Pathogenic variants in SMARCA1 cause an X-linked neurodevelopmental disorder modulated by NURF complex composition

Abstract

Pathogenic variants in ATP-dependent chromatin remodeling proteins are a recurrent cause of neurodevelopmental disorders (NDDs). The NURF complex consists of BPTF and either the SNF2H (SMARCA5) or SNF2L (SMARCA1) ISWI-chromatin remodeling enzyme. Pathogenic variants in BPTF and SMARCA5 were previously implicated in NDDs. Here, we describe 40 individuals from 30 families with de novo or maternally inherited pathogenic variants in SMARCA1. This novel NDD was associated with mild to severe ID/DD, delayed or regressive speech development, and some recurrent facial dysmorphisms. Individuals carrying SMARCA1 loss-of-function variants exhibited a mild genome-wide DNA methylation profile and a high penetrance of macrocephaly. Genetic dissection of the NURF complex using Smarca1, Smarca5, and Bptfsingle and double mouse knockouts revealed the importance of NURF composition and dosage for proper forebrain development. Finally, we propose that genetic alterations affecting different NURF components result in a NDD with a broad clinical spectrum.

Keywords: NURF complex; SMARCA1; brain overgrowth; epigenetics; exome sequencing.

Figure 1. Identification and location of pathogenic variants in the SMARCA1 gene.

A) Six generation pedigree of the index family carrying the c.271C>T [p.Arg91*] variant within SMARCA1. Genetic testing also included the maternal uncle (v5) and grandfather (iv1) who were both negative for the variant. B) Schematic diagram highlighting the position of the pathogenic variants identified and the functional domains encoded by SMARCA1. Blue text denotes variants with macrocephaly, purple text refers to previously described variants that were not identified in this study. Numbers refer to amino acid position. The motifs comprising the SNF2 ATPase/helicase domain and HSS domain are highlighted in blue and green, respectively.

Figure 3:. Identified methylation profiles for individuals with SMARCA1 variants.

Volcano plots (left) show mean methylation difference of cases and controls plotted against the −log10(p) values and highlight the selected differential probes for the distinct profiles of (A) SMARCA1 cohort, (B) male individuals carrying SMARCA1variants (SMARCA1_M), and (C) female individuals with SMARCA1 variants (SMARCA1_F). MDS plots (center) and heatmaps (right) with Euclidean clustering show clustering of cases (red for males, orange for females) and unaffected controls (blue).Columns of the heatmap correspond to the samples and rows correspond to the selected probes.

Figure 4:. Unsupervised clustering for non-discovery samples using methylation probes for SMARCA1 Cohort.

Other SMARCA1 samples not used in SMARCA1 profile discovery were investigated using unsupervised analysis and examined using the distinct profiles for (A) SMARCA1 cohort, (B) SMARCA1_M cohort, and (C) SMARCA1_F cohort. As expected, replicates cluster with respective similar cases for each profile.

Figure 5:. Smarca1 cKO mice have minor changes in brain size.

A) Schematic illustration of a mouse saggital brain section (bottom). Numbers identify the different brain regions analyzed (right) for area and length differences between Smarca1 cKO and control littermates. Graph (top) depicts the size changes observed. The light and dark yellow shading highlights the statistically significant differences (p<0.05 and p<0.01, respectively) while the grey shading denotes regions not analyzed for this study. B) Control (WT) and Smarca1 cKO (cKO) coronal brain sections isolated at E14.5 were IF-stained to detect radial glia progenitors (Pax6), intermediate progenitors (Tbr2), or mitotic progenitor cells (PH3). An EdU-click chemistry reaction was used to identify progenitor cells in S-phase. Cell nuclei were counter-stained with Hoechst dye (blue). Images were taken at the dorsomedial region of the developing cortex. Marker-positive cells were quantified as described in the text and shown in Supplemental Figure 4. Scale bars = 100 mm for PH3 and 20 mm for all other panels.

Figure 6:. Variations in cortical growth caused by ablation of different NURF components.

A) Control (WT) and Smarca1 cKO (cKO) coronal brain sections isolated at P0 and imaged at the dorsomedial region of the cerebral cortex. Sections were IF-stained for the deep layer neuronal markers Tbr1 (red; layer VI) and Ctip2 (white; layer V), and the upper layer neuronal marker Satb2 (green, layers II-V). The merged images are shown in the left panels. Cell nuclei were counter-stained with Hoechst dye (blue; right panels). Scale bars = 20 mm. B) Photographs of P20 brains dissected from transgenic mice corresponding to the genetic ablation of different genes that comprise the NURF complex (mutant) or from their control littermates (WT). Ex6DEL refers to mice with a deletion of exon 6 of the Smarca1 gene that generates an internally truncated Snf2l protein lacking part of the ATPase/helicase domain. cKO, conditional knockout; dKO, double conditional knockout. C) The CERF complex, comprising Snf2l and Cecr2, is abundantly expressed in the cerebellum. P10 cerebellar extracts were isolated from control (WT) or Smarca1 cKO animals and used for immunoblots to examine CERF protein levels and Snf2h abundance. D) Co-immunoprecipitation assays were performed from WT (upper panels) or Smarca1cKO (lower panels) cerebellar extracts with antibodies to Snf2h or rabbit IgG. Snf2h antibodies immunoprecipitated Cecr2 only when Snf2l protein was absent (Smarca1cKO). 5% of the total IP lysate was loaded as input.

Figure 7:. Normal and aberrant NURF subunit switching during mouse cortical development.

A) The NURF complex is expressed in proliferating neuroprogenitors (NPCs) and in differentiated neurons during murine corticogenesis. B) During normal cortical development (WT) the catalytic subunit within NURF during growth is SNF2H (encoded by SMARCA5). During differentiation, the catalytic subunit is switched to SNF2L (encoded by SMARCA1). C) Ablation of the Bptf gene (Bptf cKO) prevents formation of the NURF complex and results in minimal growth and a severely hypoplastic cortex. Individuals with BPTF pathogenic variants also present with microcephaly. D) When Smarca5 is inactivated (Smarca5 cKO), there is some compensation by SNF2L during growth that results in a smaller cortex but one that is slightly larger than the Bptf cKO mice. Individuals with SMARCA5 variants also present with microcephaly. E) Smarca1 knockout mice (Smarca1cKO) have a normal sized cortex which results from near complete compensation by the SNF2H protein. Individuals with SMARCA1 nonsense LOF variants have a high penetrance of macrocephaly, which is discordant from the mouse phenotype. F) In the Ex6DEL mice subunit switching occurs normally but the mutant protein lacks remodeling activity creating a dominant negative effect that delays differentiation and results in a larger brain size. Individuals with SMARCA1 variants and macrocephaly are predicted to lack remodeling activity and function in a dominant negative fashion while other missense variants that don’t abolish remodeling activity are not macrocephalic.