Delineating the molecular and phenotypic spectrum of the SETD1B-related syndrome

Abstract

Purpose: Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay, and seizures. To date, clinical features have been described for 11 patients with (likely) pathogenic SETD1B sequence variants. This study aims to further delineate the spectrum of the SETD1B-related syndrome based on characterizing an expanded patient cohort.

Methods: We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays.

Results: Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants.

Conclusion: Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome.

Fig. 1. Schematic representation of SETD1B variants in this study cohort (major circles, top labels) and in literature (minor circles, bottom labels).

The RRM (residues 94–182), coiled-coil (CC) (residues 1173–1204), N-SET (residues 1668–1821), SET (residues 1822–1948) and post-SET (residues 1949–1966) domains in respectively magenta, orange, cyan, green and brown, the largely disordered regions (residues 320–682 and residues 1338–1640) in light gray, and the LSD (exon 5, residues 577–583) and WIN (exon 12, residues 1745–1750, within N-SET) motifs both in blue.

Fig. 2. Facial images of affected individuals.

Photographs of 16 individuals (plus one affected mother) with indicated SETD1B variants. Dysmorphic features included, among others, a slightly elongated face, high anterior hairline, thick arched or straight eyebrows, deep-set eyes, a prominent nose, and thin upper lips. Lower right corner shows facial composite images for all individuals, or only those with a likely pathogenic or pathogenic variant (note: individual 13 and mother were not included in the composite, given the image angle and glasses hindering Face2Gene program analysis). LP likely pathogenic variant, P pathogenic variant, V variant of uncertain significance.

Fig. 3. Structural and functional evaluation of SETD1B variants.

(a) Homology models of SETD1B domains: RRM domain (top left), based on the crystal structure of the RRM of human SETD1A (PDB ID 3S8S, identity = 66%, QMEAN = 0.25). The segment of Asn113 to Asp121 is colored in blue. This region is known to support different protein–protein interactions in other RRM proteins. Met170 and Gly195 are shown as blue sticks. Homology model of the N-SET and catalytic SET domains of SETD1B (gray cartoon), based on the EM structure of the yeast COMPASS in a complex with a ubiquitinated nucleosome (PDB ID 6ven, identity= 40.21%, QMEAN = −5.10) (center superimposed to the template PDB, and zoom-in panels). The region containing Arg1748 was observed more accurately in the X-ray structure of the WDR5:SETD1B Win motif peptide binary complex (PDB ID 4es0 [6], top right): Arg1748 (blue sticks) is inserted into the pocket of WDR5 (yellow) and interacts with the backbone oxygen atoms of Ser91, Phe133 and Cys261 (hydrogen bonds shown as yellow dashed lines). Arg1792 (blue sticks) and the substitution by Trp (dark gray sticks) interacting with surrounding residues in the adjacent alpha helix (e.g., Glu1796, gray sticks) or with the SWD1 subunit (RBBP5 in humans, shown in violet). The insets with Arg1825 and Arg1827 show the proximity of these residues (dark blue sticks) to histone H2A (light blue cartoon). The SET domain containing Ala1901Val, Ala1901Glu, Tyr1941fs and Glu1948Lys was modeled more accurately based on the crystal structure of the yeast COMPASS catalytic module (PDB ID 6chg [40], identity=62%, QMEAN = −1.78). Ala1901 and Glu1948 are presented as blue sticks in the center figure and right insets. The Ala1901Val and Ala1901Glu substitutions (dark gray sticks) could compromise the stability of the adjacent SAM (olive sticks) binding site and the interaction with the SWD1 subunit (RBBP5 in humans, violet cartoon), which in turn contacts ubiquitin (red cartoon). Tyr1941fs alters a segment of SET and Post-SET regions involved in catalysis and cofactor binding (blue cartoon in center figure and right inset): SAM (olive sticks) and histone H3 (green sticks) binding pocket, the key Tyr1943 residue (yellow sticks), three Cys and one Arg (yellow sticks) coordinating a zinc atom (shown as a sphere). The Glu1948Lys substitution (blue/dark gray sticks in center figure and right inset) could disturb potential interactions between the flexible loops and the adjacent subunit (Bre2, homologous to human ASH2, is shown in teal cartoon). (b) Overexpression of wild-type and variant SETD1B protein in HEK293 cells 48 hours post-transfection assessed by western blot. CC cell control, lysate of mock transfected HEK293 cells (one-way analysis of variance [ANOVA] p = 0.09). (c) Nuclear localization of SETD1B variants in HEK293 cells. Upper panel—SETD1B detected by anti-Flag antibody; lower panel—overlay of nuclear staining (DAPI, cyan) and SETD1B (red); scale bar 20 μm. Images representative of 2 independent experiments are shown. (d) Colocalization of SETD1B and ASH2 in HEK293 cells. Left to right: nuclear staining (DAPI), ASH2 (anti-HA tag), SETD1B (anti-Flag tag), merge of ASH2 (green) and SETD1B (red); scale bar 20μm. Pearson’s r value (range: −1, negative correlation, 1, max correlation) calculated with coloc2 plugin (ImageJ), Z-stacks of min. 12 nuclei were used for the analysis. t-test **p = 0.005. (e) Thermal shift analysis of the SET domain. Left: T_m of GST-SETD1B proteins and GST control. Right: change in T_m of the proteins in presence of SAM substrate. Two independent protein preparations were used for the assay performed in triplicates. One-way ANOVA multiple comparison test *p < 0.05, ***p < 0.0001. (f,g) Analysis of methylation profiles. (f) Hierarchical clustering (rows represent methylation probes, columns–samples). (g) MDS plot (control samples in blue, proband samples in red, SETD1B cases from the database in pink). Sample numbers correspond to case numbers: individual 3 p.([His10Gln];[Arg927His]) (3.1 and 3.2 are the parents of individual 3), individual 4 p.([Glu94Asp];[Pro1328Ser]), individual 5 p.(Phe95*), individual 7 p.(Asn113_Asp121delins9), individual 18 p.(Arg982Gln), individual 19 p.(Ala1010Val), individual 20 p.(Ala1129Val), individual 31 p.(Ala1901Glu), and individual 33 p.(Glu1948Lys).