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Page 146 Goodman et al. J Transl Genet Genom 2020;4:144-58 I http://dx.doi.org/10.20517/jtgg.2020.23

often result in downstream DNA methylation alterations in patients carrying these mutations [10,11,15-17] .
These altered DNAm patterns reflect genome-wide transcriptional changes at the downstream target genes
that constitute the molecular underpinnings of the pathophysiology of the associated NDD. As such,
we hypothesize that through a similar cross-talk mechanism between DNAm and H3K9 methylation,
pathogenic variants in EHMT1 can affect DNAm patterns

Kleefstra syndrome or KS (MIM 610253) is caused by haploinsufficiency of the euchromatic histone lysine
methyltransferase 1 gene, EHMT1 [18,19] . EHMT1 encodes a histone methyltransferase that catalyzes mono-
and dimethylation of H3K9. As an important transcriptional repressor, EHMT1 is expressed in most
[20]
human tissues and is overexpressed in a variety of human cancers . KS is caused by either a heterozygous
9q subtelomeric deletion, which overlaps with part or all of EHMT1 (50%) or a heterozygous pathogenic
variant in EHMT1, including frameshift, missense and nonsense mutations (50%) [21,22] . Features of KS
include moderate to severe ID, childhood hypotonia, seizures, heart defects and characteristic facial
features including brachy(-micro)cephaly, synophrys, cupid bowed upper lip and prominent jaw [18,23-25] . As
well, urogenital and renal complications, psychiatric disorders, and features of autism spectrum disorder
[22]
(ASD) are often present. Males and females are affected equally . There is some evidence of genotype-
phentoype correlation in that individuals with EHMT1 pathogenic variants and those with a small 9q34.3
deletion (< 1 Mb) have similar clinical findings, whereas individuals with larger deletions (> 1 Mb) can
have more severe ID and more medical problems [19,21,26] .

Here, we report the differential DNAm patterns associated with KS. We use these patterns to derive
a predictive model to classify individuals with EHMT1 sequence variants of uncertain significance or
ambiguous clinical presentations. We found that patients with KS caused by either 9q34.3 microdeletions
or pathogenic EHMT1 variants exhibit a specific DNAm signature that is unique from both typically
developing controls and individuals with similar epigene-related disorders. As well, these differentially
methylated CpG sites map to genes enriched for heart and brain development.

METHODS
Research participants
Informed consent was obtained from all research participants according to the protocol approved by the
Research Ethics Board of the Hospital for Sick Children (REB# 1000038847). Individuals were recruited
through the Division of Clinical and Metabolic Genetics at the Hospital for Sick Children, Toronto,
Ontario; Department of Medical Genetics, Alberta Children’s Hospital Research Institute, Calgary, Alberta;
Baylor College of Medicine, Houston, Texas; Prevention Genetics, Marshfield, Wisconsin; University
Hospitals, Cleveland, Ohio; Seoul National University Children’s Hospital, Seoul, Korea; and Department of
Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario.

KS patients were divided into groups based on age, diagnosis and molecular findings [Table 1] and
availability of clinical phenotype data; importantly, the frequency of deletions and variants in this sample
is reflective of the reported frequency in the greater population of KS cases. The first group encompassed
individuals over the age of one year with a confirmed clinical diagnosis of KS and a pathogenic EHMT1
sequence variant or 9q34 deletion reported by a molecular diagnostic laboratory. These individuals were
separated into discovery and validation groups (n = 10 and n = 5, respectively), such that molecular
underpinnings, sex, and age were represented within each group. For all patients in the discovery cohort,
clinical and molecular diagnoses were defined by certified clinical and molecular geneticists, respectively.
Individuals younger than 1 year represented an additional validation group (n = 5), and individuals
with EHMT1 variants but little or no phenotypic information available at the time of data analysis (n =
5) represented an “unknown” test group. Two additional individuals who both carried duplications of
chromosome 9q34 were also included in analyses. The presence of specific KS features, such as hypotonia,