Weaver syndrome (WS), an overgrowth/intellectual disability syndrome (OGID), is caused by pathogenic variants in the histone methyltransferase EZH2 , which encodes a core component of the Polycomb repressive complex-2 (PRC2). Using genome-wide DNA methylation (DNAm) data for 187 individuals with OGID and 969 control subjects, we show that pathogenic variants in EZH2 generate a highly specific and sensitive DNAm signature reflecting the phenotype of WS. This signature can be used to distinguish loss-of-function from gain-of-function missense variants and to detect somatic mosaicism. We also show that the signature can accurately classify sequence variants in EED and SUZ12 , which encode two other core components of PRC2, and predict the presence of pathogenic variants in undiagnosed individuals with OGID. The discovery of a functionally relevant signature with utility for diagnostic classification of sequence variants in EZH2 , EED , and SUZ12 supports the emerging paradigm shift for implementation of DNAm signatures into diagnostics and translational research.

The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse’s lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as “Free radical scavenging”. We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was “Peroxisome biogenesis”; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

Summary Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein ( SRCAP ) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo ) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as “non-FLHS SRCAP -related NDD.” All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP , there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.

Background: Nicolaides-Baraitser syndrome (NCBRS) is a neurodevelopmental disorder caused by pathogenic sequence variants in SMARCA2 which encodes the catalytic component of the chromatin remodeling BAF complex. Pathogenic variants in genes that encode epigenetic regulators have been associated with genome-wide changes in DNA methylation (DNAm) in affected individuals termed DNAm signatures. Methods: Genome-wide DNAm was assessed in whole-blood samples from the individuals with pathogenic SMARCA2 variants and NCBRS diagnosis (n = 8) compared to neurotypical controls (n = 23) using the Illumina MethylationEPIC array. Differential methylated CpGs between groups (DNAm signature) were identified and used to generate a model enabling classification variants of uncertain significance (VUS; n = 9) in SMARCA2 as "pathogenic" or "benign". A validation cohort of NCBRS cases (n = 8) and controls (n = 96) demonstrated 100% model sensitivity and specificity. Results: We identified a DNAm signature of 429 differentially methylated CpG sites in individuals with NCBRS. The genes to which these CpG sites map are involved in cell differentiation, calcium signaling, and neuronal function consistent with NCBRS pathophysiology. DNAm model classifications of VUS were concordant with the clinical phenotype; those within the SMARCA2 ATPase/helicase domain classified as "pathogenic". A patient with a mild neurodevelopmental NCBRS phenotype and a VUS distal to the ATPase/helicase domain did not score as pathogenic, clustering away from cases and controls. She demonstrated an intermediate DNAm profile consisting of one subset of signature CpGs with methylation levels characteristic of controls and another characteristic of NCBRS cases; each mapped to genes with ontologies consistent with the patient's unique clinical presentation.

The additional sex combs-like (ASXL) gene family—encoded by ASXL1, ASXL2, and ASXL3—is crucial for mammalian development. Pathogenic variants in the ASXL gene family are associated with three phenotypically distinct neurodevelopmental syndromes. Our previous work has shown that syndromic conditions caused by pathogenic variants in epigenetic regulatory genes show consistent patterns of genome-wide DNA methylation (DNAm) alterations, i.e., DNAm signatures in peripheral blood. Given the role of ASXL1 in chromatin modification, we hypothesized that pathogenic ASXL1 variants underlying Bohring-Opitz syndrome (BOS) have a unique DNAm signature. We profiled whole-blood DNAm for 17 ASXL1 variants, and 35 sex- and age-matched typically developing individuals, using Illumina’s Infinium EPIC array. We identified 763 differentially methylated CpG sites in individuals with BOS. Differentially methylated sites overlapped 323 unique genes, including HOXA5 and HOXB4, supporting the functional relevance of DNAm signatures. We used a machine-learning classification model based on the BOS DNAm signature to classify variants of uncertain significance in ASXL1, as well as pathogenic ASXL2 and ASXL3 variants. The DNAm profile of one individual with the ASXL2 variant was BOS-like, whereas the DNAm profiles of three individuals with ASXL3 variants were control-like. We also used Horvath’s epigenetic clock, which showed acceleration in DNAm age in individuals with pathogenic ASXL1 variants, and the individual with the pathogenic ASXL2 variant, but not in individuals with ASXL3 variants. These studies enhance our understanding of the epigenetic dysregulation underpinning ASXL gene family-associated syndromes.