BICRA, a SWI/SNF Complex Member, Is Associated with BAF-Disorder Related Phenotypes in Humans and Model Organisms

Abstract

SWI/SNF-related intellectual disability disorders (SSRIDDs) are rare neurodevelopmental disorders characterized by developmental disability, coarse facial features, and fifth digit/nail hypoplasia that are caused by pathogenic variants in genes that encode for members of the SWI/SNF (or BAF) family of chromatin remodeling complexes. We have identified 12 individuals with rare variants (10 loss-of-function, 2 missense) in the BICRA (BRD4 interacting chromatin remodeling complex-associated protein) gene, also known as GLTSCR1, which encodes a subunit of the non-canonical BAF (ncBAF) complex. These individuals exhibited neurodevelopmental phenotypes that include developmental delay, intellectual disability, autism spectrum disorder, and behavioral abnormalities as well as dysmorphic features. Notably, the majority of individuals lack the fifth digit/nail hypoplasia phenotype, a hallmark of most SSRIDDs. To confirm the role of BICRA in the development of these phenotypes, we performed functional characterization of the zebrafish and Drosophila orthologs of BICRA. In zebrafish, a mutation of bicra that mimics one of the loss-of-function variants leads to craniofacial defects possibly akin to the dysmorphic facial features seen in individuals harboring putatively pathogenic BICRA variants. We further show that Bicra physically binds to other non-canonical ncBAF complex members, including the BRD9/7 ortholog, CG7154, and is the defining member of the ncBAF complex in flies. Like other SWI/SNF complex members, loss of Bicra function in flies acts as a dominant enhancer of position effect variegation but in a more context-specific manner. We conclude that haploinsufficiency of BICRA leads to a unique SSRIDD in humans whose phenotypes overlap with those previously reported.

Keywords: BAFopathy; CG11873; Drosophila; GLTSCR1; chromatin; developmental delay; intellectual disability; ncBAF complex; position effect variegation; zebrafish.

Figure 1

Individuals with BICRA Variants Have Dysmorphic Facial Features Individuals with variants in BICRA share a number of facial features including frontal bossing, epicanthal folds, low-set ears, prominent/rounded nasal tips, and thin upper lips.

Figure 2

BICRA Is Loss-of-Function Constrained and Mutation of bicra in Zebrafish Causes Craniofacial Defects (A) BICRA is variant constrained based upon the control population database gnomAD.BICRA has a score of 0.64 in DOMINO and is predicted to be “likely dominant.” (B) Protein structure of BICRA and its fish and fly orthologs. BICRA has two protein domains, a coiled-coil (CC) domain, and a GLTSCR1 domain. Variants identified in this study are indicated above the protein as dots (red, loss-of-function; purple, missense). BICRA’s ortholog in zebrafish has a coiled coil domain and a GLTSCR1 domain. A frameshift mutation induced by CRISPR-Cas9 is indicated by an orange dot. BICRA’s ortholog in flies has a homology score 4/16 (DIOPT v8.0⁴⁴) and also contains a GLTSCR1 domain. The location of the internal GFP tag used in Figure 4 is shown in green. (C) Zebrafish larvae from homozygous mutant parents exhibit craniofacial abnormalities, including anterior-posterior shortening of the head (brackets), an increased joining angle of the ceratohyal (ch) elements at the midline (82° ± 2.7° compared to 64.9° ± 0.4°; p < .0001), and misorientation of Meckel’s cartilage (mk) relative to the ethmoid plate (et). The bicra^b1410 allele is an 8 bp deletion in the 3^rd exon (del c.933–40, boxed region) producing a frameshift that results in a missense mutation, p.Ile312Phe, followed immediately by a stop codon (red underline).

Figure 3

Bicra Binds to Other ncBAF Complex Proteins in Drosophila S2 Cells (A) HA-tagged Bicra C terminus was transfected into Drosophila S2 and IP-MS was performed. Left, silver stain of mock and HA-Bicra purifications. Right, mass spectrometry peptide counts (see also Tables S2 and S3). (B) Left, silver stain of mock, HA-Bap60, HA-d4, and HA-CG7154 purifications. Right, mass spectrometry peptide counts (see also Tables S4, S5, S6, and S7). (C) Schematic depicting the fly versions of the SWI/SNF complexes based upon data from A and previously published work and proteomic mass-spectrometry results from purifications in (A) and (B). The compositions of human and fly complexes are very similar. Importantly, Bicra is the only ncBAF-specific member in flies.

Figure 4

Bicra Is Broadly Expressed in the Brain in Flies and Controls Telomeric Position Effect Variegation (A) Gene structure of fly Bicra. The gene region is shown in blue, exons in orange, introns as a black line, and untranslated regions (UTR) in gray. A deficiency and genomic rescue (GR) construct are shown in red and green, respectively. Blue arrows indicate available transposable element insertions. (B and C) Expression of GFP-tagged Bicra in the larval (B) and adult (C) nervous systems. Bicra::GFP is shown in green, the neuronal marker Elav in red, the glial marker Repo in white. We observe broad expression of Bicra::GFP in most neurons and glia. Bicra::GFP also localizes to the nucleus as indicated by its colocalization with Elav and Repo, which are both nuclear proteins. (D–D′′) Position effect variegation (PEV) assay using the In(1)w^m4 allele. One copy loss of Bicra with either allele does not alter variegation in the eye. (E–E′′) PEV assay using the 39C-27 w⁺ insertion in the right telomere of the 2^nd chromosome. Loss of Bicra with either allele shows a dominant lightening of eye color, suggesting that Bicra controls chromatin structure at this location. (F–F′′) PEV assay using the 39C-5 w⁺ insertion in the left telomere of the 2^nd chromosome. Loss of Bicra with either allele shows a dominant lightening of eye color, suggesting that Bicra controls chromatin structure at this location.