The clinical and molecular spectrum of the KDM6B-related neurodevelopmental disorder

Abstract

De novo variants are a leading cause of neurodevelopmental disorders (NDDs), but because every monogenic NDD is different and usually extremely rare, it remains a major challenge to understand the complete phenotype and genotype spectrum of any morbid gene. According to OMIM, heterozygous variants in KDM6B cause "neurodevelopmental disorder with coarse facies and mild distal skeletal abnormalities." Here, by examining the molecular and clinical spectrum of 85 reported individuals with mostly de novo (likely) pathogenic KDM6B variants, we demonstrate that this description is inaccurate and potentially misleading. Cognitive deficits are seen consistently in all individuals, but the overall phenotype is highly variable. Notably, coarse facies and distal skeletal anomalies, as defined by OMIM, are rare in this expanded cohort while other features are unexpectedly common (e.g., hypotonia, psychosis, etc.). Using 3D protein structure analysis and an innovative dual Drosophila gain-of-function assay, we demonstrated a disruptive effect of 11 missense/in-frame indels located in or near the enzymatic JmJC or Zn-containing domain of KDM6B. Consistent with the role of KDM6B in human cognition, we demonstrated a role for the Drosophila KDM6B ortholog in memory and behavior. Taken together, we accurately define the broad clinical spectrum of the KDM6B-related NDD, introduce an innovative functional testing paradigm for the assessment of KDM6B variants, and demonstrate a conserved role for KDM6B in cognition and behavior. Our study demonstrates the critical importance of international collaboration, sharing of clinical data, and rigorous functional analysis of genetic variants to ensure correct disease diagnosis for rare disorders.

Keywords: COMPASS; Drosophila; KDM6B; Mendelian disorders; de novo variants; missense variants; neurodevelopmental disorders.

Figure 1

Overview of the study design and the identified KDM6B variants (A) Schematic illustration of the study design. (B) KDM6B variants, and their positions, identified in independent families. JmJC, Jumonji C domain; Zn, zinc; PTVs, protein-truncating (nonsense, frameshift, canonical splice) variants; PAVs, protein altering (missense and in-frame indel) variants.

Figure 2

Analysis of KDM6B PAVs via protein 3D structure analysis and a dual Drosophila gain-of-function assay (A) KDM6B fragment (PDB: 5OY3, aa 1157–1639) bound to the H3 tail fragment (aa 17–33). The JmJC domain is shown in cyan with 2-oxoglutaric acid (purple) bound with an Fe ion (magenta), which is necessary for the enzymatic demethylation of H3K27. The Zn-containing domain is shown in blue with Zn ion (magenta). Two out of three JmJC and Zn-containing domain-stabilizing linkers are also visible in the structure (orange). The H3 tail with K27 residue positioned into the active center of the JmJC domain is shown in yellow. Amino acids affected by missense or in-frame indels are shown as balls (pathogenic, red; VUSs, gray), affecting all shown domains as well as binding to H3 tail (see Table S3 for more details on specific variants). (B) A dual Drosophila gain-of-function assay was used to assess the disruptive potential of KDM6B PAVs. Ubiquitous overexpression (left) of KDM6B^ref with the UAS/Gal4 system results in complete lethality. Percent lethality assessed for KDM6B^ΔNterm, KDM6B^ΔJmJC, KDM6B^ΔZndom, KDM6B^P415Q as a benign control, and 18 KDM6B variants were compared to KDM6B^ref (chi-squared test). n = 50–230 flies for each genotype; data are represented as mean ± 95% confidence interval. Wing-specific overexpression (right) of KDM6B^ref in the fly wings results in the formation of an extra vein protruding off the L5 vein. The length of the extra vein was compared to KDM6B^ref (Dunnet’s test). n = 18–35 flies for each sample; data are represented as mean ± SEM. PAVs are colored on the basis of the domain (JmJC, cyan; Zn-containing, blue; stabilizing linkers, orange; no domain, white; same as in Figures 1B and 2A). ^∗∗p < 0.01, ^∗∗∗p < 0.0001.

Figure 3

The Drosophila KDM6B ortholog, Utx, is required in neurons for normal memory and behavior (A) Short term (STM) and long term (LTM) courtship memory was assessed upon MB-specific expression (R14H06-Gal4) of two independent Utx RNAi lines (Utx^RNAi1 and Utx^RNAi2) and their genetic controls (control 1 and control 2). Boxplots show the distribution of courtship indices (CIs) for naive (N) and trained (T) male flies aged 5 days. Memory was observed when a significant reduction in CI occurred between naive and trained conditions of the same genotype (Kruskal Wallis test). All controls show a significant reduction in courtship in trained vs. naive groups, while Utx RNAi knockdown flies did not. At least 30 flies were tested per condition. (B) Naive courting behavior was pooled from short- and long-term memory assays and compared between MB-specific Utx RNAi knockdown flies and their genetic controls. At least 60 male flies aged 5 days were tested. (C and D) MB-specific Utx RNAi knockdown caused reduced daily activity (C) and increased sleep (D) compared to genetic controls, but these differences were only significant for Utx^RNAi1 (t test). n = 32 flies for each genotype. (E) No morphological defects were found following MB-specific knockdown of Utx compared to their genetic controls. MB morphology was consistent in at least ten brains for each genotype. Scale bar represents 50 μm. Data are represented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.0001.

Figure 4

Photos of individuals with identified KDM6B variants Individuals with (likely) pathogenic variants are shown in the red box above, and those with a VUS are shown in the gray box.