Mark M. Wurfel

Optimal Unified Approach for Rare-Variant Association Testing with Application to Small-Sample Case-Control Whole-Exome Sequencing Studies

We propose in this paper a unified approach for testing the association between rare variants and phenotypes in sequencing association studies. This approach maximizes power by adaptively using the data to optimally combine the burden test and the nonburden sequence kernel association test (SKAT). Burden tests are more powerful when most variants in a region are causal and the effects are in the same direction, whereas SKAT is more powerful when a large fraction of the variants in a region are noncausal or the effects of causal variants are in different directions. The proposed unified test maintains the power in both scenarios. We show that the unified test corresponds to the optimal test in an extended family of SKAT tests, which we refer to as SKAT-O. The second goal of this paper is to develop a small-sample adjustment procedure for the proposed methods for the correction of conservative type I error rates of SKAT family tests when the trait of interest is dichotomous and the sample size is small. Both small-sample-adjusted SKAT and the optimal unified test (SKAT-O) are computationally efficient and can easily be applied to genome-wide sequencing association studies. We evaluate the finite sample performance of the proposed methods using extensive simulation studies and illustrate their application using the acute-lung-injury exome-sequencing data of the National Heart, Lung, and Blood Institute Exome Sequencing Project.

Gene-Expression Variation Within and Among Human Populations

Understanding patterns of gene-expression variation within and among human populations will provide important insights into the molecular basis of phenotypic diversity and the interpretation of patterns of expression variation in disease. However, little is known about how gene-expression variation is apportioned within and among human populations. Here, we characterize patterns of natural gene-expression variation in 16 individuals of European and African ancestry. We find extensive variation in gene-expression levels and estimate that approximately 83% of genes are differentially expressed among individuals and that approximately 17% of genes are differentially expressed among populations. By decomposing total gene-expression variation into within- versus among-population components, we find that most expression variation is due to variation among individuals rather than among populations, which parallels observations of extant patterns of human genetic variation. Finally, we performed allele-specific quantitative polymerase chain reaction to demonstrate that cis-regulatory variation in the lymphocyte adaptor protein (SH2B adapter protein 3) contributes to differential expression between European and African samples. These results provide the first insight into how human population structure manifests itself in gene-expression levels and will help guide the search for regulatory quantitative trait loci.

Toll-like Receptor 1 Polymorphisms Affect Innate Immune Responses and Outcomes in Sepsis

RATIONALE Polymorphisms affecting Toll-like receptor (TLR)-mediated responses could predispose to excessive inflammation during an infection and contribute to an increased risk for poor outcomes in patients with sepsis. OBJECTIVES To identify hypermorphic polymorphisms causing elevated TLR-mediated innate immune cytokine and chemokine responses and to test whether these polymorphisms are associated with increased susceptibility to death, organ dysfunction, and infections in patients with sepsis. METHODS We screened single-nucleotide polymorphisms (SNPs) in 43 TLR-related genes to identify variants affecting TLR-mediated inflammatory responses in blood from healthy volunteers ex vivo. The SNP associated most strongly with hypermorphic responses was tested for associations with death, organ dysfunction, and type of infection in two studies: a nested case-control study in a cohort of intensive care unit patients with sepsis, and a case-control study using patients with sepsis, patients with sepsis-related acute lung injury, and healthy control subjects. MEASUREMENTS AND MAIN RESULTS The SNP demonstrating the most hypermorphic effect was the G allele of TLR1(-7202A/G) (rs5743551), which associated with elevated TLR1-mediated cytokine production (P < 2 x 10(-20)). TLR1(-7202G) marked a coding SNP that causes higher TLR1-induced NF-kappaB activation and higher cell surface TLR1 expression. In the cohort of patients with sepsis TLR1(-7202G) predicted worse organ dysfunction and death (odds ratio, 1.82; 95% confidence interval, 1.07-3.09). In the case-control study TLR1(-7202G) was associated with sepsis-related acute lung injury (odds ratio, 3.40; 95% confidence interval, 1.59-7.27). TLR1(-7202G) also associated with a higher prevalence of gram-positive cultures in both clinical studies. CONCLUSIONS Hypermorphic genetic variation in TLR1 is associated with increased susceptibility to organ dysfunction, death, and gram-positive infection in sepsis.

Duffy Antigen Facilitates Movement of Chemokine Across the Endothelium In Vitro and Promotes Neutrophil Transmigration In Vitro and In Vivo

The Duffy Ag expressed on RBCs, capillaries, and postcapillary venular endothelial cells binds selective CXC and CC chemokines with high affinity. Cells transfected with the Duffy Ag internalize but do not degrade chemokine ligand. It has been proposed that Duffy Ag transports chemokines across the endothelium. We hypothesized that Duffy Ag participates in the movement of chemokines across the endothelium and, by doing so, modifies neutrophil transmigration. We found that the Duffy Ag transfected into human endothelial cells facilitates movement of the radiolabeled CXC chemokine, growth related oncogene-α/CXC chemokine ligand 1 (GRO-α/CXCL1), across an endothelial monolayer. In addition, neutrophil migration toward GRO-α/CXCL1 and IL-8 (IL-8/CXCL8) was enhanced across an endothelial monolayer expressing the Duffy Ag. Furthermore, GRO-α/CXCL1 stimulation of endothelial cells expressing the Duffy Ag did not affect gene expression by oligonucleotide microarray analysis. These in vitro observations are supported by the finding that IL-8/CXCL8-driven neutrophil recruitment into the lungs was markedly attenuated in transgenic mice lacking the Duffy Ag. We conclude that Duffy Ag has a role in enhancing leukocyte recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium.

Identification of high and low responders to lipopolysaccharide in normal subjects: an unbiased approach to identify modulators of innate immunity.

LPS stimulates a vigorous inflammatory response from circulating leukocytes that varies greatly from individual to individual. The goal of this study was to use an unbiased approach to identify differences in gene expression that may account for the high degree of interindividual variability in inflammatory responses to LPS in the normal human population. We measured LPS-induced cytokine production ex vivo in whole blood from 102 healthy human subjects and identified individuals who consistently showed either very high or very low responses to LPS (denoted lpshigh and lpslow, respectively). Comparison of gene expression profiles between the lpshigh and lpslow individuals revealed 80 genes that were differentially expressed in the presence of LPS and 21 genes that were differentially expressed in the absence of LPS (p < 0.005, ANOVA). Expression of a subset of these genes was confirmed using real-time RT-PCR. Functional relevance for one gene confirmed to be expressed at a higher level in lpshigh, adipophilin, was inferred when reduction in adipophilin mRNA by small interfering RNA in the human monocyte-like cell line THP-1 resulted in a modest but significant reduction in LPS-induced MCP-1 mRNA expression. These data illustrate a novel approach to the identification of factors that determine interindividual variability in innate immune inflammatory responses and identify adipophilin as a novel potential regulator of LPS-induced MCP-1 production in human monocytes.

A Phase II Randomized Placebo-Controlled Trial of Omega-3 Fatty Acids for the Treatment of Acute Lung Injury

Objectives:Administration of eicosapentaenoic acid and docosahexanoic acid, omega-3 fatty acids in fish oil, has been associated with improved patient outcomes in acute lung injury when studied in a commercial enteral formula. However, fish oil has not been tested independently in acute lung injury. We therefore sought to determine whether enteral fish oil alone would reduce pulmonary and systemic inflammation in patients with acute lung injury. Design:Phase II randomized controlled trial. Setting:Five North American medical centers. Patients:Mechanically ventilated patients with acute lung injury ≥18 yrs of age. Interventions:Subjects were randomized to receive enteral fish oil (9.75 g eicosapentaenoic acid and 6.75 g docosahexanoic acid daily) or saline placebo for up to 14 days. Measurements and Main Results:Bronchoalveolar lavage fluid and blood were collected at baseline (day 0), day 4 ± 1, and day 8 ± 1. The primary end point was bronchoalveolar lavage fluid interleukin-8 levels. Forty-one participants received fish oil and 49 received placebo. Enteral fish oil administration was associated with increased serum eicosapentaenoic acid concentration (p < .0001). However, there was no significant difference in the change in bronchoalveolar lavage fluid interleukin-8 from baseline to day 4 (p = .37) or day 8 (p = .55) between treatment arms. There were no appreciable improvements in other bronchoalveolar lavage fluid or plasma biomarkers in the fish oil group compared with the control group. Similarly, organ failure score, ventilator-free days, intensive care unit-free days, and 60-day mortality did not differ between the groups. Conclusions:Fish oil did not reduce biomarkers of pulmonary or systemic inflammation in patients with acute lung injury, and the results do not support the conduct of a larger clinical trial in this population with this agent. This experimental approach is feasible for proof-of-concept studies evaluating new treatments for acute lung injury.

ANGPT2 Genetic Variant Is Associated with Trauma-associated Acute Lung Injury and Altered Plasma Angiopoietin-2 Isoform Ratio

RATIONALE Acute lung injury (ALI) acts as a complex genetic trait, yet its genetic risk factors remain incompletely understood. Large-scale genotyping has not previously been reported for ALI. OBJECTIVES To identify ALI risk variants after major trauma using a large-scale candidate gene approach. METHODS We performed a two-stage genetic association study. We derived findings in an African American cohort (n = 222) using a cardiopulmonary disease-centric 50K single nucleotide polymorphism (SNP) array. Genotype and haplotype distributions were compared between subjects with ALI and without ALI, with adjustment for clinical factors. Top performing SNPs (P < 10(-4)) were tested in a multicenter European American trauma-associated ALI case-control population (n = 600 ALI; n = 2,266 population-based control subjects) for replication. The ALI-associated genomic region was sequenced, analyzed for in silico prediction of function, and plasma was assayed by ELISA and immunoblot. MEASUREMENTS AND MAIN RESULTS Five SNPs demonstrated a significant association with ALI after adjustment for covariates in Stage I. Two SNPs in ANGPT2 (rs1868554 and rs2442598) replicated their significant association with ALI in Stage II. rs1868554 was robust to multiple comparison correction: odds ratio 1.22 (1.06-1.40), P = 0.0047. Resequencing identified predicted novel splice sites in linkage disequilibrium with rs1868554, and immunoblots showed higher proportion of variant angiopoietin-2 (ANG2) isoform associated with rs1868554T (0.81 vs. 0.48; P = 0.038). CONCLUSIONS An ANGPT2 region is associated with both ALI and variation in plasma angiopoietin-2 isoforms. Characterization of the variant isoform and its genetic regulation may yield important insights about ALI pathogenesis and susceptibility.

Proteomic and Computational Analysis of Bronchoalveolar Proteins during the Course of the Acute Respiratory Distress Syndrome

RATIONALE Acute lung injury causes complex changes in protein expression in the lungs. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of pathogenesis and new targets for treatment. OBJECTIVES The purpose of this study was to examine the changes in protein expression in the bronchoalveolar lavage fluid (BALF) of patients during the course of the acute respiratory distress syndrome (ARDS). METHODS Using two-dimensional difference gel electrophoresis (DIGE), the expression of proteins in the BALF from patients on Days 1 (n = 7), 3 (n = 8), and 7 (n = 5) of ARDS were compared with findings in normal volunteers (n = 9). The patterns of protein expression were analyzed using principal component analysis (PCA). Biological processes that were enriched in the BALF proteins of patients with ARDS were identified using Gene Ontology (GO) analysis. Protein networks that model the protein interactions in the BALF were generated using Ingenuity Pathway Analysis. MEASUREMENTS AND MAIN RESULTS An average of 991 protein spots were detected using DIGE. Of these, 80 protein spots, representing 37 unique proteins in all of the fluids, were identified using mass spectrometry. PCA confirmed important differences between the proteins in the ARDS and normal samples. GO analysis showed that these differences are due to the enrichment of proteins involved in inflammation, infection, and injury. The protein network analysis showed that the protein interactions in ARDS are complex and redundant, and revealed unexpected central components in the protein networks. CONCLUSIONS Proteomics and protein network analysis reveals the complex nature of lung protein interactions in ARDS. The results provide new insights about protein networks in injured lungs, and identify novel mediators that are likely to be involved in the pathogenesis and progression of acute lung injury.

Polymorphisms of the IL1-Receptor Antagonist Gene (IL1RN) Are Associated With Multiple Markers of Systemic Inflammation

Background—Circulating levels of acute phase reactant proteins such as plasma C-reactive protein (CRP) are likely influenced by multiple genes regulating the innate immune response. Methods and Results—We screened a set of 16 inflammation-related genes for association with CRP in a large population-based study of healthy young adults (n=1627). Results were validated in 2 independent studies (n=1208 and n=4310), including a pooled analysis of all 3 studies. In the pooled analysis, the minor allele of IL1RN 1018 (rs4251961) within the gene encoding interleukin (IL)-1 receptor antagonist (IL-1RA) was significantly associated with higher mean plasma log(CRP) level (P<1×10−4). The same IL1RN 1018 allele was associated with higher mean plasma log(IL-6) levels (P=0.004). In the pooled analysis, the minor allele of IL1RN 13888 (rs2232354) was associated with higher fibrinogen, (P=0.001). The IL1RN 1018 and 13888 variant alleles tag a clade of IL1RN haplotypes linked to allele 1 of an 86-bp VNTR polymorphism. We confirmed that the IL1RN 1018 variant (rs4251961) was associated with decreased cellular IL-1RA production ex vivo. Conclusions—Common functional polymorphisms of the IL1RN gene are associated with several markers of systemic inflammation.

Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells.

Lymphodepletion chemotherapy followed by infusion of CD19-targeted chimeric antigen receptor-modified T (CAR-T) cells can be complicated by neurologic adverse events (AE) in patients with refractory B-cell malignancies. In 133 adults treated with CD19 CAR-T cells, we found that acute lymphoblastic leukemia, high CD19+ cells in bone marrow, high CAR-T cell dose, cytokine release syndrome, and preexisting neurologic comorbidities were associated with increased risk of neurologic AEs. Patients with severe neurotoxicity demonstrated evidence of endothelial activation, including disseminated intravascular coagulation, capillary leak, and increased blood-brain barrier (BBB) permeability. The permeable BBB failed to protect the cerebrospinal fluid from high concentrations of systemic cytokines, including IFNγ, which induced brain vascular pericyte stress and their secretion of endothelium-activating cytokines. Endothelial activation and multifocal vascular disruption were found in the brain of a patient with fatal neurotoxicity. Biomarkers of endothelial activation were higher before treatment in patients who subsequently developed grade ≥4 neurotoxicity.Significance: We provide a detailed clinical, radiologic, and pathologic characterization of neurotoxicity after CD19 CAR-T cells, and identify risk factors for neurotoxicity. We show endothelial dysfunction and increased BBB permeability in neurotoxicity and find that patients with evidence of endothelial activation before lymphodepletion may be at increased risk of neurotoxicity. Cancer Discov; 7(12); 1404-19. ©2017 AACR.See related commentary by Mackall and Miklos, p. 1371This article is highlighted in the In This Issue feature, p. 1355.