Regulation of cell proliferation and motility is essential for normal development. The Rho family of GTPases plays a critical role in the control of cell polarity and migration by effecting the cytoskeleton, membrane trafficking, and cell adhesion. We investigated a recognized developmental disorder, Adams-Oliver syndrome (AOS), characterized by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). Through a genome-wide linkage analysis, we detected a locus for autosomal-dominant ACC-TTLD on 3q generating a maximum LOD score of 4.93 at marker rs1464311. Candidate-gene- and exome-based sequencing led to the identification of independent premature truncating mutations in the terminal exon of the Rho GTPase-activating protein 31 gene, ARHGAP31, which encodes a Cdc42/Rac1 regulatory protein. Mutant transcripts are stable and increase ARHGAP31 activity in vitro through a gain-of-function mechanism. Constitutively active ARHGAP31 mutations result in a loss of available active Cdc42 and consequently disrupt actin cytoskeletal structures. Arhgap31 expression in the mouse is substantially restricted to the terminal limb buds and craniofacial processes during early development; these locations closely mirror the sites of impaired organogenesis that characterize this syndrome. These data identify the requirement for regulated Cdc42 and/or Rac1 signaling processes during early human development.

Heterozygous germ-line mutations of BMPR2 are the major contributor to familial clustering of pulmonary arterial hypertension (PAH). To further explore the genetic basis of PAH in isolated cases we undertook a candidate gene analysis to identify potentially deleterious variation.Members of the BMP pathway, namely SMADs 1, 4, 5 and 9, were screened by direct sequencing for gene defects. Four variants were identified in SMADs 1, 4 and 9 amongst a cohort of 324 PAH cases, each not detected in a in a substantial control population. Of three amino-acid substitutions identified, two demonstrated reduced signaling activity in vitro. A putative splice site mutation in SMAD4 resulted in moderate transcript loss due to compromised splicing efficiency. These results emphasize the central role of BMPR2 mutation in the pathogenesis of 2 PAH (MIM# 178600) is a progressive vascular disorder often fatal as a result of right heart failure [Humbert et al., 2010]. Mutations of BMPR2 (MIM# 600799), encoding a bone morphogenetic type II receptor of the TGF-β family, are the major genetic determinant in familial PAH (FPAH). Idiopathic PAH (IPAH), defined as arising spontaneously in the absence of a recorded family history of disease, is indistinguishable from the familial form [Machado et al., 2009]. The detection of germ-line BMPR2 mutation in ~25% of IPAH cases, posing hereditary risk to offspring, has led to the re-classification of mutation carriers as heritable PAH (HPAH) [Deng et al., 2000;Lane et al., 2000;Machado et al., 2009]. Over 70% of HPAH mutations predict premature truncation likely leading to transcript loss via the nonsense-mediated decay (NMD) pathway [Machado et al., 2009]. Deleterious BMPR2 mutation in the IPAH population indicates a genetic basis to disease and suggests the existence of additional genetic and/or environmental factors in PAH pathogenesis yet to be fully annotated. Indeed, rare disease alleles Ethical approval for these studies was obtained by local ethical committees and all patients provided informed consent. A diagnosis of PAH was confirmed as described previously [Machado et al., 2009]. The European IPAH cohort comprised a total of 158 subjects ascertained by specialist UK and European centers and displayed a gender bias favoring females (1.9:1). The PAH panel with associated disease (APAH) (n=136) comprised cases with HIV infection (n=9), portal hypertension (n=11), congenital heart disease (n=15), thromboembolic disease (n=42) and connective tissue disease (n=59). Japanese IPAH subjects (n=30) were ascertained through a single specialist referral centre. All patients had been screened for BMPR2 mutation by direct sequencing and/or DHPLC employing primer sets previously described [Machado et al., 2001].Direct sequencing was performed on ABI377 fragment analyzer. DHPLC was performed using the Transgenomic WAVE Nucleic Acid Fragment Analysis system containing a DNASep column (Transgenomic, Crewe, UK) according to manufacturer's instruction. Due to the inherent limitations of these techni...

Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the gene SACS, encoding the 520 kDa protein sacsin. Although sacsin’s physiological role is largely unknown, its sequence domains suggest a molecular chaperone or protein quality control function. Consequences of its loss include neurofilament network abnormalities, specifically accumulation and bundling of perikaryal and dendritic neurofilaments. To investigate if loss of sacsin affects intermediate filaments more generally, the distribution of vimentin was analysed in ARSACS patient fibroblasts and in cells where sacsin expression was reduced. Abnormal perinuclear accumulation of vimentin filaments, which sometimes had a cage-like appearance, occurred in sacsin-deficient cells. Mitochondria and other organelles were displaced to the periphery of vimentin accumulations. Reorganization of the vimentin network occurs in vitro under stress conditions, including when misfolded proteins accumulate. In ARSACS patient fibroblasts HSP70, ubiquitin and the autophagy-lysosome pathway proteins Lamp2 and p62 relocalized to the area of the vimentin accumulation. There was no overall increase in ubiquitinated proteins, suggesting the ubiquitin–proteasome system was not impaired. There was evidence for alterations in the autophagy–lysosome pathway. Specifically, in ARSACS HDFs cellular levels of Lamp2 were elevated while levels of p62, which is degraded in autophagy, were decreased. Moreover, autophagic flux was increased in ARSACS HDFs under starvation conditions. These data show that loss of sacsin effects the organization of intermediate filaments in multiple cell types, which impacts the cellular distribution of other organelles and influences autophagic activity.