Whole-Genome Sequencing of a Canine Family Trio Reveals a FAM83G Variant Associated with Hereditary Footpad Hyperkeratosis

Over 250 Mendelian traits and disorders, caused by rare alleles have been mapped in the canine genome. Although each disease is rare in the dog as a species, they are collectively common and have major impact on canine health. With SNP-based genotyping arrays, genome-wide association studies (GWAS) have proven to be a powerful method to map the genomic region of interest when 10-20 cases and 10-20 controls are available. However, to identify the genetic variant in associated regions, fine-mapping and targeted re-sequencing is required. Here we present a new approach using whole-genome sequencing (WGS) of a family trio without prior GWAS. As a proof-of-concept, we chose an autosomal recessive disease known as hereditary footpad hyperkeratosis (HFH) in Kromfohrl änder dogs. To our knowledge, this is the first time this family trio WGS-approach, has successfully been used to identify a genetic variant that perfectly segregates with a canine disorder. The sequencing of three Kromfohrl änder dogs from a family trio (an affected offspring and both its healthy parents) resulted in an average genome coverage of 9.2X per individual. After applying stringent filtering criteria for candidate causative coding variants, 527 single nucleotide variants (SNVs) and 15 indels were found to be homozygous in the affected offspring and heterozygous in the parents. Using the computer software packages ANNOVAR and SIFT to functionally annotate coding sequence differences and to predict their functional effect, resulted in seven candidate variants located in six different genes. Of these, only FAM83G:c155G>C (p.R52P) was found to be concordant in eight additional cases and 16 healthy Kromfohrl änder dogs.

SrGAP2-Dependent Integration of Membrane Geometry and Slit-Robo-Repulsive Cues Regulates Fibroblast Contact Inhibition of Locomotion

Migrating fibroblasts undergo contact inhibition of locomotion (CIL), a process that was discovered five decades ago and still is not fully understood at the molecular level. We identify the Slit2-Robo4-srGAP2 signaling network as a key regulator of CIL in fibroblasts. CIL involves highly dynamic contact protrusions with a specialized actin cytoskeleton that stochastically explore cell-cell overlaps between colliding fibroblasts. A membrane curvature-sensing F-BAR domain pre-localizes srGAP2 to protruding edges and terminates their extension phase in response to cell collision. A FRET-based biosensor reveals that Rac1 activity is focused in a band at the tip of contact protrusions, in contrast to the broad activation gradient in contact-free protrusions. SrGAP2 specifically controls the duration of Rac1 activity in contact protrusions, but not in contact-free protrusions. We propose that srGAP2 integrates cell edge curvature and Slit-Robo-mediated repulsive cues to fine-tune Rac1 activation dynamics in contact protrusions to spatiotemporally coordinate CIL.

A growth factor-induced, spatially organizing cytoskeletal module enables rapid and persistent fibroblast migration

Directional migration requires robust front/back polarity. We find that fibroblasts treated with platelet-derived growth factor (PDGF) and prepolarized by plating on a fibronectin line substrate exhibit persistent migration for hours. This does not occur in the absence of PDGF or on uniformly coated fibronectin substrates. Persistent migration arises from establishment of two functional modules at cell front and back. At the front, formation of a zone containing podosome-like structures (PLS) dynamically correlates with low RhoA and myosin activity and absence of a contractile lamella. At the back, myosin contractility specifically controls tail retraction with minimal crosstalk to the front module. The PLS zone is maintained in a dynamic steady state that preserves size and position relative to the cell front, allowing for long-term coordination of front and back modules. We propose that front/back uncoupling achieved by the PLS zone is crucial for persistent migration in the absence of directional cues.

Analysis of Plasminogen Genetic Variants in Multiple Sclerosis Patients.

Multiple sclerosis (MS) is a prevalent neurological disease of complex etiology. Here, we describe the characterization of a multi-incident MS family that nominated a rare missense variant (p.G420D) in plasminogen (PLG) as a putative genetic risk factor for MS. Genotyping of PLG p.G420D (rs139071351) in 2160 MS patients, and 886 controls from Canada, identified 10 additional probands, two sporadic patients and one control with the variant. Segregation in families harboring the rs139071351 variant, identified p.G420D in 26 out of 30 family members diagnosed with MS, 14 unaffected parents, and 12 out of 30 family members not diagnosed with disease. Despite considerably reduced penetrance, linkage analysis supports cosegregation of PLG p.G420D and disease. Genotyping of PLG p.G420D in 14446 patients, and 8797 controls from Canada, France, Spain, Germany, Belgium, and Austria failed to identify significant association with disease (P = 0.117), despite an overall higher prevalence in patients (OR = 1.32; 95% CI = 0.93-1.87). To assess whether additional rare variants have an effect on MS risk, we sequenced PLG in 293 probands, and genotyped all rare variants in cases and controls. This analysis identified nine rare missense variants, and although three of them were exclusively observed in MS patients, segregation does not support pathogenicity. PLG is a plausible biological candidate for MS owing to its involvement in immune system response, blood-brain barrier permeability, and myelin degradation. Moreover, components of its activation cascade have been shown to present increased activity or expression in MS patients compared to controls; further studies are needed to clarify whether PLG is involved in MS susceptibility.