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Infinium HumanMethylation450 array (450K array; assays ~450,000 CpGs; 90% of which are represented
[42]
on the EPIC array). Therefore all 107 CpG sites in their signature are present on the 450K array . Of our
598 signature sites, approximately half (317) are not represented on the 450K array. Beyond this technical
[42]
difference, different statistical methods were employed for signature generation. The Aref-Eshghi et al.
signature was generated by initially ranking CpG sites on an interaction between P-value and effect size,
with no required minimum P-value or multiple testing correction, and the top 1000 CpG sites underwent
[42]
further analysis, including receiver operating characteristics curve, to select a final set of 100-150 CpGs .
Our signature sites were required to meet stringent significance and effect size thresholds (FDR-corrected
P-value < 0.01, absolute Δb > 10%), with no restrictions on signature size. The differences in methods
and outcomes highlight two important and overlapping applications of DNAm data, i.e., understanding
pathophysiology and use of DNAm data in diagnostics.

GO analysis of the genes identified by our KS signature sites recognized enriched functions and pathways
related to KS pathophysiology. Several processes related to neuronal and synaptic function were identified,
relevant to the high frequency of ID observed in patients with KS. The top term for GO biological processes
was “homophilic cell adhesion via plasma membrane adhesion molecules”. This term was enriched due to
signature sites mapping to CHD4, CDH5, and seven γ-protocadherin genes. Protocadherins are neuronal
cell surface proteins serving several functions including avoidance of dendritic self-synapsing by conferring
[43]
self-identity ; these genes are crucial for normal synaptic development. Aberrant epigenetic regulation of
these genes has also been associated with many NDDs, including Down syndrome and Williams-Beuren
syndrome [44,45] . Furthermore, epigenetic dysregulation of protocadherins has been previously implicated in
+/-
KS pathophysiology; brain tissue of Ehmt1 mice display increased H3K9 methylation at protocadherin
[46]
genes that exhibit dysregulated expression . While both DNA methylation loss and H3K9me2/3
gain support the role of protocadherin dysregulation in KS, we propose that these outcomes may be
paradoxically independent, since EHMT1 has been shown to silence transcription by independently
[47]
acting on both H3K9 and DNAm . In keeping with this model, loci with the greatest changes to H3K9
[46]
+/-
methylation in Ehmt1 mice, showed no consistent DNAm changes . Future studies in human cells are
necessary to show if changes to H3K9 and DNAm marks in individuals with KS are indeed uncoupled
and result from different EHMT1 functions, as proposed here. Also, such studies would greatly benefit
from expression assays in relevant tissue types to directly measure the functional consequences of these
dysregulated epigenetic patterns.

In addition to the 10 KS patients in the validation groups, including five infants, who were positively
classified by our DNAm signature, we tested an additional seven individuals of interest. Within the
validation cases, individuals did not cluster by age [Supplementary Figure 2]; this suggests that the KS
signature is not strongly affected by age and likely independent of the dynamic DNAm changes that occur
[48]
in the first year of life, which occur in part due to normal developmental shifts in blood cell composition .
The remaining seven individuals tested all carried genomic variants at 9q34.3: two individuals with partial
duplications of EHMT1 and five with single nucleotide variants but limited clinical or no information
available. Both patients with duplications were classified as negative for KS, with controls. Such copy
number variants are commonly reported as uncertain, as their impact on gene function cannot be
ascertained via cytogenetic analysis. On the basis of DNAm classifications, we suggest that the clinical
phenotypes of these individuals are likely not related to their EHMT1-asociated CNVs; however, a
functional protein assay in these cases would be valuable to confirm our findings.

Patient U1 was one of five individuals with limited clinical or no information available. This individual had
a diagnosis of ASD and had undergone whole-genome sequencing, which identified a de novo EHMT1
frameshift variant. Following positive classification of this individual, we learned that he exhibited many
features of KS, including mild ID, pulmonary stenosis, dysmorphic facial features and mild hypotonia. This