ANKRD11 pathogenic variants and 16q24.3 microdeletions share an altered DNA methylation signature in patients with KBG syndrome

Abstract

Pathogenic variants in ANKRD11 or microdeletions at 16q24.3 are the cause of KBG syndrome (KBGS), a neurodevelopmental syndrome characterized by intellectual disability, dental and skeletal anomalies, and characteristic facies. The ANKRD11 gene encodes the ankyrin repeat-containing protein 11A transcriptional regulator, which is expressed in the brain and implicated in neural development. Syndromic conditions caused by pathogenic variants in epigenetic regulatory genes show unique patterns of DNA methylation (DNAm) in peripheral blood, termed DNAm signatures. Given ANKRD11's role in chromatin modification, we tested whether pathogenic ANKRD11 variants underlying KBGS are associated with a DNAm signature. We profiled whole-blood DNAm in 21 individuals with ANKRD11 variants, 2 individuals with microdeletions at 16q24.3 and 28 typically developing individuals, using Illumina's Infinium EPIC array. We identified 95 differentially methylated CpG sites that distinguished individuals with KBGS and pathogenic variants in ANKRD11 (n = 14) from typically developing controls (n = 28). This DNAm signature was then validated in an independent cohort of seven individuals with KBGS and pathogenic ANKRD11 variants. We generated a machine learning model from the KBGS DNAm signature and classified the DNAm profiles of four individuals with variants of uncertain significance (VUS) in ANKRD11. We identified an intermediate classification score for an inherited missense variant transmitted from a clinically unaffected mother to her affected child. In conclusion, we show that the DNAm profiles of two individuals with 16q24.3 microdeletions were indistinguishable from the DNAm profiles of individuals with pathogenic variants in ANKRD11, and we demonstrate the diagnostic utility of the new KBGS signature by classifying the DNAm profiles of individuals with VUS in ANKRD11.

Figure 1

Genomic location of ANKRD11 variants. Schematic representation of the ANKRD11 protein (GenBank: ANKRD11; NM_013275; GRCh37), its functional domains and variants used in this study. The number in each lollipop represents the number of individuals with that variant. Exon structure, based on GenBank: NM_013275.5, is provided by dashed lines. ANK, ankyrin tandem repeats (167–292); repression domain I (318–611); repression domain II (2369–2663); one activation domain (amino acids 1851–2145).

Figure 2

Loss-of-function variants in ANKRD11 are associated with a distinct DNAm signature. (A) PCA and (B) heatmap showing clustering of the KBGS discovery subjects (n = 14) and control discovery subjects (n = 28) using DNAm values at the 95 CpG sites identified in the KBGS DNAm signature. The heatmap color gradient indicates the normalized DNAm value ranging from −2.0 (blue) to 2.0 (yellow). Euclidean distance metric is used in the heatmap clustering dendrograms.

Figure 3

Classification of samples using SVM machine learning models based on the KBG DNAm signature. Sample groups were scored using the KBGS SVM model. KBGS validation subject (n = 7) classified as KBG-like demonstrating 100% sensitivity of the model. Whereas validation control subjects (n = 150) classified with low probabilities demonstrating 100% specificity of the model. ANKRD11 missense variants (n = 4), two of which belong to a child–parent duo and have KBG syndrome-like probabilities, whereas the remaining two had control-like probabilities.

Figure 4

Predicting ANKRD11 protein tolerance to missense variants. The diagram on the top illustrates ANKRD11’s tolerance to missense changes landscape according to MetaDome web server. The protein structure is depicted on the bottom with ankyrin repeat domains shown. Missense variants analyzed in the present work are indicated in green. (A) Zoomed view for variants p.(Thr236Met) and p.(lys459Asn). (B) Zoomed view for p.(Ile2427Thr).