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Goodman et al. J Transl Genet Genom 2020;4:144-58 I http://dx.doi.org/10.20517/jtgg.2020.23 Page 145

Abstract
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.

Keywords: EHMT1 , Kleefstra syndrome, DNA methylation, signature, epigenetics, copy number variation,
neurodevelopmental disorder

INTRODUCTION
Epigenetic regulators, including chromatin remodelers and enzymes that write, read or erase epigenetic
[1,2]
marks, are essential to healthy human development . Chromatin states and epigenetic patterns play a key
[3,4]
role in regulating transcriptional profiles specific to cellular identity and developmental timing . This is
especially important during neurodevelopment for which developmental processes necessitate a complex
[5,6]
orchestration of gene expression and environmental signals . Recent research into the role of epigenetic
regulators in human disease has demonstrated that genes encoding this machinery, termed “epigenes”, are
[2,7]
linked to Mendelian neurodevelopmental disorders (NDDs) . To date, approximately 70 epigenes have
been implicated in 82 distinct conditions, many of which are characterized by intellectual disability (ID)
[8,9]
and growth dysregulation .
At the molecular level, our group and others have found that these NDDs are characterized by aberrant
DNA methylation (DNAm) patterns [10,11] . DNAm refers to the addition of a methyl group to cytosine,
typically in the context of a cytosine-guanine dinucleotide or CpG. CpG dense regions, known as
CpG islands, are found at 70% of gene promoters; in this context, DNAm is usually a repressive mark,
corresponding to the silencing of gene activity . DNAm found in enhancers, gene bodies and intergenic
[12]
regions have a more complex relationship with gene activity and may associate with the repression or
activation of genes. Importantly, there is cross-talk between epigenetic marks, such as DNAm and histone
3 lysine 9 (H3K9) trimethylation, exhibiting spatial and temporal co-localization [13,14] . As such, mutations
in epigenes encoding enzymes involved in regulation of histone modifications and chromatin packaging