Variants in exons 5 and 6 of ACTB cause syndromic thrombocytopenia

Germline mutations in the ubiquitously expressed ACTB, which encodes β-cytoplasmic actin (CYA), are almost exclusively associated with Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF). Here, we report six patients with previously undescribed heterozygous variants clustered in the 3′-coding region of ACTB. Patients present with clinical features distinct from BWCFF, including mild developmental disability, microcephaly, and thrombocytopenia with platelet anisotropy. Using patient-derived fibroblasts, we demonstrate cohort specific changes to β-CYA filament populations, which include the enhanced recruitment of thrombocytopenia-associated actin binding proteins (ABPs). These perturbed interactions are supported by in silico modeling and are validated in disease-relevant thrombocytes. Co-examination of actin and microtubule cytoskeleton constituents in patient-derived megakaryocytes and thrombocytes indicates that these β-CYA mutations inhibit the final stages of platelet maturation by compromising microtubule organization. Our results define an ACTB-associated clinical syndrome with a distinct genotype-phenotype correlation and delineate molecular mechanisms underlying thrombocytopenia in this patient cohort.

interface is stabilized by internal interactions of W340 1 . This residue interacts with V9, D11 and K18 of the central SD1 β-sheet and forms ring stacking interactions with P27. Our model predicts that absence of W340 in both -CYA P4 and -CYA P5 perturbs the CM-loop binding surface (Fig. 6b, top).
This view receives further support from the results of MD-simulations that compare the folding stability and behavior of β-CYA WT and β-CYA P5 .
Mutations within the supporting-loop alter the actin-activated ATPase activity, actin affinity and in vitro sliding motility of a Dictyostelium discoideum myosin 2 motor domain construct 2 . Based on biochemical results, the supporting-loop was reported to interact with actin N-terminal residues 3 .
However, this interpretation is not compatible with the structure of a human rigor actomyosin complex that was obtained using electron cryomicroscopy 1 . The structure shows two actin C-terminal glutamines (Q353, Q354) to be within the interaction range of this NM-2 loop. According to our modelling results, these residues are despite the frameshift spatially conserved in -CYA P4 , but out of the interaction range in the case of -CYA P5 (Fig. 6b, bottom).
For the actin-α-actinin complex, a low resolution cryo-EM density is available (PDB: 3LUE, described in 4 ), which shows that the binding sites for α-actinin and myosin overlap. The actin binding domain of α-actinin is commonly referred to as calponin homology domain, as it shares its actin binding domain with a large superfamily of proteins including calponin, dystonin, dystrophin, filamin, plectin, and spectrin. The interface includes the interaction of β-CYA SD1 I345 with F101 of αactinin. For both -CYA P4 and -CYA P5 the hydrophobic interactions of I345 are conserved in the form of leucine residues (Fig. 6c). However, in -CYA P4 , D349 and D351 are within the interaction range of α-actinin H102 and D95, introducing additional electrostatic interactions (Fig. 6c, left).

Transmission Electron Microscopy of ACTB-AST platelets
As cytoskeletal defects are associated with compromised platelet ultrastructure 5,6 , platelets from healthy control, P3, P4 and P5 were assessed by transmission electron microscopy (Supplementary Figure 10). Our assessment showed that all regular intracellular content in the form of dense granules, -granules, mitochondria and lysosomes is present in both healthy control and ACTB-AST patient platelets. Follow up studies are required for in depth characterization of ACTB-AST platelet ultrastructure.

3' ACTB variants listed in the gnomAD database
According to ExAC computation 7 , ACTB is intolerant to both loss-of-function and missense variants.
Out of the predicted 188 missense variants, only 12 missense changes are listed in the ExAC database and not a single loss-of-function change (pLI score 0,94; missense z-score 6,29). The number of observed variants increased to 19 in the gnomAD dataset. Of these, 7 are within the 3' region (Supplementary Table 1) and each has only been seen in a single individual. It is not currently known whether these variants listed in the gnomAD database are truly benign, not fully penetrant, or causative for ACTB-AST. One variant, p.Glu364Gln, affects the same residue as a mutation described