Karl C. Desch

Shigatoxin triggers thrombotic thrombocytopenic purpura in genetically susceptible ADAMTS13-deficient mice

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening illness caused by deficiency of the vWF-cleaving protease ADAMTS13. Here we show that ADAMTS13-deficient mice are viable and exhibit normal survival, although vWF-mediated platelet-endothelial interactions are significantly prolonged. Introduction of the genetic background CASA/Rk (a mouse strain with elevated plasma vWF) resulted in the appearance of spontaneous thrombocytopenia in a subset of ADAMTS13-deficient mice and significantly decreased survival. Challenge of these mice with shigatoxin (derived from bacterial pathogens associated with the related human disease hemolytic uremic syndrome) resulted in a striking syndrome closely resembling human TTP. Surprisingly, no correlation was observed between plasma vWF level and severity of TTP, implying the existence of TTP-modifying genes distinct from vWF. These data suggest that microbe-derived toxins (or possibly other sources of endothelial injury), together with additional genetic susceptibility factors, are required to trigger TTP in the setting of ADAMTS13 deficiency.

Detectable clonal mosaicism from birth to old age and its relationship to cancer

We detected clonal mosaicism for large chromosomal anomalies (duplications, deletions and uniparental disomy) using SNP microarray data from over 50,000 subjects recruited for genome-wide association studies. This detection method requires a relatively high frequency of cells with the same abnormal karyotype (>5–10%; presumably of clonal origin) in the presence of normal cells. The frequency of detectable clonal mosaicism in peripheral blood is low (<0.5%) from birth until 50 years of age, after which it rapidly rises to 2–3% in the elderly. Many of the mosaic anomalies are characteristic of those found in hematological cancers and identify common deleted regions with genes previously associated with these cancers. Although only 3% of subjects with detectable clonal mosaicism had any record of hematological cancer before DNA sampling, those without a previous diagnosis have an estimated tenfold higher risk of a subsequent hematological cancer (95% confidence interval = 6–18).

Linkage analysis identifies a locus for plasma von Willebrand factor undetected by genome-wide association

The plasma glycoprotein von Willebrand factor (VWF) exhibits fivefold antigen level variation across the normal human population determined by both genetic and environmental factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis, myocardial infarction, and stroke. To identify additional genetic determinants of VWF antigen levels and to minimize the impact of age and illness-related environmental factors, we performed genome-wide association analysis in two young and healthy cohorts (n = 1,152 and n = 2,310) and identified signals at ABO (P < 7.9E-139) and VWF (P < 5.5E-16), consistent with previous reports. Additionally, linkage analysis based on sibling structure within the cohorts, identified significant signals at chromosome 2q12–2p13 (LOD score 5.3) and at the ABO locus on chromosome 9q34 (LOD score 2.9) that explained 19.2% and 24.5% of the variance in VWF levels, respectively. Given its strong effect, the linkage region on chromosome 2 could harbor a potentially important determinant of bleeding and thrombosis risk. The absence of a chromosome 2 association signal in this or previous association studies suggests a causative gene harboring many genetic variants that are individually rare, but in aggregate common. These results raise the possibility that similar loci could explain a significant portion of the “missing heritability” for other complex genetic traits.

Is There a Shared Pathophysiology for Thrombotic Thrombocytopenic Purpura and Hemolytic-Uremic Syndrome?

Thrombotic microangiopathy is characterized by microvascular thrombosis coupled with thrombocytopenia, hemolytic anemia, and red blood cell fragmentation. Familiar to nephrologists and hematologists alike, classically associated with thrombotic microangiopathy are the hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP), the histories and presentations of which are closely intertwined. Not surprising, these two disorders are considered by many to be manifestations of the same disease process, whereas others consider HUS and TTP to be distinct clinical and pathologic entities. Herein are reviewed HUS and TTP along with recent progress shedding new light on possible shared pathophysiologic mechanisms for these two intriguing disorders.

Analysis of Informed Consent Document Utilization in a Minimal-Risk Genetic Study

BACKGROUND The signed informed consent document certifies that the process of informed consent has taken place and provides research participants with comprehensive information about their role in the study. Despite efforts to optimize the informed consent document, only limited data are available about the actual use of consent documents by participants in biomedical research. OBJECTIVE To examine the use of online consent documents in a minimal-risk genetic study. DESIGN Prospective sibling cohort enrolled as part of a genetic study of hematologic and common human traits. SETTING University of Michigan Campus, Ann Arbor, Michigan. PARTICIPANTS Volunteer sample of healthy persons with 1 or more eligible siblings aged 14 to 35 years. Enrollment was through targeted e-mail to student lists. A total of 1209 persons completed the study. MEASUREMENTS Time taken by participants to review a 2833-word online consent document before indicating consent and identification of a masked hyperlink near the end of the document. RESULTS The minimum predicted reading time was 566 seconds. The median time to consent was 53 seconds. A total of 23% of participants consented within 10 seconds, and 93% of participants consented in less than the minimum predicted reading time. A total of 2.5% of participants identified the masked hyperlink. LIMITATION The online consent process was not observed directly by study investigators, and some participants may have viewed the consent document more than once. CONCLUSION Few research participants thoroughly read the consent document before agreeing to participate in this genetic study. These data suggest that current informed consent documents, particularly for low-risk studies, may no longer serve the intended purpose of protecting human participants, and the role of these documents should be reassessed. PRIMARY FUNDING SOURCE National Institutes of Health.

Targeted Gene Sequencing Identifies Variants in the Protein C and Endothelial Protein C Receptor Genes in Patients With Unprovoked Venous Thromboembolism

Objective—The interaction of protein C (PC) with the endothelial PC receptor (EPCR) enhances activated PC generation. We performed targeted gene sequencing of the PC gene (PROC) and EPCR genes (PROCR) in patients with unprovoked venous thromboembolism (VTE) to determine whether mutations that impair PC–EPCR interactions are associated with an increased risk of VTE. Approach and Results—We sequenced exon 3 of PROC and exons 2 and 3 of PROCR (the exons that encode the protein–protein binding domains of PC and EPCR) in 653 patients with unprovoked VTE and in 627 healthy controls. Five single nucleotide variants, each in individual patients, were identified that result in abnormal PC (Arg9Cys, Val34Met, and Arg-1Cys) or abnormal EPCR proteins (Arg96Cys and Val170Leu). We did not detect any nonsynonymous coding variants in the controls. When the PC variants were expressed in human embryonic kidney 293 cells, all exhibited decreased synthesis, and 2 of the variants had reduced capacity for activated PC generation. When expressed on the surface of human embryonic kidney 293 cells, the EPCR variants showed reduced affinity for fluorescently labeled PC. In addition, the previously reported EPCR A3 haplotype, which promotes cellular shedding of EPCR, is over-represented in the patient group (P=0.001). Conclusions—This is the first targeted DNA sequencing analysis of PROC and PROCR in a large group of patients with unprovoked VTE. Our data suggest that mutations that impair PC–EPCR interactions may be associated with an increased risk of VTE.

Genome-wide studies of von Willebrand factor propeptide identify loci contributing to variation in propeptide levels and von Willebrand factor clearance.

Essentials Variants at ABO, von Willebrand Factor (VWF) and 2q12 contribute to the variation in plasma in VWF. We performed a genome‐wide association study of plasma VWF propeptide in 3,238 individuals. ABO, VWF and 2q12 loci had weak or no association or linkage with plasma VWFpp levels. VWF associated variants at ABO, VWF and 2q12 loci primarily affect VWF clearance rates.

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

Significance Here we report a method to rapidly examine the effect of nearly all possible single amino acid substitutions within a substrate fragment of the coagulation protein von Willebrand factor (VWF) on the efficiency of cleavage by its cognate protease, ADAMTS13. A substrate phage display library was generated containing ∼3.5 × 107 independent clones and uncleaved phages collected at multiple reaction time points after reaction with ADAMTS13. Analysis of these phages by high-throughput sequencing facilitated simultaneous calculations of kcat/KM values for multiple substitutions at each position of this protein fragment, providing a comprehensive picture of the substrate recognition landscape for the interaction between ADAMTS13 and VWF. This approach should be broadly applicable to many other protease/substrate pairs. Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (kcat/KM) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1′ substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.

Probing ADAMTS13 substrate specificity using phage display.

Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73.

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

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.