De novo mutations in the actin genes ACTB and ACTG1 cause Baraitser-Winter syndrome

Abstract

Brain malformations are individually rare but collectively common causes of developmental disabilities. Many forms of malformation occur sporadically and are associated with reduced reproductive fitness, pointing to a causative role for de novo mutations. Here, we report a study of Baraitser-Winter syndrome, a well-defined disorder characterized by distinct craniofacial features, ocular colobomata and neuronal migration defect. Using whole-exome sequencing of three proband-parent trios, we identified de novo missense changes in the cytoplasmic actin-encoding genes ACTB and ACTG1 in one and two probands, respectively. Sequencing of both genes in 15 additional affected individuals identified disease-causing mutations in all probands, including two recurrent de novo alterations (ACTB, encoding p.Arg196His, and ACTG1, encoding p.Ser155Phe). Our results confirm that trio-based exome sequencing is a powerful approach to discover genes causing sporadic developmental disorders, emphasize the overlapping roles of cytoplasmic actin proteins in development and suggest that Baraitser-Winter syndrome is the predominant phenotype associated with mutation of these two genes.

Figure 1

Craniofacial appearance and magnetic resonance imaging (MRI) for patient LP92-083. Photos of patient at 2 years (a) and 17 years (b) show prominent metopic ridge or trigonocephaly (mid-forehead in a), high-arched eyebrows, ptosis, flat philtrum and wide mouth, and a suggestion of low-set ears. Brain MRI from T1- (c) and T2-weighted (d) images show abnormally wide cerebral convolutions and thick cortex (double arrows) in all regions, with the malformation more severe in anterior than in posterior regions. We obtained written consent to publish photographs of the patient.

Figure 2

Western Blot analysis of β- and γ-actin isoforms in lymphoblastoid cells derived from an unaffected control (line 09.1359) and two patients carrying the ACTB p.Arg196His (LR04-173) and ACTG1 p.Ser155Phe (LP98-096) recurrent mutations using β-specific (Sigma clone AC-15) and γ-specific (Sigma clone 2–2.1.14.17) cytoplasmic actin antibodies. Protein extracts from cultured cells contained equal amounts of β- and γ-actin.

Figure 3

Cytoskeletal organization, morphology and F-actin stability in lymphoblastoid cells derived from an unaffected control (line 09.1359) and two patients (LR04-173 and LP98-096). (a) Cells double-labelled for F-actin (red) and α-Tubulin (green). Mutant cells contain increased accumulations of F-actin, particularly around the nucleus (yellow in merged images) and in numerous filopodia-like protrusions. The scale bar corresponds to 10 μm. (b) Elaborate protrusions significantly increase the perimeter of mutant cells by 25–30% compared to control. (c) Quantification of phalloidin fluorescence intensity shows increased F-actin content (1.8 fold to 3.4 fold higher) in mutant compared to unaffected cells. Inhibition of actin polymerization by latrunculin A reduces F-actin in all cells, but while 20% of F-actin remains in normal cells, 55% of F-actin remains in β-actin-p.Arg196His cells, indicating more stabile F-actin, and only 5% remains in γ-actin-p.Ser155Phe cells, consistent with less stabile actin filaments associated with this mutation. * Statistically significant difference compared to untreated control cells 09.1359, # statistically significant difference compared to untreated cells of the same line (p<0.05, two-tail t-test assuming unequal variance, n=28 cells in each group). Error bars indicate standard deviation.