Truncating SRCAP variants outside the Floating-Harbor syndrome locus cause a distinct neurodevelopmental disorder with a specific DNA methylation signature.

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.

DNA Methylation of the Oxytocin Receptor Across Neurodevelopmental Disorders.

Many neurodevelopmental disorders (NDDs) share common learning and behavioural impairments, as well as features such as dysregulation of the oxytocin hormone. Here, we examined DNA methylation (DNAm) in the 1st intron of the oxytocin receptor gene, OXTR, in patients with autism spectrum (ASD), attention deficit and hyperactivity (ADHD) and obsessive compulsive (OCD) disorders. DNAm of OXTR was assessed for cohorts of ASD (blood), ADHD (saliva), OCD (saliva), which uncovered sex-specific DNAm differences compared to neurotypical, tissue-matched controls. Individuals with ASD or ADHD exhibiting extreme DNAm values had lower IQ and more social problems, respectively, than those with DNAm within normative ranges. This suggests that OXTR DNAm patterns are altered across NDDs and may be correlated with common clinical outcomes.

Obsessive-compulsive disorder and attention-deficit/hyperactivity disorder: distinct associations with DNA methylation and genetic variation.

BACKGROUND: A growing body of research has demonstrated associations between specific neurodevelopmental disorders and variation in DNA methylation (DNAm), implicating this molecular mark as a possible contributor to the molecular etiology of these disorders and/or as a novel disease biomarker. Furthermore, genetic risk variants of neurodevelopmental disorders have been found to be enriched at loci associated with DNAm patterns, referred to as methylation quantitative trait loci (mQTLs). METHODS: We conducted two epigenome-wide association studies in individuals with attention-deficit/hyperactivity disorder (ADHD) or obsessive-compulsive disorder (OCD) (aged 4-18 years) using DNA extracted from saliva. DNAm data generated on the Illumina Human Methylation 450 K array were used to examine the interaction between genetic variation and DNAm patterns associated with these disorders. RESULTS: Using linear regression followed by principal component analysis, individuals with the most endorsed symptoms of ADHD or OCD were found to have significantly more distinct DNAm patterns from controls, as compared to all cases. This suggested that the phenotypic heterogeneity of these disorders is reflected in altered DNAm at specific sites. Further investigations of the DNAm sites associated with each disorder revealed that despite little overlap of these DNAm sites across the two disorders, both disorders were significantly enriched for mQTLs within our sample. CONCLUSIONS: Our DNAm data provide insights into the regulatory changes associated with genetic variation, highlighting their potential utility both in directing GWAS and in elucidating the pathophysiology of neurodevelopmental disorders.

New insights into DNA methylation signatures: SMARCA2 variants in Nicolaides-Baraitser syndrome.

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. CONCLUSIONS: Here we find that a DNAm signature of SMARCA2 pathogenic variants in NCBRS maps to CpGs relevant to disorder pathophysiology, classifies VUS, and is sensitive to the position of the variant in SMARCA2. The patient with an intermediate model score demonstrating a unique genotype-epigenotype-phenotype correlation underscores the potential utility of this signature as a functionally relevant VUS classification system scalable beyond binary "benign" versus "pathogenic" scoring. This is a novel feature of DNAm signatures that could enable phenotypic predictions from genotype data. Our findings also demonstrate that DNAm signatures can be domain-specific, highlighting the precision with which they can reflect genotypic variation.

Epigenetics of Autism Spectrum Disorder.

Autism spectrum disorder (ASD), one of the most common childhood neurodevelopmental disorders (NDDs), is diagnosed in 1 of every 68 children. ASD is incredibly heterogeneous both clinically and aetiologically. The etiopathogenesis of ASD is known to be complex, including genetic, environmental and epigenetic factors. Normal epigenetic marks modifiable by both genetics and environmental exposures can result in epigenetic alterations that disrupt the regulation of gene expression, negatively impacting biological pathways important for brain development. In this chapter we aim to summarize some of the important literature that supports a role for epigenetics in the underlying molecular mechanism of ASD. We provide evidence from work in genetics, from environmental exposures and finally from more recent studies aimed at directly determining ASD-specific epigenetic patterns, focusing mainly on DNA methylation (DNAm). Finally, we briefly discuss some of the implications of current research on potential epigenetic targets for therapeutics and novel avenues for future work.

CHARGE and Kabuki Syndromes: Gene-Specific DNA Methylation Signatures Identify Epigenetic Mechanisms Linking These Clinically Overlapping Conditions.

Epigenetic dysregulation has emerged as a recurring mechanism in the etiology of neurodevelopmental disorders. Two such disorders, CHARGE and Kabuki syndromes, result from loss of function mutations in chromodomain helicase DNA-binding protein 7 (CHD7LOF) and lysine (K) methyltransferase 2D (KMT2DLOF), respectively. Although these two syndromes are clinically distinct, there is significant phenotypic overlap. We therefore expected that epigenetically driven developmental pathways regulated by CHD7 and KMT2D would overlap and that DNA methylation (DNAm) alterations downstream of the mutations in these genes would identify common target genes, elucidating a mechanistic link between these two conditions, as well as specific target genes for each disorder. Genome-wide DNAm profiles in individuals with CHARGE and Kabuki syndromes with CHD7LOF or KMT2DLOF identified distinct sets of DNAm differences in each of the disorders, which were used to generate two unique, highly specific and sensitive DNAm signatures. These DNAm signatures were able to differentiate pathogenic mutations in these two genes from controls and from each other. Analysis of the DNAm targets in each gene-specific signature identified both common gene targets, including homeobox A5 (HOXA5), which could account for some of the clinical overlap in CHARGE and Kabuki syndromes, as well as distinct gene targets. Our findings demonstrate how characterization of the epigenome can contribute to our understanding of disease pathophysiology for epigenetic disorders, paving the way for explorations of novel therapeutics.

Genome-wide characteristics of de novo mutations in autism.

De novo mutations (DNMs) are important in Autism Spectrum Disorder (ASD), but so far analyses have mainly been on the ~1.5% of the genome encoding genes. Here, we performed whole genome sequencing (WGS) of 200 ASD parent-child trios and characterized germline and somatic DNMs. We confirmed that the majority of germline DNMs (75.6%) originated from the father, and these increased significantly with paternal age only (p=4.2×10-10). However, when clustered DNMs (those within 20kb) were found in ASD, not only did they mostly originate from the mother (p=7.7×10-13), but they could also be found adjacent to de novo copy number variations (CNVs) where the mutation rate was significantly elevated (p=2.4×10-24). By comparing DNMs detected in controls, we found a significant enrichment of predicted damaging DNMs in ASD cases (p=8.0×10-9; OR=1.84), of which 15.6% (p=4.3×10-3) and 22.5% (p=7.0×10-5) were in the non-coding or genic non-coding, respectively. The non-coding elements most enriched for DNM were untranslated regions of genes, boundaries involved in exon-skipping and DNase I hypersensitive regions. Using microarrays and a novel outlier detection test, we also found aberrant methylation profiles in 2/185 (1.1%) of ASD cases. These same individuals carried independently identified DNMs in the ASD risk- and epigenetic- genes DNMT3A and ADNP. Our data begins to characterize different genome-wide DNMs, and highlight the contribution of non-coding variants, to the etiology of ASD.

A role for glutathione, independent of oxidative stress, in the developmental toxicity of methanol.

Oxidative stress and reactive oxygen species (ROS) have been implicated in the teratogenicity of methanol (MeOH) in rodents, both in vivo and in embryo culture. We explored the ROS hypothesis further in vivo in pregnant C57BL/6J mice. Following maternal treatment with a teratogenic dose of MeOH, 4 g/kg via intraperitoneal (ip) injection on gestational day (GD) 12, there was no increase 6h later in embryonic ROS formation, measured by 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA) fluorescence, despite an increase observed with the positive control ethanol (EtOH), nor was there an increase in embryonic oxidatively damaged DNA, quantified as 8-oxo-2'-deoxyguanosine (8-oxodG) formation. MeOH teratogenicity (primarily ophthalmic anomalies, cleft palate) also was not altered by pre- and post-treatment with varying doses of the free radical spin trapping agent alpha-phenyl-N-tert-butylnitrone (PBN). In contrast, pretreatment with L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, depleted maternal hepatic and embryonic GSH, and enhanced some new anomalies (micrognathia, agnathia, short snout, fused digits, cleft lip, low set ears), but not the most common teratogenic effects of MeOH (ophthalmic anomalies, cleft palate) in this strain. These results suggest that ROS did not contribute to the teratogenic effects of MeOH in this in vivo mouse model, in contrast to results in embryo culture from our laboratory, and that the protective effect of GSH in this model may arise from its role as a cofactor for formaldehyde dehydrogenase in the detoxification of formaldehyde.

Methanol teratogenicity in mutant mice with deficient catalase activity and transgenic mice expressing human catalase.

The role of catalase in methanol (MeOH) teratogenesis is unclear. In rodents it both detoxifies reactive oxygen species (ROS) and metabolizes MeOH and its formic acid (FA) metabolite. We treated pregnant mice expressing either high (hCat) or low catalase activity (aCat), or their wild-type (WT) controls, with either MeOH (4g/kg ip) or saline. hCat mice and WTs were similarly susceptible to MeOH-initiated ophthalmic abnormalities and cleft palates. aCat and WT mice appeared resistant, precluding assessment of the developmental impact of catalase deficiency. Catalase activity was respectively increased at least 1.5-fold, and decreased by at least 35%, in hCat and aCat embryos and maternal livers. MeOH and FA pharmacokinetic profiles were similar among hCat, aCat and WT strains. Although the hCat results imply no ROS involvement, embryo culture studies suggest this may be confounded by maternal factors and/or a requirement for higher catalase activity in the hCat mice.