onset focal dystonia.…”