Recent advances in Mendelian genomics reveal the importance of variant-level characterization of allelic disorders. Non-muscle actin isoforms, encoded by the genes ACTB and ACTG1, are the most abundant intracellular proteins, but historically, they are often regarded as merely being "housekeeping" molecules. Here, we illuminate the extraordinary clinical heterogeneity and complex pathobiology of genetic non-muscle actinopathies. To do this, we combine human genomics studies with molecular biology. Strikingly, variants in ACTB and ACTG1 isoforms generate at least eight distinct clinical disorders. A subset of disease-associated missense variants causes dysregulated actin polymerization-depolymerization and neuronal migration defects. In contrast, nonsense, frameshift, and missense variants enhancing protein degradation cause milder phenotypes or are benign. These results emphasize the essential functional aspects of the non-muscle actin isoforms. Critically, they additionally constitute a template for the personalized genetic variant-level-driven management of the pleiotropic allelic single-gene disorders.

Variants in cytoskeletal actin encoding genes are associated with a broad spectrum of disorders, called non-muscle actinopathies. Among them, the Baraitser-Winter cerebrofrontofacial syndrome (BWCFF) displays the most severe symptoms, such as intellectual disability and epilepsy. We found that the BWCFF-associated R196H mutation results in reduced proliferation and migration of patient-derived fibroblast cells, and the latter is likely related to decreased fibronectin expression. The mutation causes a 50% drop in filamentous (F-) actin content, which is correlated with an approximately fourfold reduction in the stiffness of patient-derived cells probed with atomic force microscopy (AFM). We observed no significant defects either in the organization of the cellular actin cytoskeleton, analyzed by superresolution (STED) microscopy, or in the structure of purified filaments, explored with AFM. On the other hand, the more parallel orientation of the mutant actin bundles might be caused by the perturbed interaction of actin with the Arp2/3 complex. Manipulating the cells by mechanical forces through the application of the dual laser optical tweezers (DLOT) technique suggests that the mutation weakens the attachment of cytoskeletal actin to the plasma membrane. Inducing dynamic reorganization of actin by uniaxial stretching revealed that the interaction of cofilin with actin is also weakened by the mutation. Thus, the impaired function of actin to form filaments and interact with either cofilin or the Arp2/3 complex may result in the malfunction of dendritic spines, and the reduced cellular proliferation and migration might account for the lissencephaly phenotype of patients.

Actins are cytoskeletal proteins that are essential for multiple cellular processes. Mutations in the ACTB and ACTG1 genes, encoding the ubiquitous beta- and gamma-cytoskeletal actin isoforms, respectively, cause a broad spectrum of neurodevelopmental disorders, with microcephaly as the most frequent one. To investigate the pathogenesis underlying this cortical malformation, we studied patient-derived cerebral organoids from induced pluripotent stem cells of individuals with the Baraitser-Winter-CerebroFrontoFacial syndrome (BWCFF-S) carrying an ACTB/ACTG1 missense mutation. These organoids were reduced in size, showing a thinner ventricular zone (VZ) due to reduced VZ progenitor abundance. Strikingly, VZ progenitors in BWCFF-S cerebral organoids displayed a shift in the orientation of their cleavage plane from a predominantly vertical to a majoritarian horizontal orientation. The latter cleavage plane orientation is incompatible with increasing VZ progenitor abundance and instead promotes basal progenitor generation. Various cytoskeletal and morphological irregularities of BWCFF-S VZ progenitors, notably in the apical region, seemingly contribute to this change in cleavage plane orientation. Our results provide insight into the cell biological basis of the microcephaly associated with BWCFF-S caused by actin mutations.

Cellular processes such as cytokinesis, apoptosis and migration rely heavily on the myosin-based contractility of the γ-actin network in the submembrane cortex. Direct measurements of γ-actin-myosin interactions through morphological and depletion investigations remain elusive. Here, we use a synthetic nanomachine, consisting of an array of myosin motors carried on a nanopositioner and brought to interact with an actin filament attached to a bead trapped in the focus of dual laser optical tweezers. The nanomachine is able to mimic the loading conditions of γ-actin-myosin interactions in situ, allowing measurements of the maximum steady force (F0) and of the shortening velocity against loads < F0. Comparative measurements are conducted on wild-type γ-actin and γ-actin carrying the E334Q mutation, associated with non-muscle actinopathies. Our results show that the force of the single actin-myosin interaction is 2.5 pN for the wild-type actin and is halved by the mutation. The kinetics of motor attachment-detachment, underpinning the rate of isometric force rise and the force-velocity relation, are also reduced by a factor of two, resulting in a reduction of the maximum nanomachine power to one-fifth. The identification and quantitative definition of the loss of basic function caused by the E334Q γ-actin mutation serve as a starting point for understanding the chain of remodelling events leading to the pathological phenotype and demonstrate the potential of the nanomachine for targeted therapeutic interventions. KEY POINTS: Mutations in cytoskeletal actin cause rare pathologies classified as non-muscle actinopathies (NMAs), including the Baraitser-Winter cerebrofrontofacial syndrome characterized by neural cortex abnormalities leading to facial dysmorphism, developmental delay and organ malformations. Cytoskeleton dynamics control cell morphology and migration and rely on the interaction of non-muscle myosin II with cytoskeletal γ-actin, but the system's mechanical performance and its blunting by NMA-causing mutations in γ-actin have still to be defined. Here, a synthetic nanomachine is used to record the relevant mechano-kinetic parameters of the γ-actin-myosin interaction under physiological loading conditions. Quantitative estimates of these parameters for wild-type and E334Q mutant γ-actin suggest that strong defects in the actin-myosin interaction mechanics may be one of the main molecular mechanisms leading to the pathological phenotype. This paper lays the groundwork for the quantitative definition of the basic function altered by NMA-causing mutations and the evaluation of targeted therapies.

Syndromes associating both eyeball and periocular developmental anomalies, combining iris chorioretinal (ocular) coloboma and ptosis, are described in very rare clinical entities such as Baraitser-Winter cerebrofrontofacial syndrome (BWCFF). We report on six individuals from 3 unrelated families presenting with autosomal dominant eye malformations, including ocular coloboma, ptosis and craniofacial features suggesting BWCFF. However, no neurodevelopmental disorders (NDD) as usually observed in this syndrome were detected. Exome sequencing (ES) or genome sequencing (GS) was performed and allowed the identification of 3 novel heterozygous variants in the MYH10 gene, encoding the non-muscle myosin heavy chain II B. These 3 likely causative variants occur in the MYH10 tail domain required for myosin filament assembly. The MYH10 protein is mislocalized leading to abnormal actin networks in the patients' fibroblasts compared to controls. MYH10 dysfunction leads to delayed development of the eye, as well as a muscular phenotype in the zebrafish model. Heterozygous variants in MYH10 have been recently reported to be associated with an autosomal dominant NDD with other congenital anomalies, but no patients were reported with the association of ocular coloboma and ptosis as main features. Herein, we report other MYH10 variants which cause mainly an ophthalmic phenotype without NDD expanding the phenotype associated with MYH10 and representing a differential diagnosis with BWCFF. The reason for the genotype-phenotype variability with either prominent NDD or prominent ocular features will require further investigations.