Elsevier

Gene

Volume 450, Issues 1–2, 15 January 2010, Pages 70-75
Gene

A genome-wide linkage scan in German shepherd dogs localizes canine platelet procoagulant deficiency (Scott syndrome) to canine chromosome 27

https://doi.org/10.1016/j.gene.2009.09.016Get rights and content

Abstract

Scott syndrome is a rare hereditary bleeding disorder associated with an inability of stimulated platelets to externalize the negatively charged phospholipid, phosphatidylserine (PS). Canine Scott syndrome (CSS) is the only naturally occurring animal model of this defect and therefore represents a unique tool to discover a disease gene capable of producing this platelet phenotype. We undertook platelet function studies and linkage analyses in a pedigree of CSS-affected German shepherd dogs. Based on residual serum prothrombin and flow cytometric assays, CSS segregates as an autosomal recessive trait. An initial genome scan, performed by genotyping 48 dogs for 280 microsatellite markers, suggested linkage with markers on chromosome 27. Genotypes ultimately obtained for a total of 56 dogs at 11 markers on chromosome 27 revealed significant LOD scores for 2 markers near the centromere, with multipoint linkage indicating a CSS trait locus spanning approximately 14 cm. These results provide the basis for fine mapping studies to narrow the disease interval and target the evaluation of putative disease genes.

Introduction

The domestic dog has become a valuable animal model for the study of genetic disease (Castelhano, 2009, Ostrander and Kruglyak, 2000). The large litter size and well-documented parentage of purebred dogs, combined with recent developments in canine genomic resources provide the tools to perform linkage and association studies in this species. Distinct breed populations facilitate the discovery of disease genes and mutations that cause or contribute to noted “breed predispositions” to various disorders. Moreover, dogs in developed countries receive sophisticated veterinary care that includes diagnostic techniques capable of specifically defining disease phenotypes.

Numerous naturally occurring bleeding disorders have been identified in dogs (Boudreaux, 2008, Brooks, 1999). Hemophilia and von Willebrand disease are the most common of these defects in dogs and people. Molecular genetic analyses indicate that these disorders are true homologs, caused by a variety of mutations within the corresponding genes of each species. Although hereditary platelet function defects are relatively uncommon in both species, discovery of their molecular basis has proven valuable in unraveling the complex pathways of normal platelet activation (Salles et al., 2008).

Platelet procoagulant activity (PCA) refers to the ability of stimulated platelets to promote fibrin clot formation. Although not fully defined, some mechanisms of PCA include engagement of platelet membrane receptors with coagulation factors and a distinct phospholipid and calcium-dependent property that facilitates assembly of catalytic factor complexes (Bevers et al., 1991, Heemskerk et al., 2000, Zwaal et al., 1992). In response to certain stimuli, the activated platelet membrane rapidly externalizes a negatively charged phospholipid, phosphatidylserine (PS), and sheds membrane microparticles that express PS. Membrane-bound PS provides a surface for activation of coagulation complexes that generate a localized burst of thrombin sufficient to cleave soluble fibrinogen and produce a nascent fibrin clot (Dachary-Prigent et al., 1995, Sims and Wiedmer, 2001, van der Planken, 2000).

A variety of physiologic and pharmacologic agents are known to evoke platelet PCA, however the signaling pathways and molecular processes involved in rapid platelet membrane PS externalization remain to be elucidated. The rare hereditary hemostatic defect Scott syndrome (OMIM: 262890) is characterized by an inability of stimulated platelets to externalize PS (Toti et al., 1996, Weiss, 2009, Zwaal et al., 2004). Scott platelets, therefore, represent a model system to explore the biologic basis of the platelet procoagulant response. Unfortunately, the paucity of human cases has hindered genetic analyses of the trait and a disease gene capable of producing the Scott platelet phenotype has yet to be identified. A canine counterpart of Scott syndrome (CSS) has been identified that displays all the characteristic features of the human disease, i.e. platelets with normal aggregation and secretion, yet specific impairment of stimulated prothrombinase activity, PS externalization, and microvesiculation (Brooks et al., 2002). In order to further develop this model and define its hereditary basis, we undertook pedigree studies in a kindred of German shepherd dogs affected with CSS. Functional studies of platelet PCA indicated that the mode of inheritance of CSS is compatible with an autosomal recessive trait. Mapping studies, using microsatellite markers, revealed linkage of the CSS phenotype to a locus on canine chromosome 27 (CFA 27), a region demonstrating shared synteny with the centromeric region of human chromosome 12.

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Study subjects

All dogs originated from a single large colony, established from 61 foundation breeders (20 males and 41 females). The youngest study dogs represented generations 7 and 8 from colony inception. The initial diagnosis of CSS in the colony was based on a combination of abnormalities in platelet PCA assays (e.g. high residual serum prothrombin, low prothrombinase activity) and flow cytometric findings of abnormal PS externalization and lack of microvesiculation (Brooks et al., 2002). Pedigree

Pedigree samples and platelet function studies

Samples for residual serum PT determinations were analyzed from 112 dogs during the study period. Fifteen dogs had high values (> 90%), indicating a failure of platelet PCA and expression of the CSS trait. Pedigree inspection of the affected dogs demonstrated segregation of CSS through 4 generations (Fig. 1). In this pedigree, 6 matings between sires (n = 3) and dams (n = 5) with normal residual serum PT produced at least 1 CSS-affected offspring. Combined, the 6 obligate carrier matings produced 40

Discussion

Initial candidate gene studies of Scott syndrome focused on a phospholipid scramblase (PLSCR1) with a putative role in platelet PCA (Janel et al., 1999). This protein was first isolated from red blood cells and characterized as having the ability to promote rapid transmembrane lipid movement in response to elevated intracellular calcium (Zhou et al., 1997). Subsequent studies have revealed that PLSCR1 acts as a cell-signaling molecule that mediates inflammatory cell response to cytokines via

Acknowledgements

This work was supported in part by Cornell University's Vertebrate Genomics Center and The Seeing Eye Foundation. Some genotype data for markers on CFA 27 were obtained through a submission to the NHLBI Mammalian Genotyping Service (HV4814). The authors thank Jennifer Cruikshank and Peter Schweitzer for their assistance in performing Genemapper analyses.

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