Clinical and genetic delineation of autosomal recessive and dominant ACTL6B-related developmental brain disorders

Abstract

Purpose: This study aims to comprehensively delineate the phenotypic spectrum of ACTL6B-related disorders, previously associated with both autosomal recessive and autosomal dominant neurodevelopmental disorders. Molecularly, the role of the nucleolar protein ACTL6B in contributing to the disease has remained unclear.

Methods: We identified 105 affected individuals, including 39 previously reported cases, and systematically analyzed detailed clinical and genetic data for all individuals. Additionally, we conducted knockdown experiments in neuronal cells to investigate the role of ACTL6B in ribosome biogenesis.

Results: Biallelic variants in ACTL6B are associated with severe-to-profound global developmental delay/intellectual disability, infantile intractable seizures, absent speech, autistic features, dystonia, and increased lethality. De novo monoallelic variants result in moderate-to-severe global developmental delay/intellectual disability, absent speech, and autistic features, whereas seizures and dystonia were less frequently observed. Dysmorphic facial features and brain abnormalities, including hypoplastic corpus callosum, and parenchymal volume loss/atrophy, are common findings in both groups. We reveal that in the nucleolus, ACTL6B plays a crucial role in ribosome biogenesis, particularly in pre-rRNA processing.

Conclusion: This study provides a comprehensive characterization of the clinical spectrum of both autosomal recessive and dominant forms of ACTL6B-associated disorders. It offers a comparative analysis of their respective phenotypes provides a plausible molecular explanation and suggests their inclusion within the expanding category of "ribosomopathies."

Keywords: ACTL6B; Autism; BAFopathies; Epileptic-dyskinetic encephalopathy; Ribosomopathies.

Figure 1:. Overview of dominant and recessive ACTL6B-related disorder

A Bar plot showing the frequency (%) of the main clinical features associated with ACTL6B-related disorder (on the left: recessive disorder; on the right: dominant disorder). B Radar plot displaying the overlapping or distinguishable features of recessive and dominant disorder. C Degree of global developmental delay (GDD)/intellectual disability (ID) in the disorders. D Age span of achievement of developmental milestones in the recessive and dominant cohort compared to normal range. On the right, triangular radar plot showing the percentage of patients who achieved milestones at last evaluation.

Figure 2:. Dysmorphology, neuroimaging and epileptic findings in ACTL6B-related disorder

A Facial photographs of 18 newly reported patients with recessive disorder. B Facial photographs of 4 newly reported patients with dominant disorder. C Box plot displaying head circumference measurements in standard deviations (SD) to the norm. D Bar plot showing the frequency (%) of the main dysmorphic features. E Bar plot showing the frequency (%) of the main neuroimaging findings. F Epileptic phenotype of patients with recessive disorder. On the left, bar plot documenting age at onset; in the middle, sunburst chart displaying seizure types, on the right, box plots documenting seizure duration (seconds) and frequency (seizures/day).

Figure 3:. Genetic findings and genotype-phenotype correlations

A Schematic drawing illustrating the gene structure of ACTL6B and the location of the biallelic and monoallelic variants identified. Green pentagon: missense variant. Red square: stop gain. Purple triangle: splice variant. Blue diamond: in frame deletion. Grey octagon: frameshift. B Ribbon representation of a three-dimensional model of ACTL6B obtained with AlphaFold. The four missense variants identified in ACTL6B were modelled and are displayed in red. By analogy with actin, ATP is shown bound in the catalytic cleft of ACTL6B, and the four subdomains that characterize the reference actin fold are labelled (mauve-background circles). C Partially transparent surface and ribbon representations of ACTL6B, showing the four missense variants in red. ACTL6B is a subunit of the BAF chromatin remodeler related to the yeast SWI/SNF remodeler, whose three-dimensional structure has been determined using cryo-electron microscopy. ACTL6B is most closely related to actin-related protein7 (ARP7) of yeast SWI/SNF. In the structures of yeast SWI/SNF, ARP7 makes contacts with other remodeler subunits. By analogy with ARP7, we show the surface of ACTL6B predicted to be involved in inter-subunit contacts within the BAF remodeler (yellow). D Sequence of ACTL6B highlighting residues undergoing missense variants (red) and residues likely to be involved in inter-subunit contacts within the BAF remodeler (yellow). The diagrams in parts a and b were generated with the program PyMOL (Schrödinger, LLC). E Mutations mapped onto the ACTL6B sequence. F Phenotype genotype correlations in the recessive cohort. Variants are grouped in the following categories: 1) missense, 2) in frame deletion, 3) truncating, 4) frameshift, 5) pre-mRNA splice site, and 6) indel. Green: feature present; yellow: feature not present; blank: information not available. Blue rectangle: homozygous; blue triangle: compound heterozygous.

Figure 4:. ACTL6B is a nucleolar protein required for efficient pre-rRNA processing.

A Subcellular distribution analysis of ACTL6B in SH-SY5Y cells. Cells were labelled with an antibody specific to ACTL6B (in green) and co-stained with an antibody specific to the nucleolar protein Nucleophosmin (NPM1) or pescadillo (PES1) (in red). In the merge panels, the blue signal corresponds to the nucleoplasm (labelled with DAPI). Scalebar, 1 μm. B ACTL6B was immunostained (in green) with a specific antibody in U2OS cells. Blue, DAPI. Scalebar, 1 μm. C Pre-rRNA processing analysis upon ACTL6B depletion in SH-SY5Y cells. Cells were depleted with 25 nM siRNA for 3 d. Total RNA was extracted and separated on a denaturing gel, transferred to a nylon membrane, and processed for northern blotting with probes recognizing 5’ ETS, ITS1 or ITS2 sequences. Two silencers (#1 and #2) targeting a distinct region of ACTL6B transcripts were used independently. D Mature rRNA analysis of samples described in panel c. Ethidium bromide staining revealed the steady-state levels of mature 18S and 28S rRNAs. E Western blot analysis confirming efficient protein depletion. The antibody used are described in the Materials and Methods. As control for loading, the blot was probed for β-actin. F Pre-rRNA processing pathway depicting major intermediates and probes used. See doi: 10.1016/j.biochi.2012.02.001 for details.