IGSF4: A Novel Candidate Gene for Venous Thrombosis

Through a combination of genotyping and resequencing we have identified 3 SNPs (rs17564430, rs3802858 and rs6589488) in a venous thrombosis (VT) susceptibility gene encoding Immunoglobulin Superfamily 4 (IGSF4), that interacts with protein C (PC) deficiency (3363InsC) in a large thrombophilic kindred (n=516). The IGSF4 gene is located on chromosome 11q23. To verify the association with VT and to estimate the joint effect with PC deficiency, we genotyped the 3 SNPs in the 450 available kindred members, of whom 40 (32 PC deficient) had experienced VT. For each SNP we estimated the odds ratio (OR) and 95% confidence interval (CI95) for VT, adjusting for age and sex. Because earlier linkage analyses and SNP genotyping had suggested a dominant effect of the gene, we calculated the OR for carriers compared to non-carriers. Two of 3 SNPs in IGSF4 remained associated with VT. Carriers of the rs6589488 minor allele had a more than 10-fold increased risk of VT (OR 10.2, CI95 7.8–13.4), compared to those homozygous for the major allele. The effect was more pronounced in PC deficient family members (OR 17.0, CI95 13.5–21.4). PC deficient carriers of the rs3802858 minor allele had about 5-fold increased risk of VT (OR 4.7 CI95 2.2–10.0). We designed a replication study of these findings in the Leiden Thrombophilia Study (LETS) comprised of 471 VT patients and 471 control subjects. The 2 original and 11 additional SNPs were selected based on linkage disequilibrium (LD) in the locus surrounding the 2 SNPs associated with VT in the family. None of the SNPs was associated with VT across the whole LETS population. However, we observed a potential synergistic effect between some of these SNPs and the factor V Leiden (FVL) mutation, suggesting interaction with the PC system. For example, rs658948 major allele homozygotes who carry FVL have an 11–fold increased risk of VT (OR 11.2, CI95 5.1–24.3) compared to minor allele carriers who do not carry FVL. This is a 3-fold increase (CI95 0.9–9.9) over the VT risk associated with FVL alone. In LETS we took rs6589488 minor allele carriers as reference since the major allele was the higher risk allele. This is opposite of what we observed in the family. Apart from refuting associations, reverse associations in different populations might be true but caused by variation in LD patterns between the rs6589488 and other or true causal variants (Lin et al, Am. J. Hum. Genet. 80:531–8, 2007). Potential interactions between IGSF4 and the PC system in VT seem likely based on their functions. Activated protein C (APC) has important anticoagulant, anti-inflammatory and cytoprotective effects. The APC cytoprotective effect is characterized by reduction in endothelial permeability through stabilization of the endothelial cytoskeleton. This effect is mediated by protease activated receptor-1 and the sphingosine 1 phosphate receptor, S1P1, activation by APC bound to the endothelial cell PC receptor (APC-EPCR). The endothelial barrier protective effect likely improves endothelial thromboresistance. For example, exposure of endothelial cells to thrombin leads to transcellular actin stress fiber formation and cellular contraction with the formation of paracellular gaps. These gaps potentially expose plasma to subcellular tissue factor. This effect can be reversed by APC-EPCR which attenuates stress fiber formation and augments the cortical actin ring. (Finigan et al. J Biol Chem. 280:17286–93, 2005; Bae et al. Thromb Haemost. 100:101–9, 2008). IGSF4 is an immunoglobulin superfamily cell adhesion molecule with a well described role in basolateral cell-cell adhesion of pulmonary epithelial cells. We have demonstrated that IGSF4 is expressed in endothelial cells and has somewhat diminished expression in endothelial cells cultured from PC deficient kindred members compared to controls. It is likely that IGSF4 supports basolateral endothelial cell-cell adhesion. We hypothesize that a variant form or decreased expression of IGSF4, interacting with PC deficiency, impairs APC mediated endothelial barrier protection in response to thrombogenic stimuli and thus increases the risk of VT in this thrombophilic kindred.