Linkage analysis was performed on F2 intercross populations derived from chromosome substitution strains (CSS) and C57BL/6J to identify additional genetic loci involved in hemostasis and thrombosis. The CCS lines were used previously to identify Hmtb1 (hemostasis and thrombosis 1) on chromosome 5, Hmbt2 (hemostasis and thrombosis 2) on chromosome 11, and Hmbt3 (hemostasis and thrombosis 3) on chromosome 17. The CCS strains used in this study carry A/J-derived chromosomes on a C57BL/6J genetic background. Parental strain A/J displays significantly longer clotting time and clot stability compared to parental strain C57BL/6. However, bleeding time does not differ between A/J and C57BL/6J. F2 animals were phenotyped by tail bleeding/rebleeding assay at 6-8 weeks of age and genotyped for polymorphic markers spaced 10 cM - 15 cM apart on each of the CSS chromosomes.
In the (C57BL/6J-Chr5<A/J x C57BL/6J) F2 progeny a novel locus named Hmtb4 (hemostasis and thrombosis 4) was identified at 59 cM in the (C57BL/6J-Chr5
A/J x C57BL/6J)F2 cross (n=79). Hmtb4 attains peak linkage with marker D5Mit338 (LOD=3.1) with homozygous C57BL/6J-derived alleles conferring 2.9-fold increase in clot stability time compared to homozygous A/J-derived alleles. This locus explains 16% of the phenotypic variance. The 95% confidence interval for Hmtb4 spans 55 cM to 66 cM. Suggestive linkage to bleeding time mapped to 70 cM with marker D5Mit320 (LOD=1.5) and was designated Hmtb5 (hemostasis and thrombosis 5). This locus explains 8% of thevariance. Homozygosity for C57BL/6J-derived alleles at Hmtb5 confers 2.5-fold longer bleeding time compared to homozygosity for A/J-derived alleles. The 95% confidence interval for Hmtb5 spans 45 cM to 75 cM.
On mouse Chromosome 11, suggestive linkage to clot stability time mapped to 75 cM with marker D11Mit336 (LOD=1.7) in an F2 cross between C57BL/6J and a CSS line carrying A/J-derived DNA from D11Mit70 (0 cM) - D11Mit20 (20 cM) and D11Mit4 (37 cM) - D11Mit336 (75 cM) on a C57BL/6J genetic background (n=76). This locus explains 10% of the variance in clot stability time and was named Hmtb6 (hemostasis and thrombosis 6). Heterozygosity for C57BL/6J and A/J alleles at Hmtb6 confer longer clot stability time compared to homozygosity for C57BL/6J alleles at Hmtb6. The 95% confidence interval spans 56 cM to 75 cM. A previously identified QTL named Mvwf (55.7 cM) overlaps with Hmtb6.
On mouse Chromosome 17, suggestive linkage to clot stability time mapped to two regions in a (C57BL/6J-Chr17
A/J x C57BL/6J)F2 cross - at 34.3 cM with marker D17Mit20 (Hmtb8, LOD=1.7) and at 45.3 cM marker D17Mit39 (Hmtb9, LOD=1.2). Hmtb8 explains 6% of the variance for clot stability time while Hmtb9 explains 4% of the variance. Homozygosity for C57BL/6J-derived alleles at Hmtb8 and Hmtb9 confers longer clot stability time. The Hmtb8 confidence interval spans 25 cM to 52 cM and the Hmtb9 confidence interval spans 0 cM to 56 cM. The Hmtb8 interval is syntenic to a 6.4 Mb region on the short arm of human Chromosome 18 where a QTL for human protein C resistance is located. Twenty-three potential candidate genes are located in the region.
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