Recessive mutations in DOCK6, encoding the guanidine nucleotide exchange factor DOCK6, lead to abnormal actin cytoskeleton organization and Adams-Oliver syndrome

Abstract

Adams-Oliver syndrome (AOS) is defined by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). It is usually inherited as an autosomal-dominant trait, but autosomal-recessive inheritance has also been documented. In an individual with autosomal-recessive AOS, we combined autozygome analysis with exome sequencing to identify a homozygous truncating mutation in dedicator of cytokinesis 6 gene (DOCK6) which encodes an atypical guanidine exchange factor (GEF) known to activate two members of the Rho GTPase family: Cdc42 and Rac1. Another homozygous truncating mutation was identified upon targeted sequencing of DOCK6 in an unrelated individual with AOS. Consistent with the established role of Cdc42 and Rac1 in the organization of the actin cytoskeleton, we demonstrate a cellular phenotype typical of a defective actin cytoskeleton in patient cells. These findings, combined with a Dock6 expression profile that is consistent with an AOS phenotype as well as the very recent demonstration of dominant mutations of ARHGAP31 in AOS, establish Cdc42 and Rac1 as key molecules in the pathogenesis of AOS and suggest that other regulators of these Rho GTPase proteins might be good candidates in the quest to define the genetic spectrum of this genetically heterogeneous condition.

Figure 1

Pedigrees of the Two AOS Families Included in the Study and Clinical Photographs of Individuals 1 and 2 (A) Family 1 pedigree. (B) Clinical photographs of individual 1. (C) Family 2 pedigree. (D) Clinical photographs of individual 2. Note the typical ACC and TTLD in both individuals.

Figure 2

Identification of Two DOCK6 Mutations in AOS Upper panel: Diagram of DOCK6 (triangles indicate the sites of the mutations). Middle panel: Diagram of DOCK6 (note that both truncating mutations are upstream of DHR-1 and DHR-2 domains). Bottom panel: Sequence chromatogram of the two mutations with the control tracing for comparison (sequence differences are underlined in red).

Figure 3

WISH of Dock6 during Mouse Embryonic Development (A) E9.5 mouse embryo showing expression in the growing edge of the limb bud. (B) E10.5 mouse embryo showing expression in the growing edge of limb buds and heart. (C) E11.5 mouse embryo showing expression in the apical ectodermal ridge of all four limbs (arrowheads and inset) as well as the first-pharyngeal-arch-derived facial mesenchyme (triangles). (D and E) E12.5 and 13.5 embryos showing the diffuse expression of Dock6 mRNA in the fore- and hind-limbs. (F and G) Close-up view showing the expression in the limbs of an E13.5 embryo. (H and I) Sense control for comparison with (F) and (G).

Figure 4

Patient and Control Fibroblast Cells Were Visualized by Fluorescent Microscopy for Phalloidin Staining of F-actin- and DAPI-Stained Nuclei F-actin staining is in red, and DAPI staining is in blue. (A and B) Control fibroblasts (A) show the typical spindle appearance, whereas patient fibroblasts (B) show relatively high frequency of rounded (solid arrowheads) and elongated (empty arrows heads) cells. (C) A close-up view of one patient fibroblast with a rounded phenotype clearly indicates blebbing (arrow heads). (D and E) The normal appearance of lamellopodia in control fibroblasts (D) is in sharp contrast to the “blunt” edges due to severe deficiency of lamellopodia formation in patient fibroblasts (E).