Genome‐wide DNA methylation profiling confirms a case of low‐level mosaic Kabuki syndrome 1

Abstract Kabuki syndrome is a Mendelian disorder of the epigenetic machinery characterized by typical dysmorphic features, intellectual disability, and postnatal growth deficiency. Pathogenic variants in the genes encoding the chromatin modifiers KMT2D and KDM6A are responsible for Kabuki syndrome 1 (KS1) and Kabuki syndrome 2 (KS2), respectively. In addition, 11 cases of KS1 caused by mosaic variants in KMT2D have been reported in the literature. Some of these individuals display milder craniofacial and growth phenotypes, and most do not have congenital heart defects. We report the case of an infant with severe hypoplastic left heart syndrome with mitral atresia and aortic atresia (HLHS MA‐AA), pulmonary vein stenosis, and atypical facies with a somatic mosaic de novo nonsense variant in KMT2D (c.8200C>T, p.R2734*) identified on trio exome sequencing of peripheral blood and present in 11.2% of sequencing reads. KS was confirmed with EpiSign, a diagnostic genome‐wide DNA methylation platform used to identify epigenetic signatures. This case suggests that use of this newly available clinical test can guide the interpretation of low‐level mosaic variants identified through sequencing and suggests a new lower limit of mosaicism in whole blood required for a diagnosis of KS.

(including aortic coarctation, aortic stenosis, bicuspid aortic valve, and hypoplastic left heart syndrome) Murakami et al., 2020). The KS phenotype changes over time: typical craniofacial features, and especially eye and eyebrow findings, are often not evident in the newborn period and become more easily recognizable between ages 3 and 12 years (Dentici et al., 2015;Vaux et al., 2005).
KS is caused by hypomorphic or inactivating variants in either one of two genes encoding enzymatic components of the epigenetic machinery with complementary writing and erasing functions. The two forms of KS exhibit highly similar phenotypes (Fahrner & Bjornsson, 2014). Kabuki syndrome 1 (KS1, MIM #147920) is an autosomal dominant disorder resulting from heterozygous typically de novo loss-of-function variants in KMT2D, which encodes an epigenetic histone H3K4 methyltransferase writer (Ng et al., 2010). Kabuki syndrome 2 (KS2 MIM #300867) is an X-linked disorder caused by variants in the lysine demethylase KDM6A, an epigenetic eraser that removes trimethylation from H3K27 (Dentici et al., 2015;Lederer et al., 2012;Miyake et al., 2013). Pathogenic variants in KMT2D and KDM6A account for approximately 56%-75% and 5%-8% of KS cases, respectively (Bögershausen et al., 2016). Deficiency of KMT2D or KDM6A interferes with the opening of chromatin, affecting transcription of downstream targets across multiple cell types (Boniel et al., 2021;Fahrner & Bjornsson, 2019) and disrupting genomic DNA methylation (Aref-Eshghi, Fahrner & Bjornsson, 2019;Sobreira et al., 2017). Individuals with KS exhibit a highly sensitive and specific DNA methylation profile, or "episignature," in blood that can be used as a biomarker to complement standard clinical diagnostic tools in cases with ambiguous clinical phenotypes or inconclusive genetic test results (Aref-Eshghi et al., 2018;.
For example, episignatures have been used to reclassify variants of uncertain significance (VUSs) as likely benign or pathogenic in equivocal cases of KS (Aref-Eshghi et al., 2018;Aref-Eshghi, Bend, et al., 2019;Aref-Eshghi, Bourque, et al., 2019;. Somatic mosaicism-the presence of two or more populations of cells with distinct genotypes in a single organism occurring postzygotically-has been detected in a subset of patients with classical clinical features of KS. A recent retrospective study of 12,000 individuals estimated that 1.5% of all molecular diagnoses were attributed to a mosaic variant detected in proband blood samples. Of those, mosaic variants in KMT2D represented 1.6% (2/120) of all detected mosaic variants in the analyzed cohort (Cao et al., 2019). To our knowledge, 11 individuals mosaic for KMT2D variants have been reported in the literature with alternate allele frequencies ranging from 10% to 37% in blood (Table 1) (Banka et al., 2013;Lepri et al., 2017;Manheimer et al., 2018;Murakami et al., 2020). Some showed milder phenotypes including average heights and potentially less distinctive facial features, and most (7/11) do not have congenital heart defects (Banka et al., 2013;Cao et al., 2019;Lepri et al., 2017;Manheimer et al., 2018;Murakami et al., 2020). However, 18% (2/11) had hypoplastic left heart with aortic and mitral atresia (HLHS MA-AA; Table 1). Both individuals were mosaic for frameshift variants, in agreement with the 5%-13% of HLHS found among patients with KS Murakami et al., 2020).
We present a case of an infant with HLHS MA-AA, exophthalmos due to shallow orbits, and evolving facial features consistent with KS1 found to have a mosaic nonsense variant in KMT2D at a low allele fraction (11.2%). Given the somewhat nonclassical clinical presentation and low level of mosaicism in blood, we complemented trio whole exome sequencing with DNA methylation profiling, which together confirmed the diagnosis of KS1.

| DNA methylation data analysis
Methylation analysis was performed with the clinically validated Epi-Sign assay as previously described (Aref-Eshghi et al., 2020;Aref-Eshghi, Bend, et al., 2019;Aref-Eshghi, Bourque, et al., 2019;Sadikovic et al., 2021). Briefly, methylated and unmethylated signal intensity generated from the EPIC array was imported into R 3.5.1 for normalization, background correction, and filtering. Beta values ranging from 0 (no methylation) to 1 (complete methylation) were calculated as a measure of methylation level and processed through the

| DISCUSSION
The patient reported in this study presented with HLHS MA-AA, pulmonary vein stenosis, hypotonia, developmental delay, and feeding difficulties. His craniofacial features included long palpebral fissures (>2 SD above the mean for age) with eversion of the lateral third of the lower eyelid, arched, and broad eyebrows with notching and lateral sparseness, a short columella, prominent ears, and persistent fingertip and toe pads, meeting definite diagnostic clinical criteria for KS (Adam et al., 2019). His developmental delay could be attributable to being hospitalized and critically ill for most of his life. In addition, he also had exophthalmos due to shallow orbits and a short nose with depressed nasal bridge less typical of KS. Whole exome sequencing revealed low-level mosaicism for a de novo pathogenic variant in KMT2D (allele frequency of 11.2%). Congenital heart disease has been detected in 70% of patients with KMT2D pathogenic variants, including 47% with left sided obstructive lesions (mostly aortic coarctation and bicuspid aortic valve). Among patients with KS, HLHS has been reported in 5%-13% of cases Murakami et al., 2020). The severity of his heart disease and low allele frequency found in peripheral blood prompted us to confirm the diagnosis of KS1 using the EpiSign genome-wide DNA methylation assay. Such episignatures have been used to diagnose KS in individuals with suggestive phenotype but negative or inconclusive genetic testing and to reclassify variants of uncertain significance as likely pathogenic or F I G U R E 2 EpiSign DNA methylation analysis of peripheral blood from a patient with a somatic mosaic nonsense variant in KMT2D, the causative gene for Kabuki syndrome 1 (KS1). (a) Hierarchical clustering and (b) multidimensional scaling plots indicate that the patient (red) has a DNA methylation signature similar to subjects with a confirmed KS episignature (blue) and distinct from controls (green). Each row of the heatmap represents one CpG probe on the DNA methylation array, and each column represents one individual's sample. The heatmap color scale from blue to red represents the DNA methylation level (beta value) from 0 (no methylation) to 1 (fully methylated). (c) MVP score, a multiclass supervised classification system capable of discerning between multiple episignatures by generating a probability score for each episignature. The elevated patient score for KS compared to other syndromes suggests an episignature similar to the KS reference signature; however, the decreased probability compared to what is typically observed for constitutional KS (score >0.5) likely results from the low-level mosaicism of the variant benign (Aref-Eshghi et al., 2018;Aref-Eshghi, Bend, et al., 2019;Aref-Eshghi, Bourque, et al., 2019;. At this time, episignatures have only been established in blood. Mosaic variation poses a significant challenge for disease prognosis and accurate prediction of future manifestations. Whereas mosaic variation is known to cause disease, recognizing underlying mosaicism is challenging without overt cutaneous manifestation or when phenotypes deviate from what has been reported in patients with the nonmosaic variant (Biesecker & Spinner, 2013). Fortunately, the widespread use of exome sequencing has facilitated the detection of lowlevel mosaicism in blood (Gajecka, 2016). Approximately 1.5% of all molecular diagnoses from a cohort of 12,000 individuals were attributed to a mosaic variant detected in the proband (Cao et al., 2019).
Individuals with KS1 resulting from pathogenic mosaic frameshift KMT2D variants described thus far have presented with typical to more mild facial features and have varying degrees of cardiovascular involvement. Of those, HLHS has been described in 18% (2 out of 11 cases; see Table 1) (Banka et al., 2013;Cao et al., 2019;Lepri et al., 2017;Manheimer et al., 2018;Murakami et al., 2020).
The proportion of variants shared across tissues is predicted to vary according to the stage of embryonic development at which the variant arose. It is often difficult to pinpoint precisely when a particular pathogenic variant occurred during development, and different mosaic ratios across tissues can affect the phenotypic presentation.
Variants acquired later in development are predicted to have lower alternative allele frequencies in peripheral blood (Ju et al., 2017). We detected a somatic variant affecting heart development using peripheral blood, a tissue that is also derived from mesoderm. We therefore suspect that constitutional somatic mosaicism is responsible for the congenital heart defect and other systemic manifestations of KS1 in the proband, although we did not have tissue available to confirm the level of cardiac mosaicism.
We used DNA methylation profiling as an orthogonal method to confirm the diagnosis and observed a highly specific KS1 episignature, thus expanding the clinical utility of this method. EpiSign is validated to diagnose over 40 neurodevelopmental genetic conditions (including KS) and as a functional assay to resolve variants of uncertain significance in genes with a known epigenetic signature Our report adds to the published spectrum of KMT2D mosaic phenotypes and suggests that in some cases making a diagnosis of KS warrants high-depth sequencing coverage of KMT2D to uncover potential mosaicism. Ours is the first report demonstrating the utility of DNA methylation profiling as a valuable functional diagnostic test in cases of KS with ambiguous phenotypes and low-level mosaicism.
This case provides yet another example illustrating the power of utilizing combinatorial genomics and epigenomics approaches to unambiguously identify and interpret different types of genetic variation in order to optimize diagnostic capabilities.