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Page 156 Goodman et al. J Transl Genet Genom 2020;4:144-58 I http://dx.doi.org/10.20517/jtgg.2020.23

finding speaks to the potential of DNAm signatures for clinical translation, i.e., to be used in concert with
genome or exome sequencing to enhance interpretability of genome diagnostics.

A considerable strength of previously reported DNAm signatures is their utility in predicting the
pathogenicity of variants of uncertain significance (VUS) [10,11,15] . Pathogenic missense variants are rare but
[49]
present in KS , Thus, we sought to assess the power/utility of the KS signature in classifying missense
variants. We included one patient in the discovery group used to derive the DNAm signature, who carried
a pathogenic missense variant, P809R, and therefore expected the KS DNAm signature to have clinical
utility in this regard. Of note, this variant was predicted to be pathogenic/damaging in Polyphen (1.000),
Align GVGD (C65), and SIFT (0). We included three individuals with EHMT1 missense variants in the
“test” group and found that each was classified as negative for KS, with controls. Th e clinical information
for these individuals, although limited, was consistent with the DNAm signature prediction.

Although Kleefstra syndrome has a clinically recognizable phenotype, affected individuals exhibit a range
of cognitive and behavioral characteristics. Currently, there is little understanding of the relationship
between genotype and phenotype. The complex genotype-phenotype relationship in KS will require further
study using a large KS cohort with well characterized phenotypes. Our work presented here demonstrates
that EHMT1 variants and 9q34.3 deletions share a DNAm signature, further supporting the underlying
cause of KS as EHMT1 haploinsufficiency. Further epigenetic research in KS has the potential to elucidate
the relationship between genotype and phenotype by refining the DNAm signature and identifying DNAm
alterations associated with specific features of KS or specific molecular variants. Therefore, building upon
this work to identify genes with altered regulation and expression patterns in KS will provide novel insights
into the molecular pathophysiology of this disorder.

DECLARATIONS
Acknowledgments
We would like to thank all of the patients and families for participating in our research studies and the
physicians, clinical staff and research staff for their assistance with patient recruitment. We would also like
to thank Chunhua Zhang, Youliang Lou and Khadine Wiltshire for their contributions to this work.

Authors’ contributions
Analyzed and interpreted the data, generated figures/tables, and wrote the manuscript: Goodman SJ
Collected clinical data, enrolled patients, managed collaborations, integrated clinical findings: Cytrynbaum
C
Provided DNA samples and clinical data and provided input on study design and manuscript preparation:
Chung BHY, Kellam B, Keller M, Ko JM, Caluseriu O, Grafodatskaya D, McCready E, Perrier R, Yeung KS,

Ho-Ming L, Machado J, Stavropoulos DJ, Scherer SW,Innes AM, Cheung SW
Generated figures and analyzed data: Aziz C
Wrote the R scripts used, performed statistical analyses, and contributed to the manuscript: Turinsky AL
Provided intellectual contribution to the analytical pipeline: Brudno M
Assisted with study design and manuscript preparation: Choufani S
Is the principal investigator and was involved in all aspects of the study: Weksberg R
Have read and approved the manuscript: All authors

Availability of data and materials
The microarray data will be made available upon request.

Financial support and sponsorship
This research was funded by the Canadian Institutes of Health Research (CIHR), the Province of Ontario