Genetic variants in ADAMTS13 as well as smoking are major determinants of plasma ADAMTS13 levels

Abstract

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) in circulating blood, limiting the size of VWF multimers and regulating VWF activity. Abnormal regulation of VWF contributes to bleeding and to thrombotic disorders. ADAMTS13 levels in plasma are highly variable among healthy individuals, although the heritability and the genetic determinants of this variation are unclear. We performed genome-wide association studies of plasma ADAMTS13 concentrations in 3244 individuals from 2 independent cohorts of healthy individuals. The heritability of ADAMTS13 levels was between 59.1% (all individuals) and 83.5% (siblings only), whereas tobacco smoking was associated with a decrease in plasma ADAMTS13 levels. Meta-analysis identified common variants near the ADAMTS13 locus on chromosome 9q34.2 that were significantly associated with ADAMTS13 levels and collectively explained 20.0% of the variance. The top single nucleotide polymorphism (SNP), rs28673647, resides in an intron of ADAMTS13 (β, 6.7%; P = 1.3E-52). Conditional analysis revealed 3 additional independent signals represented by rs3739893 (β, -22.3%; P = 1.2E-30) and rs3124762 (β, 3.5%; P = 8.9E-9) close to ADAMTS13 and rs4075970 (β, 2.4%; P = 6.8E-9) on 21q22.3. Linkage analysis also identified the region around ADAMTS13 (9q34.2) as the top signal (LOD 3.5), consistent with our SNP association analyses. Two nonsynonymous ADAMTS13 variants in the top 2 independent linkage disequilibrium blocks (Q448E and A732V) were identified and characterized in vitro. This study uncovered specific common genetic polymorphisms that are key genetic determinants of the variation in plasma ADAMTS13 levels in healthy individuals.

Graphical abstract

Figure 1.

Meta-analysis of ADAMTS13 levels in GABC and TSS. (A) Genome-wide plot of –log10(P) for ∼5.82 million SNPs. The red line marks the 5.0E-8 threshold of genome-wide significance. (B) Quantile-quantile plot of observed vs expected –log10(P) for ADAMTS13 meta-analysis. The observed P < 5.0E-8 are shown in red. (C) Regional plot for the associated region near ADAMTS13 on Chr9. (D) Regional plot for ADAMTS13 and STKLD1 (C9orf96) genes on chromosome 9 (chr9) conditioned by the top SNP rs28673647 from meta-analysis. (E) Regional plot for ADAMTS13 conditioned by the top 2 independent SNPs, rs28673647 and rs3739893. (F) Regional plot for PRDM15 on chr21 when using the top 2 independent SNPs, rs28673647 and rs3739893, as covariates.

Figure 2.

Functional tests of ADAMTS13 variant expression secretion and activity. (A) Cell lines expressing wild-type, Q448E, and A732V ADAMTS13 were generated by using an FLP-recombinase system in 293 cells. Q448E is rs2301612 and A732V is rs41314453. (B-C) Concentrations of ADAMTS13 in (B) conditioned media and (C) cell lysate were measured by using AlphaLISA and adjusted by cell lysate glyceraldehyde-3-phosphate dehydrogenase levels to account for variations in cell number. Mean and standard deviations are plotted as well as individual values. (D) Recombinant enzyme-specific activity was measured with FRETS-VWF73 substrate. Differences in group levels were determined by the Mann Whitney U test. 1-8, thrombospondin type 1 repeats; CUB, C1r-C1s urinary epidermal growth factor bone morphogenetic protein domains; Cys-rich, cysteine-rich domain; P, propeptide; S, signal peptide.