Truncating SRCAP variants outside the Floating-Harbor syndrome locus cause a distinct neurodevelopmental disorder with a specific DNA methylation signature

Abstract

Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD." All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.

Keywords: DNA methylation signature; Floating-Harbor syndrome; SRCAP; epigenomics; genotype-phenotype correlation; intellectual disability; neurodevelopmental disorders; non-FLHS SRCAP-related NDD; nonsense-mediated decay; speech delay.

Figure 1

Spectrum and location of the SRCAP truncating variants Schematic representation of the SRCAP protein (GenBank: NP_006653.2), its functional domains, and variants used in this study. Exon structure, based on GenBank: NM_006662.3, is provided by dashed lines. Green, HSA-domain (124–196); blue, helicase ATP-binding (630–795) and C-terminal (2,044–2,197) domain; black, AT-hooks (2,857–2,869; 2,936–2,948; 3,004–3,016). Locus, predicted to escape NMD (<55 bp from the last exon/intron junction), is shown in red. Proximal and distal truncating SRCAP variants identified in this study are shown in orange and green, respectively. Floating-Harbor syndrome-causing variants are depicted in purple (recurrent and the most distant variants are specified). Black dots indicate samples used for DNA methylation analysis.

Figure 2

Loss-of-function variants in SRCAP are associated with two distinct but overlapping DNAm signatures based on variant position (A and B) FLHS DNAm signature: (A) principal components analysis (PCA) and (B) heatmap showing clustering of FLHS discovery subjects (n = 4; dark purple), FLHS validation subjects (n = 4; light purple), proximal SRCAP discovery subjects (n = 5; dark orange), proximal SRCAP validation subjects (n = 4; light orange), distal SRCAP subjects (green) and discovery control subjects (n = 35; blue) using DNAm values at 464 CpG sites in the FLHS DNAm signatures. FLHS subjects clearly segregate from all other samples, while all proximal and some distal SRCAP cases cluster intermediately. (C and D) Proximal SRCAP DNAm signature: (C) PCA and (D) heatmap showing clustering of the same subjects from (A) and (B) and matched control subjects (n = 32; blue) using DNAm values at 347 CpG sites in the proximal SRCAP DNAm signature. Proximal SRCAP discovery and validation subjects and some distal subjects clearly separate from control subjects, with FLHS subjects clustering intermediately. 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. (E) Venn diagram showing the CpG sites are shared and distinct between the proximal SRCAP and FLHS signatures.

Figure 3

Distribution of differentially methylated regions in SRCAP DNAm signatures The Manhattan plots (center) show the CpG site p values for each DNAm signature discovery comparison; sites that meet the |Δβ| > 0.20 cut off in the proximal SRCAP signature (upper) are colored orange, those that meet the cut off in the FLHS signature (lower) are colored purple. Six differentially methylated regions are boxed on the Manhattan, with the full plots for sample groups shown above and below. These six illustrate different patterns of DNAm between the proximal SRCAP and FLHS groups. Each is named for the gene body/promoter to which they map. Lines connect the average beta values for each corresponding group. Grey bars above the plots indicate CpG islands, black represent gene bodies. β values for each sample are indicated with mean lines for groups (except the distal group, since two classified positively and three negatively). The CpGs mapping to STGP2 are present in both signatures, RUFY1 and RPLP1 are in the proximal SRCAP signature, and ZBTB2, MAMSTR, and RTEL1 are in the FLHS signature. There is increased DNAm at the RTEL1 sites in the Proximal group, though not a large enough Δβ to be in the proximal SRCAP DNAm signature. In all cases, β values in the discovery and validation cohorts are concordant. RPLP1, RUFY1, and RTEL1 are encoded on the plus strand, STPG2, ZBTB22, and MAMSTR are on the minus strand.

Figure 4

Classification of samples using SVM machine learning models based on each DNAm signature Sample groups were scored using (A) the FLHS support vector machine (SVM) model and (B) the proximal SRCAP SVM model. FLHS validation subjects (n = 4) classified positively using the FLHS model; similarly proximal SRCAP validation subjects (n = 4) classified positively using the proximal SRCAP model, demonstrating 100% sensitivity of both models. Using the FLHS model, proximal SRCAP subjects (n = 9) and validation control subjects (n = 97) classified negatively, demonstrating 100% specificity of the model. Using the proximal model, FLHS subjects (n = 8) and validation control subjects (n = 97) classified negatively demonstrating 100% specificity of the model. SRCAP missense variants (n = 4) classified negatively using both models, suggesting them to be benign. Distal SRCAP subjects (n = 5) all classified negatively on the FLHS signature, suggesting these subjects do not have FLHS. Two distal SRCAP subjects classified positively on the proximal SRCAP model (distal SRCAP individual #1 and #2) demonstrating concordant DNAm profiles of these subjects with the proximal SRCAP subjects, while three classified negatively (distal SRCAP individual #3, #4, and #5). Subjects with a pathogenic variant in CREBBP (n = 10) or EP300 (n = 1) all classified negatively using both models.

Figure 5

Facial features of individuals with proximal and distal truncating SRCAP variants Phenotype of nine individuals with the proximal and three individuals with the distal truncating SRCAP gene variants. Photos at age of 2, 8, and 25 years are available for the proximal SRCAP individual #9. Shared facial (non-specific) phenotypic features of the proximal SRCAP group individuals are seen: long face and long, wide philtrum, prominent forehead, thin upper lip vermilion and everted lower vermilion, wide mouth, typical (narrow) palpebral fissures, epicanthal folds, periorbital fullness, wide nasal bridge, prominent ears, and retro- or prognathia. Distal SRCAP individual #4 has some FLHS facial features although he does not have short stature or other typical FLHS features and has Marfanoid habitus with pectus excavatum (similarly to distal SRCAP individual #1).