Functional analysis of a de novo ACTB mutation in a patient with atypical Baraitser-Winter syndrome

Abstract

Exome sequence analysis can be instrumental in identifying the genetic etiology behind atypical disease. We report a patient presenting with microcephaly, dysmorphic features, and intellectual disability with a tentative diagnosis of Dubowitz syndrome. Exome analysis was performed on the patient and both parents. A de novo missense variant was identified in ACTB, c.349G>A, p.E117K. Recent work in Baraitser-Winter syndrome has identified ACTB and ACTG1 mutations in a cohort of individuals, and we rediagnosed the patient with atypical Baraitser-Winter syndrome. We performed functional characterization of the variant actin and show that it alters cell adhesion and polymer formation supporting its role in disease. We present the clinical findings in the patient, comparison of this patient to other patients with ACTB/ACTG1 mutations, and results from actin functional studies that demonstrate novel functional attributes of this mutant protein.

Keywords: ACTB; Baraitser-Winter syndrome; Dubowitz; actin.

Figure 1

Craniofacial findings of patient. A: Frontal and B: Lateral views at 2 months of age show metopic ridging, low anterior hair line, broad nasal root, short palpebral fissures, low placed ears with prominent lobes, micrognathia. C: Frontal and D: Lateral views at 7 years of age show left ptosis, broad nasal root, prominent tip and long columella, and low placed ears with prominent lobes.

Figure 2

Effect of the p.E117K mutation on polymerization of purified actin. Purified yeast G-actin was subject to centrifugation followed by filtration as described in the methods. Polymerization was initiated by the addition of MgCl₂ and KCl as described, and the increase in light scattering was followed over time as an indication of polymerization. Shown is the net change in light scattering for each sample. Key: ● 3.5 µM; ■ 4.6 µM; ▲ 5.8 µM; ◆ 6.9 µM. B. Electron micrographs of samples taken from the 3.5 µM reaction following attainment of steady state.

Figure 3

Dynamic light scattering analyses of filtered WT and mutant G-actin samples. A: One micromolar G-actin samples were placed in a dynamic light scattering apparatus and the change in scattering was followed over time at room temperature as described in the methods. For each panel, the left vertical axis depicts the signal intensity, and the right vertical axis depicts the average radius of bodies in the sample. B: Short filaments were present in the 6.9 mM mutant G-actin sample in G-buffer after 1 hr. No filaments were present in the WT sample.

Figure 4

Latrunculin A-dependent depolymerization of 3.5 mM WT and mutant F-actin samples. A: Time course of WT and mutant F-actin depolymerization. 20 mM latrunculin A was added to each sample, and the decrease in light scattering at room temperature was followed over time. B: Electron micrographs of the WT and mutant actin samples at the end of the depolymerization reaction showing residual filament bundles in the mutant, but not WT, sample.

Figure 5

Structural model of the pathogenic helix containing residue 117 and the residues with which the helix interacts. The structure was generated based on the Oda F-actin trimer model [Oda et al., 2009] using PyMol as described.