Congenital Hyperinsulinism and Novel KDM6A Duplications -Resolving Pathogenicity With Genome and Epigenetic Analyses

Abstract

Context: Hyperinsulinemic hypoglycemia (HI) can be the presenting feature of Kabuki syndrome (KS), which is caused by loss-of-function variants in KMT2D or KDM6A. As these genes play a critical role in maintaining methylation status in chromatin, individuals with pathogenic variants have a disease-specific epigenomic profile-an episignature.

Objective: We evaluated the pathogenicity of 3 novel partial KDM6A duplications identified in 3 individuals presenting with neonatal-onset HI without typical features of KS at the time of genetic testing.

Methods: Three different partial KDM6A duplications were identified by routine targeted next-generation sequencing for HI and initially classified as variants of uncertain significance (VUS) as their location, and hence their impact on the gene, was not known. Whole-genome sequencing (WGS) was undertaken to map the breakpoints of the duplications with DNA methylation profiling performed in 2 individuals to investigate the presence of a KS-specific episignature.

Results: WGS confirmed the duplication in proband 1 as pathogenic as it caused a frameshift in the normal copy of the gene leading to a premature termination codon. The duplications identified in probands 2 and 3 did not alter the reading frame, and therefore their significance remained uncertain after WGS. Subsequent DNA methylation profiling identified a KS-specific episignature in proband 2 but not in proband 3.

Conclusion: Our findings confirm a role for KDM6A partial gene duplications in the etiology of KS and highlight the importance of performing in-depth molecular genetic analysis to properly assess the clinical significance of VUS' in the KDM6A gene.

Keywords: KDM6A; DNA methylation; Kabuki syndrome; congenital hyperinsulinism; episignature; whole-genome sequencing.

Figure 1.

Duplications of the KDM6A gene identified in 3 individuals with hyperinsulinemic hypoglycemia by targeted next-generation sequencing with the breakpoints confirmed by genome sequencing. Variants are listed according to NM_021140.3, Genome Reference Consortium Human Build 37. A tandem duplication of exons 3-26 within the KDM6A gene causes a frameshift and results in a premature stop codon in the second copy of exon 3 (proband 1). An in-frame tandem duplication of exons 3-6 within the KDM6A gene (proband 2). A tandem duplication of exons 2-29 of KDM6A and exons 5-6 of the adjacent DIPK2B gene, located next to a complete copy of KDM6A within the DIPK2B gene (proband 3). Shaded grey indicates the duplications. Single letters within boxes indicate abbreviations of amino acids. Asterisk indicates the position of an introduced frameshift.Abbreviation: STOP, premature stop codon.

Figure 2.

EpiSign (DNA methylation) analysis of peripheral blood from patient 2 and 3 with tandem duplications of KDM6A. (A) Hierarchical clustering. The plot shows clustering analysis with heatmap using probes specific to the DNA methylation of KS as compared to controls. Rows indicate probes and columns indicate samples. (B) Multidimensional scaling. The 2 dimensions represent the pairwise distance across the samples with episignatures of KMD6A-KS patients (purple), KMT2D-KS patients (blue), and controls (green). Together these results indicate that Proband 2 (red line or plot) has a DNA methylation profile similar to subjects with a confirmed KS episignature (blue or purple) and distinct from controls (green). Proband 3 (black) has a DNA methylation profile similar to controls (green). C) MVP score. A multiclass supervised classification system capable of discerning between multiple episignatures by generating a probability score for each episignature. The elevated score for Kabuki shows an episignature similar to the KS reference. MVP score >0.5 indicates positive classification. Abbreviations: KS, Kabuki syndrome; MVP, methylation variant pathogenicity.