Delineation of a KDM2B-related neurodevelopmental disorder and its associated DNA methylation signature

Abstract

Purpose: Pathogenic variants in genes involved in the epigenetic machinery are an emerging cause of neurodevelopment disorders (NDDs). Lysine-demethylase 2B (KDM2B) encodes an epigenetic regulator and mouse models suggest an important role during development. We set out to determine whether KDM2B variants are associated with NDD.

Methods: Through international collaborations, we collected data on individuals with heterozygous KDM2B variants. We applied methylation arrays on peripheral blood DNA samples to determine a KDM2B associated epigenetic signature.

Results: We recruited a total of 27 individuals with heterozygous variants in KDM2B. We present evidence, including a shared epigenetic signature, to support a pathogenic classification of 15 KDM2B variants and identify the CxxC domain as a mutational hotspot. Both loss-of-function and CxxC-domain missense variants present with a specific subepisignature. Moreover, the KDM2B episignature was identified in the context of a dual molecular diagnosis in multiple individuals. Our efforts resulted in a cohort of 21 individuals with heterozygous (likely) pathogenic variants. Individuals in this cohort present with developmental delay and/or intellectual disability; autism; attention deficit disorder/attention deficit hyperactivity disorder; congenital organ anomalies mainly of the heart, eyes, and urogenital system; and subtle facial dysmorphism.

Conclusion: Pathogenic heterozygous variants in KDM2B are associated with NDD and a specific epigenetic signature detectable in peripheral blood.

Keywords: Human Genetics; KDM2B; MDEMs; Methylation signatures; Neurodevelopmental disorders.

Figure 1. A cohort of individuals with heterozygous KDM2B variants.

A. Schematic representation of the KDM2B gene, its known domains, and the variants included in this study. Lollipops representing individual variants indicate location, classification of (predicted) effect on the transcript and/or protein (shape), and the classification based on the first analysis of the methylation arrays (color; Supplemental Figure 2). Larger deletions (ie, cases 25.1, 25.2, 29, and 30) are not shown. B. Projection of CxxC domain missense variants on the known crystal structure. Purple spheres represent Zn²⁺ ions. Side chains of relevant residues are included. C. Pedigrees depicting all cases of inherited pathogenic variants for which the pedigrees have not been published before (families 3, 4, and 25). All remaining pedigrees can be found in Supplemental Figure 1. D. Projection of the p.Val316Ile (family 4) variant on the structure of the mouse Kdm2a JmjC structure (yellow). Predicted human KDM2B JmjC structure as determined by AlphaFold is shown in green. Orange sphere indicates Fe²⁺ ion and the α-ketoglutarate cofactor is shown as yellow sticks. Purple line indicates target peptide (histon 3). ADD, attention deficit disorder; ADHD, attention deficit hyperactivity disorder; ID, intellectual disability; LD, learning difficulties; WT, wild type.

Figure 2. A KDM2B specific episignature.

After initial analysis (Supplemental Figure 2), 15 samples identified as outliers in the initial analysis were included for the training of a KDM2B specific episignature. A. Volcano plot indicating probes (red) included in the KDM2B episignature. B. Multidimensional scaling plot for selected probes, representing the pairwise distance across samples (red) and controls (blue) based on the top 2 dimensions. C. Heatmap of selected probes and unsupervised hierarchal clustering results indicating the episignature’s ability to differentiate KDM2B variants (red) from controls (blue). D. Support vector machine (SVM) classifier indicating specificity of the KDM2B episignature. Graph shows summary of 4-fold validation using all 15 case samples, using 75% of unaffected controls and other episignatures for training (blue) and the other 25% for testing (gray). Y-axis: MVP scores as determined by SVM. X-axis: different groups of samples, controls, and other known episignatures. Red arrowhead indicates the intellectual developmental disorder with seizures and language delay sample referred to in the text. MVP, methylation variant pathogenicity.

Figure 3. A CxxC-variant specific episignature.

All samples representing a CxxC-domain variant and included in the KDM2B episignature training set were used to train a CxxC-variant specific episignature. A. Volcano plot indicating all probes (red) included in the CxxC episignature. B. Multidimensional scaling plot for selected probes representing the pairwise distance across CxxC variants (orange), LoF variants (red), and controls (blue) based on the top 2 dimensions. C. Heatmap of selected probes and unsupervised hierarchal clustering results indicating the episignature’s ability to differentiate KDM2B variants (red and orange) from controls (blue) and to differentiate CxxC variants (orange) from LoF variants (red). D. Support vector machine classifier indicating specificity of the CxxC episignature. Graph is same as in Figure 2D. MVP, methylation variant pathogenicity.

Figure 4. Facial characteristics of individuals with KDM2B pathogenic variants.

A-C. Individuals of family 4 with the p.Val316Ile variant located in the JmjC-domain. D-G. Individuals with loss-of-function variants. Individual 10 (E) was also affected with Noonan syndrome. H-L. Individuals with missense variants in the CxxC domain.