Efficient estimation of SNP heritability using Gaussian predictive process in large scale cohort studies.

With the advent of high throughput genetic data, there have been attempts to estimate heritability from genome-wide SNP data on a cohort of distantly related individuals using linear mixed model (LMM). Fitting such an LMM in a large scale cohort study, however, is tremendously challenging due to its high dimensional linear algebraic operations. In this paper, we propose a new method named PredLMM approximating the aforementioned LMM motivated by the concepts of genetic coalescence and Gaussian predictive process. PredLMM has substantially better computational complexity than most of the existing LMM based methods and thus, provides a fast alternative for estimating heritability in large scale cohort studies. Theoretically, we show that under a model of genetic coalescence, the limiting form of our approximation is the celebrated predictive process approximation of large Gaussian process likelihoods that has well-established accuracy standards. We illustrate our approach with extensive simulation studies and use it to estimate the heritability of multiple quantitative traits from the UK Biobank cohort.

A Bayesian hierarchically structured prior for gene-based association testing with multiple traits in genome-wide association studies.

Although genome-wide association studies (GWAS) often collect data on multiple correlated traits for complex diseases, conventional gene-based analysis is usually univariate, and therefore, treating traits as uncorrelated. Multivariate analysis of multiple correlated traits can potentially increase the power to detect genes that affect some or all of these traits. In this study, we propose the multivariate hierarchically structured variable selection (HSVS-M) model, a flexible Bayesian model that tests the association of a gene with multiple correlated traits. With only summary statistics, HSVS-M can account for the correlations among genetic variants and among traits simultaneously and can also estimate the various directions and magnitudes of associations between a gene and multiple traits. Simulation studies show that HSVS-M substantially outperforms competing methods in various scenarios, particularly when variants in a gene are associated with a trait in similar directions and magnitudes. We applied HSVS-M to the summary statistics of a meta-analysis GWAS on four lipid traits from the Global Lipids Genetics Consortium and identified 15 genes that have also been confirmed as risk factors in previous studies.

A Bayesian hierarchically structured prior for rare-variant association testing.

Although genome-wide association studies have been widely used to identify associations between complex diseases and genetic variants, standard single-variant analyses often have limited power when applied to rare variants. To overcome this problem, set-based methods have been developed with the aim of boosting power by borrowing strength from multiple rare variants. We propose the adaptive hierarchically structured variable selection (HSVS-A) before test for association of rare variants in a set with continuous or dichotomous phenotypes and to estimate the effect of individual rare variants simultaneously. HSVS-A has the flexibility to integrate a pairwise weighting scheme, which adaptively induces desirable correlations among variants of similar significance such that we can borrow information from potentially causal and noncausal rare variants to boost power. Simulation studies show that for both continuous and dichotomous phenotypes, HSVS-A is powerful when there are multiple causal rare variants, either in the same or opposite direction of effect, with the presence of a large number of noncausal variants. We also apply HSVS-A to the Wellcome Trust Case Control Consortium Crohn's disease data for testing the association of Crohn's disease with rare variants in pathways. HSVS-A identifies two pathways harboring novel protective rare variants for Crohn's disease.

A large-scale exome array analysis of venous thromboembolism.

Although recent Genome-Wide Association Studies have identified novel associations for common variants, there has been no comprehensive exome-wide search for low-frequency variants that affect the risk of venous thromboembolism (VTE). We conducted a meta-analysis of 11 studies comprising 8,332 cases and 16,087 controls of European ancestry and 382 cases and 1,476 controls of African American ancestry genotyped with the Illumina HumanExome BeadChip. We used the seqMeta package in R to conduct single variant and gene-based rare variant tests. In the single variant analysis, we limited our analysis to the 64,794 variants with at least 40 minor alleles across studies (minor allele frequency [MAF] ~0.08%). We confirmed associations with previously identified VTE loci, including ABO, F5, F11, and FGA. After adjusting for multiple testing, we observed no novel significant findings in single variant or gene-based analysis. Given our sample size, we had greater than 80% power to detect minimum odds ratios greater than 1.5 and 1.8 for a single variant with MAF of 0.01 and 0.005, respectively. Larger studies and sequence data may be needed to identify novel low-frequency and rare variants associated with VTE risk.

Modeling the Dependence Structure in Genome Wide Association Studies of Binary Phenotypes in Family Data.

Genome-wide association studies (GWASs) are a popular tool for detecting association between genetic variants or single nucleotide polymorphisms (SNPs) and complex traits. Family data introduce complexity due to the non-independence of the family members. Methods for non-independent data are well established, but when the GWAS contains distinct family types, explicit modeling of between-family-type differences in the dependence structure comes at the cost of significantly increased computational burden. The situation is exacerbated with binary traits. In this paper, we perform several simulation studies to compare multiple candidate methods to perform single SNP association analysis with binary traits. We consider generalized estimating equations (GEE), generalized linear mixed models (GLMMs), or generalized least square (GLS) approaches. We study the influence of different working correlation structures for GEE on the GWAS findings and also the performance of different analysis method(s) to conduct a GWAS with binary trait data in families. We discuss the merits of each approach with attention to their applicability in a GWAS. We also compare the performances of the methods on the alcoholism data from the Minnesota Center for Twin and Family Research (MCTFR) study.

A robust and unified framework for estimating heritability in twin studies using generalized estimating equations.

The 'heritability' of a phenotype measures the proportion of trait variance due to genetic factors in a population. In the past 50 years, studies with monozygotic and dizygotic twins have estimated heritability for 17,804 traits;1 thus twin studies are popular for estimating heritability. Researchers are often interested in estimating heritability for non-normally distributed outcomes such as binary, counts, skewed or heavy-tailed continuous traits. In these settings, the traditional normal ACE model (NACE) and Falconer's method can produce poor coverage of the true heritability. Therefore, we propose a robust generalized estimating equations (GEE2) framework for estimating the heritability of non-normally distributed outcomes. The traditional NACE and Falconer's method are derived within this unified GEE2 framework, which additionally provides robust standard errors. Although the traditional Falconer's method cannot adjust for covariates, the corresponding 'GEE2-Falconer' can incorporate mean and variance-level covariate effects (e.g. let heritability vary by sex or age). Given a non-normally distributed outcome, the GEE2 models are shown to attain better coverage of the true heritability compared to traditional methods. Finally, a scenario is demonstrated where NACE produces biased estimates of heritability while Falconer remains unbiased. Therefore, we recommend GEE2-Falconer for estimating the heritability of non-normally distributed outcomes in twin studies.

A Bayesian hierarchical variable selection prior for pathway-based GWAS using summary statistics.

While genome-wide association studies (GWASs) have been widely used to uncover associations between diseases and genetic variants, standard SNP-level GWASs often lack the power to identify SNPs that individually have a moderate effect size but jointly contribute to the disease. To overcome this problem, pathway-based GWASs methods have been developed as an alternative strategy that complements SNP-level approaches. We propose a Bayesian method that uses the generalized fused hierarchical structured variable selection prior to identify pathways associated with the disease using SNP-level summary statistics. Our prior has the flexibility to take in pathway structural information so that it can model the gene-level correlation based on prior biological knowledge, an important feature that makes it appealing compared to existing pathway-based methods. Using simulations, we show that our method outperforms competing methods in various scenarios, particularly when we have pathway structural information that involves complex gene-gene interactions. We apply our method to the Wellcome Trust Case Control Consortium Crohn's disease GWAS data, demonstrating its practical application to real data.

A linear mixed model framework for gene-based gene-environment interaction tests in twin studies.

Interaction between genes and environments (G×E) can be well investigated in families due to the shared genes and environment among family members. However, the majority of the current tests of G×E interaction between a set of variants and an environment are only suitable for studies with unrelated subjects. In this paper, we extend several G×E interaction tests to a linear mixed model framework to study interaction between a set of correlated environments and a candidate gene in families. The correlated environments can either be modeled separately or jointly in one model. We demonstrate theoretically that the tests developed by modeling correlated environments separately are valid and present a computationally fast alternative to detect G×E interaction in families. For either strategy, we propose treating the genetic main effects as a random effect to reduce the number of main-effect parameters and thus improve the power to detect interactions. Additionally, we propose a generalization of a test of interaction that adaptively sums the interactions using a sequential algorithm. This generalized set of tests, referred to as the sequential algorithm for the sum of powered score (Seq-SPU) family of tests, can be expressed as a weighted version of the SPU. We find that the adaptive version of our test, Seq-aSPU, can outperform aSPU in cases where the interactions effects are in opposite directions. We applied these methods to the Minnesota Center for Twin and Family Research data set and found one significant gene in interaction with four psychosocial environmental factors affecting the alcohol consumption among the twins.

Genome-wide association study with additional genetic and post-transcriptional analyses reveals novel regulators of plasma factor XI levels.

Coagulation factor XI (FXI) has become increasingly interesting for its role in pathogenesis of thrombosis. While elevated plasma levels of FXI have been associated with venous thromboembolism and ischemic stroke, its deficiency is associated with mild bleeding. We aimed to determine novel genetic and post-transcriptional plasma FXI regulators.We performed a genome-wide association study (GWAS) for plasma FXI levels, using novel data imputed to the 1000 Genomes reference panel. Individual GWAS analyses, including a total of 16,169 European individuals from the ARIC, GHS, MARTHA and PROCARDIS studies, were meta-analysed and further replicated in 2,045 individuals from the F5L family, GAIT2 and MEGA studies. Additional association with activated partial thromboplastin time (aPTT) was tested for the top SNPs. In addition, a study on the effect of miRNA on FXI regulation was performed using in silico prediction tools and in vitro luciferase assays.Three loci showed robust, replicating association with circulating FXI levels: KNG1 (rs710446, P-value = 2.07 × 10-302), F11 (rs4253417, P-value = 2.86 × 10-193), and a novel association in GCKR (rs780094, P-value = 3.56 ×10-09), here for the first time implicated in FXI regulation. The two first SNPs (rs710446 and rs4253417) also associated with aPTT. Conditional and haplotype analyses demonstrated a complex association signal, with additional novel SNPs modulating plasma FXI levels in both the F11 and KNG1 loci. Finally, eight miRNAs were predicted to bind F11 mRNA. Over-expression of either miR-145 or miR-181 significantly reduced the luciferase activity in cells transfected with a plasmid containing FXI-3'UTR.These results should open the door to new therapeutic targets for thrombosis prevention.

A novel association test for multiple secondary phenotypes from a case-control GWAS.

In the past decade, many genome-wide association studies (GWASs) have been conducted to explore association of single nucleotide polymorphisms (SNPs) with complex diseases using a case-control design. These GWASs not only collect information on the disease status (primary phenotype, D) and the SNPs (genotypes, X), but also collect extensive data on several risk factors and traits. Recent literature and grant proposals point toward a trend in reusing existing large case-control data for exploring genetic associations of some additional traits (secondary phenotypes, Y) collected during the study. These secondary phenotypes may be correlated, and a proper analysis warrants a multivariate approach. Commonly used multivariate methods are not equipped to properly account for the non-random sampling scheme. Current ad hoc practices include analyses without any adjustment, and analyses with D adjusted as a covariate. Our theoretical and empirical studies suggest that the type I error for testing genetic association of secondary traits can be substantial when X as well as Y are associated with D, even when there is no association between X and Y in the underlying (target) population. Whether using D as a covariate helps maintain type I error depends heavily on the disease mechanism and the underlying causal structure (which is often unknown). To avoid grossly incorrect inference, we have proposed proportional odds model adjusted for propensity score (POM-PS). It uses a proportional odds logistic regression of X on Y and adjusts estimated conditional probability of being diseased as a covariate. We demonstrate the validity and advantage of POM-PS, and compare to some existing methods in extensive simulation experiments mimicking plausible scenarios of dependency among Y, X, and D. Finally, we use POM-PS to jointly analyze four adiposity traits using a type 2 diabetes (T2D) case-control sample from the population-based Metabolic Syndrome in Men (METSIM) study. Only POM-PS analysis of the T2D case-control sample seems to provide valid association signals.

A combination test for detection of gene-environment interaction in cohort studies.

Identifying gene-environment (G-E) interactions can contribute to a better understanding of disease etiology, which may help researchers develop disease prevention strategies and interventions. One big criticism of studying G-E interaction is the lack of power due to sample size. Studies often restrict the interaction search to the top few hundred hits from a genome-wide association study or focus on potential candidate genes. In this paper, we test interactions between a candidate gene and an environmental factor to improve power by analyzing multiple variants within a gene. We extend recently developed score statistic based genetic association testing approaches to the G-E interaction testing problem. We also propose tests for interaction using gene-based summary measures that pool variants together. Although it has recently been shown that these summary measures can be biased and may lead to inflated type I error, we show that under several realistic scenarios, we can still provide valid tests of interaction. These tests use significantly less degrees of freedom and thus can have much higher power to detect interaction. Additionally, we demonstrate that the iSeq-aSum-min test, which combines a gene-based summary measure test, iSeq-aSum-G, and an interaction-based summary measure test, iSeq-aSum-I, provides a powerful alternative to test G-E interaction. We demonstrate the performance of these approaches using simulation studies and illustrate their performance to study interaction between the SNPs in several candidate genes and family climate environment on alcohol consumption using the Minnesota Center for Twin and Family Research dataset.

A meta-analysis of 120 246 individuals identifies 18 new loci for fibrinogen concentration.

Genome-wide association studies have previously identified 23 genetic loci associated with circulating fibrinogen concentration. These studies used HapMap imputation and did not examine the X-chromosome. 1000 Genomes imputation provides better coverage of uncommon variants, and includes indels. We conducted a genome-wide association analysis of 34 studies imputed to the 1000 Genomes Project reference panel and including ∼120 000 participants of European ancestry (95 806 participants with data on the X-chromosome). Approximately 10.7 million single-nucleotide polymorphisms and 1.2 million indels were examined. We identified 41 genome-wide significant fibrinogen loci; of which, 18 were newly identified. There were no genome-wide significant signals on the X-chromosome. The lead variants of five significant loci were indels. We further identified six additional independent signals, including three rare variants, at two previously characterized loci: FGB and IRF1. Together the 41 loci explain 3% of the variance in plasma fibrinogen concentration.

Meta-analysis of 65,734 individuals identifies TSPAN15 and SLC44A2 as two susceptibility loci for venous thromboembolism.

Venous thromboembolism (VTE), the third leading cause of cardiovascular mortality, is a complex thrombotic disorder with environmental and genetic determinants. Although several genetic variants have been found associated with VTE, they explain a minor proportion of VTE risk in cases. We undertook a meta-analysis of genome-wide association studies (GWASs) to identify additional VTE susceptibility genes. Twelve GWASs totaling 7,507 VTE case subjects and 52,632 control subjects formed our discovery stage where 6,751,884 SNPs were tested for association with VTE. Nine loci reached the genome-wide significance level of 5 × 10(-8) including six already known to associate with VTE (ABO, F2, F5, F11, FGG, and PROCR) and three unsuspected loci. SNPs mapping to these latter were selected for replication in three independent case-control studies totaling 3,009 VTE-affected individuals and 2,586 control subjects. This strategy led to the identification and replication of two VTE-associated loci, TSPAN15 and SLC44A2, with lead risk alleles associated with odds ratio for disease of 1.31 (p = 1.67 × 10(-16)) and 1.21 (p = 2.75 × 10(-15)), respectively. The lead SNP at the TSPAN15 locus is the intronic rs78707713 and the lead SLC44A2 SNP is the non-synonymous rs2288904 previously shown to associate with transfusion-related acute lung injury. We further showed that these two variants did not associate with known hemostatic plasma markers. TSPAN15 and SLC44A2 do not belong to conventional pathways for thrombosis and have not been associated to other cardiovascular diseases nor related quantitative biomarkers. Our findings uncovered unexpected actors of VTE etiology and pave the way for novel mechanistic concepts of VTE pathophysiology.

Adaptive SNP-Set Association Testing in Generalized Linear Mixed Models with Application to Family Studies.

In genome-wide association studies (GWAS), it has been increasingly recognized that, as a complementary approach to standard single SNP analyses, it may be beneficial to analyze a group of functionally related SNPs together. Among the existent population-based SNP-set association tests, two adaptive tests, the aSPU test and the aSPUpath test, offer a powerful and general approach at the gene- and pathway-levels by data-adaptively combining the results across multiple SNPs (and genes) such that high statistical power can be maintained across a wide range of scenarios. We extend the aSPU and the aSPUpath test to familial data under the framework of the generalized linear mixed models (GLMMs), which can take account of both subject relatedness and possible population structure. As in population-based GWAS, the proposed aSPU and aSPUpath tests require only fitting a single and common GLMM (under the null hypothesis) for all the SNPs, thus are computationally efficient and feasible for large GWAS data. We illustrate our approaches in identifying genes and pathways associated with alcohol dependence in the Minnesota Twin Family Study. The aSPU test detected a gene associated with the trait, in contrast to none by the standard single SNP analysis. Our aSPU test also controlled Type I errors satisfactorily in a small simulation study. We provide R code to conduct the aSPU and aSPUpath tests for familial and other correlated data.

USAT: A Unified Score-Based Association Test for Multiple Phenotype-Genotype Analysis.

Genome-wide association studies (GWASs) for complex diseases often collect data on multiple correlated endo-phenotypes. Multivariate analysis of these correlated phenotypes can improve the power to detect genetic variants. Multivariate analysis of variance (MANOVA) can perform such association analysis at a GWAS level, but the behavior of MANOVA under different trait models has not been carefully investigated. In this paper, we show that MANOVA is generally very powerful for detecting association but there are situations, such as when a genetic variant is associated with all the traits, where MANOVA may not have any detection power. In these situations, marginal model based methods, however, perform much better than multivariate methods. We investigate the behavior of MANOVA, both theoretically and using simulations, and derive the conditions where MANOVA loses power. Based on our findings, we propose a unified score-based test statistic USAT that can perform better than MANOVA in such situations and nearly as well as MANOVA elsewhere. Our proposed test reports an approximate asymptotic P-value for association and is computationally very efficient to implement at a GWAS level. We have studied through extensive simulations the performance of USAT, MANOVA, and other existing approaches and demonstrated the advantage of using the USAT approach to detect association between a genetic variant and multivariate phenotypes. We applied USAT to data from three correlated traits collected on 5, 816 Caucasian individuals from the Atherosclerosis Risk in Communities (ARIC, The ARIC Investigators []) Study and detected some interesting associations.

A genetic association study of activated partial thromboplastin time in European Americans and African Americans: the ARIC Study.

Reduced activated partial thromboplastin time (aPTT) is a risk marker for incident and recurrent venous thromboembolism (VTE). Genetic factors influencing aPTT are not well understood, especially in populations of non-European ancestry. The present study aimed to identify aPTT-related gene variants in both European Americans (EAs) and African Americans (AAs). We conducted a genetic association study for aPTT in 9719 EAs and 2799 AAs from the Atherosclerosis Risk in Communities (ARIC) study. Using the Candidate Gene Association Resource (CARe) consortium candidate gene array, the analyses were based on ∼50 000 SNPs in ∼2000 candidate genes. In EAs, the analyses identified a new independent association for aPTT in F5 (rs2239852, P-value = 1.9 × 10(-8)), which clusters with a coding variant rs6030 (P-value = 7.8 × 10(-7)). The remaining significant signals were located on F5, HRG, KNG1, F11, F12 and ABO and have been previously reported in EA populations. In AAs, significant signals were identified in KNG1, HRG, F12, ABO and VWF, with the leading variants in KNG1, HRG and F12 being the same as in the EAs; the significant variant in VWF (rs2229446, P-value = 1.2 × 10(-6)) was specific to the AA sample (minor allele frequency = 19% in AAs and 0.2% in EAs) and has not been previously reported. This is the first study to report aPTT-related genetic variants in AAs. Our findings in AAs demonstrate transferability of previously reported associations with KNG1, HRG and F12 in EAs. We also identified new associations at F5 in EAs and VWF in AAs that have not been previously reported for aPTT.

Resampling-based tests for Lasso in genome-wide association studies.

BACKGROUND: Genome-wide association studies involve detecting association between millions of genetic variants and a trait, which typically use univariate regression to test association between each single variant and the phenotype. Alternatively, Lasso penalized regression allows one to jointly model the relationship between all genetic variants and the phenotype. However, it is unclear how to best conduct inference on the individual Lasso coefficients, especially in high-dimensional settings. METHODS: We consider six methods for testing the Lasso coefficients: two permutation (Lasso-Ayers, Lasso-PL) and one analytic approach (Lasso-AL) to select the penalty parameter for type-1-error control, residual bootstrap (Lasso-RB), modified residual bootstrap (Lasso-MRB), and a permutation test (Lasso-PT). Methods are compared via simulations and application to the Minnesota Center for Twins and Family Study. RESULTS: We show that for finite sample sizes with increasing number of null predictors, Lasso-RB, Lasso-MRB, and Lasso-PT fail to be viable methods of inference. However, Lasso-PL and Lasso-AL remain fast and powerful tools for conducting inference with the Lasso, even in high-dimensions. CONCLUSION: Our results suggest that the proposed permutation selection procedure (Lasso-PL) and the analytic selection method (Lasso-AL) are fast and powerful alternatives to the standard univariate analysis in genome-wide association studies.

New gene functions in megakaryopoiesis and platelet formation.

Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.

A Bayesian Partitioning Model for the Detection of Multilocus Effects in Case-Control Studies.

BACKGROUND: Genome-wide association studies (GWASs) have identified hundreds of genetic variants associated with complex diseases, but these variants appear to explain very little of the disease heritability. The typical single-locus association analysis in a GWAS fails to detect variants with small effect sizes and to capture higher-order interaction among these variants. Multilocus association analysis provides a powerful alternative by jointly modeling the variants within a gene or a pathway and by reducing the burden of multiple hypothesis testing in a GWAS. METHODS: Here, we propose a powerful and flexible dimension reduction approach to model multilocus association. We use a Bayesian partitioning model which clusters SNPs according to their direction of association, models higher-order interactions using a flexible scoring scheme and uses posterior marginal probabilities to detect association between the SNP set and the disease. RESULTS: We illustrate our method using extensive simulation studies and applying it to detect multilocus interaction in Atherosclerosis Risk in Communities (ARIC) GWAS with type 2 diabetes. CONCLUSION: We demonstrate that our approach has better power to detect multilocus interactions than several existing approaches. When applied to the ARIC study dataset with 9,328 individuals to study gene-based associations for type 2 diabetes, our method identified some novel variants not detected by conventional single-locus association analyses.

Genetic markers associated with plasma protein C level in African Americans: the atherosclerosis risk in communities (ARIC) study.

Protein C is an endogenous anticoagulant protein with anti-inflammatory properties. Single-nucleotide polymorphisms (SNPs) affect the levels of circulating protein C in European Americans. We performed a genome-wide association (GWA) scan of plasma protein C concentration with approximately 2.5 million SNPs in 2,701 African Americans in the Atherosclerosis Risk in Communities Study. Seventy-nine SNPs from the 20q11 and 2q14 regions reached the genome-wide significance threshold of 5 × 10(-8) . A missense variant rs867186 in the PROCR gene at 20q11 is known to affect protein C levels in individuals of European descent and showed the strongest signal (P = 9.84 × 10(-65) ) in African Americans. The minor allele of this SNP was associated with higher protein C levels (ß = 0.49 µg/ml; 10% variance explained). In the 2q14 region, the top SNPs were near or within the PROC gene: rs7580658 (ß = 0.15 µg/ml; 2% variance explained, P = 1.7 × 10(-12) ) and rs1799808 (ß = 0.15 µg/ml; 2% variance explained, P = 2.03 × 10(-12) ). These two SNPs were in strong linkage disequilibrium (LD) with another SNP rs1158867 that resides in a biochemically functional site and in weak to strong LD with the top PROC variants previously reported in individuals of European descent. In addition, two variants outside the PROC region were significantly and independently associated with protein C levels: rs4321325 in CYP27C1 and rs13419716 in MYO7B. In summary, this first GWA study for plasma protein C levels in African Americans confirms the associations of SNPs in the PROC and PROCR regions with circulating levels of protein C across ethnic populations and identifies new candidates for protein C regulation.