Next generation sequencing to dissect the genetic architecture of KNG1 and F11 loci using factor XI levels as an intermediate phenotype of thrombosis

Abstract

Venous thromboembolism is a complex disease with a high heritability. There are significant associations among Factor XI (FXI) levels and SNPs in the KNG1 and F11 loci. Our aim was to identify the genetic variation of KNG1 and F11 that might account for the variability of FXI levels. The KNG1 and F11 loci were sequenced completely in 110 unrelated individuals from the GAIT-2 (Genetic Analysis of Idiopathic Thrombophilia 2) Project using Next Generation Sequencing on an Illumina MiSeq. The GAIT-2 Project is a study of 935 individuals in 35 extended Spanish families selected through a proband with idiopathic thrombophilia. Among the 110 individuals, a subset of 40 individuals was chosen as a discovery sample for identifying variants. A total of 762 genetic variants were detected. Several significant associations were established among common variants and low-frequency variants sets in KNG1 and F11 with FXI levels using the PLINK and SKAT packages. Among these associations, those of rs710446 and five low-frequency variant sets in KNG1 with FXI level variation were significant after multiple testing correction and permutation. Also, two putative pathogenic mutations related to high and low FXI levels were identified by data filtering and in silico predictions. This study of KNG1 and F11 loci should help to understand the connection between genotypic variation and variation in FXI levels. The functional genetic variants should be useful as markers of thromboembolic risk.

Fig 1. Plot of the association in the KNG1 locus with plasma FXI levels.

Markers represented common variants organized by genomic position. The diamond-shaped marker represented the top SNP (rs710446) which was still statistically significantly associated after adjustment for multiple testing. The left axis shows the statistical significance of the variant-plasma FXI level variation association expressed as -log₁₀ of the p-values and colour intensities show the level of linkage disequilibrium between all variants and the top SNP. The recombination rate in the HapMap II sample [33] for this region is measured on the right axis.

Fig 2. Plot of the association between variants within the F11 locus with plasma FXI levels.

Markers represented common variants organized by genomic position. Tthe diamond-shaped marker represented the top SNP (rs56810541) with the lowest variant-plasma FXI level association p-value. The left axis shows the statistical significance expressed as -log₁₀ of the p-values and colour intensities show the level of linkage disequilibrium between all variants and the top SNP. The recombination rate in the HapMap II sample [33] for this region is measured on the right axis.

Fig 3. Plot of the collapsing method association in the KNG1 locus with plasma FXI levels.

Markers represented the mean position of low-frequency variant sets. All of the markers located above the dotted horizontal line obtained a p-value <0.05 after the collapsing method association. Diamond-shaped markers represented the five significant low-frequency variant sets with controlling FWER = 0.05. The biggest diamond-shaped marker is the top low-frequency variant set.

Fig 4. Plot of the collapsing method association in the F11 locus.

Markers represented the mean position of low-frequency variant sets. The diamond-shaped marker represented the top low-frequency variant set and markers located above the dotted horizontal line are the most significant low-frequency variant sets (p-value <0.05). None of the low-frequency variant sets were significantly associated after controlling FWER = 0.05.