Whole genome variant association across 100 dogs identifies a frame shift mutation in DISHEVELLED 2 which contributes to Robinow-like syndrome in Bulldogs and related screw tail dog breeds

Abstract

Domestic dog breeds exhibit remarkable morphological variations that result from centuries of artificial selection and breeding. Identifying the genetic changes that contribute to these variations could provide critical insights into the molecular basis of tissue and organismal morphogenesis. Bulldogs, French Bulldogs and Boston Terriers share many morphological and disease-predisposition traits, including brachycephalic skull morphology, widely set eyes and short stature. Unlike other brachycephalic dogs, these breeds also exhibit vertebral malformations that result in a truncated, kinked tail (screw tail). Whole genome sequencing of 100 dogs from 21 breeds identified 12.4 million bi-allelic variants that met inclusion criteria. Whole Genome Association of these variants with the breed defining phenotype of screw tail was performed using 10 cases and 84 controls and identified a frameshift mutation in the WNT pathway gene DISHEVELLED 2 (DVL2) (Chr5: 32195043_32195044del, p = 4.37 X 10-37) as the most strongly associated variant in the canine genome. This DVL2 variant was fixed in Bulldogs and French Bulldogs and had a high allele frequency (0.94) in Boston Terriers. The DVL2 variant segregated with thoracic and caudal vertebral column malformations in a recessive manner with incomplete and variable penetrance for thoracic vertebral malformations between different breeds. Importantly, analogous frameshift mutations in the human DVL1 and DVL3 genes cause Robinow syndrome, a congenital disorder characterized by similar craniofacial, limb and vertebral malformations. Analysis of the canine DVL2 variant protein showed that its ability to undergo WNT-induced phosphorylation is reduced, suggesting that altered WNT signaling may contribute to the Robinow-like syndrome in the screwtail breeds.

Fig 1. Phenotype of screw tail breeds.

(A) Photographs of typical representatives of breeds: 1. Boxer (brachycephalic and normal but docked tail), 2. Bulldog (brachycephalic and screw tail), 3. French Bulldog (brachycephalic and screw tail), and 4. Boston Terrier (brachycephalic and screw tail) (photographs courtesy of Nestle Purina PetCare).(B) 3D computed tomography reconstructions of 1) Boxer thoracic vertebrae, ventrodorsal view, 2) French Bulldog thoraco-lumbar vertebrae, ventrodorsal 3) French Bulldog thoraco-lumbar vertebrae lateral view. Pronounced kyphosis of the French Bulldog vertebral column is associated with multiple vertebral abnormalities including shortened vertebrae, hemivertebrae and butterfly vertebrae. 4) Lateral vertebral column radiograph of a Bulldog demonstrating the breed typical "screw tail" associated with multiple caudal vertebral malformations and truncation. (C) Computed tomography images of canine skulls showing the variation of skull morphologies: 1) German Shepherd (dolichocephalic), 2) Boxer 3) Bulldog 4) French Bulldog 5) Boston Terrier 6) Pug. White scale bar is 2 cm for each orientation.

Fig 2. Across breed genome-wide association for screw tail.

(A) Hierarchical clustering of Identity by state distance of screw tail breeds. (B) QQ plot of–log 10 of p values before (Unadj) and after genomic (GC) and statistical correction (BONF). (C) and (D) Manhattan plots with–log 10 of p values on y axis and chromosome on the x axis with horizontal blue and red lines indicating p values of 0.05 and 0.01 after correction showing all tested genotypes (C) or 90% allelic difference (D).

Fig 3. Dishevelled 2 mutation, location within the protein and amino acid alignment with comparison to Robinow Syndrome.

(A) Electropherograms of the single base pair deletion within the cDNA of DVL2. (B) Schematic representation of Dishevelled protein domains. Approximate locations of known mutations are marked by vertical grey bars within their respective Dishevelled gene (h–human; c–canine). The number of reported clinical mutations in that region, in their respective gene, is in parentheses. (C) Human wild type DVL1, DVL2, and DVL3 C-terminus protein sequences (192, 221, and 216 amino acids respectively) aligned with canine wild type DVL2 (221 amino acids). Colors of amino acids indicate their respective physiochemical property: red–small, hydrophobic; blue–acidic; magenta–basic; green–hydroxyl, sulfhydryl, amine. The location of the Dishevelled frameshift mutations (specific amino acid) are boxed in black. The highly conserved C-terminus is boxed in blue. (D) Altered amino acid sequence of human Robinow syndrome mutations in DVL1 and DVL3 and the canine DVL2 mutation identified in this work. Truncated amino acids marked by black dashes. Highly conserved region is indicated in the wild type sequence by a blue box.

Fig 4. WNT-dependent phosphorylation of the DVL2 mutant protein is reduced.

(A) Lysates from NIH/3T3 stable cell lines expressing the dog, Myc-tagged wild-type (Wt) or mutant variant (Mut) DVL2, which is 23 aa shorter than wild-type exogenous DVL2, were analyzed by western blotting using an anti-c-Myc antibody. To assess the ability of the wild-type and mutant proteins to respond to WNT stimulation, cells were treated with WNT5A or WNT3A for 6 hours. Both treatments resulted in increased gel mobility shifts of the wild-type DVL2 protein, indicative of increased phosphorylation; this effect was reduced on the mutant DVL2 protein. (B) To confirm that the DVL2 gel mobility shifts observed in (A) were due to phosphorylation, cell lysates were subjected to mock treatment (30 min incubation at 37 C), or calf intestinal phosphatase (CIP) treatment (30 min incubation at 37 C in the presence of CIP) before separation by SDS-PAGE. The DVL2 gel mobility shifts above wild-type and mutant proteins were lost after CIP treatment, confirming that they are caused by phosphorylation. (C) To test whether the DVL2 gel mobility shifts observed in (A) were driven by casein kinase 1 (CK1), cells were treated with D4476, a CK1 inhibitor, for 1 hour prior to and concurrently during the Wnt stimulation for 6 hours. The DVL2 gel mobility shifts were lost after D4476 treatment, further indicating that they are caused by CK1-dependent phosphorylation. α-tubulin was used for loading controls. Cell lysates were normalized by BCA assays for total protein.