Patients with a Kabuki syndrome phenotype demonstrate DNA methylation abnormalities

Abstract

Kabuki syndrome is a monogenic disorder caused by loss of function variants in either of two genes encoding histone-modifying enzymes. We performed targeted sequencing in a cohort of 27 probands with a clinical diagnosis of Kabuki syndrome. Of these, 12 had causative variants in the two known Kabuki syndrome genes. In 2, we identified presumptive loss of function de novo variants in KMT2A (missense and splice site variants), a gene that encodes another histone modifying enzyme previously exclusively associated with Wiedermann-Steiner syndrome. Although Kabuki syndrome is a disorder of histone modification, we also find alterations in DNA methylation among individuals with a Kabuki syndrome diagnosis relative to matched normal controls, regardless of whether they carry a variant in KMT2A or KMT2D or not. Furthermore, we observed characteristic global abnormalities of DNA methylation that distinguished patients with a loss of function variant in KMT2D or missense or splice site variants in either KMT2D or KMT2A from normal controls. Our results provide new insights into the relationship of genotype to epigenotype and phenotype and indicate cross-talk between histone and DNA methylation machineries exposed by inborn errors of the epigenetic apparatus.

Fig. 1

Facial features in patients with variants in KMT2A, a Trithorax ortholog (like KMT2D). a, b Facial features (left) in two patients identified as KS29 a and KS8 b led to a clinical diagnosis of Kabuki syndrome. The observed phenotype is summarized (right) and in more detail in Supplementary Table 1. These two patients suggest that there is sometimes phenotypic overlap between Kabuki and Wiedemann-Steiner syndromes, two disorders that are caused by variants in two independent Trithorax orthologs (KMT2A and KMT2D). c The domain structure of the two orthologous proteins. Domain structure is based on InterPro (https://www.ebi.ac.uk/interpro/)

Fig. 2

Individuals with Kabuki syndrome (KS) phenotype and a variant that alters function in KMT2D show site-specific hyper- and hypo-methylation relative to non-Kabuki syndrome controls. (a, b) Genomic loci showing significant relative loss of methylation (CIRBP, FEM1B) in samples from individuals with KMT2D variants that change function compared to controls. (c, d) Genomic loci showing significant gain of methylation (c10orf11, SOX18) in samples from individuals with KMT2D variants that alter function compared to controls. Black points denote non-KS controls. Blue points denote KS patients with KMT2D variants that alter function. Although not included in our main analysis, we also plot methylation values for individuals with Kabuki syndrome and variants of unknown significance (orange) and KMT2A variants (aqua)

Fig. 3

Examples of differentially methylated regions (DMRs) associated with Kabuki syndrome with KMT2D variants (KS-KMT2D) that alter function. (a) An example of a DMR within the MYO1F gene showing KS-KMT2D individuals have relative hypomethylation compared to non-Kabuki controls that we validated via bisulfite pyrosequencing. (b) Relative hypermethylation at the LAMB2 locus is associated with KMT2D variant that alters function that we validated with bisulfite pyrosequencing. Each of the upper panels displays genomic location on the x-axis and percent methylation on the y-axis. Inset top panel shows methylation values assessed using bisulfite pyrosequencing for the CpG sites in the region denoted by the solid red line. Black points denote non-KS controls. Blue points denote KS patients with KMT2D variants that alter function. Although not included in our main analysis, we also plot methylation values for individuals with Kabuki syndrome and variants of unknown significance (orange) and KMT2A variants (aqua) for comparison. Red asterisks denote significant differential methylation (*p < 0.05; **p < 0.01), via bisulfite pyrosequencing, between KS-KMT2D and control samples. The middle panel shows individual probe-based nominal p-values for differences in methylation between the two groups, with the dashed red line at a level of p = 0.01. The bottom panel provides gene annotation information for the differentially methylated region

Fig. 4

Unsupervised analysis reveals clustering of Kabuki syndrome cases based on variant type. Hierarchical clustering dendrogram, based on the 10% most variably methylated probes (n = 47,130), shows differences in DNA methylation patterns based on type of genetic variation within the histone machinery genes KMT2A and KMT2D. We depict loss of function KMT2D variants in red, KMT2D missense changes in green, and KMT2A missense and splice site variants in blue