EHMT1 pathogenic variants and 9q34.3 microdeletions share altered DNA methylation patterns in patients with Kleefstra syndrome

Aim: Kleefstra syndrome (KS) is a rare neurodevelopmental disorder caused by haploinsufficiency of the euchromatic histone lysine methyltransferase 1 gene, EHMT1, due to either a submicroscopic 9q34.3 deletion or a pathogenic EHMT1 variant. KS is characterized by intellectual disability, autistic-like features, heart defects, hypotonia and distinctive facial features. Here, we aimed to (1) identify a unique DNA methylation signature in patients with KS, and (2) demonstrate the efficacy of DNA methylation in predicting the pathogenicity of copy number and sequence variants.Methods: We assayed genome-wide DNA methylation at > 850,000 CpG sites in the blood of KS patients (n = 10) carrying pathogenic variants in EHMT1 or 9q34.3 deletions, as compared to neurotypical controls (n = 42). Differentially methylated sites were validated using additional KS patients (n = 10) and controls (n = 29) to assess specificity and sensitivity of these patterns.Results: The DNA methylation signature of KS demonstrated high sensitivity and specificity; controls and KS patients with a confirmed molecular diagnosis were classified correctly. In additional individuals with EHMT1 alterations, including frameshift or missense variants and partial gene duplications, DNA methylation classifications were consistent with clinical presentation. Furthermore, genes containing differentially methylated CpG sites were enriched for functions related to KS features, including heart formation and synaptic activity.Conclusion: The KS DNA methylation signature did not differ in patients with deletions and variants, supporting haploinsufficiency of EHMT1 as the likely causative mechanism. Beyond this finding, it provides new insights into epigenetic dysregulation associated with KS and can be used to classify individuals with uncertain genomic findings or ambiguous clinical presentations.